Nanovesículas de Tamaño Controlado para Aplicaciones Biotecnológicas Y Bioanalíticas

UNIVERSIDAD DE OVIEDO
Departamento de Química Física y Analítica
Programa de Doctorado de Análisis Químico, Bioquímico y Estructural y

Modelización Computacional
NANOVESÍCULAS DE TAMAÑO CONTROLADO PARA

APLICACIONES BIOTECNOLÓGICAS Y BIOANALÍTICAS
TESIS DOCTORAL
PABLO GARCÍA MANRIQUE
Oviedo, 2019
UNIVERSITY OF OVIEDO
Departament of Physical and Analytical Chemistry
Ph.D. program: Chemical, Biochemical and Structural Analysis and
Computational Modelling
SIZE CONTROLLED NANOVESICLES FOR

BIOTECHNOLOGICAL AND BIOANALYTICAL
APPLICATIONS
Ph.D. THESIS
PABLO GARCÍA MANRIQUE
Oviedo, 2019
RESUMEN DEL CONTENIDO DE TESIS DOCTORAL
1.- Título de la Tesis

Español/Otro Idioma: “Nanovesículas de Inglés: “Size controlled nanovesicles for
tamaño controlado para aplicaciones biotechnological and bioanalytical
biotecnológicas y bioanalíticas” applications”
2.- Autor
Nombre: Pablo García Manrique DNI/Pasaporte/NIE: -Q
Programa de Doctorado: Análisis químico, bioquímico y estructural, y modelización

computacional
Órgano responsable: Departamento de Química Física y Analítica, Facultad de Química
RESUMEN (en español)

FOR-MAT-VOA-010 (Reg.2018)
Las nanovesículas, un tipo particular de coloides orgánicos, está generando un interés

creciente en el ámbito de la biotecnología, y particularmente en el campo del bioanálisis. La
versatilidad de sus características morfológicas y funcionales hace de ellas un biomaterial
muy interesante para vehículo de liberación de fármacos, marca de inmunoensayos, o como
sistema biomiético de membranas o vesículas extracelulares. Sin embargo, estas nuevas
aplicaciones imponen una serie de requisitos sobre los métodos de preparación que suponen
un reto tecnológico y han generado un campo de investigación muy activo. El control sobre el
tamaño de partícula y su monodispersidad son características esenciales que se deben
controlar mediante la mejora de los métodos actuales y la propuesta de nuevas tecnologías
diseñadas para tal fin. En este escenario científico-tecnológico se enmarca la presente Tesis
Doctoral. El objetivo general es desarrollar métodos eficaces de síntesis con tamaño
controlado. Con las vesículas obtenidas se han mimetizado exosomas (vesículas
extracelulares) con el fin de investigar su uso como standard analítico en inmunoensayos.
Estas vesículas están despertando un gran interés en el campo de la salud como nuevos
biomarcadores, y son necesarias nuevas aproximaciones para poner a punto métodos de
análisis.
Tras una introducción sobre las generalidades de las nanovesículas, el quinto capítulo de
la tesis presenta una profunda revisión del estado del arte del concepto de exosoma artificial,
así como la revisión crítica de las diferentes aproximaciones metodológicas para su
producción y una nueva clasificación de estos nuevos biomateriales en función de los
mismos. Además, se han expuesto los usos biotecnológicos de dichas partículas, como son
la distribución y liberación controlada de fármacos, su papel en diagnóstico, y su posible
empleo en el desarrollo y estudio de métodos bioanalíticos. Para ello, es necesario procesos
adaptados para la preparación de exosomas puramente artificiales mediante técnicas nano-
biotecnológicas. Esta temática es precisamente sobre la que versa el trabajo experimental
expuesto en los siguientes capítulos.
El sexto capítulo presenta el desarrollo de un modelo matemático multivariante de Diseño

de Experimentos y su aplicación al estudio de un método clásico de preparación de
nanovesículas, como es la inyección de etanol. Con dicho estudio se pretende controlar los
parámetros para la obtención de partículas con propiedades morfológicas deseadas y
controladas. Para llevar a cabo el trabajo se han escogido dos formulaciones clásicas: una
de liposoma y otra de niosoma. Además, se ha realizado un estudio de parámetros
adicionales como la estabilidad coloidal y la eficacia de encapsulación de compuestos de
diferente naturaleza química.
En el séptimo capítulo se ha abordado la producción de niosomas de tamaño controlado y

monodispersos mediante un dispositivo de microfluídica a temperatura controlada. El sistema
ha sido diseñado para su acoplamiento sobre un microscopio invertido con el fin de observar
los fenómenos de interfase acaecidos en su interior (como por ejemplo fenómenos de
difusión molecular en flujo laminar). Además, se ha estudiado el efecto de parámetros
metodológicos, poniendo especial énfasis en la temperatura. También se han tenido en
cuenta otros parámetros como la longitud de la cadena hidrocarbonada del tensioactivo. Este
sistema miniaturizado es idóneo para etapas de optimización de formulaciones y para la
evaluación de la eficacia de encapsulación de las mismas, especialmente en los casos en
que se disponga de poco volumen de reactivos.
En el octavo capítulo se estudia la mejora de la encapsulación de compuestos hidrófilos

mediante el empleo de disoluciones acuosas con poli-oles (glicerol y polietilenglicol), y el
efecto de éstas sobre el tamaño de vesícula. Por otra parte, se evalúa el impacto sobre
dichos parámetros que puede tener el peso molecular de los compuestos encapsulados.
Este estudio se ha llevado a cabo con el empleo de una formulación basada en una mezcla
de surfactantes no iónicos en presencia y ausencia de dodecanol como estabilizante de
membrana.
Finalmente, en el noveno capítulo se parte de los modelos obtenidos para niosomas en

etapas anteriores, y han sido empleados para obtener partículas de tamaño y
monodispersidad similar a exosomas naturales. Además, mediante la funcionalización
superficial de los mismos con estreptavidina y posteriormente con péptidos biotinilados
recombinantes de tetraspaninas (principal marcador molecular de exosomas), se han
desarrollado exosomas artificiales. Dichos exosomas han sido testados mediante
immunoensayos de tipo ELISA como un posible nuevo estándar analítico para el desarrollo
y/o mejora de métodos bioanalítcos basados en el reconocimiento molecular para la
determinación de vesículas extracelulares, como son los exosomas.
RESUMEN (en Inglés)
Nanovesicles, a particular type of organic colloids, is generating a growing interest in the field
of biotechnology, and particularly in the field of bioanalysis. The versatility of its morphological
and functional characteristics makes them a very promising biomaterial. Some of these
aplications are as drug delivery vehicle, multilabel system for immnunoassays or biomietic
membrane or extracellular vesicles. However, these new applications impose a series of
requirements on preparation methods that carry along a technological challenge and have
triggered a very active field of research. Control over the particle size and its monodispersity are
essential characteristics that must be controlled by improving current methods and proposing
new technologies designed for such purpose. Withing this framework, the general objective of
this Doctoral Thesis is the development of efficient methods for controlled size syntesis. The
nanovesicles obtained have been used to mimic exosomes (extracelular vesicles) with the aim
of investigating their use as analytical standard at immunoassays. These nanovesicles are
attracting high interest as novel biomarkers at the biomedical field, and novel approaches are
necessary in order to develop analytical methods.
After an introduction about the general concepts of nanovesicles, the fifth chapter presents
a deep review of the state of the art of the artificial exosome concept, as well as the critical
evaluation of the different approaches for its production. A systematic classification of these
new biomaterials was carried out, with the aim of providing some reference guides for
standardization the field. In addition, the biotechnological application of these particles has been
discussed. This includes the distribution and controlled release of drugs, their role in diagnosis,
and their possible use in the development of bioanalytical methods. For that purpose, novel
methods are required for the preparation of fully artificial exosomes by using nano-
biotechnological techniques. This topic is the core of the experimental work presented in the
following chapters.
The sixth chapter includes de development of a mathematical model of Design of

Experiments (DoE) for nanovesicles preparation by the ethanol injection method (EIM.) The
objective of the study is to control the parameters for obtaining particles with controlled
morphological properties. For the DoE set up, two classical formulations have been chosen: one
representative of liposomes and the other of niosomes,. In addition, a study of additional
parameters such as colloidal stability and the encapsulation efficiency of compounds with
different chemical nature has been carried out.
In the seventh chapter, the production of controllable-size and monodispersed niosomes has
been approached by means of a microfluidic device at controlled temperature. This system was
designed in a way to be coupled to an inverted microscope. This allowed to visualize the
interphase phenomena occurring inside (such as molecular diffusion in laminar flow). In
addition, the effect of methodological parameters has been studied, with special emphasis on
temperature. Other parameters such as the length of the surfactant alkyl chain have also been
taken into account. This miniaturized system is ideal for formulations optimization and for the
evaluation of their encapsulation efficiencies. It is especially useful when small volume of
reagents is available.
In the eighth chapter the enhacement of hydrophilic compounds encapsulation is studied

through the use of aqueous solutions with polyols (glycerol and polyethylene glycol), and their
effect of these over vesicle size. On the other hand, the effect of cargo molecular weight over
these parameters has been evaluated. This study has been carried out with the use of a
formulation based on a mixture of non-ionic surfactants in the presence and absence of
dodecanol as a membrane stabilizer.
Finally, in the nineth chapter the models obtained for niosomes in previous stages have been
used to obtain particles with an average size and monodispersity similar to that of natural
exosomes. In addition, by surface functionalization first with streptavidin and in a second step
with recombinant biotinylated tetraspanin peptides (classical molecular biomarkers of
exosomes), fully artificial exosomes have been developed. These particles have been tested by
ELISA immunoassays as a potential new analytical standard for the development and/or
enhacement of bioanalytical methods based on molecular recognition for the determination of
extracellular vesicles, such as exosomes.
SR. PRESIDENTE DE LA COMISIÓN ACADÉMICA DEL PROGRAMA DE DOCTORADO EN

ANÁLISIS QUÍMICO, BIOQUÍMICO Y ESTRUCTURAL, Y MODELIZACIÓN
COMPUTACIONAL
Índice de contenidos
Índice de Figuras ................................................................................................................. 11
Índice de Tablas ................................................................................................................... 19
Lista de Abreviaturas ......................................................................................................... 21
1. Introducción ....................................................................................................................... 27
2. Objetivos/Objetives ............................................................................................................ 37
3. Consideraciones básicas ..................................................................................................... 43
3.1. Sistemas vesiculares: generalidades .......................................................................... 45
3.1.1. Moléculas anfifílicas.............................................................................................. 45
3.1.2 Clasificación de los sistemas vesiculares ............................................................ 52
3.1.3. Tipos morfológicos de vesículas ......................................................................... 59
3.2. Métodos de preparación de vesículas ....................................................................... 60
3.2.1. Métodos que implican cambio de polaridad en el medio ............................... 61
3.2.2. Métodos directos ................................................................................................... 62
3.2.3. Métodos de nueva generación............................................................................. 63
3.3. Procesos asociados al uno biotecnológicoy bioanalítico de los sistemas

vesiculares .................................................................................................................................. 66
3.3.1. Encapsulación ........................................................................................................ 67
3.3.2. Bioconjugación....................................................................................................... 68
3.3.3. Purificación ............................................................................................................ 70
3.4. Exosomas: ejemplo de sistema vesicular natural con interés biotecnológico ...... 73
3.4.1. Exosomas como modelo de inspiración de vesículas mejoradas ................... 75
3.4.2. Retos tecnológicos asociados a los exosomas y soluciones biomiméticas..... 75
3.5. Referencias bibliográficas............................................................................................ 77
4. Experimental ........................................................................................................................ 93
4.1. Dynamic Light Scattering (DLS): tamaño y potencial ζ .......................................... 95
4.2. Microscopía Electrónica de Trasmisión (TEM): morfología................................... 97
5
4.3. Turbiscan (Formulaction): estabilidad coloidal ....................................................... 98
4.4. HPLC fase reversa: eficacia de encapsulación ......................................................... 99
4.5. Lector de placas .......................................................................................................... 100
4.6. Analizador para la medida de Western Blot .......................................................... 101
4.7. Equipo de Differential Scanning Calorimetry ........................................................ 101
5. Therapeutic biomaterials based on extracellular vesicles: classification of bio-

engineering and mimetic preparation routes .......................................................................... 103
5.1. Extracellular vesicles in nanomedicine: possibilities and limitations ................. 105
5.2. Bio-engineered and mimetic EVs for nanomedicine: classification of artificial

EVs ............................................................................................................................................. 106
5.3. Impact of the artificial EVs classification at the design of new therapeutic agents
based on EVs ............................................................................................................................ 108
5.4. Semi-synthetic exosomes: biotechnological modification of naturally released

exosomes ................................................................................................................................... 109
5.4.1 Selection of the EVs cellular origin .................................................................... 109
5.4.2 Obtaining a good substrate for modification: isolation procedures. ............ 111
5.4.3 Strategies for biochemical modification ............................................................ 113
5.5. Top-down and bottom-up methods for the development of full synthetic EVs122
5.5.1. Production of artificial EVs by generation of plasma membrane fragments: a

top-down inspired methodology ...................................................................................... 122
5.5.2 Bottom-Up methodologies: artificial membranes decorated with functional

proteins to mimic EVs functions ....................................................................................... 125
5.6. Conclusions and future perspectives ...................................................................... 131
5.7. Referencias bibliográficas .......................................................................................... 133
6. Using factorial experimental design to prepare size-tuned nanovesicles ................. 142
6.1. Introduction ................................................................................................................ 143
6.2. Materials and methods .............................................................................................. 146
6.2.1 Materials ................................................................................................................ 146
6.2.2 Factorial design of experiments ......................................................................... 147
6
6.2.3 Vesicles preparation ............................................................................................. 149
6.2.4. Vesicles characterization .................................................................................... 149
6.3. Results and discussion............................................................................................... 151
6.3.1. Effects of variables on morphological characteristics .................................... 151
6.3.2. PC liposomes ....................................................................................................... 153
6.3.3. S60:Cho niosomes................................................................................................ 160
6.3.4. Vesicles characterization .................................................................................... 162
6.4. Conclusions ................................................................................................................. 166
6.5. Bibliographic references ............................................................................................ 167
7. Continuous flow production of size-controllable niosomes using a thermostatic

microreactor ................................................................................................................................. 172
7.1. Introduction ................................................................................................................ 173
7.2.1. Materials ............................................................................................................... 176
7.2.2. Thermostatic system fabrication ....................................................................... 176
7.2.3. Microfluidic devices manufacturing and channel characterization ............ 178
7.2.4. Niosomes production and morphological characterization ......................... 179
7.2.5. Mixing efficiency visualization ......................................................................... 179
7.3.1. High resolution 3D-printing of master moulds for microfluidic devices

fabrication ............................................................................................................................. 180
7.3.2. Production of nanoparticles with temperature control for formulations with

high Tm non-ionic surfactants ............................................................................................ 182
7.3.3. Production of niosomes at different temperatures to study potential

tailoring effect over particle morphology ........................................................................ 189
7.3.4. Effect of surfactant acyl chain length over particles size and monodispersity
................................................................................................................................................ 191
7.4. Conclusions ................................................................................................................. 192
7
8. Particle size and encapsulation efficiency dependence with cargo molecular weight
and film hydration solution for niosomes formulated with mixture of surfactants .......... 199
8.1. Introduction ................................................................................................................ 201
8.2.1. Materials ............................................................................................................... 203
8.2.2 Niosomes preparation ......................................................................................... 204
8.2.3. Niosomes purification ........................................................................................ 204
8.2.4. Niosomes size and distribution analysis ......................................................... 205
8.2.5. Niosomes encapsulation efficiency (EE) .......................................................... 206
8.2.6. Differential Scanning Calorimetry (DSC) ........................................................ 208
8.3. Results and discussion ............................................................................................... 208
8.3.1. Niosomes size vs hydration solution composition and cargo molecular

weight.................................................................................................................................... 208
8.3.2. Encapsulation Efficiency vs hydration solution composition and cargo

molecular weight ................................................................................................................. 211
8.3.3. Thermal behavior of formulation in the hydration solutions ....................... 216
8.4. Conclusions ................................................................................................................. 217
9. Selected tetraspanins functionalized niosomes as potential standards for exosomes

detection based on immunoassays ........................................................................................... 221
9.1. Introduction ................................................................................................................ 223
9.2. Material and methods ................................................................................................ 226
9.2.1. Niosomes preparation and size measurement ................................................ 227
9.2.2. Streptavidin conjugation to niosomes surface ................................................ 227
9.2.3. Tetraspanins (CD9/63) large extracellular loops (LELs) production .......... 228
9.2.4. Vesicles functionalization with tetraspanins LELs constructions ................ 228
9.2.5. Immunoassays for artificial EVs detection ...................................................... 229
8
9.3.1. Streptavidin-coated niosomes development as generic scaffold for artificial

EVs production .................................................................................................................... 230
9.3.2. Artificial EVs production using Nio_Str functionalized with tetraspanin LEL
................................................................................................................................................ 233
9.3.3. Development of ELISA assays using artificial exosomas .............................. 236
9.3.4. Commercial potential of our artificial exosome model ................................. 240
9.5. Conclusions and future work ................................................................................... 242
10. Conclusiones/Conclusions ............................................................................................ 249
ANEXO I. Publicaciones derivadas de la Tesis Doctoral ................................................ 259
ANEXO II. Publicaciones relacionadas con la Tesis Doctoral ........................................ 261
9
Índice de Figuras
Figura 3.1. Diversidad química y morfológica de las vesículas artificiales creadas por
autoensamblaje de moléculas anfifílicas. Figura adaptada de la referencia 1. SUV: small
unilamelar vesicles; LUV: large unilamelar vesicles; MLV: multilamelar vesicles.............. 45
Figura 3.2. Estructura típica de una molécula anfifílica, en la que se señalan sus dos
porciones principales: la cabeza polar y la cola apolar, entre las cuales se establece la
interfase cuando son puestas en disolución acuosa. ................................................................ 46
Figura 3.3. (A) Representación gráfica del concepto del parámetro de
empaquetamiento crítico o CPP sobre un fosfolípido (superior) y dos surfactantes no
iónicos como son el Span® 60 y Span® 20 (medio e inferior respectivamente) que difieren
únicamente en longitud de cadena apolar. (B) Implicaciones geométricas de los valores de
CPP sobre la morfología del agregado de compuestos anfifílicos, figura adaptada de la
referencia 6. .................................................................................................................................... 48
Figura 3.4. Factores que condicionan la curvatura de la bicapa, como el parámetro de
empaquetado crítico que deriva de la forma de las moléculas anfifílicas, y como éste afecta
al radio de curvatura R durante la etapa de crecimiento de la bicapa discoidal. Figura
compuesta y adapta de la referencia 7. ...................................................................................... 49
Figura 3.5. Simulación informática del remodelado que sufre la micela inicial de
moléculas anfifílicas durante su auto-ensamblado en bicapas para formar vesículas. Estos
cambios en la estructura de la micela se producen como fruto de interacciones cabeza-
cabeza en la superficie y cola-cola en el interior al interactuar las moléculas con las de
agua de la fase contínua. Este reordenamiento lleva a la creación de poros por los que
circula el agua (B1 y B2), y este paso de disolvente acaba por reordenar la estructura hasta
formar un disco plano (D1). En rojo se representan las moléculas de agua, mientras que en
azul la cabeza polar y en negro la cola apolar. Figura tomada de la referencia 11. ............. 50
Figura 3.6. Surfactantes no iónicos más frecuentes en formulaciones de niosomas. Se
representan de arriba abajo en función del valor creciente de HBL, que surge de la
diferente longitud de cadena apolar que poseen los miembros de una misma familia de
surfactante. Figura tomada de la referencia 114. ...................................................................... 54
Figura 3.7. Diversidad morfológica de las vesículas, y su relación con los métodos de
preparación. Figura adaptada de la referencia 9. ..................................................................... 59
11
Figura 3.8. Diferentes estrategias de mejora de las propiedades básicas de las vesículas
artificiales, basadas en la incorporación de biomoléculas bioactivas, para el desarrollo de
biomateriales multifuncionales. Figura adaptada de la referencia 14. .................................. 67
Figura 3.9. Esquema ilustrativo de los grupos funcionales involurados en las
principales estrategias de conjugación basadas en el establecimiento de un enlace químico
entre la superficie de una vesícula y una biomolécula. (a) Entrecruzamiento de aminas
primarias mediante glutaraldehido, (b) enlace amida entre un carboxilo y una amina
primaria, (c) enlace amida mediante reacción de para-nitrofenilcarbonil con una amina
primaria, (d) puente disulfuro, (e) enlace tioester mediante reacción con maleimidetiol, y
(f) enlace hidrazona. Figura adaptada de la referencia 118. .................................................... 68
Figura 3.10. (A) Estrategia de bioconjugación basada en la interacción Estreptavidina-
Biotina, donde la Estreptavidina se conjuga previamente sobre la superficie de la bicapa
mediante alguno de los métodos mostrados en la figura 10, y el otro elemento se
encuentra biotinilado. (B) Conjugación de biomoléculas mediante el método de la
carbodiimida empleando EDC/NHS como reactivos de conjugación. Este método puede
ser empleado para la conjugación de Estreptavidina como se indica en el caso A.............. 70
Figura 3.11. Estrategias de purificación de vesículas para la eliminación del material
no encapsulado o conjugado. (A) Diálisis, (B) (Ultra)centrifugación, y (C) Cromatografía
de Exclusión de Tamaño (SEC) por filtración en gel. ............................................................... 71
Figura 3.12. Representación gráfica de la estructura y componentes de un exosomas
natural. Figura adaptada de la referencia . ................................................................................ 74
Figura 3.13. Ventajas (verde) y desventajas (rojo) de los dos tipos de aproximaciones
metodológicas basadas en bionanotecnología para la producción de exosomas puramente
artificiales con fines teranósticos. ................................................................................................ 76
Figura 4.1. Equipo Zetasizer NANO-ZS, Malvern Instruments. ...................................... 96
Figura 4.2. Microscopio TEM JEOL-2000 ExII, Jeol. ........................................................... 97
Figura 4.3. Equipo Turbiscan LAB Expert con estación de envejecimiento,
Formulaction. ................................................................................................................................. 98
Figura 4.4. Equipo de HPLC HP serie 1100 con módulo de detección UV/Vis HP
G1315A UV/ 281, Hewlett-Packard. ........................................................................................ 100
Figura 4.5. Lector de placas y absorbancia TECAN Genios, Tecan Trading AG. ........ 100
Figura 4.6. Analizador de imágenes de luminiscencia para geles y membranas
LAS4000 mini Image System analyser de, GE Healthcare. ................................................... 101
Figura 4.7. Equipo de DSC 822e, Mettler-Toledo. ............................................................ 102
12
Figure 5.1. Artificial EVs landscape: explored routes up-to-date for the preparation of
artificial EVs for specific purposes.*EBSSNs (ref. 19)............................................................. 107
Figure 6.1. Pareto chart of the standardized effects of independent variables (factors)
on (A) Z-average size and (B) PDI of PC liposomes for the Plackett-Burman fractional
factorial design............................................................................................................................. 151
Figure 6.2. Pareto chart of the standardized effects of independent variables (factors)
on the (A, C) Z-average size and (B,D) PDI of (A,B) PC liposomes and (C,D) S60:Cho
niosomes (1:0.5, w/w) for the 23 full factorial design............................................................ 155
Figure 6.3. Three-dimensional (3D) response surface plots for the factors O/A
(organic/aqueous phase volume ratio), C (lipid or surfactant/stabilizer concentration,
g/L), and A (sonication amplitude, %) for the (A,C) Z-average size and (B,D) PDI of (A,B)
PC liposomes and (C,D) S60:Cho niosomes (1:0.5, w/w)...................................................... 156
Figure 6.4. Contour Plot for the factors O:A (organic:aqueous phase volume ratio), C
(lipid or surfactant/stabilizer concentration, g/L) and A (sonication amplitude, %) on Z-
average size, nm (A) and PDI (C) of PC-liposomes, and Z-average size, nm (B) and PDI
(D) of S60:Cho niosomes (1:0.5, w/w). ..................................................................................... 157
Figure 6.5. Optimization plot and values of individual (d) and composite (D)
desirability provided by the response optimizer (Minitab, version 17) for an example of
size-tuned PC liposome (desired size = 70 nm, with a minimum PDI value). ................... 163
Figure 6.6. Optimization plot and values of individual (d) and composite (D)
desirability provided by the response optimizer (Minitab, version 17) for an example of
size-tuned S60:Cho niosome (1:0.5 w/w) (desired size = 240 nm, with a minimum PDI).
........................................................................................................................................................ 164
Figure 6.7. (A,B) BS profiles and (C,D) TEM micrographs of empty vesicles designed
with a controlled size and PDI values by applying the models obtained from experimental
design: (A,C) PC liposomes and (B,D) S60:Cho niosomes (1:0.5, w/w). ............................ 165
Figure 7.1. Schematic diagram of a continuous flow microreactor based on
hydrodynamic flow focusing for vesicular systems production. The reduction of focused
stream width under laminar flow conditions makes possible the mixing of chemical
species by molecular diffusion, since time for mixing decreases with the square root of
distance. By changing flow rates, the kinetics and extension of mixing can be modified,
and then, the size of particles. Amphiphilic molecules are self-assembled into bilayers
once critical concentration of solvent is reached, and molecules acquired an ordered state
13
to minimize the interaction with water molecules. At a certain size bending modulus
induce planar bilayer to be closed into vesicles. ..................................................................... 175
Figure 7.2. Pictures composition showing the whole setup (central) and detailed
components (sides) used in this work for niosomes production by Hydrodynamic Flow
Focusing with controlled temperature. .................................................................................... 177
Figure 7.3. Calibration plot (A) and temperature stability (B) of the in-house designed
thermostatic chamber for microfluidics chips, fabricated by 3D-printing technology with
PLA filaments. Values represented are the average of three independents measurements.
........................................................................................................................................................ 180
Figure 7.4. Figure 2. Precipitation of Span® 60 (Tm=45 °C) at room temperature (upper
arrow) at the focusing region (left), 0.5 and 1.0 cm downstream (centre and right). At a
temperature above surfactant Tm, focusing is complete and vesicles formation could be
checked by negative staining (Phosphotungstic acid 2%) and Transmission Electron
Microscopy (TEM). ...................................................................................................................... 183
Figure 7.5. Size (nm) and size distribution (PDI, a.u.) measured by DLS in undiluted
samples from niosomes formulated with Span® 60:Cholesterol (1:0.5 molar ratio) at 5mM
(A,C) and 20mM (B,D) in a continuous flow microreactor based on hydrodynamic flow
focusing at controlled temperature (50 °C). Each condition was tested twice, and each
batch was measured by triplicate. ............................................................................................. 184
Figure 7.6. Effect of bilayer components concentration for niosomes formulated with
Span® 60:Cholesterol (1:0.5 molar ratio) and produced under the same flow conditions
(QT and FRR) for size (upper row) and size distribution (lower row). ............................... 186
Figure 7.7. Images (4X) of focusing region and end of the mixing channel evidencing
hydrodynamic flow focussing of a central ethanol stream at different FRR values for
QT=100 μL/min and 50 °C. Yellow colour indicates acid pH (pure EtOH, no mixing),
while blue colour indicates basic pH (complete mixing by codiffusion of solvent and no
solvent). Bromoxylenol blue dye was dissolved in EtOH (acidified with acetic acid), and
PBS was adjusted with NaOH to pH 10. .................................................................................. 187
Figure 7.8. Values of ethanol focused stream (Wf, μm) as flow rate ratio (FRR)
increased for different values of volumetric rates (QT) at constant temperature (50 °C),
and different temperatures at constant QT (100 μL/min). Values represent the average of
two independent measurements, taken at approx. at 100 μm from the end of focussed
region. ........................................................................................................................................... 188
14
Figure 7.9.Size (nm) (left) and size distribution (PDI, a.u.) (right) measured by DLS in
undiluted samples from niosomes formulated with Span® 20:Cholesterol (1:0.5 molar
ratio) at 5mM in a continuous flow microreactor based on hydrodynamic flow focusing at
different controlled temperatures (30, 40, 50, and 60 °C). Each condition was tested twice,
and each batch was measured by triplicate. ............................................................................ 190
Figure 7.10. Influence of acyl chain length (C12 and C18 for Span® 20 and Span® 60
respectively) of two different sorbitan sters used in niosomes formulation (surfactant:
cholesterol 1:0.5 molar ratio, 5 mM), and produced under different conditions by
hydrodynamic flow focusing at controlled temperature (50 °C) and a flow rate QT=100
μl/min. .......................................................................................................................................... 192
Figure 8.1. Chemical structures of the encapsulated compounds ................................. 202
Figure 8.2. (A) Size values of empty vesicles. Tw80: tween® 80; Sp80: span® 80; Dc:
dodecanol. (B) Effect of hydration solution and cargo molecular weight over particle size,
for niosomal formulations Tw80:Sp80 (1:1 molar ratio) and Tw80:Sp80:Dc (1:1:1 molar
ratio) prepared by Thin Film Hydration method (TFH)........................................................ 209
Figure 8.3. Effect of hydration solution and cargo molecular weight over encapsulation
efficiecy, for niosomal formulations (A)Tw80:Sp80 (1:1 molar ratio) and (B) Tw80:Sp80:Dc
(1:1:1 molar ratio) prepared by Thin Film Hydration method (TFH). Tw80: tween® 80;
Sp80: span® 80; Dc: dodecanol. Dialysis (10 MWCO membranes) and gel filtration
(Sepahdex G25 or Seharose CL-4B, depending on the compound) were used as
purification methods................................................................................................................... 212
Figure 8.4. Naparticle Tracking Analysis (NTA) of the three different hydration
solution based niosomes after and before SEC purification. MQ and Gly vesicles were
purified using Sephadex G25 and PEG vesicles by Sepharose CL-4B. ................................ 215
Figure 8.5. DSC curves acquired in heating mode for the formulation without
dodecanol in the three different hydration solutions: ultrapure water (MQ), water:glycerol
60:40 v/v (Gly), and water:PEG-400 55:45 v/ v (PEG). ......................................................... 216
Figure 9.1. Schematic fully artificial exosome produced by bottom-up bio-
nanotechnological methods based on supramolecular chemistry. The different molecular
components are detailed with their functions......................................................................... 226
Figure 9.2. Figure 2. (A) Elution profile of a solution of streptavidin from a Sepharose
CL-4B SEC gravity elution column. Signal quantification of each 0.5Ml fraction was
measured by BCA total protein assay according with manufacturer instruction. Graph
insight shows (left) the first 3.5 mL collected of a suspension of red dye loaded niosomes
15
to allow their visualization, after their elution from the SEC column; (right) the SEC
column after the elution of the 3.5 mL of dyed niosomes. Both elements, niosomes and
free protein, eluted from the column enough separate to allow their separation based on
Sepharose CL-4B gravity elution coloumns. (B) Dot-Blot assay for checking the effectively
of streptavidin bioconjugation to niosomes through carbodiimide method (EDC/NHS).
Standards of different concentrations allows the semiquantification of the process by
comparison of spot intensity. The result shows the 5 different batches. Biotinylated-HRP
(4µg/mL) was used as detecting agent. (C) Size distribution by DLS of bared niosomes (or
NIO) and streptavidin-conjugated niosomes or (NIO_Str). The average size increment
(2nm) shown as peak displacement demonstrate the effective conjugation of the protein.
(D) Size distribution by DLS of the 5 different batches of NIO_Str (152 ± 3 nm), to
demonstrate the reproducibility of the process. ..................................................................... 232
Figure 9.3. (A) Dot-blot assays for revelation of LEL, CD9 or CD63, positive fractions
collected from a Sepharose CL-2B gravity elution SEC column. Mon- and double-
functionalized Nio_Str have been produced. (B) Size distribution measured by NTA
(Nanosight, Malvern Instruments) of previous described fully artificial exosomas
(NIO_LEL). A sample of natural exosomas isolated from mesenchymal triple-negative
breast cancer cell line SUM159 was also measured for comparison purposes. .................. 235
Figure 9.4. Dot-blot assay for the screening and selection of α-tetraspanin antibodies
for their future use in ELISAs for the detection of fully artificial exosomes (NIO_LEL).
Secondary antibodies labelled with HRP were appropriate selected. Different negative
controls were also introduced (bared niosomes, niosomes functionalized with streptavidin
with and without biotin saturation), as the use of isolated exosomas from mesenchymal
triple-negative breast cancer cell line SUM159 as positive control. Values are uniplicate
assays. (PU) monoclonal purified antibody; (biot) Biotynilated monoclonal antibody;
(poli) polyclonal antibody. ......................................................................................................... 236
Figure 9.5. (A,B) Cross-reactive responses for mono-functionalized LEL-tetraspanin
niosomes (fully articial exosomas) for different configuration of capture (c) and detection
(d) anti-tetraspanin antibodies. (C) ELISA assays for the detection of NIO_LEL9 (N9) and
NIO_LEL63 (N63) fully artificial exosomes using different combination of capture (c) and
detection (d) antibodies. Monoclonal α-CD9 or α-CD63 antibodies were used for capture,
whereas polyclonal α-CD9 or α-CD63 antibodies were used for detection. (D) ELISA
assays for the detection of NIO_LEL9/63 using different combinations of capture (c) and
16
detection (d) antibodies. The graphs shows the mean ± SD of 3 independent experiments.
*** p˂0.005, Student’s t-test. ....................................................................................................... 238
Figure 9.6. Dose-response graphs for different types of fully artificial exosomas
detected by ELISA assay using monoclonal and polyclonal antibodies α-tetraspanins CD9
y CD63 for capture and detection respectively. As secondary appropriate α-IgG-HRP was
used. N9 and N9/63 are NIO_LEL9 and NIO_LEL9/63 respectively, which are mono-
and double-functionalized LEL-tetraspanin niosomes. ......................................................... 240
17
Índice de Tablas
Tabla 3.1. Usos de los surfactantes no iónico en función de su valor HLB. ................... 55
Tabla 3.2. Ejemplos de aplicaciones biotecnológicas de los diversos tipos de sistemas
vesiculares según sector industrial y tipo de NVs.................................................................... 58
Tabla 3.3. Ventajas y desventajas de los métodos de purificación de vesículas. ........... 73
Table 5.1. Classification of techniques for the production of artificial EVs, mainly
exosomes, according to type of final product (semi- or fully synthetic) and the principle of
the obtention mechanism ........................................................................................................... 113
Table 5.2. Pre-isolation methods for cargo incorporation into EVs ............................... 115
Table 5.3. Post-isolation methods for cargo incorporation into EVs ..................................... 119
Table 5.4. Summary of the published work about the generation of mimetic EVs
nanovesicles by Top-Down bionanotechnology. Cell source and type of cargo are
encapsulated, and main characteristics are given. ................................................................. 123
Table 5.5. Advantages and disadvantage of most frequently used methods for Small
Unilamellar Vesicles (SUVs) preparation ................................................................................ 127
Table 5.6. Summary of published work about the development of mimetic EVs
nanovesicles by bottom-up bionanotechnology. Formulation of the vesicles, molecules for
the surface functionalization and main physical characteristic (size) are given. ............... 129
Table 6.1. Plackett-Burman Fractional Factorial Design: Responses, Levels, and Factors
........................................................................................................................................................ 148
Table 6.2. Full factorial design (23) with center point repetitions (n=5): factors, levels
and responses............................................................................................................................... 148
Table 6.3. ANOVA results (coded units) for Z-average size of PC-liposomes for the 23
full factorial design; results for S60:Cho niosomes are also given (cursive numbers) ...... 153
Table 6.4. ANOVA results (coded units) for PDI of PC-liposomes for the 23 full
factorial design; results for S60:Cho niosomes are also given (cursive numbers) ............. 154
Table 6.5. COOK's distance and DFITS values obtained for each response in the full
factorial designs ........................................................................................................................... 155
Table 6.6. Estimated coded coefficients for the considered effects on Z-average size
and PDI of PC liposomes and S60:Cho niosomes (1:0.5, w/w) ............................................ 162
Table 7.1. Morphological characteristics of mixing channel for original Solidworks®
CAD 2016 design and 3D-Printed positive moulds (3D-PM) onto VeroClearTM resin with
19
the 3D printer Objet350 ConnexTM (Stratsys). Average and standard deviation values are
given for the parameters............................................................................................................. 181
Table 7.2. Correlation factor between flow focusing parameters (FRR and Wf) and
particle size at different concentration of bilayer components and variable QT (A), and at
fixed concentration and QT for different working temperatures (B). .................................. 189
Table 8.1. Chromatography mediums used for gel filtration based purification of
loaded niosomes .......................................................................................................................... 206
Table 9.1. Some commercial available kits based on ELISA for exosomes quantification
in biological samples. Most of them are based on colorimetric signal quantification based
on HRP substrates, with a typical format of 96 well microtiter plate. ................................. 241
20
Lista de Abreviaturas
A
AA Ácido ascórbico
AFM Acronimo anglosajón para atomic force microscopy
ANOVA Acronimo anglosajón para analysis of the variance
ATMPs Acronimo anglosajón para advanced therapy medicinal products
a.u. Acronimo anglosajón para arbitrary units
B
B12 Vitamina B12, conocida también como cobalamina
BBS Acrónimo anglosajón para borate buffer saline
BCA Acrónimo anglosajón para bicinchoninic acid
BirA Acrónimo anglosajón para biotin ligase A
BSA Acrónimo anglosajón para bovine serum albumin
BS Acrónimo anglosajón para backscattering
C
CRY2 Acronimo anglosajón para cryptochrome 2
CPP Acrónimo anglosajón para critical packing parameter
CD9/63/81 Tetraspanina 9/63/81
D
DBCO Acrónimo anglosajón para dibenzocyclooctyne
Dc Dodecanol
DCs Acrónimo anglosajón para dendritic cells
DF Acrónimo anglosajón para degrees of freedom
DLS Acrónimo anglosajón para dynamic light scattering
DoE Acrónimo anglosajón para design of experiments
DOGS-NTA Acrónimo anglosajón para 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-

carboxypentyl)iminodiacetic acid)succinyl]
DSC Acrónimo anglosajón para differential scanning calorimetry
21
E
EBSSNs
ECL Acrónimo anglosajón para enhanced chemiluminescence
EDC 1-etil-3-(3-dimetilaminopropil)carbodiimida
EE Eficacia de encapsulación
EGFR Acrónimo anglosajón para epidermal growth factor receptor
EIM Acrónimo anglosajon para ethanol injection method
ELISA Acrónimo anglosajón para enzyme-linked immunosorbent assay
EXPLORs Acrónimo anglosajón para exosomes for protein loading via optically reversible
protein–protein interactions
EVs Acrónimo anglosajón para extracellular vesicles
F
FRR Acrónimo inglés para flow rate ratio
G
Gly Glicerol, aunque también se refiere a una disolución agua:glicerol 60:40 v/v
H
HEK293 Acrónimo anglosajón para human embryonic kidney cell line 293
HER2 Acrónimo anglosajón para human epidermal growth factor receptor 2
hFC Acrónimo anglosajón para high-resolution flow cytometry
HLA Acrónimo anglosajón para human leukocyte antigen
HLB Acrónimo anglosajón para hydrophilic-lipophilic balance
HPLC Acrónimo anglosajón para high performance liquid chromatography
HR Acrónimo anglosajón para high resolution
HRP Acrónimo anglosajón para Horseradish peroxidase
I
IDO Acrónimo anglosajón para indoleamine 2,3-dioxygenase
L
LEL Acrónimo anglosajón para large extracelular loop
22
LUV Acrónimo anglosajón para large unilamelar vesicles
LUV-TRAIL Acronimo anglosajón para vesículas sintéticas recuviertas de TRAIL
M
miRNA Acrónimo anglosajón para micro interference ribonucleic acid
MFLs Acrónimo anglosajón para membrane fusogenic liposomes
MLVs Acrónimo anglosajón para multilamelar vesicles
MS Acrónimo anglosajón para mean of squares
MSCs Acrónimo anglosajón para mesenchymal stem cells
MQ Acrónimo anglosajón para Mili-Q water, agua ultrapura o de Tipo I
MVBs Acrónimo anglosajón para multivesicular bodies
MW Acrónimo anglosajón para molecular weigth
MWCO Acrónimo anglosajón para molecular weigth cut-off
N
(sulfo)NHS Sulfo N-hyidroxyisulfosuccinimida
Nio niosomas
Nio_Str Niosomas con streptavidina conjugada en superficie
NLCs Acronimo anglosajón para nanostructured lipid carriers
NPMs Nanopartículas metálicas
NTA Acrónimo anglosajón para nanoparticle tracking analysis
NVs Nanovesículas
O
O/A relación de volúmenes de fase orgánica/acuosa
OVA Acrónimo anglosajón para chicken egg ovalbumin
P
PB Acronimo anglosajón para phosphate buffer
P-B Plackett-Burman
PBS Acrónimo anglosajón para phosphate buffer saline
PC Acronimo anglosajón para Phosphatidylcholine
PDI Acrónimo anglosajón para polydispersity index
23
PEG Poly(Ethylene) glicol, aunque también se refiere a una disolución de
polietilengligol-400 55:45 v/v.
R
RB Rodamina B
RM acrónimo anglosajón para reference material
RNA Acronimo anglosajón para ribonucleic acid
RT acrónimo anglosajón para room temperature
RP-HPLC Acrónimo anglosajón para Reverse-Phase High Perfomance Liquid

Chromatography
S
SDS-PAGE Acronimo anglosajón para sodium dodecyl sulfate polyacrylamide gel
electrophoresis
SEC Acrónimo anglosajón para size exclusion chromatography
siRNA Acronimo anglosajón para small interference ribonucleic acid
Sp80 Span® 80, o monooleato de sorbitano
SPIONs Acronimo anglosajón para superparamagnetic iron oxide nanoparticles
SS Acrónimo anglosajón para sum of squares
Str Estreptavidina
SUV Acrónimo anglosajón para small unilamelar vesicles
T
TAMEL Acronimo anglosajón para targeted and molecular extracelular vesicles loading
TEM Acrónimo anglosajón para transmission electron microscopy
TFH Acrónimo anglosajón para thin film hydration method
TFR Acrónimo anglosajón para total flow rate
TMB 3,3’,5,5’-Tetrametilbenzidina
tRPS Acrónimo anglosajón para tunable resistive pulse sensing
Tw80 Tween® 80, o monooleato de polioxietilen(20)sorbitano
U
UC Ultracentrifugación
Uv/vis Ultravioleta/visible
24
V
VE Vesículas extracelulares
v/v Relación de volúmenes
w/v Relación peso/volumen
Símbolos y unidades
λ Longitud de onda
ζ Potencial zeta
A Amplitud de sonicación (%)
C Concentración de compuestos de membrana
F Fischer’s ratio (u.a.)
I Fuerza iónica (mM)
NE Rotación del matraz en evaporación (rpm)
NS Velocidad de agitación durante la inyección (rpm)
P p-valor (u.a.)
QT Flujo volumétrico (mL)
QV Flujo de inyección (mL)
Re Número de Reynolds
t Tiempo de sonicación (min)
T Temperatura
Tc Temperatura de transición gel-líquido cristalino
TE Temperatura de evaporación (°C)
TI Temperatura de inyección (°C)
V0 Volumen muerto
25
1. Introducción
Capítulo 1. Introducción
En la actualidad existen numerosos productos basados en coloides orgánicos como

elemento funcional: 1 limpiadores cosméticos basados en soluciones micelares, cremas y
alimentos con vesículas para la administración y liberación controlada de principios
activos, nuevas formas de administración de fármacos basadas en vehículos
moleculares, detergentes con aromas encapsulados en partículas orgánicas, o aceites
lubricantes con aditivos para la mejora de sus propiedades. A su vez, los coloides
orgánicos también forman parte de procesos químicos, como son la fabricación de
nanomateriales 2 y los métodos bioanalíticos. 3
Un tipo particular de coloide orgánico cuyo espectro de aplicaciones es elevado, y

en expansión, son los sistemas vesiculares o nanovesículas (NVs): partículas
generalmente de unos pocos nanómetros a unas pocas de micras, con una estructura
molecular basada en una bicapa de compuestos anfifílicos 4 que encierra en su interior
una cavidad acuosa. Esta estructura ofrece la capacidad de incorporar tanto
compuestos de naturaleza hidrofílica como de carácter hidrofóbico. 5 La bicapa ofrece
una gran versatilidad, ya que puede presentarse en diversos tamaños y grado de
lamelaridad, lo que les confiere multitud de aplicaciones en los campos de la
medicina, 6 cosmética, 7 alimentación, 8 medioamiente 9 y de la química, especialmente la
química analítica.3
Por otra parte, la naturaleza química de dichos compuestos, permite su asociación a

macromoléculas 10 (como enzimas, anticuerpos, ácidos nucleicos, polisacáridos, etc.) o
su fijación a superficies 11 o nanomateriales inorgánicos, tales como nanopartículas
metálicas. 12 Las NVs fueron desarrolladas inicialmente para el campo de la cosmética,
siendo posteriormente expandido su uso a otros sectores tecnológicos.
Sin embargo, es en el campo de la biomedicina donde han encontrado mayor

aceptación y auge, siendo numerosas las formulaciones aprobadas y comercializadas
para uso terapéutico humano y animal, 13 habiendo sido Doxil/Caelyx® el primer
fármaco basado en liposomas (en el mercado desde 1995). Por otra parte, es el campo
donde existe mayor diversidad de tipos de vesículas empleadas.
Este elevado interés clínico ha llevado al desarrollo de nanovesículas con

propiedades mejoradas, como una mayor estabilidad coloidal, distribución
organotrópica, o liberación inteligente del componente encapsulado. 14 Estas
características funcionales se consiguen mediante la incorporación de elementos
activos como son polímeros hidrófilos (ej. Poli(Etilenglicol)), 15 anticuerpos 16 o ligandos
de receptores de membrana, 17 o la cuidadosa selección del tipo de lípidos empleados 18
así como la incorporación de nanopartículas metálicas (NPMs) que ejercen de
interruptores de la liberación. 19
En el campo de la biomedicina este tipo de partículas no sólo han sido empleados

como vehículos moleculares de por sí, sino que han sido empleados para la mejora y
aumento de propiedades de sistemas vesiculares naturales como son los exosomas. 20,21
Éstos son un sub-tipo de vesículas extracelulares (VE) secretadas por cualquier célula,
y con un papel muy importante en comunicación celular, así como en procesos fisio-
patológicos. 22 Este rol les ha puesto en el foco de atención de nuevas aplicaciones
terapéuticas y de diagnóstico. 23 Y a su vez, éstos han servido de inspiración para el
desarrollo de nuevos agentes teranósticos, 24 denominados exosomas artificiales. Este
tema será abordado con mayor profundidad en capitulos siguientes.
Por último y no menos importante, el sector químico se ha beneficiado de estas

partículas. En concreto, la química analítica y el sector de los biosensores
especialmente, han explorado su uso en el desarrollo y mejora analítica de varios
métodos. 25,32 El campo de los immunoensayos es un claro ejemplo, y numerosos
trabajos sobre su uso como sistema de amplificación de señal pueden encontrarse en la
literatura, 26 tanto en formatos tradicionales como ensayos ELISA, 27 en test
immunocromatográficos, 28 o en sistema de flujo. 29 La amplificación de señal puede
también producirse no por la vesícula en sí, si no que ésta puede adquirir un rol de
soporte 30 y potenciación de fenómenos espectroscópicos como es la fluorescencia
mejorada por metales. 31 A su vez, nanovesículas como los liposomas pueden ser
empleados como elemento de reconocimiento, 32 como por ejemplo de lipasas (enzimas
implicadas en procesos de inflamación). Este reconocimiento puede ser empleado para
su detección, y mediante una correcta transducción, su cuantificación en fluidos
biológicos mediante test rápidos. 33
La semejanza estructural y química de estos biomateriales con las vesículas

naturales (como los exosomas), han inspirado su empleo como herramientas de estudio
de los mismos. 34 Para ello, es necesario su producción con características morfológicas
lo más parecidas posibles, especialmente de tamaño y monodispersidad, ya que son
30
parámetros importantes en el bioanálisis de las VE. Por otra parte, su modificación
biotecnológica para incroporar moléculas bioactivas de diversa naturaleza química en
el interior o en superficie, hace necesario la adaptación o diseño de procesos adaptados
a tal fín, con la idea de producir estructuras biomiméticas de una forma adecuada y
asequible. Si bien los liposomas han destacado en estas aplicaciones, este campo en
auge, ofrece elevadas posibilidades para los niosomas también, y su empleo en
bioanalítica de exosomas está ofreciendo los primeros resultados. 35
La efectividad de los sistemas vesiculares en sus aplicacione está íntimamente

ligada a una propiedad esencial como es el tamaño promedio de partículas y a otro
parámetro relacionado como es la monodispersidad de tamaño (cuán grande es la
diversidad de tamaños de partícula en una suspensión). 36
Su empleo como vehículos de distribución y liberación de fármacos, se asocia a un

tamaño inferior a 150-200 nm para no ser reconocidos por el sistema monocítico
nuclear, 37 y como consecuencia ser eliminados de la circulación (proceso de
aclaramiento plasmático). Por ello, se reduce su tiempo de vida útil, requiriéndose
mayores dosis, lo que puede conllevar efectos secundarios asociados a su transporte no
específico.
Debe existir un compromiso, ya que a mayor tamaño mayor es la carga de principio

activo que pueden portar. Sin embargo, un tamaño pequeño (≤150 nm) les confiere una
capacidad intrínseca para permear por la red de capilares que nutren los tumores, ya
que el tamaño de las fenestraciones vasculares permite un filtrado no selectivo de las
vesículas al espacio intersticial. 38 Pero por otro lado, deben poseer un tamaño mínimo
que evite su aclaramiento plasmático a nivel renal (≥10-15 nm). De este modo, este
compromiso de tamaño permite una distribución del fármaco con características
pseudo-dirigidas. En relación con la distribución de biocompuestos, otro aspecto
importante es la incorporación celular, es decir, su paso al interior de las mismas. Este
proceso se sabe que es dependiente de la forma que presenta la partícula, pero también
del tamaño de la misma. 39
Puesto que ha quedado evidenciado la importancia de tamaño promedio de

partícula, se puede llegar a la conclusión que una homogeneidad en el mismo es igual
de importante. Una distribución de tamaño monodispersa, asegura un
comportamiento homogéneo de la población de partículas, lo que permite observar
31
respuestas diferenciadas atribuidas al material, y en cierta forma predecible. En otros
casos, como es su papel como marca en immunoensayos, es una característica casi más
importante que el propio tamaño. Puesto que a mayor tamaño, mayor es su superficie,
la capacidad de portar más elementos de reconocimiento aumenta en proporción, lo
que tiene importantes repercusiones sobre las características analíticas como puede ser
el límite de detección. Distribuciones de tamaños amplias introducen heterogeneidad
en el comportamiento de las partículas en cuanto a capacidades de reconocimiento y
generación de señal, ya que pueden producirse impedimentos estéricos que reduzcan
la señal analítica y generen un aumento en el límite de detección, siendo contrario a la
finalidad de su empleo en esta aplicación, como es una amplificación de la señal
analítica.
Por todo esto, estrategias de producción de partículas con control de tamaño y de

su monodispersidad son esenciales para sus aplicaciones bioanalíticas. En este
escenario socio-tecnológico se enmarca la presente Tesis Doctoral. El objetivo general
de la misma, será el estudio de metodologías de preparación de sistemas vesiculares
(liposomas y niosomas), con especial énfasis en la producción controlada de tamaño de
partícula, para su uso en aplicaciones biotecnológicas, fundamentalmente bioanalíticas.
Referencias bibliográficas
[1] Willen Norden (editor) (2011). Colloids and interfaces in Life Sciences and
Bionanotechnologies, segunda edición, CRC Press, Taylor & Francis Group, New York, USA.
[2] De S., Kundu R., Biswas A. (2012). Synthesis of gold nanoparticles in niosomes. Journal of
Colloids and Interface Science, 386:9-15.
[3] Gómez-Hens A., Fernández-Romero J.M. (2005). The role of liposomes in analytical
process. Trends in Analytical Chemistry, 24:9-19.
[4] Chen I.A., Walde P. (2010). From self-assembled vesicles to protocells. Cold Spring Harbor
Perspectives in Biology, 2:a002170.
[5] Damera D.P., Krishna Venuganti V.V., Nag A. (2018). Deciphering the role of bilayer of a
noisome towards controlling the entrapment and release of dyes. Chemistry Select, 3:3930-3938.
32
[6] Caddeo C., Diez Sales O, Valentia D., Ruiz Saurí A., Fadda A.M., Manconi M. (2013).
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36
2. Objetivos/Objetives
Capítulo 2. Objetivos
La presente Tesis Doctoral se ha centrado en el estudio de metodologías de

preparación de sistemas vesiculares (liposomas y niosomas), con especial énfasis en la
producción controlada de tamaño de partícula, para su uso en aplicaciones
bioanalíticas. En concreto, se emplearán para el desarrollo de un modelo mimético de
exosomas para su empleo como estándar analítico. Este objetivo general se ha divido
en cuatro objetivos específicos, que se exponen a continuación:
• Estudio de la influencia de parámetros de operación sobre las características

morfológicas finales de vesículas, tanto niosomas como liposomas, preparadas
mediante una técnica comúnmente utilizadas como es la inyección con etanol.
• Desarrollo y puesta a punto de un dispositivo de microfluídica con control de

temperatura para la preparación de sistemas vesiculares, lo que permitirá el
uso de pequeños volúmenes de muestra.
• Estudio de la influencia de las variables medio de hidratación y compuesto a

encapsular, en la caracterización final de vesículas preparadas con el método
de hidratación de la película fina.
• Diseño y producción de sistemas vesiculares para la producción de un analito

modelo: partículas biomiméticas de exosomas, denominados Exosomas
Artificiales, y su aplicación en bioanálisis.
39
Chapter 2. Objetives
The main objective of this Ph.D. Thesis is the study of methodologies for the
preparation of vesicular systems (liposomes and niosomes), with special emphasis on
the controlled production of particle size for their use in bioanalytical applications.
Specifically, they will be used for the development of a mimetic model of exosomes for
their use as an analytical standard. This main objective has been divided into the
following specific objectives:
• Study of operating parameters influence over the final morphological

characteristics of vesicles, niosomes and liposomes, prepared by a commonly
used technique such as ethanol injection method.
• Development and optimization of a microfluidic device with temperature

control for the preparation of vesicular systems, which will allow the use of
small sample volumes.
• Study of the influence of the variables hydration solution and encapsulated

compound, over the final characterization of vesicles prepared by the thin film
hydration method
• Design and preparation of vesicular systems for the production of a model

analyte: exosomes biomimetic particles, called Artificial Exosomes, and their
application for bioanalysis.
41
3. Consideraciones básicas
Capítulo 3. Consideraciones básicas
3.1. Sistemas vesiculares: generalidades
Las vesículas son un tipo de coloide orgánico artificial, formado por la agregación
supramolecular de moléculas anfifílicas que se disponen en una organización de
bicapa, que puede ser unilamelar o multilamelar. 1 Ésta, separa una cavidad acuosa
interior de la solución en la que se encuentran. La naturaleza química y la disposición y
tamaño de la misma establecen los tipos de vesículas artificiales que se pueden
encontrar en la literatura. La Figura 3.1 representa esta diversidad química y
morfológica que se pueden encontrar en este tipo de sistemas.
Figura 3.1. Diversidad química y morfológica de las vesículas artificiales creadas por autoensamblaje de
moléculas anfifílicas. Figura adaptada de la referencia 1. SUV: small unilamelar vesicles; LUV: large
unilamelar vesicles; MLV: multilamelar vesicles.
3.1.1. Moléculas anfifílicas
La estructura en bicapa responde al comportamiento físico-químico de ciertos tipos

de moléculas anfifílicas, ya que no todas son capaces de agregarse en este tipo de
45
estructura supramolecular. 2 La característica por la que pueden o no formar bicapa se
comentará más adelante.
Para entender primero el proceso de agregación, es necesario considerar

previamente la físico-química básica de las moléculas anfifílicas. Como su propio
nombre indica, se trata de moléculas con un comportamiento simultáneo hidrofílico e
hidrofóbico, derivado de poseer en su estructura una región polar y otra apolar,
denominadas de forma general cabeza y cola respectivamente (ver Figura 3.2). 3 Esta
última, puede estar presente en una, dos, o varias repeticiones, siendo a su vez iguales
o diferentes entre sí en cuanto a longitud o grado de saturación como se ha indicado en
la sección anterior.
Figura 3.2. Estructura típica de una molécula anfifílica, en la que se señalan sus dos porciones
principales: la cabeza polar y la cola apolar, entre las cuales se establece la interfase cuando son puestas en
disolución acuosa.
La red de moléculas de agua que se establece gracias a los puentes de hidrógeno de

las mismas se ve interrumpida por la existencia de esas porciones apolares de las
moléculas anfifílicas en disolución, en contraposición a las interacciones positivas que
establecen las cabezas polares (debido a esto, se habla entonces de la existencia de un
balance de fuerzas que no se encuentra en equilibrio). 4 Este hecho establece pues una
interfase en este tipo de moléculas, con una consecuente entropía negativa que tiende a
minimizarse por la agregación de las mismas en un intento de reducir la exposición de
dicha interfase. La existencia de fuerzas de repulsión entre las cabezas polares hace que
no se produzca una separación total de fases, sino que se las moléculas agreguen en
estructuras de orden superior.
Esta agregación es un fenómeno cooperativo, donde una concentración de

moléculas cercana a un punto concreto favorece la incorporación al estado agregado.
46
Este punto se conoce generalmente como concentración crítica de agregación o CCA, que
en el caso de la formación de micelas recibe el nombre de concentración micelar crítica o
CMC, valor de extremada importancia en la predicción de fenómenos de interfase en
disoluciones acuosas de tensioactivos.
En este punto de agregación inicial, la geometría de las moléculas juega un papel

importante para la formación final de una bicapa u otra estructura como puede ser una
micela. La proporción entre ambas porciones de la molécula es un valor crítico, y su
valor se mide por un parámetro denominado balance hidrofílico-lipofílico, conocido por
su acrónimo anglosajón como HBL.
El parámetro de empaquetamiento del tecton 5,*1 o CPP (siglas en ingles de Critical

Packing Parameter) es la expresión geométrica del valor HBL, y se define como la
relación entre el volumen de la porción hidrofílica (v) y el producto entre el área
interfacial (a) y la longitud de la cadena hidrofóbica (l) como muestra la ecuación 1
(Ecuación 1) y se muestra gráficamente en la Figura 3.3.A.
𝑣𝑣
𝑐𝑐𝑐𝑐𝑐𝑐 = Ecuación 1
𝑎𝑎𝑎𝑎
Determinados valores de este parámetro sirven para predecir la estructura que

pueden formar las moléculas. 6 De acuerdo a este parámetro, valores próximo a 1 darán
como resultado bicapas, mientras que a medida que se reduce hasta 1/3 se formarán
cilindros y esferas (micelas) respectivamente (Figura 3.3.B). Esto es debido a las
implicaciones geométricas que conllevan estos valores, y al radio de curvatura que
permiten, produciéndose por consiguiente las diversas estructuras mencionadas. Esta
agregación de una forma u otra es la consecuencia final del intento de reducir la
energía libre de la interfaz por unidad de volumen. Esta agregación se ve aún más
forzada cuando la parte apolar es rígida (aporta comportamiento nemático, con
orientaciones moleculares preferenciales) como en el caso de los lípidos, y otro tipo de
interacciones moleculares secundarias (interacciones dipolares y π-π) que colaboran en
la estabilidad del agregado. Debido a esto, estructuras como la bicapa son muy
estables.
*1 Término general para referirse a la porción apolar de la molécula anfifílica que actúa como monómero
de la estructura.
47
Figura 3.3. (A) Representación gráfica del concepto del parámetro de empaquetamiento crítico o CPP
sobre un fosfolípido (superior) y dos surfactantes no iónicos como son el Span® 60 y Span® 20 (medio e
inferior respectivamente) que difieren únicamente en longitud de cadena apolar. (B) Implicaciones
geométricas de los valores de CPP sobre la morfología del agregado de compuestos anfifílicos, figura
adaptada de la referencia 6.
La formación de una vesícula se puede dividir en dos etapas: la formación de una

micela plana, y su posterior crecimiento como bicapa hasta cerrarse en forma de
vesícula (Figura 3.4). 7 A su vez, la primera etapa se divide en una fase de nucleación
(formación del agregado primigenio), y una fase de crecimiento del agregado por
fusión entre agregados, pero también por la incorporación a los mismos de moléculas
anfifílicas en disolución.1 Estos procesos de nucleación y crecimiento ocurren en el
orden de microsegundos y nanosegundos respectivamente. Inicialmente, este modelo
fue propuesto para el método de preparación de vesículas mediante detergentes, 8 pero
su corroboración experimental ha hecho que se extienda a casi todos los métodos que
ocurren con una mezcla de disolventes polares y apolares. 9 Recientemente, un modelo
de dispositivo microfluídico especial ha demostrado mediante crio-capturas la
existencia de las bicapas discoidales. 10
48
Figura 3.4. Factores que condicionan la curvatura de la bicapa, como el parámetro de empaquetado crítico
que deriva de la forma de las moléculas anfifílicas, y como éste afecta al radio de curvatura R durante la
etapa de crecimiento de la bicapa discoidal. Figura compuesta y adapta de la referencia 7.
La planicidad de la micela proviene de esa disposición preferencial de las

moléculas que se ha comentado anteriormente, y es la consecuencia del proceso de un
reordenamiento molecular que tiene lugar también como resultado de la interacción
con el medio acuoso en el que se encuentran. 11 Brevemente, estos reordenamientos
ocurren mediante interacciones cabeza-cabeza en la parte externa de la micela, cola-
cola en la porción interior, y a consecuencia de ambos, la creación de canales de paso
del agua (ver Figura 3.5). 12 Como resultado final, se forma la micela plana en forma de
disco cuyo extremo es la parte activa donde se incorporan nuevas moléculas en
disolución que permanecen individualizadas, y por donde se fusionan las micelas
como segundo mecanismo de crecimiento.
Cuando la elasticidad de la bicapa es reducida, la tensión superficial aumenta, lo

que provoca el aumento del módulo de bending y de la energía responsable del cierre
de la bicapa para formar la vesícula. Cuando el tamaño de la micela es tal que el
módulo de bending supera la tensión lineal, ésta se cierra espontáneamente en forma de
esfera. 13
49
Figura 3.5. Simulación informática del remodelado que sufre la micela inicial de moléculas anfifílicas
durante su auto-ensamblado en bicapas para formar vesículas. Estos cambios en la estructura de la micela
se producen como fruto de interacciones cabeza-cabeza en la superficie y cola-cola en el interior al
interactuar las moléculas con las de agua de la fase contínua. Este reordenamiento lleva a la creación de
poros por los que circula el agua (B1 y B2), y este paso de disolvente acaba por reordenar la estructura
hasta formar un disco plano (D1). En rojo se representan las moléculas de agua, mientras que en azul la
cabeza polar y en negro la cola apolar. Figura tomada de la referencia 11.
Un aspecto importante en esta etapa es el papel de la propia formulación de las

vesículas. Como se ha visto es importante la contribución geométrica de las
moléculas 14 y las interacciones entre ellas. La formulación pues, introduce cambios en
la fluidez de la bicapa y la posibilidad de la existencia de asimetría en la misma, que
favorece la curvatura. 15 Por ejemplo, se ha observado que longitudes de cadena
reducidas (C12) generan bicapas más fluidas que longitudes mayores (C18) incluso en
presencia de colesterol como aditivo de membrana, 16 cuya presencia suele asociarse a
menor fluidez y por ende, una menor tasa de liberación pasiva de compuestos
50
encapsulados. Esa presencia de colesterol inhibe la transición gel-líquido de los
compuestos de membrana, 17 y por lo tanto permite alcanzar rápidamente valores de
módulos de bending que induzcan el cierre de la bicapa con prontitud, por lo que las
vesículas formadas son pequeñas al reducirse la etapa de crecimiento.
El proceso de autoensamblaje requiere de energía como se comentó anteriormente,

y esa energía generalmente se aporta en forma de calor. Ese calor debe ser por una
parte el necesario para sobrepasar ligeramente la temperatura de transición gel-líquido
(Tm) y así permitir el cierre de las vesículas. La temperatura provoca estados más
relajados en el reordenamiento molecular (una reducción en el valor del módulo de
compresión superficial Cs-1), 18 y una disminución de las interacciones inter-
moleculares. Así pues, el tamaño final de la vesícula dependerá de los parámetros de
tasa de crecimiento de las bicapas planas y la tasa de cierre en vesículas de las mismas.
Esto se integra en uno de los modelos de formación más aceptados, el denominado
modelo de la cinética en no equilibrio. 19 En este modelo, ambos parámetros se ven
influenciados por la temperatura como se ha explicado anteriormente. Cabe destacar,
que las moléculas de disolvente empleado para dispersar los compuestos anfifílicos
estabilizan los bordes activos de las membranas planas, por lo que contribuyen en ese
crecimiento. De forma similar, las moléculas de detergentes empleadas en ciertos
métodos cumplen la misma función. 20
Por otra parte, pueden producirse subdominios como son las balsas lipídicas
(reordenamientos de lípidos que se caracterizan por un mayor grado de
empaquetamiento y por ende, una reducida fluidez).2 También se han descrito la
existencia de micro-dominios en estados fluidos. 21 Es comprensible pues que esto
modifique las energías involucradas en el cierre de la vesícula y en la duración de la
etapa de crecimiento. Así mismo, la incorporación de heterogeneidad en la formulación
y la posibilidad de reordenamientos intra-moleculares de los compuestos anfifílicos
con cadenas no rígidas, es la base de la existencia de vesículas estables y altamente
deformables como son los transfersomas. 22
Como conclusión de esta sección, se puede afirmar que parámetros como la

concentración de componentes de bicapa, estructura y composición molecular, así
como homogeneidad o heterogeneidad en la composición son parámetros de la
formulación con un claro efecto en la morfología final de la vesícula. Por otra parte,
aspectos como la temperatura, composición del medio acuoso o aporte de
51
componentes libres durante la formación de las vesículas son aspectos metodológicos
que también poseen impacto sobre las características de las vesículas. Este hecho
conforma la base del control del proceso e introduce la posibilidad de obtener sistemas
vesiculares con características deseadas. Para ello, el estudio de procesos y el diseño de
los mismos teniendo en cuenta estos aspectos son un campo de estudio que permite la
ampliación del empleo de estos biomateriales en innovadoras aplicaciones.
3.1.2 Clasificación de los sistemas vesiculares
Los tipos de moléculas anfifílicas que han sido empleadas en formulaciones para la
preparación de sistemas vesiculares son numerosos, y con naturaleza química muy
diversa: fosfolípidos (neutros o cargados), esfingolípidos, copolímeros, ácidos grasos,
surfactantes no iónicos y/o de amonio cuaternario, o incluso mezclas de dos o más de
estos tipos de compuestos.
Estos compuestos representan la unidad básica de las bicapas que forman, y

frecuentemente son denominados componentes de membrana. Adicionalmente en muchas
formulaciones vesiculares es necesario la adición de un aditivo de membrana, con el fin
de ofrecer mayor estabilidad y rigidez a la membrana vesicular, o para introducir
modificaciones químicas que permitan nuevas características empleadas para ciertas
aplicaciones.
El tipo de compuesto utilizado en la formulación de la membrana vesicular da

nombre a los diversos tipos químicos de vesículas que existen, y que de manera
resumida se exponen a continuación.
Liposomas
Descubiertos por Bangham y colaboradores hace 50 años, 23 inicialmente estaban

compuestos por lípidos de origen natural, aunque en la actualidad muchos de ellos se
encuentran formulados con lípidos sintéticos, muchos de ellos con modificaciones de la
cabeza polar con la finalidad de introducir nuevas propiedades (al emplearse
generalmente en pequeñas cantidades, se pueden considerar como aditivos). 24
Sin lugar a dudas se trata del tipo de vesícula más empleado y sobre el que existe
un mayor número de publicaciones tanto experimentales como de revisión. Parte de
esta popularidad se debe a las ventajas que poseen, como son un alto grado de bio-
compatibilidad, su capacidad de portar tanto moléculas hidrófilas como hidrófobas en
52
su interior o bicapa respectivamente y su carácter biodegradable. 25 Han sido
formulados tanto con composiciones muy sencillas 26 como con composiciones
miméticas de la composición natural de membranas biológicas, 27 y la recreación de
dominios de membrana han sido descritas con éxito. 28 Fosfatidilcolina, fosfatidilserina,
fosfatidiletanolamina, esfingomielina, y colesterol son algunos de los tipos de lípidos
naturales más comunes en liposomas.
Sin embargo, poseen una clara desventaja como es una reducida estabilidad en
suspensiones acuosas, donde los fenómenos de hidrólisis degradan los lípidos y
reducen la vida útil de los liposomas. Esta característica fue la fuerza impulsora para
explorar otros compuestos a la hora de preparar vesículas.
Niosomas
La primera alternativa a los lípidos surgió en el uso de los tensioactivos no iónicos

(compuestos química- y estructuralmente análogos a los lípidos,) en el campo de la
cosmética. 29 Los ternsioactivos no iónicos poseen unas claras ventajas como son su
elevada estabilidad físico-química, elevada diversidad química, bajo coste y fácil
derivatización para introducir nuevas posibilidad químicas y físicas en las bicapas. 30
Los surfactantes no iónicos son compuestos sintéticos que pueden prepararse a

partir de aminoácidos, ácidos grasos, amidas, ésteres alquílicos y éteres alquílicos de
surfactantes.6 Estos últimos pueden crearse con azúcares, óxidos de etileno o
repeticiones de moléculas de glicerol. Sin embargo, no todos los surfactantes son
idóneos para la formación de vesículas. Los surfactantes más comúnmente utilizados
para la formulación de niosomas son los comercializados con el nombre de Span®,
Tween® y Brij®, ésteres de sorbitan y ácidos grasos, de poli-oxietilen sorbitan y ácidos
grasos, y éteres de cadenas alquiladas y repeticiones de poli-oxietilen respectivmente
(Figura 3.6).
53
Figura 3.6. Surfactantes no iónicos más frecuentes en formulaciones de niosomas. Se representan de
arriba abajo en función del valor creciente de HBL, que surge de la diferente longitud de cadena apolar
que poseen los miembros de una misma familia de surfactante. Figura tomada de la referencia 114.
Un parámetro que sirve como predictor de su idoneidad para la formulación de

niosomas es el HLB (acrónimo anglosajón de Balance Hidrofílico-Lipofílico) y que
representa una estimación de las proporciones de ambas porciones químicas en el
anfífilo. La Tabla 3.1 resume los principales usos de los tensioactivos según su HLB.
Como puede comprobarse, valores entre 4-8 son los adecuados para la formulación de
niosomas, siendo necesario incorporar en las formulaciones aditivos de membrana
para valores cercanos a 10. Esta adición compensa el exceso de peso que posee la
porción hidrofílica de la molécula, e interacciones con el colesterol y entre moléculas
del mismo permiten establecer bicapas estables. 31
Estas formulaciones pueden manejarse como las de los liposomas, por lo que los
mismos tipos morfológicos de vesículas pueden obtenerse, ya que los métodos de
preparación empleados son los mismos en ambos tipos de vesículas.
54
Tabla 3.1. Usos de los surfactantes no iónico en función de su valor HLB.
Valor de HLB Aplicación general
1-3 Antiespumantes
3-6 Emulsiones agua en aceite
7-9 Agentes humectantes y dispersantes
8-18 Emulsiones aceite en agua
13-15 Detergencia
15-18 Solubilización
Transfersomas
Los dos tipos de formulaciones anteriores se caracterizan por la rigidez de la bicapa

que se produce por el empaquetamiento de las moléculas gracias a las fuertes
interacciones inter-moleculares. Esta rigidez puede llegar a ser una desventaja en
ciertas vía de administración como puede ser la vía tópica, ya que provoca la
incapacidad para llegar a capas profundas de la piel y pasar al torrente sanguíneo. 32
Para evitar este inconveniente, se propusieron formulaciones que contenían a la vez

ambos tipos de componentes de membrana: lípidos y surfactantes, denominados
Transfersomas por su principal uso transdérmico. 33 En este caso, el surfactante no
iónico adquiere un papel muy importante, ya que es el encargado de producir la
elasticidad de la bicapa. Para ello, se suelen emplear compuestos como Tween® 80 y
Span® 80, con altos valores de HLB (≥15), que rompen la cohesión molecular de los
lípidos, permitiendo un mayor radio de curvatura y elasticidad de membrana. 34 Otro
compuesto que actúa de forma similar y es frecuentemente empleado es el colato de
sodio.
Esfingosomas
Otro tipo de vesículas con base lipídica son los esfingosomas, nombre recibido en
alusión al tipo de lípido empleado en su formulación: los esfingolípidos. 35 El empleo de
estos lípidos tiene su origen en la búsqueda de una mayor estabilidad por ausencia de
fenómenos de oxidación en fase acuosa. 36 Como estabilizante de membrana para la
formación de una bicapa estable y con buenos valores de eficacia de encapsulación se
suele emplear colesterol. 37
55
Ufasomas
Otro tipo de vesículas con base mixta lipídica y de surfactantes son los ufasomas, 38
vesículas formuladas a base de ácidos grasos insaturados (oleico y linoleico) e
insaturados (octanoico y decanoico). De nuevo, su composición y propiedades de
membrana los hacen buenos candidatos para la preparación de vehículos de
administración de biocompuestos por vía tópica. 39
Etosomas
Este tipo de vesículas recuerda a los transfersomas, ya que poseen de nuevo una
base lipídica y otro componente encargado de modular la elasticidad mediante
relajación molecular de la bicapa. En este caso, esa función es llevada a cabo por
moléculas de alcoholes como el etanol e isopropanol en elevadas proporciones. 40 De
forma análoga a los casos anteriores, son de gran utilidad en la administración tópica. 41
Quatsomas
Existe otra familia de surfactantes no iónicos capaces de formar vesículas

únicamente en presencia de esteroles, y son los surfactantes de amonio cuaternario
(que dan nombre al tipo vesicular). 42 Estos compuestos son de reducido coste, y
fácilmente disponibles en calidad farmacéutica, además presentan una característica
adicional como es un carácter antimicrobiano. 43
Polimersomas
Por último, se han empleado copolimeros en la formulación de vesículas

denominadas polimersomas. 44 Estos copolimeros tienen un peso molecular alto, y
existen diversos tipos que facilitan la versatilidad química de este tipo de vesículas. 45
Idealmente, los polímeros deben estar formados por bloques hidrofóbicos con
temperaturas de transición cristalina bajas. Un ejemplo son los polímeros peptídicos
sintetizados por la polimerización anionica de N-carboxianhidros. 46 Estas vesículas han
sido funcionalizadas por la incorporación de proteínas funcionales en su estructura de
bicapa, 47 y representan un tipo vesicular con elevado potencial tanto en estudios de
biología básica como aplicada.
56
3.1.2.1. El papel de los aditivos en las formulaciones
Mejora de la estabilidad coloidal
Existen dos mecanismos básicos que permiten a un coloide mantenerse en

suspensión de forma individual: 48 que existan impedimentos estéricos (físicos) que
impidan aproximarse y coalescer dos partículas adyacentes, o que dicho impedimento
se deba a la repulsión por poseer ambas una carga del mismo signo, es decir, una
repulsión electroestática.
Uno de las moléculas más empleadas para fines estéricos es el polímero hidrofílico
poli(etilenglicol) o PEG. 49 Su incorporación post-preparación o la incorporación en
forma de lípido modificado aumenta considerablemente la vida de la vesícula, ya que
no solo aporta el citado impedimento estérico, si no que les hace “invisibles” al sistema
monocitico-nuclear. 50
Compuestos con carga negativa como dicetilfosfato y ácido fosfatídico, o con carga
positiva como estearilamina y cloruro de cetilpiridinio, son empleados frecuentemente6
para aportar carga superficial neutra diferente de cero (parámetro medido por el
potencial ζ). Esta carga, ayuda a mantener la estabilidad coloidal mediante repulsiones
electrostáticas, siendo ±30 mV un valor de ζ lo suficiente grande como para asegurar
dicha estabilidad. 51
Propiedades nuevas, tales como fluorescencia
En la actualidad, existen numerosos lípidos sintéticos comerciales conjugados a

fluoróforos orgánicos como la rodamina o NBD. 52 La existencia tanto de fosfolípidos
como de esteroles, como el colesterol, modificados con este tipo de marcadores permite
la preparación de casi cualquier tipo de vesícula capaz de ser monitorizada mediante
fluorescencia molecular. Por otro lado, existen otro tipo de fluoróforos capaces de
incorporarse a bicapas, por lo que pueden ser añadidos a posteriori, y marcar las
vesículas para fines tanto analíticos como biomédicos. Un ejemplo de este tipo de
marcadores es la carbocianina lipofílica DilC18.
Mejora de la penetrabilidad dérmica
La mejora de la penetrabilidad dérmica de las vesículas ha sido explorada también

con la incorporación en las formulaciones de aditivos como el ácido oleico y linoleico. 53
57
A pesar de existir vesículas formuladas con ellos como son los ufasomas, en este caso la
proporción en la que se añaden hace que sean clasificados como aditivos.
Incorporación de puntos de unión a otras biomoléculas
Los lípidos y los esteroles son fácilmente modificables de forma química para
introducir nuevos grupos funcionales que permitan ser empleados en reacciones de
bioconjugación de moléculas o a superficies. 54 Son numerosos los ejemplos de su uso
para tal fin con liposomas y niosomas. 55- 57 Estos compuestos presentan un elevado
coste, aunque pueden ser preparados en el laboratorio,30 pero su empleo en relaciones
molares pequeñas respecto a los componentes mayoritarios permite su empleo con
facilidad y de una forma económicamente viable.
3.1.2.2 Principales aplicaciones de los tipos de sistemas vesiculares
Como se ha citado en la introducción, los sistemas vesiculares poseen un amplio

espectro de aplicaciones. A modo de ejemplo de esta versatilidad, la Tabla 3.2 resume
dichas aplicaciones por tipo de sector tecnológico y tipo de sistema vesicular
empleado.
Tabla 3.2. Ejemplos de aplicaciones biotecnológicas de los diversos tipos de sistemas vesiculares según
sector industrial y tipo de NVs.
Sector Industrial Tipo de nanovesícula Aplicación Ref.

Farmaceútico Liposomas Solubilización de compuetos hidrofóbicos, sensibles a la 58, 33
Niosomas luz y al pH, y su posterior distribución y liberación 40, 45
Transfersomas controlada por diferentes vías de administración (oral, 59
Etosomas nasal, ocular, tópica y parenteral)
Incoporación de nanopartículas metálicas para el 60
Polimersomas
desarrollo de agentes de contraste en diagnóstico por
Esfingosomas
imagen
Ufasomas Modificación de vesículas extracelulares teranósticas 61, 62
Quatsomas
Cosmético Liposomas Encapsulación de compuestos con reducida 63,64
Niosomas biodisponibilidad y sensibilidad ambiental
Alimentario Liposomas Encapsulación de compuestos con reducida 65,127
Niosomas biodisponibilidad y sensibilidad ambiental
Ambiental Liposomas Bio-remediación de vertidos de combustibles fósiles 66
Textil Liposomas Fabricación y tinción del tejido 67
Químicio Liposomas Amplificación de señal en immunoensayos y biosensores 68, 69
(Bioanalítico) 70, 71
Reconocimiento específico de biomoleculas 72
Fase estacionaria en Cromatografía Electrocinética 73
Capilar
Nanocristalizadores para estudios de Difracción de Rx 74
(DRX)
Soporte de macromoléculas de membrana para estudios 75
por DRX
Niosomas Síntesis de nanopartículas metálicas 76, 77
Desarrollo de estándares analíticos 122
58
3.1.3. Tipos morfológicos de vesículas
Como se comentaba al principio de esta sección, existen diversos tipos morfológicos

de vesículas en cuanto a tamaño, grado de lamelaridad, y número de cavidades
acuosas internas (partículas multivesícula). Existe una dependencia del tipo de vesícula
con el método de preparación, que deriva de los procesos de formación de vesícula que
ocurren durante su preparación. En la Figura 3.7 se representa la diversidad de tipos
morfológicos vesiculares y su relación con el método de formación.
Figura 3.7. Diversidad morfológica de las vesículas, y su relación con los métodos de preparación. Figura
Por un lado tenemos vesículas unilamelares (una única bicapa separa el medio
acuoso de la cavidad interior), que se dividen de acuerdo en el tamaño en pequeñas
(conocidas como SUVs, Small Unilamelar Vesicles), o grandes (conocidad como LUVs,
Large Unilamer Vesicles). Estos tipos se relacionan con métodos de cambio de
polaridad del medio, como la inyección de disolventes, y con métodos que implican
hidratación de películas finas mediante descargas eléctricas o flujos de fase acuosa en
microcanales respectivamente. Estos métodos se comentan en la sección 3.2.
Por otro lado, existen vesículas con más de una bicapa, y éstas se encuentran de
forma concéntrica como si fuesen las capas de una cebolla (vesículas multamelares o
59
MLV). Generalmente presentan un tamaño grande, aproximadamente entorno a media
micra, y este tipo morfológico se asocia al método de hidratación de capa fina, donde la
película se hidrata de forma progresiva liberando bicapas que geman de la película
hasta cerrarse en vesículas.
En ocasiones, y dependiendo de las condiciones de la hidratación se ha visto la

generación de multivesículas (vesículas con más de una cavidad acuosa en su interior,
o vesículas que contiene a su vez vesículas en su interior). Este tipo morfológico se ha
observado también en métodos de hidratación directa o en tecnología de Proniosomas.
Por otro lado, existen metodologías para reducir el tamaño o cambiar el tipo
morfológico, como son la extrusión 78 y la sonicación. 79 En el primero, el paso por una
membrana de tamaño de poro reducido hace que las vesículas se rompan en
fragmentos que se cierran en nuevas vesículas con un tamaño similar al del poro. En el
segundo, la aplicación de ultrasonidos en las suspensiones crea fenómenos de
cavitación con igual resultado que la extrusión. En este caso, el tamaño final va en
función de la amplitud de la frecuencia de los ultrasonidos.
3.2. Métodos de preparación de vesículas
Desde el descubrimiento de los liposomas hace 50 años, se han descrito numerosos

métodos de preparación de vesículas aplicables a casi todas las formulaciones
comentadas en la sección 3.1.2., y éstos han sido revisados en la literatura, 80
encontrándose incluso revisiones monográficas sobre tipos particulares de métodos. 81
Es importante remarcar que no existe un método mejor que otro, o particularmente

idóneo para un tipo específico de formulaciones, pero ciertos aspectos han de tenerse
en cuenta a la hora de seleccionarlo. Posiblemente lo más importante a la hora de la
selección del método de preparación sea la aplicación final para la que se desean
preparar las vesículas. Como se ha comentado en la sección 3.1.3. existen diversos tipos
morfológicos de vesículas que se encuentran íntimamente ligados a los métodos de
preparación y a los principios que rigen el autoensablaje. Estos métodos influencian de
manera importante las características finales de las partículas, 82 como son el tamaño, el
grado de lamelaridad, y la eficacia de encapsulación, todas ellas características a
controlar en el desarrollo de una aplicación biotecnológica.
60
A continuación se hace una breve descripción de los métodos de preparación
encontrados en la literatura. Se han agrupado en base al principio general en el que se
sustentan.
3.2.1. Métodos que implican cambio de polaridad en el medio
Los métodos que se describen a continuación tienen en común el empleo de un

disolvente orgánico en el que se disuelven los componentes de la bicapa y los aditivos.
Posteriormente esta fase es dispersada en una fase acuosa cuya proporción en la
mezcla final es muy superior, lo que provoca el cambio de polaridad del medio en el
que se encuentran las moléculas anfifílicas.
Inyección de etanol
En este método, los componentes de membrana son disueltos en etanol, y esta

disolución es dispersada en el seno de una disolución acuosa con agitación constante,
mediante un sistema continuo de bombeo (generalmente una bomba de jeringa). 83 La
fase acuosa se mantiene a una temperatura superior a la de transición Tc del sistema
(formulación), y tras finalizar la fase de dispersión el disolvente es eliminado mediante
evaporación a vacío. Como ventajas posee la baja toxicidad del etanol, la sencillez de
los dispositivos involucrados y la facilidad de escalado a nivel industrial. Dependiendo
de la polaridad del compuesto a encapsular, éste se incorpora en la fase orgánica o
acuosa si es apolar o polar respectivamente. Método frecuentemente empleado para la
preparación de SUVs.
Inyección de éter
Conceptualmente es idéntico al anterior, pero se emplea como disolvente dietil éter,

que no es miscible con la fase acuosa, y la temperatura de la fase acuosa se ajusta para
estar por encima del punto de ebullición del disolvente. Debido a esto, no es
recomendable para la encapsulación de compuestos termolábiles, y generalmente
requiere de etapas adicionales para la reducción del tamaño promedio de partículas, ya
que por lo general produce LUVs. 84
Evaporación de fase reversa
Similar al anterior, los componentes de membrana se disuelven en un disolvente no

miscible en agua, pero previo a la evaporación del mismo, la mezcla se somete a
agitación mecánica para formar una emulsión, donde las gotas harán de molde para la
61
formación de las vesículas en la etapa de eliminación del disolvente. 85 Únicamente se
emplea en la encapsulación de compuestos apolares, ya que la encapsulación pasiva de
compuestos hidrófilos no es compatible.
Existe una variedad, donde se evapora el primer disolvente tras la disolución de los
compuestos de membrana, y se forma una capa fina que se re-disuelve en un segundo
disolvente que se mezcla con la fase acuosa, y se procede como en la versión anterior. 86
En ambos casos, se forman LUVs y MLVs, por lo que se requieren etapas de post-
procesado para reducir el tamaño o cambiar el tipo de vesícula.
Eliminación de detergentes
En este método los componentes de membrana se disuelven en una solución

micelar de detergentes, que posteriormente se somete a un proceso de eliminación de
los mimos (por ejemplo por diálisis), lo que fuerza la formación de las vesículas. 87
Hidratación de capa fina
Este método es el más antiguo, con el que se descubrieron los liposomas. Se parte
de una disolución orgánica que contiene los componentes de membrana y que
mediante rotavapor es desecada para forma una película final en las paredes del
matraz. Esta capa fina es hidratada por la fase acuosa a temperatura adecuada a la Tc
del sistema, y una fuerza mecánica en forma de agitación o ultrasonidos asiste en la
homogenización e hidratación. Debido a este proceso, MLVs es el producto
mayoritario, por lo que se requieren de etapas siguientes para obtener SUVs. Es
adecuada a todo tipo de compuestos, polares y apolares, e incluso para la
encapsulación de NPMs.12
3.2.2. Métodos directos
Sonicación directa
La disolución de los componentes de membrana se realiza directamente en fase

acuosa, que es sonicada directamente a temperatura elevada, hasta la homogenización
y formación de vesículas. 88
Método de calentado de disoluciones
En este método, los componentes se disuelven en fase acuosa directamente en

presencia o ausencia de poliol (3%v/v), sometida posteriormente a altas temperatura y
62
a agitación contínua a medida que se reduce la temperatura. 89 En ocasiones, los
diferentes componentes de membrana son disueltos por separado y puesto en común
en la etapa de agitación. 90
Método del burbujeo
Se trata de otro método directo en el que los componentes en disolución acuosa se

someten a calentamiento directo en presencia de un flujo burbujeante de N2, que da
lugar a una suspensión de LUVs. 91
Microfluidización
Flujos acuosos conteniéndolos compuestos de membrana son propulsados a alta

velocidad y presión por canales de reducidas dimensionas hacia una cámara donde
interacciones entre ellos en un espacio confinado, produciéndose la mezcla y formación
de las vesículas, 92 que poseen un tamaño uniforme, unilamelaridad y reducido tamaño.
Proniosomas
Este método implica el recubrimiento de partículas solubles (tipo cristales de

azúcares) por los surfactantes no iónicos, por lo que es exclusivo de niosomas. 93 Los
complejos formados en estado seco son posteriormente hidratados en el momento de la
encapsulación y de uso, por lo que este método permite tener lotes pre-preparados que
pueden almacenarse y transportarse con facilidad.
3.2.3. Métodos de nueva generación
Esta categoría de métodos engloba técnicas no convencionales desarrolladas

fundamentalmente en los últimos 15 años, y que se clasifican en dos grandes bloques:
3.2.3.1. Métodos basados en fluidos supercríticos
Este conjunto de métodos se basa en el empleo de fluidos supercríticos 2* para la

formación de vesículas 94 en un proceso que se caracteriza por: (i) reducción del estrés
mecánico y de la temperatura a la que se someten las vesículas, por lo que mejora la
posible degradación de lípidos y compuestos termolábiles, (ii) una reducción en el
2* Compuestos que se encuentran a una presión y temperatura superiores a su punto crítico,
lo que provoca un comportamiento híbrido entre líquido y gas, por lo que puede difundir como
un gas y disolver sustancias como un líquido.
63
consumo de disolventes orgánicos y de la toxicidad residual de su uso, (iii) facilidad
del proceso que se lleva a cabo en una única etapa, y (iv) esterilidad de la producción
inherente al empleo de fluidos supercríticos). 95
El fluido más empleado es CO2, por ser inerte, no tóxico, barato y no inflamable.
Este gas se mezcla con el disolvente que contiene los componentes de membrana a una
alta temperatura y presión. Esta mezcla se inyecta sobre la fase acuosa, y en ese
contacto se forman las vesículas mientras se evapora el disolvente al burbujear el gas.
Existen números ejemplos del empleo de estas técnicas para producir niosomas 96 y
liposomas. 97
3.2.3.2. Métodos basados en microcanales (microfluídica)
Las ventajas que aportan los microcanales en dispositivos de microfluídica han

hecho que este conjunto de técnicas haya ganado mucha importancia en la preparación
de vesículas para bio-aplicaciones.98 De forma resumida, estas características son: (i)
manipulación precisa de los flujos, lo que permite un control elevado sobre los
procesos de mezcla de reactivos, (ii) Mezclado rápido y homogéneo, (iii) temperatura
homogénea y de rápido intercambio frio/calor, (iv) reducción del consumo de
reactivos, (v) producción de pequeños lotes para procesos de optimización, (vi) rapidez
en la obtención de partículas, (vii) capacidad de observar la formación in situ de las
partículas, (viii) oportunidad de integrar on line etapas de post-procesado y/o
caracterización, (ix) fácil escalado mediante combinación en paralelo de varis
dispositivos.
Existe gran diversidad de técnicas microfluidicas 99,100 ampliamente descritas en

excelentes revisiones bibliográficas. A continuación se hace un breve resumen de los
mismos, en función del mecanismo de acción del dispositivo microfluídico.
Electroformación
Aunque descrita a nivel macroscópico inicialmente, 101 la electroformación en

microcanales fue descrita por Kuribayashi et al. en 2006, 102 y consiste en la aplicación de
corriente alterna en el interior de microcanales con fase acuosa sobre los que
previamente se ha depositado una capa fina de lípidos por evaporación de una
solución orgánica con los mismos. Se podría asemejar al método de hidratación de capa
fina, pero en este caso la fuerza eléctrica sustituye a la agitación mecánica o
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ultrasonidos. Este método suele generar vesícular unilamelares gigantes (más de una
micra).
Mejoras de este método han ido encaminadas al uso de micro-patrones sobre los
electrodos, para conseguir un mayor control de la hidratación de la capa fina
depositada sobre ellos, y así, controlar el tamaño de partícula generada. 103
Hidratación directa de capas lipídicas
Similar al anterior, en este método es el flujo de la fase acuosa a través del canal la
fuerza que desprende las bicapas de la capa fina previamente depositada, 104 por lo que
se consiguen vesículas MLVs y altamente polidispersas.
Micro-extrusión
En esta técnica, se deposita una capa fina sobre una membrana de nitruro de silicio
con un tamaño de poro nanométrico (≤ 300 nm), y es impulsada por un flujo de fase
acuosa para atravesando la membrana, fragmentarse y general pequeñas vesículas de
tamaño controlado y monodisperso. 105
Enfoque hidrodinámico de fluidos
Aunque recuerda a la inyección de disolvente, esta técnica no es comparable con el

método convencional. En este caso, un flujo de disolvente orgánico que contiene los
precursores de membrana es enfocado por dos flujos adyacentes de fase acuosa. 106 Este
enfoque provoca la reducción del diámetro del flujo orgánico, y gracias a ello, el
completo mezclado de ambas corrientes se produce por difusión molecular
únicamente, al encontrarse en un régimen de flujo laminar debido a los bajos valores
de Reynolds que se obtienen al trabajar en la microescala.
De esta forma, controlando los flujos y su relación, 107 se puede controlar la

extensión y eficacia del mezclado por su efecto sobre el tiempoo necesario para
alcanzar la total mezcla al igual que se puede llevar a cabo el proceso en canales de
longitud variable, con o sin elementos que favorezca la total mezcla adicional. 108 El
enfoque puede llevarse a cabo bidimensional o tridimensionalmente. 109
Propulsión de gotas o burbujas
Esta sofisticada técnica recuerda a soplar por el aro de formación de pompas de

jabón como sus autores describen. 110 Una bicapa se establece entre dos compartimentos
65
acuosos, y el empleo de micro pulsos de aire o fluido sobre la misma efectúa su
deformación hasta formar una vesícula que emerge de la bicapa cuando ésta se
deforma en exceso y se fragmenta.
Empleo de emulsiones dobles W/O/W como micropatrones
En este caso, micro-elmulsiones dobles son creadas en microcanales 111 o mediante

emulsificación con membranas, 112 para su posterior empleo como patrones en la
creación de vesículas a partir de componentes de membrana disueltos en la fase
orgánica (generalmente un disolvente no miscible en agua). La eliminación de dicho
disolvente por evaporación fuerza la creación de bicapas en la interfaz O/W, que se
acaban convirtiendo en vesículas.
3.3. Procesos asociados al uno biotecnológicoy bioanalítico de los

sistemas vesiculares
Como se ha comentado en la introducción, la versatilidad que ofrecen estos

sistemas vesiculares deriva de su capacidad para encapsular moléculas tanto
hidrofílifcas como hidrofóbicas, y de la posibilidad de crear estructuras complejas con
otras biomoléculas, y así sumar propiedades hasta crear auténticos biomateriales multi-
funcionales (Figura 3.8). En este apartado se explican brevemente los procesos de
asociación a otras moléculas (encapsulación y bioconjugación) y el conjunto de
procesos que implican la separación de los elementos que no han interactuado con las
vesículas de las nuevas estructuras generadas (purificación).
66
Figura 3.8. Diferentes estrategias de mejora de las propiedades básicas de las vesículas artificiales,
basadas en la incorporación de biomoléculas bioactivas, para el desarrollo de biomateriales
multifuncionales. Figura adaptada de la referencia 14.
3.3.1. Encapsulación
La encapsulación se define como la incorporación de moléculas al interior acuoso

de la vesícula o a la bicapa, en función de la polaridad de las mismas. Este proceso
puede ser pasivo o activo, en función del momento en el que se incorporan las
moléculas a la vesícula. 113 En el primer caso, pasivo, éstas se incorporan durante la
formación de las vesículas añadiéndose dichas moléculas, o bien junto a los
componentes de membrana en la fase orgánica, o bien disueltas en la fase acuosa. Este
es el mecanismo que se ha empleado en la presente Tesis Doctoral.
En el segundo de los casos el proceso es activo puesto que se realiza una vez se han
creado las vesículas, por lo que es necesario mecanismos físico-químicos que
incorporen las moléculas a dichas estructuras. Estos mecanismos se basan en la
creación de gradientes transmembrana, bien sean de pH o iónicos. 114
La eficacia de encapsulación depende en gran medida de la naturaleza química de

la especie a encapsular. Compuestos hidrofílicos suelen ofrecer eficacias de
encapsulación inferiores, especialmente por encapsulación pasiva. Este hecho suele ir
asociado a una elevada tasa de fuga por permeación através de las bicapas, a pesar de
la existencia de aditivos como el colesterol, cuya presencia suele reducir este
fenómeno.31
67
Por otro lado, la composición de la bicapa condiciona la incorporación de moléculas
en la misma, y el peso molecular del compuesto a encapsular suele ser un parámetro
crucial. 115 Recordemos que las interacciones moleculares en la bicapa es parte de la
estabilidad de la misma, por lo que la llegada de nueva moléculas puede desestabilizar
el sistema. Por último, el método de preparación de vesículas puede jugar un papel
importante, 116 especialmente para la encapsulación pasiva ya que como se ha
mencionado, los mecanismos de formación de las vesículas son complejos y altamente
influenciables por variables metodológicas.
3.3.2. Bioconjugación
La bioconjugación es un proceso químico por el que se unen dos elementos, donde

al menos uno de ellos es una biomolécula. 117 Esta unión puede ser de diversa
naturaleza: de afinidad molecular, de coordinación o mediante enlace covalente. Como
se comentaba en la sección de aditivos de membrana, existen numerosos lípidos y
surfactantes modificados con diversos grupos funcionales que permiten establecer los
citados tipos de unión (Figura 3.9). A modo de ejemplo de esta diversidad, la figura 10
muestra el elevado número de grupos funcionales que se pueden emplear con
vesículas.
Figura 3.9. Esquema ilustrativo de los grupos funcionales involurados en las principales estrategias de
conjugación basadas en el establecimiento de un enlace químico entre la superficie de una vesícula y una
biomolécula. (a) Entrecruzamiento de aminas primarias mediante glutaraldehido, (b) enlace amida entre
un carboxilo y una amina primaria, (c) enlace amida mediante reacción de para-nitrofenilcarbonil con
una amina primaria, (d) puente disulfuro, (e) enlace tioester mediante reacción con maleimidetiol, y (f)
enlace hidrazona. Figura adaptada de la referencia 118.
68
Las diversas estrategias de bioconjugación de vesículas han sido revisadas 118.
Debido a esto, únicamente se comentarán aquellas estrategias que han sido empleadas
en la presente Tesis Doctoral.
Bioconjugación por afinidad: interacción Biotina-Estreptavidina
Esta estrategia emplea la elevada afinidad que existe entre la biotina y la

estreptavidina/avidina/neutravidina, la mayor constante de afinidad descrita en la
naturaleza 119. Este método es muy popular, ya que existen formas recombinantes de
estas proteínas con un precio muy reducido, y por otro lado se pueden encontrar kits
comerciales de biotinilización que permite marcar con esta molécula casi cualquier otra
biomolécula. 120 Para ello, estos kits emplean muchas de las reacciones que se muestran
el Figura 3.9.
En la bibliografía se pueden encontrar ejemplos de bioconjugación de moléculas de

estreptavidina sobre la superficie de vesículas, y su posterior conjugación con
elementos biotinilados, como por ejemplo anticuerpos 121 o proteínas recombinantes. 122
La elevada constante de afinidad hace este proceso pasivo (únicamente hay que poner
ambos elementos en común disolución, Figura 3.10.A) rápido, eficaz y muy sencillo.
Bioconjugación por enlace covalente tipo amida mediante el método de la

Carbodiimida
Este método se basa en el establecimiento de un enlace covalente de tipo amida

entre una amina primaria y un grupo carboxilo activado. 123 Esta activación está llevada
a cabo por molécula que reacciona con este grupo a pH ácido, como son las
carbodiimidas (EDC). Esta activación es estabilizada por moléculas de N-
hidroxisuccinimida (NHS), que sustituye a la carbodiimida de forma estable, ya que ésta
tiende a hidrolizarse del –COOH en medio acuoso. Posteriormente, el éster al entrar en
contacto con la amina primaria forma un enlace de tipo amida, cuya eficacia de
establecimiento es mejor a pH neutro. El proceso se recoge en la Figura 3.10.B.
Puesto que muchas macromoléculas poseen ambos grupos funcionales, este

proceso se lleva a cabo por etapas, eliminando el exceso de EDC/NHS mediante
alguna de las técnicas que se comentan en el siguiente apartado. Esto a su vez favorece
que se pueda ajustar el pH de cada etapa para maximizar la conjugación. Esta
estrategia se ha empleado tanto en liposomas 124 como en niosomas. 125 Para evitar
69
reacciones cruzas e interacciones inespecíficas, los ésteres que quedan libres pueden ser
bloqueados con una amina primaria como puede ser la hidroxilamina o la glicina. 126
Figura 3.10. (A) Estrategia de bioconjugación basada en la interacción Estreptavidina-Biotina, donde la

Estreptavidina se conjuga previamente sobre la superficie de la bicapa mediante alguno de los métodos
mostrados en la figura 10, y el otro elemento se encuentra biotinilado. (B) Conjugación de biomoléculas
mediante el método de la carbodiimida empleando EDC/NHS como reactivos de conjugación. Este método
puede ser empleado para la conjugación de Estreptavidina como se indica en el caso A.
3.3.3. Purificación
Diálisis
Se trata de uno de los métodos más empleados en la literatura 127-129. Este proceso
está basado en los flujos osmóticos que se establecen a favor de gradiente de
concentración. La suspensión de nanovesículas se contiene en un saco o dispositivo
comercial separado del medio por una membrana, cuyo tamaño de poro deja pasar
únicamente las moléculas de compuesto no encapsulado o conjugado (ver Figura
3.11.A). Como inconvenientes de esta técnica destacan: el tiempo requerido para
completar la purificación y la necesidad de optimizarse para cada compuesto de forma
70
individual y la dilución de la muestra por entrada de medio de purificación hacia el
interior del cartucho o bolsa. Como ventajas destacan la capacidad de purificar grandes
lotes y la sencillez de la técnica.
Figura 3.11. Estrategias de purificación de vesículas para la eliminación del material no encapsulado o
conjugado. (A) Diálisis, (B) (Ultra)centrifugación, y (C) Cromatografía de Exclusión de Tamaño (SEC)
por filtración en gel.
Centrifugación/Ultracentrifugación
Se trata de otro método muy frecuente, 130,131 y no solo para sistemas vesiculares.
Este método emplea la mayor densidad de las vesículas, debido a su tamaño, respecto
a los otros elementos en disolución, ya que al someter a una suspensión de vesículas a
la fuerza centrífuga, éstas se irán al fondo del tubo pudiendo decantarse con facilidad
el sobrenadante (ver Figura 3.11.B). Este método permite a su vez una concentración
de la suspensión, ya que el pellet puede reconstituirse en un volumen menor. En
función de la fuerza aplicada en el proceso, se habla de centrifugación o
ultracentrifugación. A menudo este proceso se lleva a cabo a baja temperatura, para
compensar el exceso de temperatura que genera el rotor a tales revoluciones.
Existe una modificación del método donde se lleva a cabo en un gradiente de

densidad, 132 lo que permite separar los componentes en función de su densidad y así
conocer este valor para elementos desconocidos.
71
Filtración por membranas
Se trata de una versión de la técnica anterior, en la que la centrifugación a cabo

empleando dispositivos que poseen una membrana de tamaño selectivo por la que
pasan los compuestos libres así como el medio de la suspensión, por lo que se puede
emplear para una concentración simultánea a la purificación. 133 Presenta las mismas
ventajas y desventajas que la técnica anterior.
Filtración en gel o Cromatografía de Exclusión por Tamaño (SEC)
Este método es una técnica cromatográfica, donde los compuestos se separan en

función de una característica diferencial entre ellos, como es el tamaño o peso
molecular en este caso. 134 El nombre de la técnica deriva del empleo de geles formado
por polímeros entrecruzados que se emplean como fase estacionaria.
En este tamiz, los compuestos libres y las vesículas avanzan de forma diferente, ya
que el bajo peso molecular respecto a las vesículas hace que éstos pueden seguir
caminos más largos al ser capaces de penetrar por los poros de las partículas. Al
contrario, las vesículas avanzan por el especio muerto que dejan las partículas entre sí,
por lo que el orden de elución es: primero las vesículas y luego los compuestos libres
en orden de mayor a menor peso molecular (ver Figura 3.11.C).
Los tipos de fase estacionaria más empleados para la purificación de vesículas son
aquellos basados en partículas poliméricas de dextrano o agarosa entrecruzado que
generan mallas de tamaño de poro diferentes. Sephadex® G25, G50 o G75; 135,136 o
Sepharosa CL-2B o CL-4B 137,138 suelen ser los medios más empleados. En función del
peso molecular del compuesto encapsulado/conjugado se emplea uno u otro, siendo
CSephadex® empleado para moléculas de bajo/medio peso molecular y Sepharosa lo
indicado más macromoléculas tipo proteínas o polímeros. En algunos casos, este
método se emplea con fuerza centrífuga para una mayor rapidez y eficacia. 139
72
Tabla 3.3. Ventajas y desventajas de los métodos de purificación de vesículas.
Método Ventajas Desventajas
Optimización del tiempo

Sencillez
necesaria para cada compuesto
Diálisis Equipamiento barato
y medio
Escalable a nivel industrial
Proceso largo
(Ultra)centrifugación Posibilidad de aglomerar las
Elevada eficacia vesículas al ser sometidas a
Rapidez del proceso fuerzas elevadas
Filtración por membranas Escalable mediante Posibilidad de que se rompan
multiplicación de los tubos las vesículas
Equipos costosos
Muy eficiente
Versátil con amplio rango de
fases estacionarias en No escalable a nivel industrial
Filtración en gel o SEC función de los pesos Tiempos largos si se usa elución
moleculares de los cargos por gravedad
Dispositivos reutilizables
Rápido
3.4. Exosomas: ejemplo de sistema vesicular natural con interés

biotecnológico
Durante la última década, la comunidad científica ha presenciado una revolución

en el conocimiento de los procesos fisiológicos, especialmente en cuestión de
comunicación celular a nivel tisular y sistémico. Este hecho se debe en gran parte en los
avances sobre cómo se producen y se desarrollan ciertas patologías gracias al mayor
conocimiento sobre el papel fisio-patológico de las vesículas extracelulares, y en
especial sobre los exosomas. 140
Los exosomas son un tipo de vesículas extracelulares liberados al medio

extracelular por todo tipo de células, y que pueden por ello encontrados en todo tipo
de fluidos biológicos, además de en el medio de cultivo de células y tejidos cultivados
in vitro. 141
73
Figura 3.12. Representación gráfica de la estructura y componentes de un exosomas natural. Figura
Estas vesículas naturales que derivan de los cuerpos multivesiculares (orgánulos

celulares encargados de la importación-exportación entre la célula y su entorno, poseen
por ello una membrana similar a la plasmática, pero que difiere en términos de
composición lipídica y proteica. 143 Este perfil bioquímico es definitorio pues de ellos, y
sirve para su identificación y clasificación (entendiendo este proceso como la
asignación de su origen y destino) (ver Figura 3.12). 144
El aumento de la información sobre la composición, biogénesis y los roles de los

exosomas en procesos fisióligcos y patológicos ha introducido nuevas posibilidades en
el campo del diagnóstico y de la terapia. 145 Los exosomas poseen características únicas
que resultan de su origen celular y que los hace atractivos como nuevos biomarcadores
de diagnóstico, estratificación de la enfermedad, o incluso como parámetro de medida
de la respuesta y eficacia de un tratamiento.
Su composición molecular, tanto en términos de composición de membrana y de

cargo, que incluye información lipidómica, proteómica y genómica, ofrece una fuente
importante de conocimiento sobre su origen celular y el estado fisiológico del mismo.
El hecho de poder analizar exosomas en fluidos biológicos fáciles de tomar (saliva,
sangre, orina) que provengan de tejidos de difícil acceso (cerebral, medular) hace aún
más atractivo su bioanálisis.
74
3.4.1. Exosomas como modelo de inspiración de vesículas
mejoradas
Los exosomas combinan las ventajas de los vehículos de distribución de fármacos y

de los agentes terapeúticos. De hecho, son considerados como los sistemas de
distribución de compuestos más prometedores, especialmente en terapia génica para
desórdenes genéticos o como agentes anti-tumorales. 146 Durante estos últimos años se
han publicado números estudios sobre la modificación de elementos diana y sobre la
encapsulación de elementos endógenos y exógenos en estas biopartículas, bien sea
antes o después de su aislamiento de biofluidos o cultivos celulares. 147
Estos trabajos han introducido el concepto de exosomas semisintéticos, un tipo de

exosomas artificiales (modificados o creados en un laboratorio) que incluye a todos los
exosomas que han sido modificados de alguna manera para su posterior uso en
aplicaciones específicas.
3.4.2. Retos tecnológicos asociados a los exosomas y soluciones

biomiméticas
Sin embargo, a pesar del desarrollo de estas metodologías, existen una serie de
limitaciones relativas a su uso a nivel clínico, y que incluyen su aislamiento y
purificación a escala de producción con un grado aceptable según estándares clínicos.
Por otro lado, estas desventajas impiden a día de hoy la existencia de un material de
referencia con el que poner a punto y comparar estrategias bioanalíticas que ayuden a
profundizar en su estudio y permitan su plena instauración a nivel clínico. 148
Estas desventajas han llevado a la comunidad científica a desarrollar estrategias

para el diseño y producción de exosomas totalmente artificiales, creados en el laboratorio
siguiendo metodologías propias de bionanotecnología. Estas metodologías se agrupan
en dos grupos clásicos de la nanotecnología como son las metodologías top-down y
bottom-up. La Figura 3.13 muestra gráficamente el concepto de ambas, y resume
brevemente sus ventajas y desventajas. En las secciones siguientes se explica
brevemente en que consiste cada una. Información más detallada de cada una así como
ejemplos encontrados en la literatura se expondrán ampliamente en el capítulo 1.
75
Figura 3.13. Ventajas (verde) y desventajas (rojo) de los dos tipos de aproximaciones metodológicas
basadas en bionanotecnología para la producción de exosomas puramente artificiales con fines
teranósticos.
Soluciones basadas en métodos Top-Down
Este conjunto de técnicas se basa en la producción de partículas en la nanoescala

mediante la fragmentación de material de mayor tamaño y complejidad. La producción
de exosomas artificiales en este caso comienza con el cultivo de células que serán
empleadas como precursoras de fragmentos de membrana para la preparación de
nanovesículas.
Esta “reducción” mediante fragmentación puede llevarse a cabo extrusión por

membranas 149,150, o mediante dispositivos microfluídicos. 151,152 Ambos métodos han
permitido encapsular material terapeútico (moléculas o nanoparticulado) o para
producir partículas con propiedades regenerativas.
Ambas metodologías permiten la producción de cantidades suficientes para

estudios clínicos, con una pureza aceptada, y lo que es más importante, con un perfil
molecular y estructural muy similar al de los exosomas. Sin embargo, la encapsulación
es un proceso poco selectivo, y las etapas de purificación siguen siendo largas,
laboriosas y requieren de personal altamente cualificado.
76
Soluciones basadas en métodos Bottom-Up
Por contrario, esta aproximación crea elementos complejos y de tamaño

nanométrico mediante el ensamblado molecular, empleado para ello principios físico-
químicos como los mencionados en la sección 2.1 de la química supra-molecular. Es la
aproximación en la que se basan los métodos de preparación de sistemas vesicular
comentados en la sección 2.2. Por esto, la unidad básica en esta aproximación es la
creación de una vesícula artificial que es funcionalizada con elementos típicos de
exosomas mediante técnicas de encapsulación y bioconjugación. 153
Estos métodos ofrecen la posibilidad de crear elementos muy puros, ya que la

composición queda determinada por los precursores empleados, y por la pureza
química de los compuestos empleados (tanto lípidos como proteínas son generalmente
adquiridos en formas de elevada pureza, cercana al 100%). Por otra parte, muchos de
estos procesos ya han sido adaptados y optimizados 154 a la encapsulación de ácidos
nucleicos, moléculas caras y de difícil manejo.
Es por ello, un campo de trabajo muy interesante, donde la información que nos
aporta el estudio de los exosomas naturales puede ayudar en la creación de nuevos
liposomas mejorados, pero a su vez, éstos nos pueden permitir en el diseño y
desarrollo de nuevos biomateriales complejos que puedan ser empleados en clínica y
bionálisis.
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91
4. Experimental
Capítulo 4. Experimental
Para una mejor comprensión de los resultados, tanto los materiales utilizados como
los reactivos y los procedimientos empleados se van a detallar en cada uno de los
capítulos experimentales de manera individual. De esta manera, el apartado
experimental de la Tesis Doctoral recoge de manera general los principios de las
técnicas y los detalles relacionados con la instrumentación empleada en la
caracterización de las nanovesículas producidas a lo largo de la misma.
4.1. Dynamic Light Scattering (DLS): tamaño y potencial ζ
La dispersión dinámica de luz o Dynamic Light Scattering (DLS) es una técnica

analítica no invasiva que se emplea para las medidas del tamaño de partícula (radio
hidrodinámico) en suspensión, y de la distribución de tamaños. Esta técnica permite la
medida de tamaños desde 0.3 nm hasta 10 µm aproximadamente.
Emplea un haz de luz láser que se dirige hacia la suspensión de partículas, siendo
la luz reflejada por las mismas en múltiples direcciones. Este reflejo no posee ni una
intensidad ni una direccionalidad fija, si no que debido al movimiento browniano de
las partículas (por choques con moléculas del disolvente), éste fluctúa en el tiempo en
proporción al tamaño de las partículas. Partículas pequeñas se mueven más rápido,
mientras que partículas grandes se mueven despacio, por lo que se observan
fluctuaciones que se producen de acuerdo a ese movimiento.
En un instrumento de DLS, la muestra se ilumina con un rayo láser y las

fluctuaciones de la luz dispersada se detectan en un ángulo de dispersión conocido (θ)
mediante un detector rápido de fotones. Las fluctuaciones de intensidad se detrminan
mediante el cálculo de la función de correlación de intensidad g2(t). De esta correlación
se obtiene coeficiente de difusión D de las partículas, que se relaciona con el radio
hidrodinámico (R) de las partículas mediante la ecuación de Stokes-Einstein
(D=K(B)T/6πηR), Donde k(B) es la constante de Boltzmann, T la temperatura y η la
viscosidad.
El movimiento browniano de las macromoléculas depende de su tamaño, pero

también de la temperatura y viscosidad del solvente, por lo que dichos datos son
requeridos en un análisis por DLS.
95
Figura 4.1. Equipo Zetasizer NANO-ZS, Malvern Instruments.
En el caso de la presente Tesis Doctoral se ha utilizado un instrumento Zetasizer

NANO-ZS (Malvern Instruments Ltd, Malvern, Reino Unido). Este intrumento (ver
Figura 4.) es un analizador de tamaño de partícula con dos ángulos de detección (173°
y 13°), utilizando la dispersión de luz dinámica con óptica NIBS (retrodispersión no
invasiva o Non-Invasive Backscatter), lo que permite medir tanto suspensiones muy
concentradas como diluidas. Opera con un láser de tipo He-Ne laser (633 nm, max.
4mW), a una temperatura por defecto de 25 °C (si bien es un parámetro que puede
modificarse, hasta 120 °C). En esta Tesis, todas las medidas han sido realizadas a 25 °C.
Este equipo permite medir también potencial ζ a través de la luz dispersada. El

potencial ζ se define como la carga que adquiere una partícula en un determinado
medio, y que proviene de la carga superficial y de la concentración y tipo de iones en la
solución. Estos iones son lo que se “ordenan” sobre la superficie de la partícula y
generan la carga de la misma. Por tanto, El potencial zeta es una medida de la
magnitud de la repulsión o atracción electrostática (o de carga) entre las partículas, y es
uno de los parámetros fundamentales que afectan a su estabilidad coloidal. Su medida
ofrece información de las causas de la dispersión, agregación o floculación, y se puede
emplear en la mejora de las formulaciones.
Dicho parámetro se mide mediante una técnica denominada microelectroforesis de

láser Doppler. En esta técnica, se aplica un campo eléctrico a una solución de moléculas
o a una dispersión de partículas, que se mueven con una velocidad proporcional a su
potencial ζ. Esta velocidad es medida mediante una técnica interferométrica de láser
patentada denominada M3-PALS (Dispersión de luz para análisis de fase). Esta medida
permite el cálculo de la movilidad electroforética, y a través dedicho valor se calcula el
valor de potencial ζ.
96
4.2. Microscopía Electrónica de Trasmisión (TEM): morfología
El análisis morfológico de las vesículas se ha realizado mediante microscopía

electrónica de transmisión por tinción negativa (NS-TEM), utilizando un microscopio
electrónico de transmisión JEOL-2000 Ex II (Tokio, Japón). Es un instrumento que
aprovecha los fenómenos espectroscópicos que se producen cuando un haz de
electrones suficientemente acelerado colisiona con una muestra delgada
convenientemente preparada, o una suspensión de partículas depositada sobre
portamuestras adecuados.
Figura 4.2. Microscopio TEM JEOL-2000 ExII, Jeol.
Cuando los electrones colisionan con la muestra, en función de su grosor y del tipo
de átomos que la forman, parte de ellos son dispersados selectivamente, es decir, hay
una gradación entre los electrones que la atraviesan directamente y los que son
totalmente desviados. Todos ellos son conducidos y modulados por unas lentes para
formar una imagen final sobre una cámara CCD que puede tener miles de aumentos
con una definición inalcanzable para cualquier otro instrumento. La información que
se obtiene es una imagen con distintas intensidades de gris que se corresponden al
grado de dispersión de los electrones incidentes.
El caso concreto de la tinción negativa, es una técnica de microscopía que permite

contrastar las muestras mediante una sustancia opaca a los fotones (microscopía
óptica) o a los electrones (microscopía electrónica). En caso de microscopía electrónica
de transmisión, se emplean metales pesados (de alto número atómico) que, por tanto,
resultan opacas a los electrones transmitidos. Típicamente, estas sustancias son acetato
de uranilo, citrato de plomo, ácido fosfotungstico, molibdato de amonio o tetraóxido
de osmio.
97
En el caso particular de la presente Tesis Doctoral, el microscopio utilizado (ver
Figura 4.2) está especialmente indicado para la nano-caracterización estructural y
analítica de materiales. Posee 200 kV de aceleración lo que permite alcanzar una
resolución de 3,4 Å entre líneas. El equipo dispone además de una cámara CCD de alta
resolución (2048x2048 pixels). Se ha empleado ácido fosfotungstico (2% w/v, en agua
ultrapura).
4.3. Turbiscan (Formulaction): estabilidad coloidal
La estabilidad coloidal de lad nanovesículas se ha determinado midiendo los

perfiles de retrodispersión en un aparato Turbiscan Lab Expert (Formulaction,
L’Union, Francia) provisto de una estación de envejecimiento (Formulaction, L’Union,
Francia). Dicha estación de envejecimiento (ver Figura 4.3) permite el análisis, a largo
plazo, de la estabilidad de todo tipo de dispersiones líquidas, de forma automatizada.
Figura 4.3. Equipo Turbiscan LAB Expert con estación de envejecimiento, Formulaction.
Este equipo emplea la técnica denominada dispersión estática de luz múltiple o

Static Multiple Light Scattering (S-MLS). En esta técnica, la suspensión de partículas es
iluminada con una fuente de fotones (880 nm, NIR), que son reflejados por las mismas
en múltiples direcciones y en numerosas ocasiones (al ir siendo dispersada múltiples
veces al ir encontrando varias partículas en su trayectoria de rebote). Finalmente, esta
luz es detectada por dos detectores sincronizados, uno a 135° para luz dispersada y
otro a 0° para luz trasmitida. La relación entre ellas y la intensidad individual será
proporcional al tamaño de partícula y a su concentración. Esta medida se realiza a lo
largo de la altura de la celda de medida, lo que crea un patrón de señal que permite
monitorizar fenómenos de agregación, floculación, sedimentación o cremado, ya que
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éstos se verán reflejos en un cambio de la señal detectada por ambos detectores y por
consiguiente un cambio en ese patrón a lo largo del tiempo de medida.
4.4. HPLC fase reversa: eficacia de encapsulación
La cromatografía líquida de alta eficacia o high performance liquid

chromatography (HPLC) de fase reversa es un tipo de cromatografía que emplea la
polaridad de los analitos como característica en base a la cual se produce la separación.
La separación cromatográfica se produce como consecuencia del avance diferencial de
los componentes de la mezcla a través de una fase estacionaria de carácter hidrofóbico,
de tal forma que los compuestos polares no interaccionan con la misma, y eluyen con el
volumen muerto. Sin embargo, los compuestos apolares interaccionan con la fase
estacionaria, y esta interacción es más fuerte cuanto mayor es la apolaridad del
compuesto, avanzando más lento por la columna y eluyendo a tiempos de retención
superiores.
La fase estacionaria está compuesta de partículas de sílica químicamente

modificadas con hidrocarburos saturados, insaturados o aromáticos de diferentes tipos.
Los compuestos apolares se eluyen de forma selectiva a medida que se incrementa la
apolaridad de la fase móvil que los impulsa por la columna, de tal forma que a
determinado porcentaje de disolvente orgánico (metanol, acetonitrilo o isopropanol), el
compuesto pasa a la fase móvil y éste eluye de la columna.
Por lo tanto, para este tipo de cromatografías se emplean mezclas dinámicas de

solventes polares, tales como agua, y acetonitrilo, acetato de etilo, acetona y alcoles
alifáticos cuya proporción cambia con el tiempo, generando lo que se denomina
gradiente cromatográfico. Alternativamente, se puede usar el modo isocrático, en el
que la composición de la fase móvil permanece sin alterarse.
En el caso concreto de la presente Tesis Doctoral se ha utilizado un cromatógrafo

HP serie 1100, (Hewlett-Packard, Palo Alto, CA), con un módulo de detección UV/Vis
HP G1315A UV/ 281 (Agilent Technologies, Palo Alto, CA, USA), tal y como se puede
observar en la Figura 4.4.
Se ha utilizado la siguiente columna cromatográfica: Zorbax Eclipse Plus C18

(Agilent Technologies, 284 Palo Alto, CA), con un tamaño de partícula de 5 μm y unas
dimensiones de 4,6 mm × 150 mm. En todos los casos, se han empleado gradientes
99
analíticos cuyas espefificaciones se encuentran detalladas en las secciones
experimentales de cada capítulo donde se ha empleado esta técnica.
Figura 4.4. Equipo de HPLC HP serie 1100 con módulo de detección UV/Vis HP G1315A UV/ 281,
Hewlett-Packard.
4.5. Lector de placas
Un lector de placas (ver Figura 4.5) es un instrumento que permite detectar eventos
biológicos, químicos o físicos en muestras contenidas en placas de microtitración. Los
métodos comunes de detección para los ensayos de microplacas son: absorbancia,
intensidad de fluorescencia, luminiscencia, fluorescencia resuelta en el tiempo y
polarización de fluorescencia.
En el caso particular de esta Tesis, se ha empleado medidas de absorbancia del

producto enzimático de la catálisis de dihidrocloruro de o-fenilendiamina (OPD) por
parte de la enzima HRP, con una longitud de onda de detección λ = 492 nm, en un
equipo Tecan Genios (Tecan Trading AG, Suiza)
Figura 4.5. Lector de placas y absorbancia TECAN Genios, Tecan Trading AG.
100
4.6. Analizador para la medida de Western Blot
Para la realización de las medidas por quimiluminiscencia en dot-blot se ha

utilizado el analizador LAS4000 mini Image System analyser de GE Healthcare (ver
Figura 4.6). Es un analizador específico para geles y blots, realizando la detección o
bien por quimiluminiscencia o por fluorescencia. Posee una cámata CCD de
FujifilmTM, con una resolución de imagen de 6.3 Mpixels.
En el caso particular de esta Tesis, se ha empleado quimiluminiscencia mejorada,

proveniente de la reacción enzimática de un producto luminiscente que se forma por
catálisis de la enzima HRP que se encuentra conjugada a los anticuerpos empleados
para detección molecular específica.
Figura 4.6. Analizador de imágenes de luminiscencia para geles y membranas LAS4000 mini Image
System analyser de, GE Healthcare.
4.7. Equipo de Differential Scanning Calorimetry
La calorimetría diferencial de barrido es una técnica analítica empleada en la

caracterización de fenómenos que involucran cambios térmicos como consecuencia de
procesos físico-químicos. Estos fenómenos producen cambios en la entalpía de las
muestras, que son registrados a lo largo del tiempo y de un gradiente de temperatura.
Dicho gradiente puede realizarse en modo de aumento de temperatura o en modo de
descenso. Frecuentemente se suelen emplear ambos, para conocer la reversibilidad del
proceso y así identificar su naturaleza.
Estas fluctuaciones térmicas se registran tanto para la muestra como para un

material de referencia, y los cambios de flujos de calor son registrados como señal
analítica. De esta forma se detectan procesos endo- y exotérmicos en forma de picos,
cuya morfología y posición informan a cerca del fenómeno que los produce. En el caso
101
particular de los sistemas vesiculares, el cambio de estado gel a líquido cristalino de las
bicapas se manifiesta como un pico endotérmico, ya que dicho cambio se produce
gracias a energía en forma de calor. Su posición y forma de pico ofrece información
acerca de la estabilidad y grado de empaquetamiento de los compuestos que forman la
bicapa, y resulta en una técnica muy útil para la caracterización de formulaciones.
Figura 4.7. Equipo de DSC 822e, Mettler-Toledo.
En la presente Tesis Doctoral se ha empleado un equipo de DSC (ver Figura 4.7)

modelo 822e (Mettler-Toledo, España). Las muestras han sido cargadas en crisoles
sellados de aluminio, con flujo de nitrógeno para evitar procesos de degradación por
oxidación.
102
5. Therapeutic biomaterials based on
extracellular vesicles: classification of bio-
engineering and mimetic preparation routes
Capítulo/Chapter 5
Therapeutic biomaterials based on
extracellular vesicles: classification of bio-
engineering and mimetic preparation routes
5.1. Extracellular vesicles in nanomedicine: possibilities and

limitations
Extracellular vesicles (EVs) represent an important portion of the secretome. An

overview of their functions in physiological conditions of EVs was compiled by a
recent position paper of the International Society of Extracellular Vesicles (ISEV). 1
Some of the described properties can be used for therapeutic uses, and their testing has
been transformed sometimes into several registered clinical trials. 2 Exosomes are being
applied in antitumour immunotherapy, 3 as therapeutic agents against infectious
diseases, 4 unmodified exosomes for immune-modulatory 5 and regenerative therapies, 6
and modified ones for targeted drug delivery, 7 especially in gene therapy. 8 Although
some of the mechanisms behind their properties remains undescribed, some general
characteristic of EVs make them advantageous over other therapeutic strategies.
Both the structure of the membrane and the formation route are the origin of the
following advantageous aspects: (1) high selective targetability and minimum off-
target effect, thanks to a set of molecules involved in targeting, signalling and receptor-
mediated uptake, complete with all the co-receptors needed for the internalisation
process; (2) capacity of extravasation due to a gel-state core derived from the presence
of hydrated macromolecules (proteins and nucleic acids) combined with a minimum
cytoskeleton that allows deformability while keeping the whole integrity of the vesicle;
(3) Size distribution; (4) great stability in the blood due to the evasion of the innate
immune system; (5) adaptative responses that cause clearance from the blood, with the
corresponding decrease in bioavailability.
EVs can be used as therapeutic agent by themselves or as delivery systems. The

great potential of EVs as drug delivery vehicles has been acknowledged in the
literature.7-12 In most cases, the encapsulated drug acts in collaboration with elements
naturally present in the EVs, creating a synergetic effect. In other cases, EVs serve only
105
as vehicles to reach specific target population, sometimes highly protected from
conventional administration routes.
Nevertheless, limitations of EVs as therapeutic agents have also been reported,

including the absence of a good definition from a pharmaceutical point of view, 13 an
incomplete understanding of their role in the development and spread of pathology,
the absence of methods for the isolation of homogeneous populations and
subpopulations of EVs, and cost-ineffective technology for the availability of sufficient
quantities for clinical trials with constant characteristics. Moreover, it has been
acknowledged that there is little understanding on how biological barriers are crossed
by EVs, and a need for loading methods with scalable properties in clinical translation
has been identified. 14
5.2. Bio-engineered and mimetic EVs for nanomedicine:

classification of artificial EVs
EVs have been modified in the search for broader therapeutic capability.
Sometimes, this included the incorporation of new elements for targeted puporses, in
vitro or in vivo traceability, or the material to be delivered. In other cases, modification
was aimed at the enhancemt of colloidal stability, or change in surface charge to
increase their uptake rate. These new approaches have generate new terms such as
bioengineered exosomes, artificial exosomes, 15 exosome-mimetic nanovesicles, 16
exosome-like nanovesicles, 17,18 and exosome-based semi-synthetic vesicles. 19 These
expressions have been used with different meanings in the literature, but to date, there
has not been yet a clear criterion for their classification. One example is the term
“exosome-like nanovesicles”. In some works, this concept is used to name artificial
vesicles made from cells through different techniques to mimic exosomes.17-18
However, cell-derived vesicles with morphological and biochemical characteristics
similar to exosomes were also named exosome-like nanovesicles by other authors.19-20
Other authors working with non-animal research models used this term to refer to
vesicles with size and flotation density values similar to exosomes. For example,
Regente et al. 21 described the presence of exosome-like vesicles in sunflower plant
fluids, and Prado et al. 22 showed evidence of vesicles quite similar to exosomes during
pollen germination.
106
In order to provide a systematic classification to move around in this new emerging
field, we suggest the nomenclature given in Figure 5.1. This Artificial EVs landscape is
based on the concept behind the term. This way, “artificial EVs” will be used as general
concept to designate all vesicles, modified or manufactured (from natural or synthetic
sources) with the aim to mimic EVs (mainly exosomes) for therapeutic uses. Behind
this term, two categories of artificial EVs could be discerned: “semi-synthetic EVs” and
“fully synthetic” or “EVs mimetic vesicles”, corresponding to modified natural EVs (pre-
or post-isolation) and artificial structures, lab-made or generated from cultured cells.
Type of artificial EVs
ARTIFICIAL EVs
SEMI-SYNTHETIC FULLY SYNTHETIC

EVs-based semi-synthetic nanovesicles* EVs mimetic nanovesicles
Bio-engineering Techniques
Bio-Nanotechnology
SURFACE MEMBRANE CARGO TOP-DOWN BOTTOM-UP

Type of modification
and technology
+ + +
Modification pre- or post EVs isolation From cultured cells From individual molecules
Express new target Enhance bioavailabity Exogenous cargo Increasing release Preparation of a well
and functional of poor soluble drug, delivery to cytosol in yields, while keeping known product, with
Purposes
molecules, or make without off-target target cells constant ability to only desirable elements
EVs traceables effects. New physical incorpórate cargo
and chemical
properties
Figure 5.1. Artificial EVs landscape: explored routes up-to-date for the preparation of artificial EVs for
specific purposes.*EBSSNs (ref. 19)
The former generate semi-synthetic products, as they start from a natural substrate,
which can be subsequently modified before or after their isolation. The modification
affects the structures of the outer surface of the vesicles, the membrane or the cargo
that travels within, and could also include hydrophobic molecules at the membrane.
The term fully synthetic, on the other hand, stresses the artificiality of the product.
We have recently briefly commented their potential in therapeutics. 23 These techniques
can be classified on the basis of their manufacturing route: those starting from larger
substrates (cells) that are reduced to units for the creation of small size vesicles (top-
down nanotechnology) or those taking individual molecules (lipids, proteins, etc.) that
self-assemble in higher order structures with tunable composition (bottom-up
107
nanotechnology). Top-down products differ from natural EVs in terms of micro-
structure and biochemical composition, since they are formed from cell fragments: the
characteristical membrane microdomains 24 (lipid rafts) and associated pools of surface
markers (especially tetraspanins) are absent, and the minimal cytoskeleton is not
present.
5.3. Impact of the artificial EVs classification at the design of new

therapeutic agents based on EVs
The preparation route chosen is important for the final purpose of the artificial EVs,
but it could be critical for the clinical trials and subsequent commercialization. The
extense manipulation of EVs during the bio-engineering methods is the reason for their
classification as advanced therapy medicinal products (ATMPs),2 with a particular
regulatory framewok. Following the same criteria, fully artificial EVs produced from
cultured cells (top-down) should be also incorporated into this category.
On the other hand, botton up artificial mimetic EVs are more difficult to assign to
one or another category. To date, a set of proteins has been fixed on lipidic vesicles
with undefined purpose. While there is some evidence that the EVs membrane is
important for the uptake process,1 the role of the artificial membranes is not clear yet.
Comparative studies of the effects over target cell lines with conventional liposomes
and exosome-mimetic nanovesicles would be very useful to clarify this. The work in
this field is reviewed in the section “Bottom-up methodologies: artificial membranes
decorated with functional proteins to mimic EVs functions.”
A critical evaluation of the fully synthetic EVs concept would imply providing an
answer to questions such as: What we are trying to mimic from EVs?; Is the biochemical
composition, the morphology or the whole entity?; Is it worth mimicking a specific function?.
This is still a challenge in the field, since some of these questions are being answered at
the same time for natural EVs. The best approach would involve an extensive
biochemical characterization of natural EVs and a detailed description of their
functions. Regarding functionality, other populations and not only the target cell lines
should be assayed. This could give information about possible side effects. In the same
way, multiple parameters should be registered as output factors, not just the process
targetted by the EVs-based treatment. Proteins, nucleic acids, and lipid composition
from specific types of exosomes are registered in specialized databases such as
108
ExoCarta, 25 EVpedia 26 and Vesiclepedia. 27 But this is not enough: a database with
assays performed with EVs reporting treated cell lines, EVs type as therapeutical
agents, tipe of assay (in vitro or in vivo) and experimental conditions could be of great
interest to the scientific community. The combination of both sources of information
would be the perfect scenario for the design of future artificial EVs-based therapies.
In any case, both types of artificial EVs should meet product specifications related
to “purity, identity, quantity, potency and sterility” in concordance with the
pharmaceutical market regulations. 28 Once more, there are some important differences
in the definition of these parameters depending on the semi- or fully synthetic
character of EVs. These key points will be considered and discussed in the following
sections.
5.4. Semi-synthetic exosomes: biotechnological modification of

naturally released exosomes
The simplest idea to manufacture EVs would be to use the natural mechanisms for
the formation of vesicles, that is, the cellular machinery itself. It is known that the
composition of the EVs at all levels responds to a high degree of control at very
selective cellular mechanisms. 29 Therefore, the composition of the EVs could be
controlled by intentional alteration of the cellular environment.
This method would lead to the creation of EVs with a composition profile adapted
to a specific purpose. The technological methods used for bioengineering EVs are
explained in the following sections. Two key aspects are the selection of producer cells
(and their in vitro harvesting conditions) and the EVs isolation/enrichment procedures.
Both choices will condition subsequent uses.
5.4.1 Selection of the EVs cellular origin
Cell lines could potentially release EVs vesicles, but there are great differences in
release rate and biochemical composition and their susceptibility to modifications. 30 It
is also accepted that before translation to clinical use of EVs, limitations regarding
biocompatibility, economic viability, harvesting methods, and immunotolerance must
be overcome. A summary of cell lines used for the production of EVs for clinical
purposes, especially drug delivery, can be found in the literature. 31 Dendritic cells and
cancer cell lines, such as melanoma, are the most used lines for EVs production.
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Mesenchymal stem cells (MSCs) are one of the most promising sources of EVs,
especially exosomes. 32 Yeo et al. 33 defended their use for mass production of exosomes
with future therapeutic purposes based on some important facts related to their
advantages over other cell lines. Mainly, MSCs are easy to obtain from all human
tissues (even those considered as medical waste), and they have great ex vivo expansion
capacity compatible with immortalization without compromising their therapeutic
efficacy. These two facts are essential to establish a scalable and long-term source of
well-characterised EVs, particularly exosomes.14,28 In addition, their
immunomodulatory effect gives them and their derived EVs important features in
autologous and allogenic therapeutic applications.
Dendritic Cells (DCs) have important roles in immunity (both innate and
adaptative). Some authors have paid attention to this cell line in order to enhance the
production of clinical grade DC-derived exosomes for immunotherapy.33 Properties of
DCs have even been enhanced with different nanoparticles. 34 Exosomes from DCs
modified to express indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading
enzyme that is important for immune regulation and tolerance maintenance, have been
used in the treatment of rheumatoid arthritis. 35 In another study, DC-derived exosomes
modified to express FasL on the surface, (ligand involved in apoptosis induction), were
tested as inflammatory and autoimmune therapy. 36 Other recent works are related to
the role of tumour-released exosomes to load antigens into DCs for the therapy of
malignant mesothelioma. 37
To overcome the problem of low release rate, some authors lowered the micro-
environmental pH, mimicking the natural cancer mechanism. 38 By culturing HEK293
cell lines at different pH, these authors found that low values of pH were the best to
isolate high amounts of exosomes. In spite of the impact of these results, it would be
necessary to test similar effects in non-cencerous cell lines and to determine how the
change in the harvesting conditions affects EV composition (membrane components
and cargo).
Not only human cell have been explored as EVs source: exosomes isolated from
bovine milk and loaded with different drugs were a promising strategy for mass
production of therapeutic EVs. 39 They can be easily isolated by differential
centrifugation. The biocompatibility of milk-derived exosomes was checked by clinical
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biochemical analysis in an animal model by oral gavage during 6 h (short-term
toxicity) and 15 days (medium-term toxicity).
In recent years, some attention has been drawn to non-animal (especially plants)
EVs and their potential use in therapy. 40 In particular, fruit-derived exosomes (lemon 41
and grapes 42) have been isolated, characterized and tested as beneficial products.
Perhaps this new source of EVs could be used in the near future for the development of
EBSSNs following modifications to express the desired targeting molecules against
specific cell lines. Evidences about immunotolerance should also be provided in order
to avoid any interference in the results.
In any case, studies involving the encapsulation of the same drug into different cell
lines-derived EVs would be desirable in order to clarify whether the beneficial effect is
due to the drug or the combination of drug/type of carrier.
5.4.2 Obtaining a good substrate for modification: isolation

procedures.
Since the final destination of artificial EVs would be the administration for
therapeutic purposes, the highest level of standards would be required in order to
preserve patient safety.27,43 A key point in artificial EVs development is the enrichment
from different biological samples, from cell-culture supernatants to several body fluids.
There are different reviews43- 45 and technique-comparative papers 46- 49 about isolation
procedures. They involve ultracentrifugation, filtration, immunoaffinity isolation,
polymeric precipitation and microfluidics techniques, with different degrees of purity
for the final product. 50 In this chapter, we have focussed on scalability, reproducibility
and synthetic EVs potential damage or physical modification.
Since the efficient function of EVs depends on their size distribution, aggregation
and size changes after isolation are important. Lane et al.47 studied these parameters in
four isolation methods: two aggregation kits, a density-based method and
ultracentrifugation. These authors found that some methods kept a constant vesicle
size, but large differences were observed regarding yield of isolation (the two
sedimentation methods gave recovery values two order of magnitude higher than the
other methods). Another reported obstacle is the co-purification of material with
similar physical characteristics to EVs.42,51,52
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Scalability is also important, since the batch size is correlated- with the homogeneity
of the final product; sometimes low-size batches are more prone to being susceptible to
bias during the process, but on other occasions, higher batches yield more
heterogeneous populations due to microenvironments during procedures.
The scale up step with ultracentrifugation (UC) is limited by the number of rotor
positions and the maximum sample volume. On the other hand, methods such as Size
Exclusion Chromatography (SEC) are easy to scale up by using large columns, but with
the associated longer separation time. Pressure application to reduce processing time
can disturb EVs.43 Other methods, such as immunoaffinity isolations, are only used for
small amounts of original sample because of the high price of the reagents required.
Finally, microfluidic methods45,53 are promising, with possibility to be coupled to
online analysis. 54
Reproducibility is crucial when the product is going to be used in the clinical field.
Comparative results of UC are difficult to obtain due to the high number of models
available in the market, and this could affect the quality of the isolated product.43 The
use of a fixed instrument would keep low variability between batches.47
Welton et al. 55 reported that ready-to-use SEC columns could overcome some
problems related to homemade poured columns,52 thereby avoiding variations from
column to column, and facilitating robust protocols to be used routinely. The main
problem associated with this method is dilution of EVs in the final sample and the
subsequent need for concentration using precipitating agents or UC. This increases
retention time and the possibilities of co-precipitation of other molecules with the same
size and physical properties.
Considerable effort has been made in the field of EV isolation methods, which are
still limiting the expansion of this field. There is not yet a perfect and universal
method, and it is also accepted that selection of the isolation method could impact
downstream steps. 56
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5.4.3 Strategies for biochemical modification
5.4.3.1 Pre-isolation modification using own cellular machinery
The advantages and disadvantages of the methods applied to incorporate proteins

of nucleic acids are shown in Table 5.2 and Table 5.3. Based on the study of exosome
biogenesis-related mechanisms, several methods have been developed [50, 57-62].50,57-
62 The following criteria have been identified for their classification (see Table 5.1):
(I) location of exosomal functional entities, such as transmembrane proteins, and

the use of their natural tropism to co-localized the exogenous element;
(II) strategies using molecular mechanisms for the introduction of exogenous

molecules into EVs for cytosolic delivery;
(III) increasing the amount of molecules into the cellular plasma to be encapsulated
by passive mechanism during MVB formation.
Table 5.1. Classification of techniques for the production of artificial EVs, mainly exosomes, according to
type of final product (semi- or fully synthetic) and the principle of the obtention mechanism
Semi-synthetic exosome production: modification of vesicles naturally produced by cells

Pre-isolation modification
Class I Co-localization of cargo and exosomal carrier moiety thanks to the natural tropism of the
second
Class II Use of sequences (i.e. nucleic acid-based sequences) for the exosomal biogenesis pathway
signaling
Class III Take the advantage of passive loading via increment of their presence, by genetic over-
expression or active loading of producer cells
Post-isolation modification
Class IV or passive Methods that use passive adsorption of molecules into external surface of EVs, thanks to
methods their hydrophobicity nature
Class V or active V.a (Physical methods), based on the creation of transitional alteration in the integrity of
methods EVs that allows cargo to enter into the vesicles by concentration gradient or by passive
incorporation during subsequent restoring of initial status post-stimuli
V.b (Chemical methods), based on induced chemical reactions between EVs and cargo
with or without previous introduction of functionalization agents into vesicles.
Creation of artificial mimetic structures of the natural exosomes
Type I or Top-down Starting from larger substrates (cells) that are reduced to units for the creation of vesicles
bionanotechnology with reduced size
Type II or Bottom-up Starting with individual molecules (Lipids, proteins, etc.) that are assembled in a
bionanotechnology controlled way for generating complex structures of higher order
Class I methods (Table 5.2) involve the design of chimeric constructions of proteins
by genetic engineering. In this case, the fusion between the gene of a protein to be
incorporated and the gene of an exosomal-localized protein can be used for the
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expression of the former on the outer surface of exosomes. This has been referred to in
the literature as Exosome Display technology, 63 and it enables the manipulation of the
protein content of exosomes and the subsequent tailoring of activities. The potential of
the method was successfully demonstrated by the production of specific antibodies
against human leukocyte antigen (HLA), a low immunogenic antigen. 64
Lactadherin C1C2 domain was also used for similar purposes by Zeelemberg et al. 65
and Hartman et al. 66 to induce expression of chicken egg ovalbumin (OVA) peptide
and the human epidermal growth factor receptor 2 antigen (HER2), respectively.
Álvarez-Erviti et al. 67 described a method of inducing surface expression of the central
nervous system–specific rabies viral glycoprotein (RVG) peptide on exosomes isolated
from immature dendritic cells derived from mice. Complementarily, these brain-
targeted exosomes were loaded with siRNA for the first time by electroporation. The
delivery of GAPDH-siRNA specifically to neurons, microglia, and oligodendrocytes in
the brain, resulted in a specific gene knockdown. This was considered the first example
of EV-based genetic therapy. One of the most important facts of this work was the
successful treatment of a highly protected tissue, the brain, in spite of the existence of
brain-blood-barrier selectivity.
Tian et al. 68 used lysosome-associated membrane glycoprotein 2b to target

electroporated doxorubicin loaded exosomes (20% of loading efficacy) produced in
dendritic cells. Ohno et al. 69 used platelet-derived growth factor receptor
transmembrane domain to anchor GE11 peptide, a ligand of the epidermal growth
factor receptor (EGFR). This construction was transfected to Human Embryonic
Kidney cell line 293 (HEK293) using pDisplay vector and FuGENE HD transfection
reagent. As a model cargo, siRNA let-7 was selected for its ability to alter cell cycle
progression and reduce cell division in cancer cells. This siRNA was introduced into
EV producer cells by lipofection method. Modified exosomes (15-21 % of total released
exosomes) were isolated by centrifugation and intravenously injected into an animal
model with induced breast cancer. GE11 peptide as targeting moiety was selected by
the elevated expression of his receptor (EGFR) in tumors of epithelial origin.
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Table 5.2. Pre-isolation methods for cargo incorporation into EVs
Cargo incorporation previous to the release of exosomes

Method EVs Modified Category of Advantages Disadvantages Molecules
component modification incorporated
(according with
table 1)
Genetic Surface Class I -Efficient exposure -Only successfully Peptides, small
fusion of of targeting moiety explored with proteins such as
cargo gene on the surface of exosomal GE11 peptide
with an EVs membrane [69], HLA [63],
exosomal -By selecting the proteins OVA [65], HER2
protein gene EVs protein to be [66], and RVG
fusioned with, [67]
different expression
rate can be
achieved.
Exosome Surface Class I -Suitable to induce -Not tested with -Fluorescent
Surface expression of high molecular proteins [62]
Display protein in both, weight proteins such as GFP and
Technology extravesicular and RFP
intravesicular sides
at the same time
RNA zipcodes Cargo Class II -Alternative to -Applicable only mRNA [70] and
electroporation of to mRNA and miRNA [71]
miRNA miRNA modified with
-Influence of zipcodes
mRNA size not
tested
EXPLORE Cargo Class II -Excellent platform -Limited loading -mCherry, Bax,
platform to load proteins to capacity due to SrIκB and Cre
be delivered to the the presence of recombinase
cytosol of the target fluorescence proteins [61]
cell. reporter proteins
-Expected better in future work,
results than this protein can
commercial be omitted
solutions available.
TAMEL Cargo Class II -By selection of one -High-cost RNA [59] with
platform component of the effective method variable length
platform, the EV- -Required highly
enriched protein experimented
loading efficiency personal
can be controlled -EE values
depending on
RNA molecule
size
Over- Cargo Class III -Used in all types of -Nonspecific RNA and
expression of exosomes loading Proteins by
RNA cargo -Applicable to all mechanism expression of
into types of RNA -Low efficiency, RNA into cell
producing especially for producer cytosol
cells mRNA
Fusion with Surface Class III -High efficiency -Cellular uptake -Hydrophobic
liposomes and/or cargo -Both, hydrophilic rate can be and hydrophilic
and hydrophobic decreased compounds,
compounds can be -Better such as DiI and
loaded efficiencies for calcein
hydrophobic respectively [72]
molecules -Photosensitizer
drugs, such as
ZnPc [72]
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More recently, Stickney et al.62 developed another genetic engineering method for
surface expression of proteins in human cells, called surface display technology. In this
case, tetraspanin CD63 was used as a scaffold for the presentation at both
extravesicular and intravesicular orientations.
Class II methods englobe a heterogeneous group of strategies that have in common

the use of specific molecular interaction between two elements and can be used to
transport the complex into the exosomes.
One example of this strategy is the interaction between specific sequences in RNA
molecules and proteins that are present in the route of exosomes formation.16 Highly
observed sequence motifs into RNA types studied into EVs, called EXOmotifs, was
found in mRNA 70 and miRNA. 71 Exosomes for Protein Loading via Optically
Reversible protein–protein interactions (EXPLORs) and Targeted and Molecular EV
Loading (TAMEL) are technologies based on the molecular interaction between certain
types of proteins or between RNA special structures and specific proteins. Proteins of
interest can also be loaded into the inner compartment of exosomes for their direct
delivery to the cytosol of the target cell as an alternative for therapeutic target
locations. EXPLORs61 has recently presented for that purpose. The system integrates
two elements: one is composed by the genetic fusion of the photoreceptor
cryptochrome 2 (CRY2) to the protein to be loaded, and the other is made by the fusion
of a truncated version of the CRY-interacting basic-helix-loop-helix 1 (CIBN) to
tetraspanin CD9. Both elements can be transitory attached by exposure to blue light,
which induces the interaction between CRY and CIBN, and the interaction can be
stopped once the blue light is not present.
TAMEL is another genetic engineering tool that has recently been published59 for
the active cargo of RNA. This platform is a fusion between an engineered EV-loading
protein and the RNA to be loaded. Engineered EV-loading protein is also a fused
product between an EV-enriched protein and an RNA-binding domain. This
construction is transfected into EV producer cells to make his function. Different
loading degrees can be obtained by selecting the EV-enriched protein. This is related to
the natural expression of different proteins into EVs.
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Class III methods includes the simplest method: passive loading into vesicles
through their biogenesis. There can be two different approximations to this objective.
First, the overexpression of RNA cargo in the producer cells. The major disadvantage
of this method is the lack of selectivity in the loading process since it is gradient-driven
(the higher the concentration in the cytoplasm, the higher its possibility of being
trapped into exosomes during invagination of MVBs formation). On the other hand,
the great advantage of the method is that by translation of mRNA into receptor cell
cytoplasm, codified proteins can also be passively loaded into EVs.
The second approximation explores the active loading of the producer cells, i.e. by
nanocarriers such as fusogenic liposomes. This strategy is based on physico-chemical
properties that govern the type of mechanism (the fusiogenic properties of the two
elements that take place in the method, cells and liposomes). Second, they modify the
whole cell and not only the exosomes.
As an alternative method for the incorporation of exogenous molecules (specially

designed for hydrophobic compound) into EVs, Lee et al. 72 presented the use of
membrane fusogenic liposomes (MFLs). By the treatment of cells with MFLs loaded
with a hydrophobic compound (DiI) and a hydrophilic molecule (calcein), these
authors obtained EVs modified with both molecules. Only slight differences in the
efficiency of incorporation into EVs were found, since a hydrophobic compound would
remain in the plasma membrane after liposome fusion and in the subsequent formation
of EVs membrane through their routes of biogenesis. In contrast, a hydrophilic
molecule would be released into the cytosol. Intercellular transport of both molecules
mediated by EVs was successfully observed in vitro in a multicellular tumor spheroid
model.
A similar method was used to modify the composition of EVs, with special focus on
the modification of the properties of the EV membrane.57 Dyes, fluorescent lipids with
different length and saturation grade of acyl chains, and chemotherapeutics were
loaded into cells by means of EVs.
These authors carried out too a membrane surface modification, with the possibility
of conjugation with molecules for targeting purposes. They first prepared MFLs
containing azide-modified lipids which were fusioned with cells. By simple incubation
with dibenzocyclooctyne (DBCO)-modified peptides, a covalent bond was created due
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to the fast and selective reaction between DBCO and the azide group. 73 This strategy
will allow new possibilities of surface ligand decoration on EVs for targeting purposes
(such as peptides, aptens or antibodies) or for the introduction of molecules with
therapeutic properties via interaction with selective receptors. By the combination of
different head-group modified lipids, several different ligands could be incorporated
into EVs outer membrane, including receptors and co-receptors.
5.4.3.2 Physical and chemical post-isolation modifications
These are the methods that require an external force (chemical or physical) to
incorporate new molecules on previously isolated exosomes (Table 5.3).
Passive Methods. Incubation of EVs and cargo
The simplest way to incorporate any cargo into cell culture or body fluid isolated
EVs is the co-incubation of both elements. This strategy was explored by Sun et al. 74
who found that curcumin exosome-loaded exhibited better stability and higher
bioavailability in serum in an animal model. For these therapeutic-modified exosomes,
an improvement at in vivo anti-inflammatory and septic-shock was observed.
In another study, 75 two anti-inflammatory compounds were loaded into exosomes

and microparticles, and they were administrated intranasally, opening up new
therapeutic possibilities. The effects of solvents and drug release kinetics from loaded
exosomes by dialysis have been studied.39,76
Active Methods. Physical methods: electroporation and other temporary membrane

disruptive methods
The most commonly used method for cargo incorporation into EVs after their
release is electroporation. 77 This technique involves the temporary permeabilization of
membranes through the creation of pores due to the application of high-voltage
electricity. Some authors have pointed out that this method is not suitable for siRNA
cargo into EVs due to technical problems, and an overestimated encapsulation into EVs
could be observed. It has been reported that electroporation induces siRNA
aggregation and co-pelletization with EVs during purification by ultracentrifugation,
without dependence on electroporation buffer composition. 78 They also postulated that
slight differences could be found between different EVs attending their cellular origin
(e.g. primary cells).
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Table 5.3. Post-isolation methods for cargo incorporation into EVs
119
Another relevant problem concerning electroporation is exosome aggregation and
subsequent decrease in functionality. To avoid these problems, Hood et al. 79
electroporated exosomes from mouse B16-F10 melanoma cells by incorporating 5 nm
superparamagnetic iron oxide nanoparticles (SPIONs) as model exogenous cargo.
Other authors compared the loading efficiency of different porphyrins with different
hydrophobicities into EVs with various origins by passive loading (co-incubation), and
by active loading, such as electroporation, extrusion, saponin-assisted drug loading
and hypotonic dialysis.58 The best results were obtained for hydrophobic compounds
and for electroporation. Interestingly, zeta potential (ζ) related to the chemical
composition of EV membranes seems to play a role in loading efficiency, since higher ζ
values led to higher EE. The chemical properties of cargo are also relevant, since their
charge will condition the final outcome. Electroporation did not induce drug
precipitation.
In contrast, extrusion over polycarbonate membranes altered the morphology of

vesicles and, subsequently, their delivery efficacy. Other authors used the sonication of
EVs in the presence of drug solutions.60 The loading efficacy was found to follow the
order: incubation at RT < electroporation << mild sonication. A similar trend was
observed for size changes after the loading procedure. On the other hand, surface
charge and protein profile were similar after loading, evidencing no alteration in
exosome stability. These authors explained the results concerning sonication as a
decrease in bilayer rigidity after sonication, which allowed a better incorporation of the
hydrophobic drug. Therefore, mild sonication should be considered as an enhancement
of co-incubation. Additionally, loaded exosomes were stable over large periods of time
at different temperatures.
A similar comparative study was carried reported, with the enzyme catalase. 80 For
the preparation of exosomes modified with this oxidative stress-protecting agent used
for the treatment of Parkinson’s disease, these authors selected four methods:
incubation at RT in the presence/absence of saponin, freeze/thaw, sonication, and
extrusion. Sonication yielded the higher EE (26.1 %) and the more stable product. In
contrast, this method also produced the higher increment in size, from 105 nm naïve
exosomes to 183.7 nm in catalase-loaded exosomes. Associated with size increment,
AFM observation also revealed a change in morphology, with a final non-spherical
shape.
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Despite these promising results regarding the encapsulation of different molecules
into exosomes, standardization in systematic conditions followed by the study of
several cell lines is still necessary to strongly support the use of these methods as
routine practice in the clinical field.
Chemical methods: click-chemistry mediated functionalization and other targeted

drug delivery strategies
The chemical copper-catalyzed reaction between an alkyne and an azide that forms
a triazole linkage (click-chemistry) has been used for the surface functionalization of
exosomes. 81 These were first chemically modified by the incorporation of alkyne
groups into amine groups from proteins by carbodiimide chemistry. 82 These authors
conjugated azide-Fluor 545 (a fluorescent compound) to activated EVs. Since no
differences in morphological and functional properties were found, it was concluded
that modification by click chemistry does not alter exosome characteristics, and allows
the incorporation of exogenous molecules to the surface of EVs.
Finally, there is another type of cargo modification that has been applied into
artificial vesicular systems (liposomes) with potential applicability to EVs. This method
is based on the ability of some peptides to be incorporated into lipidic membranes
causing disruption. 83,84 By fusion of the peptide D1-7 to an adhesion molecule
expressed in cells, targeted lipidic carriers with therapeutic cargo were produced and
successfully tested in vitro and in vivo. Another interesting application of this strategy
is its ability to insert peptidic cargo into live cell membranes, giving possibilities of
imaging live cells and modifying cell surface. This last property could be explored for
the modification of plasma membrane in EV producer cells.
The modification of EVs membrane results in changes in surface charge, fusiogenic

properties, immunogenicity decrease, and colloidal stability. 85 Engineered hybrid
exosomes were prepared by membrane fusion with liposomes formulated with
different types of lipids (i.e. zwitterionic, anionic, cationic and PEGlilated). Fusion
properties with cell culture-derived exosomes were studied according to the chemical
nature of liposomes lipids. 86 It was found that zwitterioninc and anionic lipids did not
alter the uptake rate, while the introduction of cationic lipid greatly decreased the
phenomena, and PEGilated lipid increased it by two-fold. Therefore, it can be
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concluded that functional properties could be tuned by modifying the membrane
composition.
5.5. Top-down and bottom-up methods for the development of

full synthetic EVs
5.5.1. Production of artificial EVs by generation of plasma

membrane fragments: a top-down inspired methodology
Approaches based on top-down nanotechnology have been developed for the

production of EVs mimetic nanovesicles using cells as precursors of plasma membrane
fragments. Those strategies rely on the principle of self-assembly of lipids and lipid
membranes into spherical structures and the encapsulation of surrounding material
into the aqueous cavity of generated nanovesicles. Current methods include extrusion
over membrane filters,16,17,87 hydrophilic microchannels 88 or cell slicing by SixNy blades
(see Table 5.4).18
5.5.1.1. Extrusion over polycarbonate membrane filters
Jang et al.16 used a serial extrusion through polycarbonate membrane filters with a
decreasing pore sizes (10 μm, 5 μm, and 1 μm) in a mini-extruder, similar to those
commonly used for the preparation of liposomes. Human monocytes were chosen as
precursors for membrane fragments. The yield production of NVs was 100 fold in
comparison with the production of exosomes by using the same number of cells.
Morphological studies of these NVs by crio-TEM and NTA showed many similarities
with the exosomes, round shape and a peak diameter around 130 nm. Even the
exosomal protein marker profile (CD63, Tsg101, moesin and beta-actin) checked by
Western blot was identical for the NVs and exosomes. The chemotherapeutic drugs,
doxorubicin, 5-FU, gemcitabine and carboplatin were added to the buffer where cells
were resuspended. The encapsulation efficiency in the final purified NVs was found to
be dependent on the initial amount of added drug.
122
Table 5.4. Summary of the published work about the generation of mimetic EVs nanovesicles by Top-
Down bionanotechnology. Cell source and type of cargo are encapsulated, and main characteristics are
given.
Looking for a scaled-up process using extrusion as generation procedure of NVs, Jo

et al.87 developed a device that uses centrifugal force to extrude cells over
polycarbonate filters (10 μm and 5 μm of pore sizes). The device has a central piece
where filters are located and connected to two syringes where the sample is dispensed
by the centrifugal force. Uniform-sized 100 nm NVs with a yield 250-fold higher than
123
that of exosome from the same number of cells was achieved. Analysis of the filters by
TEM showed that many cells remained trapped in the structure.
The same device previously cited was employed by Jeon et al.17 to produce
exosome-mimetic NVs from murine embryonic stem cells for the treatment of mice
isolated skin fibroblasts. These authors wanted to explore the potential of mimetic
exosomes to induce proliferation and recovery after injury in an in vitro model.
Genomic and proteical profiles similar to original cells were assessed by PCR and
Western blotting of specific markers, and it was confirmed that successful delivery of
genetic material by NVs was reached.
5.5.1.2 Pressurization, extrusion, and slicing over hydrophilic parallel

microchannels in a microfluidic device
Jo et al.87 produced exosome-mimetic nanovesicles by extruding cells over

hydrophilic microchannels, with the aim of delivering endogenous RNA across plasma
membrane with high efficiency and low toxicity. These authors developed a
microfluidic device made of PDMS by soft lithography. This device had an array of 37
parallel microchannels, with a common inlet and outlet connection for the pumping
with a syringe pump and the collection of NVs, respectively. The higher amount of
nanovesicles generated by extrusion over hydrophilic channels with a similar size to
exosomes was obtained with a length of 200 μm and a width of 5 μm. These results
showed that an appropriate total shear force induced by the channel has to be reached
in order to produce NVs with acceptable results. This force is responsible for NV
generation due to elongation of the plasma membrane on the microchannel surface.
When the elongated membrane reaches a certain value, it breaks into small portions
that directly form nanovesicles, thanks to the self-assembly property of lipids in
aqueous media.
With the appropriate channel morphology, these authors produced 100 nm

nanovesicles similar in composition (proteinical and nucleic acid profile) to naturally
produced exosomes. The analysis of these NVs88 revealed that the formation of
exosome-like NVs through hydrophilic channels produced a delivery system of
endogenous material with identical results as exosomes.
More recently, Yoon et al.18 reported the production of exosome-mimetic

nanovesicles by the slicing of cells through SixNy blades aligned to the flow direction
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over hydrophilic microchannels. These authors combined the induced shear stress
formation on NVs with the fragmentation of plasma membrane by the blades to obtain
fragments for the generation of exosome-like nanovesicles and the co-encapsulation of
exogenous material, (polystyrene latex beads as a model substance). It was found that
NV diameter increased as the width channel increased. This is because channel
morphology is proportional to Reynolds number (Re). In this particular case, Re is
proportional to the hydraulic diameter and, therefore, to the inertial force, which
directly increases with the channel width. In other words, NVs travelling through wide
channels have higher inertial force when they reach the blades, generating larger sliced
fragments that produce bigger NVs. These have a similar composition to that of
parental cells and naturally released exosomes by those cell lines.
One of the most interesting works in the literature18 describes the encapsulation of
22 nm fluorescent polystyrene latex beads as an exogenous simulated material, adding
these nanoparticles to the media where cells were diluted before slicing. With a final
corrected EE of 30 %, these NVs containing exogenous material were given to
fibroblasts in an in vitro experiment. After a period of time, red dots corresponding to
fluorescent beads were detected in the cytoplasm of fibroblast by confocal microscopy.
The delivery efficiency of encapsulated beads into exosome-like NVs was higher than
that of bead-aggregated NVs, revealing that exogenous material delivery with these
NVs was possible, but the efficiency was still lower than that achieved with parental
cell-components generated NVs.
5.5.2 Bottom-Up methodologies: artificial membranes decorated

with functional proteins to mimic EVs functions
The third option for obtaining artificial EVs is their construction in a fully synthetic
way by assembling individual molecules (lipids, proteins and cargo) into complex
structures, such as a bilayer structure resembling EVs membranes functionalized with
proteins for mimicking EVs function. This could be achieved by assuming that not all
components in natural exosomes are essential for specific and efficient delivery,13
including the transport of a message through direct contact with target cell receptors.
Another assumption that encourages researchers to explore this route is that, from a
structural and biochemical point of view, exosomes are liposomes with attached
proteins. Therefore, this type of vesicular systems could be an ideal substrate to
125
develop exosome-mimetic structures. The main functional components of exosomes to
be incorporated in mimetic materials have been reviewed.13 The three main
components of exosomes reported were lipids, membrane proteins and therapeutic
cargo.
One of the main advantages of fully artificial EVs over previous strategies is the
production of pure and well-defined biomaterial. In addition, production strategies of
artificial EVs based on liposomes are more sustainble and easier to scaled up.11,89 This
fact is quite important for preclinical and clinical studies and in order to manufacture
products ready to be sent to the market.2
Liposome preparation techniques have been extensively reviewed,89- 101 but not all
the methods yield vesicles suitable to becoming an artificial exosome. It could be
considered that only small unilamellar vesicles (SUVs) are ideal precursors due to their
similarities to natural exosomes (size range and membrane disposition).
Methodologies for SUV preparation (Table 5.5) can be classified according to

different criteria.92 For example, number of steps to reach SUV. Another classification is
based on the physical principle applied to prepare vesicles: mechanical processes,
organic solvent replacement, detergent removal and other techniques as microfluidic-
based methods. Reverse-phase evaporation, ethanol injection method, ether injection
method (EtIM), thin-film hydration method (TFH), homogenization techniques, French
press cell extrusion, microfluidization, extrusion over membranes, ultrasound and
membrane contactors are some of the techniques developed for SUV preparation.
All these techniques rely on the self-assembly of amphiphilic molecules, such as

lipids, in ordered structures due to their physicochemical behavior in aqueous
media.93,94 This principle is at the basis of bottom-up nanotechnology. Vesicles with a
size-range close to that of natural EVs could be obtained95 when operational variables
were oprtimized by design of experiments. In addition, a wide spectrum of molecules
with biological activity, independently of their physicochemical nature (hydrophilic or
hydrophobic, low molecular weight or macromolecules) can be incorporated into
liposomes, during or after their formation.96
126
Table 5.5. Advantages and disadvantage of most frequently used methods for Small Unilamellar Vesicles
(SUVs) preparation
Functionalization of liposomes with biomolecules is possible, owing to the different

headgroup-modified lipids that are available.97 Headgroup modification usually
includes a molecule of polyethylene glycol as a spacer between the functional group
and the polar region of the lipid. This avoids the sterical hindrance caused by the
proximity of biomolecules and liposome surface. The chemical modification includes
the introduction of different types of functional groups, such as biotin, amine,
maleimide, carboxylic acid, folate, cyanur, DBCO, azide, and succinyl. These groups
127
determine the crosslinking strategy98-101 which should not compromise the biological
function. Bioconjugation should ideally show ideally be carried out under mild
conditions, aqueous media, chemoselectivity, and with a high yield.
Successful conjugation of peptides/proteins with liposomes can be checked using

conventional molecular biology techniques such as dot-blot,15 SDS-PAGE, or even flow
cytometry. 102 A preliminary purification step is required in order to remove
unconjugated biomolecules. For this purpose, authors have used classical separation
methods, such as ultracentrifugation102 or gel filtration15,103 (SEC) with high exclusion
limit resins (Sepharose CL-2B, 4B mainly). Dyalisis, however, is not used due to the
high molecular weight of biomolecules selected for mimicking exosomes.
Undecorated liposomes have also been used in the EVs research field, as EV model
for comparing isolation efficacy and physical integrity,47 detection by flow
cytometry, 104 and EV refractive index study. 105 However, their use as scaffold for
artificial EV development could offer new possibilities in basic research about EVs and
theranostic applications. To date, there are been few examples of this approximation
for the development of mimetic exosomes, and no comparative results are available
owing to the great differences between the methods used. A summary of the main
experimental work on mimicking exosome by bottom-up nanotechnology is given at
Table 5.6 and briefly commented below.
The most frequent preparation technique for SUVs as templates for EVs mimicking
is the TFH method combined with extrusion over polycarbonate membranes and
with102 or without15 previous freeze-thaw cycles. Martínez-Lostao et al.102 had a
formulation that included lipids and stoichiometry inspired in natural exosomes. The
introduction of only 5% (w/w) of an iminodiacetic acid derivative or DOGS-NTA
allowed the binding of APO2L/TRAIL-His10 to liposomes in a single step. Its
bioactivity was a higher activity than that of the soluble ligand. Moreover, a treatment
based on these synthetic exosomes achieved 60% of disease improvement in a
rheumatoid arthritis-induced animal model. In another study, liposome-bound
Apo2L/TRAIL overcame the resistance to the soluble ligand exhibited by
chemorresistive tumor cell mutants. 106 The mechanism of action of LUV-TRAIL in
hematologic cells 107 was also studied by using mimetic structures of exosomes.
128
Table 5.6. Summary of published work about the development of mimetic EVs nanovesicles by bottom-up
bionanotechnology. Formulation of the vesicles, molecules for the surface functionalization and main
physical characteristic (size) are given.
Another approximation to artificial EVs (exosomes) for therapeutic purposes was

carried out by De la Peña et al.15 using a reported formulations. 108,109 The main
components were phosphatidylcholine and cholesterol, and headgroup-modified
lipids such as DSPE-PEG-MAL. In order to make traceable NVs, both in vivo and in
vitro, a fluorescent lipid was included into the formulation, and magnetic nanoparticles
(SPIONs) were encapsulated during thin-film hydration step. After optimization of
chemical-activated ligands binding, mimetic SNVs simulating DCs derived exosomes
were successfully tested as new tools in basic and clinical immunology. A T-Cell
expansion rate higher than that with previously reported experiences using
conventional methods was achieved.
129
An innovative methodology for the production of protein encapsulated
nanoliposomes was also reported.103 They produced 82 ± 4 nm antibody-coated
liposomes with aprox. 93 % EE of BSA. The preparation route that combined a micro-
emulsion containing the protein to be encapsulated, with micelles, in order to create a
lipid bilayer formed through layer-by-layer assembling. In this work, authors selected
a Box-Behnken experimental design to optimize (maximize) the EE by adjusting some
formulation parameters. The final optimized formulation is summarised in Table 4.9.
In this particular case, researchers selected mimetic exosomes for the potential
transmission of antigen to DCs by a controlled target delivery using a conjugated
monoclonal antibody anti-DEC205, a highly expressed receptor on the surface of DCs.
The introduction of cholesterol succinate in the outer layer of the liposomes allowed
the bioconjugation of the Ab by EDC/NHS chemistry.
Despite the promising results and the advantages of these methods for the
development of liposome-based artificial EVs, there are some limiting aspects that
hinder the transfer to the clinical field. While technological progress has allowed the
design, development and production of nanomedicines with high pharmaceutical
grade, their clinical impact has been smaller than expected due to a lack of sufficient
information about in vivo interactions and fates inside the human body.11 Specific
challenges13 are related to the functionalization of vesicle surface with a combination of
functional proteins at the same time because actual methodologies are time-consuming
and because of the incorporation of nucleic acids with acceptable efficiencies. The
dependence of vesicle formulation on parameters such as fusion properties and
stability could be another limit of special relevance to immunotolerance. Finally, the
knowledge about key components in exosomes is not yet complete, since they may
vary from one cell line to another. They could even be health-state-dependent and
sensitive to harvesting conditions.
Despite attempts to mimic the exosome natural lipid composition, there is a need
for actually checking whether the formulation is active and involved in the expected
functions or is just a passive element involved only in the scaffolding of true functional
elements. Comparison of the efficiency of the intended integrated component in
differently formulated vesicles could be an interesting experiment to elucidate the role
of membrane components. Parameters of uptake route and incorporation efficiency
could also be measured with different cell lines, in order to assess the role of the target
130
cell. Other compounds as an alternative to lipids, with a high grade of
biocompatibility, could also be used for the formulation of artificial exosome bilayers.
One option is to use non-ionic surfactants 110 for the preparation of niosome-based
artificial exosomes. These compounds offer some advantages 111 over lipids, such as
price, versatility and sustainability. On the other hand, their chemical structure offers
enhanced stability from both a chemical and physical points of view. Niosomes with
size range close to EVs can also be produced.95
In recent years, microfluidics has been playing an important role in the

development of enhanced vesicular systems, enabling robust and highly controlled
preparation routes of vesicles 112 and allowing rapid characterization of products. 113
Another important aspect in the development of exosome-based therapy, regarding
any preparation route, is the creation of reduced systems for the study of traffic and
delivery into in vivo microenvironments.19 Again, microfluidic-based systems are
opening new possibilities by the development of organ-on-a-chip platforms that enable
the study of these processes in an innovative and highly efficient way. 114
It is expected that microfluidic synthesis of nanovesicles will open the path for new
artificial EVs routes, with the required control of size and EE, and minimal
consumption of reagents.
Other recently explored drug-delivery systems have developed platelet-mimetic

nanoparticles by also using bottom-up technology. These authors have produced
unilamellar polymeric nanoparticles functionalized with immunomodulatory and
adhesion antigens, and they have tested them as another approach to diseasetargeted
delivery. 115
5.6. Conclusions and future perspectives
Knowledge about all the biological aspects related with EVs, especially exosomes,
has opened up new frontiers in the clinical field. After an explosion of publications in
recent years about the role of EVs in physiological and pathological conditions, novel
opportunities for the development of enhanced therapeutic biomaterials have arisen.
These observations could help in the production of new materials inspired by natural
vesicles, without the classical inconveniences associated with up-to-date synthetic
alternatives (liposomes, polymersomes, inorganic nanoparticles, etc.). EV-based
therapies include tissue regeneration or immunomodulation, but drug delivery is one
131
of the most promising applications. Production, isolation, modification, and
purification at large-scale clinical grade are the main limitations of EVs becoming a
true clinically settled therapeutic agent.
These limitations have promoted the development of mimetic material inspired by

EVs, the so-called artificial EVs. In this chapter we have introduced a systematic
classification of the types of artificial EVs according to their preparation routes. Two
well-defined strategies have been developed: semi-synthetic or fully synthetic
products. The first strategy uses natural exosomes as precursors that are modified at
the moment of their biogenesis (pre-isolation modifications), whereas the second
strategy modifies the vesicles after their release by cells and their isolation from
cultured media or biological fluids. Genetic engineering-based modifications, active
loading platforms, specific signaling sequences for selective sorting or precursor cell
modifications with nanomaterials are some of the methods developed for exosome-
based semi-synthetic nanovesicle production.
Fully synthetic vesicles with EVs mimetic properties can be produced by bio-
nanotechnology. Top-down techniques that produce vesicles made of membrane
fragments obtained from the extrusion or slicing of cells, or bottom-up techniques that
take advantage of supra-molecular chemistry (mainly self-assembly) to produce
vesicles from individual molecules, represent the technology developed for that
purpose.
Despite the great potential of artificial EVs, some limitations to their development
as therapeutic tools have been identified. There is not a perfect technique, and,
depending on the final purpose of artificial EVs, combinations of procedures could
offer new insights in the field. Systematic studies with different cellular origins and
target cell lines would expand and consolidate the applications of artificial exosomes.
Comparative work including the encapsulation into different artificial vesicles would
be interesting in order to identify effects due to the carrier.
Multidisciplinary teams with complementary actions in the fields of applied

biology, pharmacology, chemical engineering, material sciences and medicine, would
allow the definitive consolidation of these therapeutic biomaterials in clinical routines.
132
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6. Using factorial experimental design to
prepare size-tuned nanovesicles
Capítulo/Chapter 6
Using factorial experimental design to prepare
size-tuned nanovesicles
6.1. Introduction
Controlled preparation of nanoparticles has attracted great interest in recent years. 1

Nanovesicles are an important family of organic nanoparticles, produced by bottom-up
nanotechnology, with relevant applications in biomedicine, 2 food science, 3 analytical
chemistry, 4,5 or biosensors. 6 They are considered soft nanoparticles because interactions
among their molecular components are similar to those arising from biological systems. 7
Most of the work describing their preparation for specific uses focuses on the
optimization of their composition with the aim of maximizing encapsulation efficiency
or delivery or delivery control.
However, size is one of the most critical properties (together with shape and surface
chemistry) for understanding cell-uptake process and, therefore, bioavailability and
targetability.7 Several studies have focused on the optimization of the drug
encapsulation efficiency, while considering size as just a property for controlling
administration parameters, such as penetration kinetics in topical formulations. For
example, Padamwar et al. 8 studied the encapsulation of Vitamin E in liposomes and
found that the amount of lipids yielded a positive correlation with size, which was, in
turn, negatively correlated with penetration efficiency into the skin. Sometimes, size has
been found to increase with higher amounts of membrane components, such as
cholesterol, whereas it decreased with higher amounts of surfactants (e.g., Tween® 80).
Simultaneously, cholesterol or surfactants could affect encapsulation efficiency (EE).
Optimal situations can be reached as a compromise at intermediate levels of both factors.
In that case, Taha 9 also reported an interaction between membrane components
concentration and size reduction by ultrasounds, making factor optimization an essential
task. In other cases, an opposite effect was observed, and higher concentrations of
membrane components (such as Span® 60 and cholesterol) produced larger sizes and
increased EEs. It is useful to deliver efficient amounts of a selected drug into superficial
143
skin layers without systemic absorption. 10 On this basis, the goal of our work was to set
up a bulk-method for producing nanovesicles of controlled size that could be
subsequently modified for specific applications.
Vesicles are colloidal particles in which a concentric bilayer made up of amphiphilic

molecules surrounds an aqueous compartment. These vesicles are commonly used to
encapsulate both hydrophilic and lipophilic compounds, for food, cosmetic,
pharmaceutical or medical applications, such as diagnosis or therapy. 11 Hydrophilic
compounds are entrapped into the aqueous compartments between bilayers, whereas
lipophilic compounds are preferentially located inside these bilayers. 12,13 The most
common types of vesicles are liposomes and niosomes.
Liposomes were first described by Bangham et al. in 1965, 14 and they are basically
spherical bilayer vesicles formed by the self-assembly of phospholipids. This self-
assembly process is based on the interactions occurring between phospholipids and
water molecules, where the polar head groups of phospholipids are exposed to the
aqueous phases (inner and outer), and the hydrophobic hydrocarbon tails are forced to
face each other in a bilayer. 15 Because of the presence of both lipid and aqueous phases
in liposomes structure, they can be used for encapsulation, delivery, and controlled
release of hydrophilic, lipophilic, and amphiphilic compounds.15,16
On the other hand, niosomes are vesicles formed by the self-assembly of nonionic
surfactants in aqueous media resulting in closed bilayer structures.13,17,18 As liposomes,
their formation process is a consequence of unfavorable interactions between surfactants
and water molecules, and they can also entrap hydrophilic, lipophilic, and amphiphilic
compounds. 19,20
Niosomes exhibit a number of advantages over liposomes, such as higher stability,

easy access to raw materials, lower toxicity, high compatibility with biological systems,
non-immunogenicity, and versatility for surface modification.20
Cholesterol is commonly used as a membrane additive for nanovesicle preparation in

order to improve vesicles stability, entrapment efficiency, and release under storage.20 It
increases vesicle size and rigidity, improving encapsulation efficiency, but at high
concentrations, it could adversely affect the encapsulation rate. 21,22 Cholesterol also plays
a fundamental role in niosomes formation when hydrophilic surfactants are
used (hydrophile/lipophile balance around 10).20
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More than 20 different methods have been identified for nanovesicle preparation,
and these methods were recently reviewed. 23,24 In this work, a modified ethanol injection
method (EIM) is used, because it offers some advantages over other methods, such as
simplicity, absence of potentially harmful chemicals, and suitability for scaleup. 25
The conventional EIM was first described in 1973. 26 In this technique,

lipids/surfactants and additives are first dissolved in an organic solvent, such as diethyl
ether or ethanol, and then injected slowly through a syringe into an aqueous phase
containing the compound of interest. Then, the organic solvent is removed using a
vacuum rotary evaporator. When ethanol is used as organic solvent, the spontaneous
formation of vesicles occurs as soon as the organic solution is in contact with the
aqueous phase, 27 but vigorous agitation is needed to obtain narrower size distributions.
For this purpose, a final sonication stage was applied in this study after organic phase
removal by vacuum evaporation.
However, there is large number of variables involved in this modified EIM, and
selection of the most important of them (screening) is a crucial step in rationally
preparing vesicles by this versatile method. In this work, Z-average size and
polydispersity index (PDI) were selected as the dependent variables. They are
considered to be of great importance in nanovesicles design because most of the final
applications of these vesicular systems are directly related to these two parameters.
Factorial experimental design and analysis of variance (ANOVA) methodology are
appropriate and efficient statistical tools that permit the effects of several factors that
influence responses to be studied by varying the factors simultaneously in a limited
number of experiments.
In the recent past years, Design of Experiments (DoE) has been extensively used for
the study and optimization of vesicles and other similar organic materials. Different
designs can be applied to reduce the number of factors involved in the preparation
techniques 28 and, therefore, to minimizing the number of experiments without losing
valuable information. Plackett-Burman desing is a type of fractional design involving
relatively few runs, 29 commonly used for the screening of variables.
Another important role of DoE is the optimization of nanovesicles composition for

the enhancement of intended purposes. For instance, it has been applied to the
formulation of liposomes (phospholipid and cholesterol ratio) for topical delivery of
145
vitamin E,8 hybrid liposomes (with both low- and high-transition-temperature
phospholipids) to improve the encapsulation and delivery of silymarin, 30 and niosomes
for topical delivery applications.10,31 DoE has also been used to enhance transdermal flux
of raloxifene hydrochloride 32 or diclofenac diethylamine 33 loaded transfersomes, and of
other polymeric nanoparticles encapsulating an anticancer drug. 34 Moreover, the
interaction between vesicles and proteins, such as pectin, to improve drug-delivery
properties has been studied by DoE. 35 Nanostructured lipid carriers (NLCs) loaded with
flurbiprofen were also produced under optimal conditions using full factorial design. 36
In this work, an initial fractional factorial design with two levels (Plackett-Burman)
was used to screen the most important factors in vesicles preparation by the EIM. Then,
a 23 two-level full factorial design using center-point replicates was applied to study the
influence of the main factors and their interactions on Z-average size and PDI. Once the
appropriate operating conditions were determined, vesicles stability was studied by
using multiple light scattering technology and by measuring the encapsulation
efficiencies (EE) of different compounds.
6.2. Materials and methods
6.2.1 Materials
Phosphatidylcholine (PC) (predominant species: C42H80NO8P, MW= 775.04 g/mol)

from soybean (Phospholipon 90G) was a kind gift from Lipoid (Ludwigshafen,
Germany). Sorbitan monostearate (Span® 60, S60) (C24H46O6, MW=430.62 g/mol) and
Cholesterol (Cho) (C27H46O, MW=386.65 g/mol) were purchased from Sigma Aldrich (St.
Louis, MO, USA). All membrane components were dissolved in absolute ethanol
(Sigma-Aldrich).
Methanol, acetonitrile, 2-propanol, and acetic acid of high-performance liquid

chromatography (HPLC) grade were supplied by Sigma-Aldrich.
A phosphate buffer (PB) solution (10 mM, pH 7.4) was used in all experiments as the
aqueous phase. The buffer solution was prepared in Milli-Q water by dissolving proper
amounts of sodium dihydrogen phosphate and sodium hydrogen phosphate, supplied
by Panreac (Barcelona, Spain). Sodium chloride from Panreac was added to increase the
ionic strength when it was required according to experiments listed in Table 1. For the
encapsulation experiments, Fat Red Bluish or Sudan Red 7B dye (C24H21N5, MW= 379.46
146
g/mol) and cholecalciferol or Vitamin D3 (C27H44O, MW=384.64 g/mol) were purchased
from Sigma-Aldrich.
6.2.2 Factorial design of experiments
Factors that could potentially affect the size of vesicles produced by the EIM were
classified in four groups, according to the different steps involved in this preparation
method: formulation (organic/aqueous phase volume ratio, phospholipid concentration,
and ionic strength), injection (injection flow, temperature, and stirring speed), evaporation
(temperature and rotation speed), and sonication (amplitude and time of sonication).
To identify the relative effect of variables on the response, a two-level fractional

factorial design was used. A Plackett-Burman (P-B) resolution III design with n=16 runs
was proposed of the initial factors screening. Two levels were selected for each variable.
Table 6.1 list the factors and levels involved in the P-B fractional factorial design
used, where O/A is the organic/aqueous phase volume ratio, C is the concentration of
phospholipid, I is the ionic strength, QV is the injection flow, TI is the injection
temperature, NS is the stirring speed during injection, TE is the evaporation temperature,
NE is the evaporator rotation speed, A is the sonication amplitude, and t is the sonication
time.
In a second step, a 23 full factorial design with center-point repetitions (n=5) was
carried out to study the main effects and interactions between factors previously selected
by the screening design (Table 6.2). All the other factors were fixed at a certain value.
147
Table 6.1. Plackett-Burman Fractional Factorial Design: Responses, Levels, and Factors
Table 6.2. Full factorial design (23) with center point repetitions (n=5): factors, levels and responses
148
In both designs, mean diameter (Z-average size) and PDI were selected as response
variable. MINITAB statistical software (version 17) was used for all data analysis.
Analysis of variance (ANOVA) was used for this purpose.
Once the models were obtained taking into account significant factors and
interactions, a set of selected size-tuned vesicles were prepared and characterized.
6.2.3 Vesicles preparation
For liposomes preparation, appropriate weighed amounts of PC were dissolved in

different volumes of absolute ethanol (5-20 mL range). The same procedure was applied
to niosomes preparation by weighing and dissolving S60 and Cho in 1:0.5 weight ratio.
Then, the organic solution was injected, with a syringe pump (KD Scientific, Holliston,
MA, USA) at a flow rate of 120 mL/h, into Milli-Q water that was kept at 60ºC and
stirred at 500 rpm. Once vesicles were formed, ethanol was removed at 40°C under
reduced pressure (90 kPa) in a rotary evaporator. The resulting vesicular systems were
further sonicated for 15 min (CY-500 sonicator, Optic Ivymen System, Biotech SL,
Barcelona, Spain), using 30-55% amplitude, 500 W power, and 20 kHz frequency. The
sonication probe was placed in a 100 mL glass beaker at a constant depth, 1.5 cm over
the container bottom.
6.2.4. Vesicles characterization
6.2.4.1. Vesicles size
Z-average size and PDI of vesicles were determined via Dynamic Light Scattering
(DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Three
independent samples were taken from each formulation, and measurements were
performed three times at room temperature without dilution.
6.2.4.2. Vesicles morphology
Morphological analysis of vesicles was carried out by negative staining transmission

electron microscopy (NS-TEM), using a JEOL-2000 Ex II TEM (Tokyo, Japan). A sample
drop was placed on a carbon-coated copper grid and sample excess was removed with
filter paper. Then, a drop of 2% (w/v) phosphotungstic acid solution (PTA) was applied
to the carbon grid and allowed to stand for 1 minute. Once the excess of staining agent
149
has been removed with filter paper, the sample was air-dried, and the thin film of
stained and fixed vesicles was observed with the transmission electron microscope.
6.2.4.3. Vesicles stability
Vesicles stability was determined by measuring backscattering (BS) profiles in a

Turbiscan Lab Expert apparatus (Formulaction, L’Union, France) provided with an
Ageing Station (Formulaction).
Samples were placed in cylindrical glass test cells, and backscattered light was
monitored at 30 °C as a function of time and cell height every 2 h for 7 days.
The optical reading head scans the sample in the cell, providing BS data every 40 μm
in percentages relative to standards as a function of the sample height (in millimeters).
These profiles build up a macroscopic fingerprint of the sample at a given time,
providing useful information about changes in size distribution or appearance of a
creaming layer or a clarification front with time.3,37,38
6.2.4.4. Encapsulation Efficiency (EE)
EE also provides useful information related to the stability of the vesicle membrane.
Hydrophilic compounds are entrapped in aqueous compartments between bilayers,
whereas lipophilic compounds are preferentially located within surfactant or lipid
bilayer. 39 Substances as drugs, bioactive compounds, dyes, and nanomaterials
incorporated into vesicles can also affect the morphology and stability of the final
dispersion.
For the purpose of determining EEs, Sudan Red 7B and vitamin D3 (hydrophobic
compounds) were encapsulated in the two different formulations.
Each compound was analyzed by Reverse-Phase High Performance Liquid

Chromatography (RP-HPLC) (HP series 1100 chromatograph, Hewlett Packard, Palo
Alto, CA, USA). Before RP-HPLC analysis could be performed, the non-encapsulated
compound had to be removed by passing the sample through a Sephadex G-25 column
(GE Healthcare Life Sciences, Wauwatosa, WI, USA). Then, both filtered and non-
filtered samples were diluted 1:10 (v/v) with methanol to facilitate vesicle rupture and
to extract the encapsulated compound. EE was calculated according to Equation 2.
150
peak area of filtered sample
EE = x 100
peak area of non-filtered sample
Equation 2
The RP-HPLC system was equipped with a HP G1315A UV/VIS absorbance detector
(Agilent Technologies, Palo Alto, CA, USA). The column was a Zorbax Eclipse Plus C18
with a particle size of 5 µm, 4.6 mm × 150 mm (Agilent Technologies). The mobile phase
consisted of a mixture of (A) 100% Milli-Q-water and (B) 100% methanol with gradient
elution at 0.8 mL/min. The step gradient started with a mobile phase of 80% A, running
100% mobile phase B in minute 5 for 10 minutes. The mobile phase B was fed for 2
minutes after each injection to prepare the column for the next sample. The separation
was carried out at 30°C. Different wavelengths were used for UV/VIS detector, namely,
533 nm for Sudan Red 7B and 270 nm for Vitamin D3.
6.3. Results and discussion
6.3.1. Effects of variables on morphological characteristics
The responses (Z-average size and PDI) of each batch from P-B design were
measured by DLS. The relative importance of the main effects on Z-average size and PDI
of PC liposomes are shown in the Pareto chart given in Figure 6.1.
Figure 6.1. Pareto chart of the standardized effects of independent variables (factors) on (A) Z-average size
and (B) PDI of PC liposomes for the Plackett-Burman fractional factorial design
Researchers must be aware of the confusion of main effects with two-factor

interactions in this type of design (resolution III), where the alias structure is too
151
complex. However, we decided to use the initial Plackett−Burman design only for
screening purposes and selection of the main factors from the Pareto chart, as is usually
accepted. Effects were selected by applying the hierarchical ordering principle, known
sometimes as the sparsity-of-effects principle, where higher-order effects (three- or four-
way interactions) are sacrificed to study lower-order effects (main effects first and two-
way interactions next). This principle suggests that priority should be given to the
estimation of lower-order effects, especially when resources (time and money) are scarce.
This postulate is an empirical principle whose validity has been confirmed by the
analysis of many real experiments.
According to these data, the most important variables on both responses are
organic/aqueous phase volume ratio, the (final aqueous phase) phospholipid
concentration, and the sonication amplitude. These results are in good agreement with
previous studies carried out by Kremer et al., 40 who evaluated the effect of some
preparation variables on the size and polydispersity of liposomes made from two
different natural phosphatidylcholines. Their experimental results showed that the most
important factor in the final size of liposomes was the lipid concentration in the alcohol
injected into the buffer solution. This factor corresponds to the interaction of lipid
amount and flow rate of organic solvent injected, two factors present in the Pareto chart
in Figure 6.. The same explanation was postulated by other authors,8,41,42 confirming that
the lipid concentration clearly affects the liposome size. This factor was found to be the
most relevant one for controlling morphological characteristics of phosphatidylcholine
liposomes. Szoka et al. 43 found that stirring, ionic strength, and temperature of aqueous
phase could also contribute to final size, but the effects of these factors were smaller than
those observed for lipid concentration, organic/aqueous phase ratio, and chemical
nature of the organic solvent (a parameter not included in our study). Therefore,
experimental results in Figure 6.1 confirm the previously reported observations.43
The ethanol injection method is usually chosen because it avoids the sonication step,
which is needed in several other methods of liposome preparation, such as the thin-film
hydration method. Preliminary experiments (data not shown) indicated that sonication
is a crucial step for reducing the size of both liposomes and niosomes. Alternatively,
small vesicles can be produced without sonication by using low concentration of
lipids/surfactants, but with low yield. This is why we decided to include this step as a
factor at the present study.
152
6.3.2. PC liposomes
The first three main effects from the Pareto chart obtained for the P-B design were
selected for the 23 full factorial design. The ANOVA results for Z-average size and PDI
values are listed in Table 6.3 and Table 6.4, respectively, whereas the corresponding
Pareto chart is shown in Figure 6.2. Mean sizes in the range of 55-156 nm with PDI
values between 0.173 and 0.378 were obtained for PC liposomes (with standard
deviations ranging from 0.304 to 4.40 nm for size and from 0.003 to 0.053 for PDI).
Similar size ranges were also obtained using the EIM in other previously reported
studies.22,27,41,43,44
Table 6.3. ANOVA results (coded units) for Z-average size of PC-liposomes for the 23 full factorial design;
results for S60:Cho niosomes are also given (cursive numbers)
ANOVA indicates analysis of variance; DF, degrees of freedom; SS, sum of squares; MS, mean of squares;
F, Fischer’s ratio; P, p-value.
153
Table 6.4. ANOVA results (coded units) for PDI of PC-liposomes for the 23 full factorial design; results
for S60:Cho niosomes are also given (cursive numbers)
ANOVA indicates analysis of variance; DF, degrees of freedom; SS, sum of squares; MS, mean of squares;
F, Fischer’s ratio; P, p-value.
The normality, variance homogeneity, and randomness assumptions were tested

with the normal probability plot, frequency histogram, and residuals versus fits plot and
residuals versus order plots, respectively.
No clear aberrant tendencies were observed, because the residuals tended to form a
line, no typical cornet pattern was observed, and no time-based pattern was detected.
Only some outliers values were detected (Cook's distance and DFITS values are given in
Table 6.5).
154
A 2.18
B 2.18
A A
C C* A
O:A*C O:A*C
O:A O:A*C*A
C*A C
O:A*C*A O:A
O:A*A O:A*A
C 2.18
D 2.18
C* A C
C A
O:A*C*A O:A
A C*A
O:A O:A*A
O:A*A O:A*C*A
O:A*A O:A*C
Figure 6.2. Pareto chart of the standardized effects of independent variables (factors) on the (A, C) Z-
average size and (B,D) PDI of (A,B) PC liposomes and (C,D) S60:Cho niosomes (1:0.5, w/w) for the 23 full
factorial design.
Table 6.5. COOK's distance and DFITS values obtained for each response in the full factorial designs
155
The ANOVA results allowed for an analysis of the contributions of the effects of the
independent variables on the response function (mean size of PC liposomes). In this case,
significant two-way interactions were identified: (O/A)×C and C×A (see Figure 6.3).
Larger sizes are reached when the organic solution has higher lipid concentration (more
than 20 g/L). On the other hand, C×A interaction reveals that the degree of size
reduction upon application of a higher amplitude depends on the total lipid
concentration present in the medium (referred to final volume of dispersion).
Figure 6.3. Three-dimensional (3D) response surface plots for the factors O/A (organic/aqueous phase
volume ratio), C (lipid or surfactant/stabilizer concentration, g/L), and A (sonication amplitude, %) for the
(A,C) Z-average size and (B,D) PDI of (A,B) PC liposomes and (C,D) S60:Cho niosomes (1:0.5, w/w).
All of the main effects are significant (p-value ˂ 0.05), with a positive effect on mean
size (a higher response value with an increase in the factor level) for the lipid total
156
concentration and a negative effect (a lower response value with a decrease in factor
level) for organic/aqueous phase volume ratio and the sonication amplitude.
Figure 6.4. Contour Plot for the factors O:A (organic:aqueous phase volume ratio), C (lipid or
surfactant/stabilizer concentration, g/L) and A (sonication amplitude, %) on Z-average size, nm (A) and
PDI (C) of PC-liposomes, and Z-average size, nm (B) and PDI (D) of S60:Cho niosomes (1:0.5, w/w).
These effects can be explained according to a previously reported vesicle formation

model. 45- 47 This model relies on the formation of vesicles through intermediate
structures, such as phospholipid bilayer fragments and sheet-like micelles. These
intermediates are the result of amphiphilic self-assembly because of their characteristic
physical-chemical properties. 48
During the injection of ethanol droplets into the aqueous phase, lipids reorganization
inside these dispersed droplets to form bilayers is favored by the fact that lipids
energetically prefer a parallel molecular arrangement.45 These planar structures give rise
157
to closed vesicles when their size induces enough surface tension to close the structure
and to minimize the bending energy.
The size of these intermediates depend directly on the number of lipid molecules
(concentration) and the dispersion degree (solubilization) in the organic phase. It is
obvious from the previous assessment that higher concentration of lipids in the droplets
will form higher membrane fragments, as our experimental results and previous
observations confirm.8,40-42
It is also important to know how easily lipid droplets are dispersed, as well as their
size and homogeneity. Lipids of higher solubility will then form smaller lipid droplets
and, consequently, short membrane fragments (and ultimately tiny vesicles).40 This
explains, in a simplified way, why higher organic/aqueous phase ratio yields smaller
liposomes.
The negative effect of sonication amplitude is explained by vesicles rupture, which

takes place when an excess of energy is applied to vesicles during the sonication process
as a result of the effect of induced cavitation. 49,50 The final effect of ultrasounds can be
controlled by varying the input power, ultrasound frequency, sonication time, and probe
depth into the container. As frequency increases, liposomes of smaller size are produced
as a result of stronger acoustic cavitation events. This assumption was confirmed by our
results, in accordance with previous studies.49,50 It is important to point out that, to
minimize the effects of variations in the probe depth, this factor was kept constant at 1.5
cm above the container bottom.
Another aspect to be taken into consideration is the effect of sonication time. It has
been reported by Silva et al.49 that sonication time plays an important role in decreasing
vesicles size, although they observed that this effect reached a plateau at about 21 min.
Our P-B design revealed a positive effect of sonication time on Z-average size (from 15 to
30 min), although it was weaker than the effects of the other variables selected for the 23
full factorial design (especially sonication amplitude). A similar influence is observed for
PDI response, but with a stronger effect. We preferred to select sonication amplitude
instead of sonication time because one of the goals of controlling factors is to obtain a
narrow size distribution.
As the design included a center point with several repetitions (n=5), the presence of
curvature in the response variables could be tested (Tabla 9.). Because curvature seemed
158
to be significant (p-value ˂ 0.05), a term involving center point (Ct Pt) was included in
the equations for its estimation.
With all this information about the effects and their estimated coefficients, an
equation (R2 = 96.69 %) for Z-average size value of PC liposomes (Y1) was generated in
Equation 3.
Y1 = 62.8 + 2.55 (O/A) + 0.449 C – 0.185 A – 0.0185 (O/A)×C – 0.00555 C×A –

9.26 (Ct Pt) (2)
Equation 3
Different behavior was observed regarding PDI, which was strongly affected by the
sonication amplitude as the only significant main effect and its interaction with total
lipid amount. There is also another interaction (O/A)×C, but with a lower effect on the
PDI response.
To understand the C×A interaction, it is important to take into account the effect of
the sonication amplitude as the main effect. An increase in this factor level leads to less
monodisperse size distribution, that is, higher PDI values. However, according to the
interaction, this response depends highly on the total amount of lipids present in the
sample. At a low level of lipids amount, the reduction in size is more effective (as
previously mentioned), but the size distribution is large. However, at a high level of the
lipids amount, this enlargement of the size distribution is significantly lower.
Curvature in the response was also tested, again revealing a significant presence (p-
value ˂ 0.05). For PDI response (Y2), the following equation with a R2 value of 89.35 %
was obtained in Equation 4.
Y2 = -0.160 + 0.00939 A – 0.0000420 (O/A)×C – 0.0000250 C×A – 0.0425 (Ct Pt)

(3)
Equation 4
These equations are formulated with uncoded coefficients, making it easier to use
them to predict selected target size and PDI values.
159
6.3.3. S60:Cho niosomes
To investigate whether the selected factors in the P−B design for PC liposomes (a
reference model for vesicular systems) produced similar effects with other different
formulations, the same 23 full factorial design using center-point replicates was
performed for a typical noisome formulation, in this particular case, S60:Cho niosomes
(1:0.5, w/w). The main variables were the organic/aqueous phase volume ratio (O/A),
the total concentration of surfactant and stabilizer (C), and the sonication amplitude (A).
The ANOVA results for Z-average size and PDI values are listed in Table 6.3, and the
corresponding Pareto chart and three-dimensional surface plot are shown in Figure 6.2
and Figure 6.3, respectively. Mean sizes in the range 224-362 nm with PDI values
between 0.032 and 0.291 were obtained for S60:Cho niosomes (with standard deviation
ranging from 1.05 to 7.28 nm for size and from 0.009 to 0.052 for PDI). Similar size and
PDI ranges were reported for niosomes prepared by EIM using Span 60 as membrane
component.17
Two-way interactions ((O/A)×A, C×A) and a three-way interaction (O/A)×C×A)

were detected, with sonication amplitude (A) as the common factor in these interactions
(see Figure 6.2.C). Therefore, it can be postulated that sonication amplitude is the key
factor in the niosomes size response. The response depends on both the O/A and C
factor levels, with a higher interaction between sonication amplitude and total amount of
membrane components. Differences in the magnitude of the coefficient of this factor
between liposomes and niosomes can be attributed to the initial size before sonication
(smaller for liposomes) and vesicles stability.50
The three main effects are significant, but in contrast to the case for liposomes, the
organic/aqueous phase volume ratio (O/A) shows a positive effect on niosomes size.
This behavior could be due to different molecular features of the surfactant and
stabilizer that result in different interactions with the organic phase and, therefore, poor
or not enough solubility.
The other two variables (C, A) have effects similar to those described above for
liposomes. Therefore, the same explanation regarding surfactant concentration and
sonication amplitude can be applied here to justify their effects on niosomes size. In this
160
case, the stronger effect of C is explained by the influence of cholesterol on the final size
of vesicles, as reported by Padamwar et al.8
Once again, curvature was detected for Z-average size response. The following
equation (Equation 5) was obtained to model this case, with an adjusted correlation
coefficient (R2) of 91.27 %.
Y3=236.9– 4.31 (O/A) – 0.012 C – 0.56 A + 0.0461 (O/A)×C + 0.00363 C×A – 0.00114 (O/A)×C×A
+ 44.00 (Ct Pt) (4)
Equation 5
On the other hand, a completely different behavior was observed regarding the PDI
response. Only the three main effects (O/A, C, A) were found to be significant, and no
interactions were found. Two positive effects on the niosome PDI were detected:
surfactant/stabilizer concentration and sonication amplitude. In this case, the total
concentration of membrane components seemed to have an important role in the vesicle
size distribution, as can be seen in the correspondent Pareto chart (Figure 6.2). This
observation once again can be attributed to the solubilization of membrane components
in the organic phase. Higher concentration of these components requiere better
solubilization in dispersed droplets to reach small membrane fragments.
It is important to note that some combinations of factors yield narrow size

distributions, namely, PDI ≤ 0.100, a value frequently obtained by other preparation
methods, such as microfluidic hydrodynamic focusing 51 also using also S60/Cho as the
formulation.
A negative effect was detected for organic/aqueous phase volume ratio (O/A). As
the final concentration of ethanol increased during the injection process, a smaller size
distribution was obtained. As previously mentioned, no interaction between this factor
and the total concentration of membrane components was observed.
The Equation 6 with a R2 value of 84.73 % was obtained for the niosome PDI
model response (Y4):
Y4 = 0.053 - 0.00392 (O/A) + 0.000039 C + 0.00067 A + 0.0597 (Ct Pt)

(5) Equation 6
161
The estimated coded coefficients for the considered effects on the Z-average sizes and
PDIs of PC liposomes and S60:Cho-niosomes are listed in Table 6.6, as a summary of the
factorsínfluence. Coded coefficients were used to maintain the orthogonality of the
designs and to allow for a direct comparison between coefficients.
Table 6.6. Estimated coded coefficients for the considered effects on Z-average size and PDI of PC
liposomes and S60:Cho niosomes (1:0.5, w/w)
6.3.4. Vesicles characterization
Size-tuned vesicles were prepared under selected operating conditions by applying

the models obtained from the experimental design (Equation 3-6) and the assistance of
response optimizer and response prediction at Minitab statistical software (version 17).
This tools can be applied to the simultaneous optimization of several responses only
when the same set of factors are studied separately, because a common experimental
region is needed.
The operating conditions were selected to prepare PC liposomes with a mean size of
70 nm and the minimum PDI value (predicted value of Y1= 67 ± 4, and Y2= 0.317 ± 0.013),
and S60-Cho niosomes with a mean size of 240 nm and the minimum PDI value
(predicted value of Y3= 239 ± 11, and Y4= 0.120 ± 0.025). These sizes and PDI values were
selected only as an example. The factor output values were O/A= 5:50 , C= 2 g/L and
162
A= 55 % for the liposomes and O/A= 5.9:50, C= 2 g/L and A= 55 % for the niosomes.
Figure 6.5 and Figure 6.6 show optimization plots and values of individual and
composite desirability for size-tuned liposomes and size-tuned niosomes, respectively.
The experimental results showed that the models obtained with the experimental
design were accurate, since mean sizes of 69 ± 0.5 nm (PDI= 0.245 ± 0.005) and 233 ± 3
nm (PDI= 0.112 ± 0.004) were obtained for PC liposomes and S60:Cho niosomes,
respectively. The relative error was low for the experimental results regarding mean size
(3% for Y1 and Y3), but higher for the size distributions (22% for Y2 and 7% for Y4).
Figure 6.5. Optimization plot and values of individual (d) and composite (D) desirability provided by the
response optimizer (Minitab, version 17) for an example of size-tuned PC liposome (desired size = 70 nm,
with a minimum PDI value).
163
Figure 6.6. Optimization plot and values of individual (d) and composite (D) desirability provided by the
response optimizer (Minitab, version 17) for an example of size-tuned S60:Cho niosome (1:0.5 w/w)
(desired size = 240 nm, with a minimum PDI).
The sizes and morphologies of the vesicles were investigated by TEM, using a
negative contrast. Figure 6.7 shows black-stained vesicles, as a result of the interactions
of the electron beam with PTA, which produces a selective deposit of metal ions that
enhances morphological details. Themicrographs show spherical structures of
approximately 80 nmfor the liposomes (Figure 6.7.C) and about 250 nm for the niosomes
(Figure 6.7.D). These values agree with the DLS measurements.
164
A B
CA DB
C D
Figure 6.7. (A,B) BS profiles and (C,D) TEM micrographs of empty vesicles designed with a controlled size
and PDI values by applying the models obtained from experimental design: (A,C) PC liposomes and (B,D)
S60:Cho niosomes (1:0.5, w/w).
Figure 6.7.D shows clusters of niosomes that are all similar in size. Aggregation arose
during the drying step prior to TEM measurements, because no flocculation phenomena
were monitored with Turbiscan apparatus.
Slight differences were noticed in zeta potential measurements, exhibiting low values
for both types of vesicles. Niosomes had values of about -16.8 ± 0.7 mV, whereas the
liposomes had values of -6.9 ± 0.3 mV. This small value for liposomes could be due to
neutralization of the negative charge from phosphate groups by sodium cations present
in the medium (from sodium chloride in the PBS buffer).
The formulated vesicles exhibited a high stability after 1 week of monitoring time. BS
profiles obtained for PC liposomes are given in Figure 6.5, where a variation of 4.5% in
the middle part of the cell (from 10 to 30 mm) is noticed. A simultaneous slight
clarification process was observed in the middle and top parts of the cell in the
corresponding transmission profile (results not shown). This was promoted by some
movement of PC liposomes towards the bottom of the cell, resulting in a slight increase
165
in BS (sedimentation). However, this was a reversible process, caused by differences in
concentration, with the sample remaining stable and maintaining its initial properties
(size and PDI). The vesicles were again characterized after gentle agitation of the cell at
the end of the monitoring time with analogous results.
For S60:Cho niosomes (Figure 6.7.B), the BS profile remained nearly constant
(variations of approximately 0.5%) with time, showing high stability. Some variation was
also observed in transmission profile profile all along the cell height, since the sample
was not translucent.
6.3.4.1. Encapsulation efficiency (EE)
Vesicles containing Sudan Red 7B and Vitamin D3 as model compounds (both

lipophilic) were also prepared and characterized. No differences were observed
regarding mean size and PDI values or TEM, zeta potential or Turbiscan measurements,
meaning that entrapped compounds did not affect vesicle´s behavior.
High EE values were obtained for both Sudan Red 7B and Vitamin D3, as expected
taking into account their hydrophobic character. EE values up to 90.1% and 88.0%
corresponded to Sudan Red 7B encapsulated in PC liposomes and S60:Cho niosomes,
respectively. Experiments carried out with Vitamin D3 led to EE values of 99.2% for PC
liposomes and 73.9% for S60:Cho niosomes. These results are in good agreement with
those previous studies, where compounds with similar chemical properties were
encapsulated.12,13,27
6.4. Conclusions
In this work, an adequate approximation using DoE was applied to study the
influence of experimental factors of the EIM on the mean size and size distribution of PC
liposomes and S60:Cho niosomes (1:0.5, w/w).
An initial screening design enabled a reduction of the number of variables. This was
a necessary step before carrying out a full factorial design. Finally, response models were
applied to prepare selected size-tuned nanovesicles, which were characterized from a
stability point of view.
This was achieved with a low number of experiments (58 runs). This methodology
enabled two different formulations (liposomes and niosomes, the most common types of
166
nanovesicles) to be studied in a comparative way. Stable liposomes and niosomes of the
targeted sizes were successfully prepared with the model equations obtained, with
encapsulation efficiencies higher than 73.9 % in all cases for selected hydrophobic
compounds.
The most important variables identified by ANOVA were the organic/aqueous

phase volume ratio, the (final aqueous phase) phospholipid concentration and the
sonication amplitude.
These results offer new insights into the mechanism and effects of the factors
involved in nanovesicles preparation by the EIM, one of the most easily scaled-up
methods for preparing vesicles for several fields of interest.
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171
7. Continuous flow production of size-
controllable niosomes using a thermostatic
microreactor
Capítulo/Chapter 7
Continuous flow production of size-
controllable niosomes using a thermostatic
microreactor size-tuned nanovesicles
7.1. Introduction
A precise control over local environment during production of colloids is essential to

minimise perturbations in chemical characteristics that could lead to heterogeneous
populations, and then, differences in particle properties. To achieve such homogeneity
and uniform properties, a strict control of particle size is necessary. 1,2
Nanovesicles (organic colloids) are particles formed by self-assembled amphiphilic

molecules into closed bilayered structures with an inner aqueous core. Depending on the
chemical nature of bilayer constituents, these particles are categorised into liposomes
(lipids), niosomes (non-ionic surfactants) or polymersomes (block copolymers), as most
frequently found in the literature. 3- 5
Niosomes exhibit unique advantages over the other types of vesicular systems due to
their inherent characteristics of non-ionic surfactants. 6,7 These advantages include; (i)
better chemical and physical stability of suspensions due to the absence of oxidation-
related degradation, (ii) easy derivatization to introduce different functional groups for
stability enhancement or bioconjugation, (iii) wide range of surfactant types available
(with single or double acyl chain, with different length or saturation), (iv) high
immunological tolerance, and (v) cost effectiveness. Firstly introduced in the cosmetic
industry by L’Oreal for dermal bioactive compounds delivery, 8 over the last 15 years
their applications have expanded to many fields. Food fortification, 9 diagnostic agents, 10
analytical chemistry, 11 nanomaterial synthesis, 12 and drug delivery 13 are just some of
examples. For all of these applications, a product with specific characteristics,
homogeneity and reproducibility is desired, and in particular, controlled size and
monodispersity are essential.
Effort has been made to the production of niosomes by traditional methods with tight
control over size and size distribution 14 for some specific applications. 15 For example, in
173
our previous work, 16 we have used experimental design to study the influence of
variables in the ethanol injection process, in order to improve particle size tunability.
One of the most popular chemical families for niosome production involves sorbitan
esters (commercially available as Span®). Span family members differ in terms of acyl
chain length and saturation, with a big range of hydrophilic-lipophilic balance values
(HLB), where HLB is an important parameter with implications in drug encapsulation
efficiency and morphological characteristics of particles. This parameter is also related to
the physical state at room temperature (RT), and influences the minimum temperature
(together with gel-to-liquid transition temperature, or Tc) that is required at the very stage
of the particle formation. On the other hand, some of the compounds used in
formulations with great loading capacity, low release rate and stability in solution are
solid at RT. For these reasons, a higher and controlled temperature level is mandatory
for this process.
Microfluidics technology is very promising for precise control over input variables
when mixing chemical species. 17 Other advantages include low consumption of
chemicals (relevant in formulation optimization), scale-up possibilities for industrial
production, on-line coupling to other processes (such as purification steps), and efficient
control over temperature if required. 18 Jahn et al. 19 reported for the first time the
hydrodynamic flow focussing (HFF) technique (Figure 7.1) for liposomes production.
Following that, other researchers have used this method to examine various liposomes
formulations and for encapsulating either hydrophobic or hydrophilic molecules. 20,21
Under laminar flow conditions within the HFF configuration, a stream of lipids in
organic phase is focussed between two aqueous streams in microchannels, allowing the
mixing of chemical species by molecular diffusion. At the two organic/aqueous
interfaces, bilayers can be formed and self-assembled into liposomes once a critical
concentration is reached. By controlling the flow, the extension of mixing and hence the
size of liposomes, could also be controlled. However, the production of niosomes
through microfluidic routes remains less explored, and limited attention has been paid
to using HFF technique. 22-24
174
Figure 7.1. Schematic diagram of a continuous flow microreactor based on hydrodynamic flow focusing for
vesicular systems production. The reduction of focused stream width under laminar flow conditions makes
possible the mixing of chemical species by molecular diffusion, since time for mixing decreases with the
square root of distance. By changing flow rates, the kinetics and extension of mixing can be modified, and
then, the size of particles. Amphiphilic molecules are self-assembled into bilayers once critical concentration
of solvent is reached, and molecules acquired an ordered state to minimize the interaction with water
molecules. At a certain size bending modulus induce planar bilayer to be closed into vesicles.
At present, the high temperature required for the preparation of niosomes has not
been well taken into account in microfluidics routes. For example, the previous work
that firstly explored microfluidics assembly of niosomes faced such temperature related
challenge, thus only included Span® 20 and Span® 80 (Tm = 25 °C and -30 °C,
respectively) in the study.22
Along with the wide application of continuous flow microreactors for organic
colloids preparation18 is the development of microreactor itself, including design and
manufacturing of such microdevices, with simpler and more affordable production
methods. 25 As a result, some traditional fabrications methods which stem from the
photo-electronics field, such as photolithography, 26 are being substituted by new
processes that require less expensive equipment and can be performed in common labs
with no need for clean rooms facilities. 27 Among the techniques explored, additive
manufacturing, especially 3D-printing, has emerged as a promising method for
microfluidic device manufacturing. 28 The rapid development of 3D-printing technology
and the commercialization of desk printers have enabled researchers to explore its utility
175
in microfluidic prototyping and manufacturing, 29-31 that generally use low cost raw
materials and can print objects with desired resolution.
The aim of the present work was to develop a thermostatic microreactor platform for
the continuous flow production of niosomes in a size-controllable manner. The
microfluidic reactor was designed with a hydrodynamic flow focusing configuration,
and fabricated in order to allow visualization of the dynamic process including
molecular diffusion, with the aid of an inverted microscope and a digital image
acquisition system. 3D-printing technology was used for fabricating the microfluidic
device (positive mould) and thermostatic system. The effect of operational parameters
was investigated on the final morphological characteristic of niosomes. Niosomes were
formulated with non-ionic surfactants with different transition temperatures (Tm) with
controlled temperature as a tailoring parameter to tune the size and homogeneity of
particles.
7.2.1. Materials
Sorbitan monostearate or Span® 60 (Sigma-Aldrich), sorbitan monolaureate or

Span® 20 (Sigma-Aldrich), cholesterol from lamb wool (Akros Organics), Phosphate
Buffer Saline (10 mM, pH 7.4) prepared from tablets according to manufacture
instructions (Sigma-Aldrich), Bromoxylenol blue (Sigma-Aldrich), and technical grade
solvents such as ethanol absolute, 2-propanol (or isopropyl alcohol, IPA), and acetone
(all from J.T. Baker, Avantor, USA) were used in this work. Ultrapure water was used for
all experiments. Poly(dimethylsiloxane) monomer Sylgard® 184 or PDMS was
purchased from Dow Corning Corporation (Auburn, AL, USA). Other materials used for
devices fabrication are specified in the following respective sections.
7.2.2. Thermostatic system fabrication
Thermostatic chamber was design in Autodesk® Inventor® and 3D-printed with

PLA filaments using a special printer for fused deposition modelling (Ultimaker 2+ 3D
printer, Ultimaker B.V., The Netherlands). Main chamber and cap of the device were
produced separately. A microscope glass slide of 50 x 70 mm (Corning® microscope
slides, Sigma-Aldrich, Gillingham, UK) was sealed to the chamber with a 2-phase
176
adhesive glue special for plastic materials, bought in a local store. A transparent piece of
plastic was glued to the cap aperture with the same adhesive used with the other piece.
Teflon tape was used to enhance the closure of both elements in a removable way. Holes
for the inlets and outlet pipes of the microfluidic device were manually prepared with a
sharp tool.
The previously described chamber was connected to a temperature-controllable

recirculation system (F12-MC, Julabo GmbH, Germany) through the inlet, and a
peristaltic pump (MasterFlex®, Cole-Parmer Instruments Company, USA) through the
outlet. The plastic pipes were those from the recirculator, and connections to the
chamber were made with common plastic adapters (see supplementary material).
External supply of the recirculator was set approximately at a flow rate of 55 % of the
total volume, while peristaltic pump revolution rate was adjusted to remove water from
the chamber at a rate that allowed a continuous and constant flow through it.
Temperature inside the chamber was monitored with a digital temperature probe (Testo
110, Testo SE & Co., Germany). The sensor probe was introduced into the chamber
through a hole placed in one side of plastic window of the cap (see Figure 7.2).
Figure 7.2. Pictures composition showing the whole setup (central) and detailed components (sides) used in
this work for niosomes production by Hydrodynamic Flow Focusing with controlled temperature.
177
7.2.3. Microfluidic devices manufacturing and channel
characterization
Master mould of devices was designed in Solidworks® CAD 2016 software and 3D-
printed onto VeroClearTM resin with the HR-3D printer Objet350 ConnexTM (Stratasys
Ltd., USA). A post-printing process was also needed. First, mould was flushed with (I)
IPA, (II) deionized water, (III) acetone, and finally compressed air. Then, it was cured
overnight at 60 °C, and on the following day a treatment of the inner surface was carried
out with Aquapeel® (to avoid interference of the resin with PDMS curing process).
Three individual moulds were printed.
Once the positive mould was ready, a mixture of degassed PDMS curing agent (1:10
w/w) was poured into it, and left overnight in an oven at 40 °C. For degassing the PDMS
mixture, a bench centrifuge was used at 3000 rpm for 10 min. It should be noted that
pouring into master mould must be done slowly to minimise bubble formation. On the
following day, the replica of the mould was carefully peeled off from the mould, and
inlets and outlet holes were prepared with a 1.5 mm biopsy punch with plunger
(Miltex®, Fischer Scientific, UK).
Oxygen plasma (PVA-TePla 300 plasma cleaner, Wettenberg, Germany) treatment

was applied to bond a microscope glass slide (50 x 70 mm; Corning® microscope slides,
Sigma-Aldrich, Gillingham, UK) to the PDMS replica to complete the microfluidic
channel. Four pieces of thermic resistant plastic (Ø 8 mm and 3 mm height) were glued
in each corner at the bottom of the glass slide, to elevate the device allowing a flow of hot
water under the channels.
Polytetrafluoroethylene (PFTE, 0.5 mm I.D.) pipes (Cole-Parmer, UK) were inserted

into the holes, and the other end was attached to a syringe needle to create a connection
for introduction of the fluids from syringe pumps (NE-300, NEW ERA Pump Systems
Inc., USA). Luer lock syringes (Becton, Dickinson and Company, UK) of 1, 10 or 20 mL
were used depending on the selected Flow Rate Ratio (FRR), i.e. volumetric flow rate of
total aqueous phase/ volumetric flow rate of organic phase.
The mixing channel (23 mm long) on the 3D-printed positive mould was
characterized in terms of morphology, accuracy and reproducibility by mechanical
profilometry (Talysurf-120L, Taylor-Hobson, United Kingdom). Three equidistance
178
measurements were taken (2 mm across the channel, perpendicular to it), and data were
processed with OriginPro 18 (OriginLab Corporation, USA) software.
The whole setup (microfluidic device inside the thermostatic chamber with
respective inlets and outlets) was placed over the stage of an inverted microscope
(IN200TAB series, AmScope, USA) with a digital imaging system to capture images
(5M.P USB CCD camera, AmScope, USA) supported with the software supplied by the
camera manufacturer. The entire experimental setup is illustrated in Figure 7.2.
7.2.4. Niosomes production and morphological characterization
Working solutions of 5 and 20 mM of Span® 60:cholesterol and Span® 20:cholesterol

(1:0.5 molar ratio) were prepared by dilution from a 50 mM stock solution. Ethanol
absolute was used as organic solvent, since it is miscible in aqueous buffer (PBS, 10 mM
pH 7.4). Aqueous and organic phases were pumped into microfluidic device once
appropriate temperature was reached. Three different total flow rates (QT) were studied
(50, 100 and 200 µL/min), and aqueous:organic flow rates were adjusted to five different
flow rates ratios (FRR) (5, 15, 25, 35 and 50). Span® 20:cholesterol formulation was
injected at 30, 40, 50 and 60 °C; while Span®60: cholesterol was only injected at 50 °C. All
the combination of membrane components concentration, QT, FRR, and temperature was
conducted by duplicate.
A total volume of 2.5 mL was collected from the outlet of the device for each
experimental condition in a glass vial. Size (z-average or peak value, depending on the
number of peaks in the size distribution) and homogeneity (PDI) of particles were
measured by Dynamic Light Scattering (DLS) in a Zetasizer NANO-ZS equipment
(Malvern Instruments Ltd, Malvern, UK). Samples were measured undiluted by
triplicate, with the 173° backscatter detector in disposable low volume cuvettes (Malvern
Instruments Ltd, Malvern, UK).
7.2.5. Mixing efficiency visualization
Solvent and no-solvent diffusion by hydrodynamic flow focusing was monitored by

an adaption of a previous published methodology. 32 Briefly, a change in colour of a pH
indicator dye (bromoxylenol blue) was used, since this dye exhibits a strong yellowish
colour at pH below 6.0 and blue at pH above 7.6. A saturated solution of dye in absolute
ethanol acidified with acetic acid was focused by PBS adjusted to pH 10.0 with 2M
179
NaOH solution. Once focused, a change in colour of the stream from yellow blue
indicated a molar fraction of aqueous phase close to one, and then, completes mixing by
diffusion.
With the microreactor platform developed, systematic characterisation and operation

were conducted in terms of 3D printing outcomes and nanoproduction, as detailed
below. (The performance and optimization of the thermostatic system are shown in
Figure 7.3).
Figure 7.3. Calibration plot (A) and temperature stability (B) of the in-house designed thermostatic
chamber for microfluidics chips, fabricated by 3D-printing technology with PLA filaments. Values
represented are the average of three independents measurements.
7.3.1. High resolution 3D-printing of master moulds for

microfluidic devices fabrication
As a key element of the device, mixing channel morphology was characterized by

mechanical profilometry onto 3D-printed positive moulds. A considerable difference in
nominal dimensions between Computer Aided Design (CAD) and printed object was
observed (Table 7.). With an original squared cross sectional geometry of 100 µm width
and 100 µm height, printed features onto VeroClear® resin showed a curved morphology
five times wider and approximately half of the height. At the same time, variations in
width and height of the mixing channel were found between the three 3D-printed
positive moulds (see Table 7.1) even following the same fabrication procedure.
180
However, these dimensions were reasonably constant along the mixing channel length,
especially for channel height.
Table 7.1. Morphological characteristics of mixing channel for original Solidworks® CAD 2016 design
and 3D-Printed positive moulds (3D-PM) onto VeroClearTM resin with the 3D printer Objet350
ConnexTM (Stratsys). Average and standard deviation values are given for the parameters.
Width Height Cross sectional area

Mould
(µm) (µm) (µm)
CAD 100 100 10000

3D-PM1 535 ± 40 47 ± 2 13090 ± 485
3D-PM2 423 ± 14 51 ± 1 11605 ± 262
3D-PM3 507 ± 28 68 ± 3 18007 ± 1092
A possible explanation for these variations in channel dimensions could be related to

printer operational parameters. Objet350 Connex3 printer used PolyjetTM inkjet-head
patented technology for a layer-by-layer process based on Stereolithography. 33 The
jetting head dispensed a proper amount of a photopolymer resin onto a build tray and
instantly cured them with UV light. The process took place in XY-axes to create a 2D
sheet (down to 16 microns thickness), and by lowering the build tray, another layer was
created over the previous one. The cycle was repeated until the whole design was
completed. With a resolution of 600 x 600 x 1600 dpi (X-Y-Z-axes respectively) and an
accuracy of 20-85 microns for features below 50 mm (up to 200 microns for full model
size), the final features depended on geometry (proximity between elements), build
parameters (exposure time, printing speed) and model orientation.29 Comina et al.29
reported the successful printing of positive moulds for microfluidics devices with
elements from 50 µm to 2 mm, however, some artefacts were described between close
elements with 50 µm in dimension differences, though working with optimized
parameters. Unfortunately, no details about cross section geometry were given for these
channels. Some other authors 34 have reported differences between CAD and printed
designs with efforts in resin formulation optimization.
In our recent work,31 we found that 3D printed channels with the Objet350 Connex3
printer were smoother than channels printed with a conventional desk 3D printer
(Ultimaker 2+). However, for the same dimensions and aspect ratio, accuracy in cross
sectional shape was lower for the HR-3D printer even at large dimensions (1 mm
181
squared channels). It suggested that further studies are needed to understand this effect
with the scale and for different materials in order to inform printing parameters
optimization in terms of element dimensions, geometry, and printing materials. Apart
from the difference between CAD and 3D-PMs, the cross sectional area of Mould 3 was
similar to that previously used by Lo et al.,22 on which the selected operational
parameters of the present work were based.
7.3.2. Production of nanoparticles with temperature control for

formulations with high Tm non-ionic surfactants
The use of non-ionic surfactants for the formulation of organic colloids, especially for
NVs preparation, exhibits numerous advantages.4,6 However, a strict control of the
temperature is necessary if Span®60 (Tm = 45 °C), one of the most commonly used
surfactant in niosome formulation) is involved. Figure 7.4 shows its precipitation at RT
in microchannels once reaching the focusing region, highlighting the significance of
temperature effect.
In Figure 7.4 surfactant precipitation was observed at the focussing region and
persists along the channel length when Span® 60 is used at 25 °C. However, at 50 °C a
complete mix of both phases were produced without the presence of any surfactant
precipitation. Moreover, the production of niosomes at this temperature conditions were
observed using Transmission Electron Microspcopy (TEM) and negative staining
protocol.
This technique has been less explored than traditional bulk preparation routes,18 and
with important advantages such as better control over particle preparation and the
subsequent final characteristics (size and monodispersity, i.e.). This is important for
biomedical,1 food 35 and analytical chemistry2 applications. In this regard, the influence of
operational conditions over particles physical properties was tested by analysing the
results of 3 total flow rates (QT), two different concentrations of bilayer components, for
5 different FRR. Particle size (nm) and size distribution (PDI) were measured by DLS as
output variables. All the combinations were conducted at 50 °C, a temperature over
surfactant Tm.
182
Figure 7.4. Figure 2. Precipitation of Span® 60 (Tm=45 °C) at room temperature (upper arrow) at the
focusing region (left), 0.5 and 1.0 cm downstream (centre and right). At a temperature above surfactant
Tm, focusing is complete and vesicles formation could be checked by negative staining (Phosphotungstic
acid 2%) and Transmission Electron Microscopy (TEM).
In general terms, smaller particles were produced as the FRR increased (Figure 7.5.A
and Figure 7.5.B) for both concentrations (5 and 20 mM), and for all the QT levels. At a
concentration of 5 mM (Figure 7.5.A), the particle size decreased from 278, 298 and 358
nm (when FRR = 5) to 155, 129 and 143 nm (when FRR = 50), where QT = 50, 100 and 200
µL/min, respectively. At a concentration of 20 mM (Figure 7.5.B), similarly, the particle
size reduced from 342, 361 and 386 nm (when FRR = 5) to 164, 147 and 151 nm (when
FRR = 50) at the three QT levels of 50, 100 and 200 µL/min, respectively. Size reduction
was rapidly reached with an increment in FRR from 5 to 15, and this reduction became
less pronounced from FRR 15 to 50. It is important to take into account that when FRR
increased the total amount of bilayer components decreased, not only producing vesicle
with smaller size, since particle concentration was also reduced.
No significant effect of different QT was observed, while only some differences were
noticed in some particular combinations of parameters at low surfactant concentration (5
mM), as seen in Figure 7.5.A. These observations were in accordance with previous
studies22 carried out with identical chip configuration for the production of niosomes
formulated with other sorbitan esters (Span®20 and 80), and also for the production of
liposomes.19,20,36,37 At lower QT, also the linear velocity was lower (hence larger residence
time) what can counteract the effect of the bilayer components concentration.
183
Figure 7.5. Size (nm) and size distribution (PDI, a.u.) measured by DLS in undiluted samples from
niosomes formulated with Span® 60:Cholesterol (1:0.5 molar ratio) at 5mM (A,C) and 20mM (B,D) in a
continuous flow microreactor based on hydrodynamic flow focusing at controlled temperature (50 °C). Each
condition was tested twice, and each batch was measured by triplicate.
The increase in FRR, and the subsequent decrease in initial focused width (Wf),
reduced the time needed for a complete mixing between solvent and no-solvent (tmix,),
thus the critical concentration to induce molecules self-assembly was reached faster. This
led to smaller vesicles since the total amount of bilayer components was reduced. 38 On
the other hand, the reduction of solvent introduced in the mixing channel also decreased
the possibility of particle fusion into bigger unities by Ostwald-ripening phenomena.20,39
A reduced tmix also led to complete mixing in limited length channels. In other cases, no
diffused solvent containing amphiphilic molecules self-assembled out of the channel
under entirely different conditions (outlet pipes, with no laminar flow characteristics).
Regarding size distribution of particles (Figure 7.5.C and Figure 7.5.D), PDI value
reduced as FRR increased from 5 to 15, (for 5 mM: from 0.119, 0.141 and 0.163 nm at FRR
= 5 to 0.092, 0.071 and 0.093 nm at FRR = 50; for 20 mM: from 0.080, 0.131 and 0.118 nm
at FRR = 5 to 0.061, 0.057 and 0.071 nm FRR = 50; for both concentration values are
184
indicated for QT = 50, 100 and 200 µL/min respectively), and remained without
significant changes at higher FRR for both concentrations. Some authors19,20,32 reported a
significantly increase in PDI with the increment of FRR for an identical chip
configuration, but for liposomes production instead. However, our observation was in
line with that of Bottaro et al. [32] in a “Y”-shaped device, while Joshi et al.21 described
also a reduction in PDI as FRR increase during liposome formation. No significant
differences on PDI were observed for all QT levels applied.
The use of microreactors with different channel configurations, and the use of static
mixing enhancers, 40 could be the reason of different results among published works.
Some of them have highlighted the influence of channel dimensions and configurations
over mixing efficiency and particle properties.19,22,36 The preparation of solvent mixture
containing bilayer precursors can also influence the extension and homogeneity of
solubilisation, and in consequence, nanoprecipitation process. In the present work,
ethanol was used as solvent for microfluidic-based preparation of niosomes for the first
time, and this limited the possibility for comparison with other studies.
We have noticed that at high FRRs some transitory perturbations of the focused fluid
were recorded, especially at 50 µL/min. The focused stream exhibited a “beating pulse”
like effect that was likely produced by the syringe pump due to its own pumping
mechanism. These pulses created really short increments in the width of the focused
fluid that introduced alteration in solvent exchange kinetics and the subsequent changes
in the local concentration of bilayer precursors and solvent concentration.
Surprisingly, lower PDI values were obtained at 20 mM for all FRRs at the three
different QT. Indeed, these differences were higher at 50 µL/min. At low concentration,
those mentioned instabilities can induce more pronounced local changes in bilayer
precursor’s abundances, with the corresponding effect in particle monodispersity. To
gain insights into these observations further studies are needed.
On the other hand, larger particles were obtained when a higher concentrated
ethanolic solution of bilayer components was used (20 mM vs. 5 mM). This was observed
at all QT and FRR levels (see in Figure 7.6). The same observation was also reported by
other authors when producing liposome using microchannels,37 and in agreement with
the mechanism of vesicle formation under microfluidic flow dynamic mixing.
185
Figure 7.6. Effect of bilayer components concentration for niosomes formulated with Span® 60:Cholesterol
(1:0.5 molar ratio) and produced under the same flow conditions (QT and FRR) for size (upper row) and
size distribution (lower row).
The efficiency of mixing under the assayed working conditions was studied
following a published methodology.32 With this method, mixing efficiency was
measured through the change in colour of a pH indicator dye (bromoxylenol blue), that
changed from yellow (acidic ethanolic solution containing bilayer precursors) to blue
(basic aqueous phase, PBS pH= 10). A shift in focused fluid colour from yellow to blue
indicated that molar fraction of water into the stream was close to 1 and the subsequent
molar fraction of EtOH became close to 0, evidencing a complete mixing by solvent and
aqueous effluents. This change in colour was easily detected in the inverted microscope,
and recorded with the digital camera. As an example, results for QT = 100 µl/min at
several FRRs are shown in Figure 7.7.
186
Figure 7.7. Images (4X) of focusing region and end of the mixing channel evidencing hydrodynamic flow
focussing of a central ethanol stream at different FRR values for QT=100 μL/min and 50 °C. Yellow colour
indicates acid pH (pure EtOH, no mixing), while blue colour indicates basic pH (complete mixing by
codiffusion of solvent and no solvent). Bromoxylenol blue dye was dissolved in EtOH (acidified with acetic
acid), and PBS was adjusted with NaOH to pH 10.
Complete mixing was only reached at high values of FRR (35 and 50) for QT = 50
µl/min and QT = 100 µl/min, and only at the high FRR (50) for QT = 200 µl/min. As QT
increased, residence time of the fluid inside mixing channel reduced (from 0.5 s at 50
µl/min to 0.13 s at 200 µl/min), preventing to stay the necessary time to reach complete
mixing. Only at high FRR value, tmix was short enough to be compatible with low values
of residence time for our channel dimension (tmix = 17 ms and 8 ms for FRR = 35 and 50,
respectively, predicted according to a theoretical model (Equation 7, 41). In this model Wf
represents the width of focused stream, where w is the channel width and D is solvent
diffusion coefficient.
187
Wf2 w2 1 Equation 7
tmix ~ ≈
4D 9D (1+FRR)
As seen in Figure 7.5, Wf decreased as FRR increased, and a dependence of Wf with

QT was observed at lower FRR values (Figure 7.8.A). It was also observed that a lower
QT generated wider focused streams probably due to the lower pressure exercised by the
lateral aqueous flows to the middle solvent flow, but these differences became less
pronounced at higher FRRs. A similar trend was observed by Bottaro et al.32 in an identic
channels configuration, but contrary to Jahn et al.19 who reported a non-variation in Wf
with modifications in QT.
Figure 7.8. Values of ethanol focused stream (Wf, μm) as flow rate ratio (FRR) increased for different
values of volumetric rates (QT) at constant temperature (50 °C), and different temperatures at constant QT
(100 μL/min). Values represent the average of two independent measurements, taken at approx. at 100 μm
from the end of focussed region.
Moreover, an intense inverse correlation (potential) between FRR and Wf was

observed at all the QT levels (Table 7.2). However, a strong negative correlation (linear)
between particle size and Wf was observed at the two different concentrations studied.
These correlations reflect that particle size is governed by focusing parameters. It is clear
that niosomes size can be tuned with the selection of the appropriate FRR and QT values,
which are key parameters for Wf and residence time.
188
Table 7.2. Correlation factor between flow focusing parameters (FRR and Wf) and particle size at different
concentration of bilayer components and variable QT (A), and at fixed concentration and QT for different
working temperatures (B).
(A)
R2 (T = 50 °C) 50 µl/min 100 µl/min 200 µl/min
(*) FRR vs Wf 0.99 0.97 0.92

(**) Wf vs Particle size 0.86 (5 mM) 0.98 (5 mM) 0.97 (5 mM)
0.98 (20 mM) 0.99 (20 mM) 0.93 (20 mM)
FRR, Flow Rate Ratio; QT, Total volumetric rate; Wf, initial width of focused stream; (*) potential
correlation; (**) linear correlation
(B)
R2 (QT = 100 µl/min) 30 °C 40 °C 50 °C 60 °C
(*) FRR vs Wf 1.00 0.99 0.97 0.98

(**) FRR vs Particle size 0.24 0.56 0.91 0.65
FRR, Flow Rate Ratio; QT, Total volumetric rate; Wf, initial width of focused stream; (*) potential
correlation; (**) linear correlation
7.3.3. Production of niosomes at different temperatures to study

potential tailoring effect over particle morphology
In this part of work QT of 100 µL/min and 5 mM of components concentration were

selected, since these have been the best operating conditions in terms of smaller particles
with narrower size distributions.
The effect of temperature was examined in a rage of 30 °C and 60 °C as another

operating parameter on size-tuned niosomes formation through flow-focused based
microfluidics. For this purpose, a non-ionic surfactant with low Tm was needed that
allowed to test a wide range of working temperatures. Sorbitan monolaureate or Span®
20 (Tm = 25 °C and HLB 8.6) was selected, another common surfactant used for niosomal
formulations, 42 and surfactant:cholesterol molar ratio was kept in 1:0.5 as for Span® 60 in
order to allow formulations comparisons.
189
Figure 7.9.Size (nm) (left) and size distribution (PDI, a.u.) (right) measured by DLS in undiluted samples
from niosomes formulated with Span® 20:Cholesterol (1:0.5 molar ratio) at 5mM in a continuous flow
microreactor based on hydrodynamic flow focusing at different controlled temperatures (30, 40, 50, and 60
°C). Each condition was tested twice, and each batch was measured by triplicate.
Figure 7.9 depicts the results of particle size at the same FRRs previously used for
Span® 60 niosomes at different working temperatures: 30, 40, 50 and 60 °C. At 30 °C, a
reduction in particle size from FRR=5 to FRR=15 was observed. As FRR increased size
became also larger (even higher that those particles produced at FRR = 5). This
phenomenon could be related to the observation of cholesterol precipitates inside the
mixing channel that were formed immediately after focusing region. The low solubility
of cholesterol in water at nearly room temperature induced its precipitation as crystals.
Those precipitates modified the flow properties and introduced turbulences that induced
micro domains in the fluid with different concentrations of bilayer components, and
particles with different morphologies. Also the depletion of cholesterol could generate
different particles than those produced in their presence. These perturbations were
magnified at higher FRR, since as seen in Figure 7.9.B the width of focused fluid became
smaller with the increment of FRR, and this stream was relatively smaller than the
formed crystals (around 100 µm structures).
For the rest of temperatures, a similar behaviour as for Span® 60 niosomes was
observed. Particles size became smaller with an inverse correlation with FRR. At higher
temperatures, focused ethanol stream was wider, and these differences were reduced
with the increment in FRR. Only slight differences in particle size could be detected
(Figure 7.9.A).
190
Regarding temperature effect some authors reported an increase in particle size as
temperature increased24 which were attributed to the bilayer expansion at higher
temperature. 43 In our case, such increase in particle size was not observed. It is know that
collapse pressure and surface compressional moduli decrease with temperature for all
surfactants, and this implies that Span monolayers are more expanded with increments
in temperature. However, as temperature increases planar bilayer precursors are less
rigid, which could be easily bended to closed structures, and this effect could lead then
to smaller particles.38
Regarding size distribution and temperature, it was observed that the increment of
temperature yielded more monodisperse particles, especially at 50 °C. Complete mixing
can be reached at FRR = 35 and FRR = 50 at any temperature. Only at 50 and 60 °C PDI
values remained nearly constant (after a first reduction from FRR=5 to 15) with the
increment in FRR.
7.3.4. Effect of surfactant acyl chain length over particles size and
monodispersity
Another interesting finding resulted from the comparison between niosomes

formulated with different non-ionic surfactants under identical preparation conditions.
In this work niosomes with sorbitan esters with different saturated acyl chain lengths
(C12 and C18 for Span® 20 and 60 respectively) were prepared. As seen in Figure 7.10,
shorter chains generally yielded larger niosomes. That was contrary to what would be
expected; it is generally understood that shorter chains increase the curvature radius of
the bilayer, according to the critical packing parameter (cpp) of the molecules,6 allowing
smaller particles. However, if was taken into account the higher hydrophilic character of
Span® 20 compared to Span® 60 (higher HLB value) the higher hydrophilicity could
enhance water soak into the inner core of the vesicle, resulting in larger vesicles size.
Similar results were reported by Gutierrez et al. 44 when niosomes were prepared by
mechanical agitation. Regarding niosome size distributions, no differences between both
types of surfactants were observed.
191
Figure 7.10. Influence of acyl chain length (C12 and C18 for Span® 20 and Span® 60 respectively) of two
different sorbitan sters used in niosomes formulation (surfactant: cholesterol 1:0.5 molar ratio, 5 mM), and
produced under different conditions by hydrodynamic flow focusing at controlled temperature (50 °C) and
a flow rate QT=100 μl/min.
7.4. Conclusions
Novel prototyping and additive manufacturing techniques with such as (HR)3D-

printing have been applied for the fabrication of a microfluidic continuous flow reactor
for hydrodynamic flow focusing at controlled temperature compatible with commercial
inverted microscopes. Despite some alteration in cross sectional dimensions and
morphology accuracy with respect to the original CAD design, high resolution 3D-
printed positive moulds allow us to create functional microreactors for organic colloid
production under different working conditions, and to study their effect on
aqueous/solvent mixing efficiency through molecular diffusion, and its relationship
with particles morphology.
This work shows that temperature is an essential parameter that must be taken into
consideration when formulating niosomes with surfactants with Tm over RT. Also it can
be used to modify the properties of particles (size and dispersity) produced with non-
ionic surfactants with Tm above RT.
We have found that flow focussing at controlled temperature follows the same
patterns as for RT, with the ratio between aqueous and solvent streams being the main
parameter to control focused stream width and hence, mixing efficiency and kinetics.
However, total flow rate only has insignificant effect when FRRs are set to low values,
whilst it can influence residence time, and subsequently, mixing efficiency. In general
192
terms, an increase in FRR yields a focused stream being narrower, and then, smaller
particles due to the reduction in residual solvent and the introduction of less amount of
bilayer components. This reduction allows complete mixing, even at high total flow rate,
resulting in the size distribution of generated particles being more homogeneous. The
counterpart is that production yield is reduced, since particles are generated in a less
concentrated suspension. Another variable found to be relevant is the component
concentration in ethanol feeding solution, with a direct effect on particle size and
monodispersity. A more concentrated solution induces an increment in particle size at
any total flow rate, but surprisingly, better size distribution. Complementary, we have
checked the influence of acyl chain length over particles morphology, and the versatility
that introduces this parameter into the properties and functionalities of this type of
biomaterial.
The effect of ethanol stratification due to differences in density was not taken into
account, which need further investigation in for future work, in particular in its
relationship with focusing temperature.
The findings in this works provide valuable information about microfluidics-based

production of niosomes at different operational conditions, and are expected to support
the expansion of this technique for the preparation of a wider range of organic colloids
with important characteristics for related industries with growing interest in different
application fields.
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8. Particle size and encapsulation efficiency
dependence with cargo molecular weight and
film hydration solution for niosomes
formulated with mixture of surfactants
Capítulo/Chapter 8
Particle size and encapsulation efficiency
dependence with cargo molecular weight and
film hydration solution for niosomes
formulated with mixture of surfactants
8.1. Introduction
Vesicles are commonly used as drug delivery systems for different active compounds.
Vesicles show special promise as drug carries due to their unique properties such as
nanometric size, high surface-volumen ratio, and ease of drug-release modulation. 1
Niosomes are a specific type of vesicles formed by the self-assembly of non-ionic
surfactants in aqueous media that leads to closed bilayers. 2 This structure enables them to
encapsulate aqueous solutions leading either the encapsulation of hydrophilic and
hydrophobic compounds. 3
Niosomes offer some advantages over other encapsulation technologies such as their
low cost, chemical stability, biocompatibility, among others. 4 Furthermore, non-ionic
surfactants self-assemblies are easily derivatized, which provides functional versatility to
their structure. 5
In recent years, niosomes have been used for encapsulating drugs, 6 nutraceuticals, 7
antioxidants, 8,9 micronutrients, 10 etc. There are still many challenges in the development of
delivery systems that could encapsulate hydrophilic compounds effectively. There are
colors, nutraceuticals and vitamins of industrial interest that need to be protected of
chemical degradation, to inhibit adverse interaction with other components, to mask off-
flavors, or to obtain a particular release profile. For example, some water soluble colors are
susceptible to chemical degradation under certain conditions, e.g., pH, light or
temperatura. 11 Water soluble vitamins are not stored in the body, and could be washed
out during food processing making necessary to replenish them daily. 12
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In this work, three different bioactive hydrophilic compounds with different molecular
weight and industrial interest were encapsulated (Figure 8.1): ascorbic acid (Vitamin C),
rhodamine B (Fluorescent organic dye) and cobalamin (Vitamin B12). Ascorbic acid, found
in citrus fruits, berries and vegetables, acts mainly as antioxidant, but also promotes the
production of noradrenaline, collagen, bile acids, and increases the intestinal absorption of
non-heme iron.12,13 Rhodamine B is a synthetic and highly soluble molecule used as
pigment in drug and cosmetic formulations due to its absorption and emission
properties. 14,15 Finally, cobalamin belongs to the B-complex vitamins and can be found in
cheese, fish, milk or eggs. It functions as coenzyme and as an important intermediate in
the metabolism of folic acid, a compound really important to prevent congenital disorder
during the first stage of pregnancy.12
Figure 8.1. Chemical structures of the encapsulated compounds
On the other hand, it is important to consider the effect of the composition of aqueous
solution used to hydrate the film, over particles morphology (size and PDI) and functional
characteristics such as encapsulation efficiency (EE), with special focus on hydrophilic
compounds encapsulation. The use of different co-solvents in the self-assembly process of
the niosomes, expands their application through the possibilities of tune particle size and
EE values, depending on selected applications. For example, glycerol has been used to
enhance the solubility of bioactive compounds, 16 but also as cryoprotector agent for
202
liophilization, 17 or to enhance the drug carriers penetration for transdermal
administration.16,18 In addition, polyethylene glycol (PEG), a water soluble polymer with
several molecular weight versions, has been used to prepare highly stable niosomes, 19 and
as sterical stabilizer of liposomes, 20 allowing longer circulation times in blood. 21
In this work, we prepared niosomes with a formulation based on an equimolar mixture

of surfactants Tween® 80 and Span® 80. Moreover, in some formulations dodecanol was
used as membrane additive, and using aqueous solutions of water, glycerol and
polyethylene glycol solutions as hydration media. We found that these prepared niosomes
are able to encapsulate hydrophilic compounds of different molecular weights. We were
able to compare two different purification methods, dialysis and gel filtration (SEC
Chromatography) used to separate encapsulated from the free compounds in solution.
8.2.1. Materials
Niosomes were formulated by the use of non-ionic surfactants Tween® 80 (Tw80, MW

1310 g/mol, HLB 15.0) and Span® 80 (Sp80, MW 428.60 g/mol, HLB 4.3), from Sigma
Aldrich (San Luis, MO, USA) and Fluka Analytical (Bucharest, Romania) respectively. 1-
Dodecanol 98 % (Dc, Mw 186.34 g/mol) from Sigma Aldrich was used as optional
membrane additive. L-(+)-ascorbic acid (AA, MW 176.12 g/mol) was obtained from J.T.
Baker (), while Rhodamine B purity ≥ 95 % (RB, MW 479.02 g/mol), and Vitamin B12
purity ≥ 98.5 % (B12, MW 1355.38 g/mol) were also purchased from Sigma Aldrich.
For the film hydration solution, polyethylene glycol 400 (PEG-400, MW 380-420 g/mol,
density 1.128 g/cm3, VWR International LLC, BDH PROLABO), glycerol bidistilled 99.5 %
(Gly, MW 92.09 g/mol, density 1.261 g/cm3, VWR International LLC, BDH PROLABO)
and, ultrapure water were used.
Absolute ethanol from J.T. Baker was used for bilayer components stock solutions.
Methanol HPLC grade from VWR International LLC, BDH PROLABO), and acetic acid
solution (49-51 %, HPLC grade) from Sigma Aldrich were used for liquid chromatography
(HPLC) performance.
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8.2.2 Niosomes preparation
Niosomes were prepared by a modified Thin Film Hydration method (TFH). The
corresponding amount of surfactants and membrane additives were placed into a 100 mL
round bottom flask in an equimolar ratio (from ethanolic stock solutions). The organic
solvent was removed using a rotary evaporator (Buchi Labortechnik AG, Flawil,
Switzerland), until get a homogeneous dried film. This film was then hydrated using
different aqueous-based solutions at 60 ºC, and agitated at 100 rpm during 30 minutes.
Suspension on vesicles were let acquire room temperature prior to purification and/or
characterization.
Niosomes containing the active principle inside, were prepared by the hydration of the
thin films formed after the evaporation with the corresponding aqueous solutions:
ultrapure water (or MQ), water:glycerol (60:40, v/v) or GLY, and water:PEG-400 (55.3:44.7,
v/v) or PEG. Both solutions have the same density.
Two different formulations of niosomes were studied. The first one contained an
equimolar ratio of Tw80 and Sp80, while the second was a mixture of Tw80, Sp80, and Dc
as bilayer stabilizer in a 1:1:1 molar ratio. The total concentration of membrane
components was kept constant at 10 mM (final concentration in vesicles suspension). The
molar ratio between both surfactants were selected to yield an HLB value of 10.
8.2.3. Niosomes purification
The purification of niosomes suspension was carried out by using two different
methods: dialysis and gel permeation chromatography.
8.2.3.1. Dialysis
Loaded niosomes suspensions were placed in a dialysis bag (dialysis tubing, 10 K

MWCO, Thermo Scientific, Waltham, MA, USA), and let floating in the corresponding
hydration media in a 1:100 v/v ratio. Dialysis time was adjusted depending on the
encapsulated compound (3h, 4h and 8h for AA, RB and B12, respectively).
Dialysis times were optimized by using a control solution containing the encapsulated
compound at the same concentration used for encapsulation experiments. Samples were
204
collected for the external phase, once the concentration on the external media was more
than the 99.8% of the original concentration, it was considered that the dialysis time was
enough for the purification.
HPLC was used to determine the concentration of free compound in the collected
samples.
8.2.3.2. Gel permeation chromatography
AA and RB loaded niosomes (with the exception of RB-niosomes in PEG) were

purified using a Sephadex G-25 Superfine column (HiTrapTM desalting columns, GE
Healthcare Life Sciences, UK); while B12 and RB loaded niosomes in PEG were purified
using a gravity elution PD Column (V0 = 2.5 mL) packed with Sepharose CL-4B (both
from, GE Healthcare Life Sciences). Sepharose CL-4B was used since in optimization steps,
we realized that RB and B12 PEG solution was not properly retain in Sephadex G25. Table
8.1 summarize the purification conditions based on gel filtration. Control solutions of the
corresponding compounds in the selected media were utilized to assess the efficiency of
the used method.
HPLC was used to determine the absence of free compound in the first collected
fractions from the column, were niosomes eluted, to assess the suitability of the SEC
column.
8.2.4. Niosomes size and distribution analysis
Mean diameter (z-average, nm) and Polydispersity Index (PDI, a.u.) for the prepared
niosomes were measured by Dynamic Light Scattering (DLS) on a Zetasizer NanoZS
Series (Malvern Instruments Ltd., Malvern, UK). Measurements were performed by
triplicate with undiluted samples, and at 25 °C. Low volume plastic disposable cuvettes
were used during size characterization (Malvern Instruments Ltd., Malvern, UK).
205
Table 8.1. Chromatography mediums used for gel filtration based purification of loaded niosomes
Component Hydration media Gel filtration

Encapsulated medium used
Ascorbic acid MilliQ Sephadex G25
Ascorbic acid MilliQ/Glycol Sephadex G25
Ascorbic acid MilliQ/PEG Sephadex G25
Rodamine MilliQ Sephadex G25
Rodamine MilliQ/Glycol Sephadex G25
Rodamine MilliQ/PEG Sepharose CL-4B 25
Vitamin B12 MilliQ Sephadex G25
Vitamin B12 MilliQ/Glycol Sephadex G25
Vitamin B12 MilliQ/PEG Sepharose CL-4B 25
8.2.5. Niosomes encapsulation efficiency (EE)
Encapsulation efficiency was calculated as the ratio between the quantity of

encapsulated compound (after proper purification), and the total amount in the unpurified
suspension according to Equation 8.
206
[𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐]𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 Equation 8
% 𝐸𝐸𝐸𝐸 = × 100
[𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐]𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖
Purified niosomes were diluted 1:10 (v/v) using methanol in order to break the
niosomal bilayers and release the encapsulated compounds. The quantification of the
cargo molecules was carried out by reverse phase liquid chromatography, or RP-HPLC
(HP series 1100 chromatograph, Hewlett Packard, Agilent Technologies), with a Zorbax
Eclipse Plus C18 column (4.6 mm x 150 mm, 5 μm, Agilent Technologies, Santa Clara,
California, USA). UV/vis (HP G1315A detector, Agilent Technologies) and fluorescence
(1260 Infinity A detector, from Agilent Technologies), were used as detection coupled to
the chromatographic separation. The following HPLC programs were used:
Ascorbic Acid
A linear gradient was performed with 0.1 % (v/v) acetic acid in MQ (mobile phase A)
and methanol (mobile phase B). The gradient started with 95 % of A, reaching 20 % of A at
min. 15, and kept constant for 5 min. The flow rate was 0.9 mL/min. Retention time for
AA was 2.28 min at λ = 278 nm.
Rhodamine B
A linear gradient was used with MQ (mobile phase A) and methanol (mobile phase B).
The gradient started with 2 % of B, running 100 % of B at min. 21, and kept constant for 5
min. The flow rate 1 mL/min. Retention time for RB was 19 min at λ = 554 nm.
Vitamin B12 (B12)
A linear gradient was used with MQ (mobile phase A) and methanol (mobile phase B).
The gradient started with 20 % of B, obtaining 100 % of B at 5 min and kept constant for 10
min. The flow rate was 0.8 mL/min. Retention time for B12 was 4.35 min at λ = 361 nm.
207
8.2.6. Differential Scanning Calorimetry (DSC)
Differential Scanning Calorimetry measurements were conducted in aluminum sealed

pans (10mg sample), heating mode (5 °C/min), from – 40 °C to 25 °C, under N2
atmosphere, in a DSC Mettler Toledo model 821e (Mettler Toledo International Inc.,
Barcelona, Spain).
In the present work, the effect of hydration solution composition in the thin film
hydration method for niosomes preparation has been studied. More precisely, glycol
compounds such as glycerol and polyethylene glycol 400 have been tested to prepare
niosomes for the encapsulation of hydrophilic compounds. A mixture of surfactant has
been used in the formulation, with and without a bilayer stabilizer, such as dodecanol.
8.3.1. Niosomes size vs hydration solution composition and cargo

molecular weight
Hydration solution composition seems to have an important role in particle size. In

particular, bigger particles were obtained with PEG solution than with GLY solution, and
the last compared to hydration with MQ (see Figure 8.2). This observation was reported
for Tw80-Sp80 and Tw80-Sp81-Dc formulations, with an influence with compound
molecular weight, since for the same hydration solution, different size values are observed
depending on the compound, but without any clear tendency. However, these differences
in size are less marked for RB and B12 with Tw80-Sp80-Dc formulation.
The influence of dodecanol over particle size has a discrete effect. For example, it
highly influenced niosomes size when encapsulated AA and PEG was used as hydration
solution. On the other hand, Dc seems to reduce particle size for RB and B12 loaded
niosomes when Gly and PEG are used for hydration.
208
Figure 8.2. (A) Size values of empty vesicles. Tw80: tween® 80; Sp80: span® 80; Dc: dodecanol. (B) Effect of
hydration solution and cargo molecular weight over particle size, for niosomal formulations Tw80:Sp80 (1:1
molar ratio) and Tw80:Sp80:Dc (1:1:1 molar ratio) prepared by Thin Film Hydration method (TFH).
Empty vesicles allows to study the effect of the hydration solution used and the
presence of Dc on the final particle size. The presence of Dc increases the drop size
independently of the hydration medium used, this is due to their location at the
membrane layer and their reduction on the surfactant curvature on the presence of
alcohol. In order hand the use of Gly and PEG solutions as hydration media increases
significantly the particle size of the vesicles formed. It is important to point that these
compounds can be attached to the membrane compounds producing higher stability to the
system but also could increase the particle size.16,28
Molecular weight of encapsulated compounds into the vesicles has not a clear effect
over particle size. For vesicles formulated with Tw80 and Sp80 the particle size seems to
increase when larger molecular weight compounds are encapsulated, these effect is
especially noticeable for the B12, probably due to their significantly major molecular
weight value.
The addition of polyethylene glycol with different MW in aqueous solution for vesicles
(especially niosomes preparation has been also studied.19,22-24 PEG-400 and PEG-6000 are
the most popular options, frequently used for surfactant mixture based formulations,
209
where Tween® and Span® (60 and 80) are widely used. The concentration of PEG-400 in
aqueous solutions was reported to influence the niosomal formation in a formulation¡Error!
Marcador no definido. based on Tween® 80. Similar, PEG-6000 concentration influenced the
particle size of niosomes formulated with Tw80:Sp80 1:0.3 mass ratio.¡Error! Marcador no definido.
Similar size values to the ones reported in this work have been found by other authors
when Span® 60 is combined with PEG-400.¡Error! Marcador no definido.
These glycol compounds alter the packing of the bilayer and increase their curvature,
which is manifested as an increment in particle radius. For example, glycerol has the
property to change the dielectric constant of the bilayer interior, and bilayer components
can re-arrange with different interactions.18 PEG molecules can interact by H bonds with
Tween® 80 molecules, so al low concentration can be part of the bilayer, which is less rigid.
However, al high concentration it can decrease the stability of the membrane until the
disruption. In that case, the instability of niosomes could led to fusion of particles and
large vesicles can be obtained. This may be the mechanism that explains our size results
when PEG is used as hydration solution.
Some works16 have checked the influence of chemical composition of the film
hydration solution for niosomal formulation, with special focus on the presence of poly-ol
compounds (alcohols). These authors reported the influence alcohol type and
concentration over particle size, and they found differences. Particularly, they reported
that glycerol concentration in hydration solution has positive effect: as glycerol
concentration increases (up to 40%, like our study) bigger particles were obtained without
any impact over monodispersity.
Some other works18 using aqueous-glycerol solutions for vesicles preparation by direct
hydration with sonication, have reported that empty and loaded vesicles did not differ in
terms of size, however other hydration solutions such as propylene glycol (1:1, v/v), more
similar to PEG, yields bigger particles compared to MQ or GLY solutions. These authors
have attributed this phenomena to the interaction of this compound with bilayer
components.¡Error! Marcador no definido.
210
Surprising the size of encapsulating RB and B12 vesicle is reduced around 30% when
Dc is used as a membrane component and PEG solution as a hydration method. This
phenomena indicates that even encapsulated dompounds are hydrophilic and hence are
encapsulated at the iner part of the vesicle they have some interacions with the membrane
compounds.
Regarding the monodispersity of the suspensions, in all the cases, both empty and
loaded vesicles showed a PDI value from 0.3 ± 0.1 for empty Gly niosomes (with Dc) to 0.7
± 0.1 for AA loaded MQ niosomes (with Dc). Encapsulation seems to not influence the
value, since in some cases loaded vesicles exhibit better monodispersity than loaded
niosomes.
8.3.2. Encapsulation Efficiency vs hydration solution composition

and cargo molecular weight
Regarding the EE of the different compounds we observed differences related with the
molecular weight of the cargo, and for both formulations. The best EE values were
obtained for AA when MQ is used as hydration solution, with independence of the
purification method (Figure 8.3). For this compound it seems that hydration solution
composition is not a key factor for encapsulation, since in almost all the cases the EE
values were similar. Only some particular differences are observed in the formulation
where Dc is used as membrane stabilizer (Gly yields the lowest EE value, 68%, using
dialysis as purification method, and MQ the higher EE value, 100%, using gel filtration as
purification technique).
However, RB and B12 seems to have an important dependence with hydration solution
composition, since a great difference in EE values can be observed depending on medium
chemistry. In the case of RB, EE ranges between 8.4% (Gly) and 60% (PEG) when dialysis
is used as purification method, and from 9% (MQ) to 22% (PEG) in the case of Dc
containing formulation with same purification method.
211
Figure 8.3. Effect of hydration solution and cargo molecular weight over encapsulation efficiecy, for niosomal
formulations (A)Tw80:Sp80 (1:1 molar ratio) and (B) Tw80:Sp80:Dc (1:1:1 molar ratio) prepared by Thin
Film Hydration method (TFH). Tw80: tween® 80; Sp80: span® 80; Dc: dodecanol. Dialysis (10 MWCO
membranes) and gel filtration (Sepahdex G25 or Seharose CL-4B, depending on the compound) were used as
purification methods.
It seems that PEG greatly improve RB encapsulation. Besides these good results, when
gel filtration is used EE remains with similar values and in general, offering lower values
212
than dialysis purified vesicles for both types of formulation. A possible explanation is
given into the next section.
A similar pattern of results is described for B12 encapsulation. Again, the use of PEG as
hydration solution yields the higher EE values (67%) for dialysis purified vesicles for both
formulation, but a not clear effect of medium composition can be observed for gel-filtrated
niosomes. Dc addition into the formulation is not an important parameter for his
encapsulation.
Muzzalupo et al.16 reported that 40% of glycerol yields the higher EE% compared to
other alcohols for sulfadiazine, and hydrophilic drug. They attributed this effect to the
presence of multiple –OH groups in the alcohol that could help the drug to be totally
solubilized thanks to H bonds between both molecules. A similar effect could be related to
B12 EE% without Dc, but may be not useful for RB encapsulation. However, PEG increase
EE of both, RB and B12, for both formulations. PEG has been tested to enhance the
solubility of amphiphilic compounds such as ellagic acid,¡Error! Marcador no definido. quercetin, 25
paclitaxel, 26 however no data about hydrophilic compounds have been found.
Interestingly, our formulation offers better AA EE% than liposomes found in the
literature, where only 10% was reached. 27 Regarding vitamin B12, PEG niosomes
with/without Dc offer better encapsulation than liposomes, 28 however there is a great
difference in size (2 µm for niosomes vs less than 100 nm for liposomes). On the other
hand, EE% values for RB are unusual, since the papers only report the encapsulation,
without interest in maximize this parameter.
These results evidence that exists and interaction between cargo molecular weight and
hydration solution composition without any clear influence of niosomes formulation, at
least for the introduction of Dc as bilayer stabilizer. Only in the case of MQ and dialysis, a
clear influence of cargo MW is observed, with a reduction in EE as MW increase. The
composition of film hydration solution could be a key parameter for some compounds EE
optimization, with a possible relationship with their molecular weight (medium or high
molecular weight compounds), and particular dependence of chemical nature of the
compounds, since a not general pattern was observed. In our opinion, the enhancement of
213
EE% values using Gly, and especially PEG, is an interaction of solubility enhancement and
the effect of increase particle size, which for hydrophilic compounds is translated into a
bigger capacity of niosomes to carry more cargo molecules due to physical (volume)
capacity.
Regarding the purification strategies, dialysis is the most popular option chosen to
purified vesicles encapsulating hydrophilic and amphiphilic compounds18,19,¡Error! Marcador no
definido.,¡Error! Marcador no definido.. However, if the permeating through the bilayer is not clear
understood for a specific formulation, realease and purification can be overlapped, and
EE% could be underestimated. To assess this for our formulation we selected SEC
chromatography as alternative method, 29 to be able to compare the performace of both
techniques.
As described previously, important differences in EE values are obtained when the two
purification methods are compared, and those discrepancies seems to be magnified with
hydration medium composition. The general trend is to observe a lower EE value for
niosomes suspensions purified by gel filtration.
The greatest differences are in all the cases observed for the use of PEG in both
formulations, and in the majority of them, MQ offers no differences between methods.
Also, a positive interaction with molecular weight is described for GLY and PEG.
To assess if this result relies on physical forces involved in the purification methods,
empty niosomes prepared in the three different aqueous solutions were subjected to gel
filtration. Particle size, size distribution and concentration were measured by Nanoparticle
Tracking Analysis (NTA) before and after the process (Figure 8.4).
214
Figure 8.4. Naparticle Tracking Analysis (NTA) of the three different hydration solution based niosomes
after and before SEC purification. MQ and Gly vesicles were purified using Sephadex G25 and PEG vesicles
by Sepharose CL-4B.
The results showed a reduction in mean size, and particle concentration for PEG based
niosomes suspension. It seems that a retention of vesicles of a mechanical disruption of
particles may occur during their flow through the column. During the process, the bed of
the column experiments a visible compaction due to the high viscosity of the fluid even at
low flow rate recommended by the manufacturer for viscous solutions. This phenomena
could yield to mechanical stress of the vesicles due to shear forces as they pass through the
compacted gel. Subsequently, loaded vesicles could collapse and their content could be
release to the medium where is trapped by the effect of the gel even when is compacted. A
reduction in EE value could be then observed for this case as reported.
Interestingly, particles in GLY solutions seems to keep their integrity, since no changes
in measured parameters have been reported. And also curiously, MQ suspended niosomes
experiment a slight reduction in size and concentration, what could be related to
entrapment phenomena of the smallest and highest niosomes by capture and clogging
with the stationary phase respectively.
Besides being a popular choice for vesicles purification, SEC has some drawbacks that
must be taken into consideration. For example, it has been reported that vesicles can be
retained by stationary phase 30, and this retention is dependent of particles pores and not
by particles size itself. Sepharose CL-40 contains particles with 20 nm pore size, which is
enough to allow the flow of small vesicles through them. However, large flexible particles
215
like PEG-hydrated may clog the pores and disturb the process, leading to underestimation
of EE% by vesicles loss and vesicles disruption into the columns. This fact could be
potentiated by viscous solution, such as 45% PEG in water.
8.3.3. Thermal behavior of formulation in the hydration solutions
Differential Scanning Calorimetry (DSC) is a useful technique carried out to study the
phase behavior of lipids and surfactant based bilayers, and gives information about
molecular interactions of the structure, which allows getting information about stability
and fluidity of the vesicle bilayer. 31 Also, this technique has been applied to measure the
EE% of hydrophobic compounds, 32 since they are loaded into the structure and then, alter
the cohesion of the bilayer.
Figure 8.5. DSC curves acquired in heating mode for the formulation without dodecanol in the three different
hydration solutions: ultrapure water (MQ), water:glycerol 60:40 v/v (Gly), and water:PEG-400 55:45 v/ v
(PEG).
Figure 8.5 represents the DSC curves obtained for empty vesicles prepared into the
three different hydration solutions. It is clear, that the incorporation of PEG and Gly
changes the stability of the bilayer, since Tc is decreased and the morphology of the peaks
shows a less ordered structure, evidenced for broad peak transition and loss of symmetry.
Gly and PEG transitions from gel-to-liquid seems to be in two steps, showing
216
heterogeneity of the bilayer. As consequence, bilayer becomes more fluid, and bigger
particles could be formed.
8.4. Conclusions
The preparation of niosomes encapsulating different hydrophilic compounds with

differences in molecular weight and by thin film hydration method performed with
different aqueous based solutions, allowed us to check the existence of the influence of
these parameters over particle size and encapsulation efficiency, two important and
related characteristics for niosomes aimed to bioactive compound protection and/or
delivery bio-applications.
Hydration solution composition has been correlated with particle size for a niosomal
formulation based on surfactant mixture with/without dodecanol as membrane stabilizer.
In addition, a possible interaction with cargo molecular weight could exist, since in
particular cases, particles become slightly bigger as molecular weight increase. This
increment in size is correlated with an increase in EE for these compounds. Since bigger
particles can encapsulated more compound molecules than smaller ones, especially for
hydrophilic compounds where they are loaded into the aqueous inner cavity of vesicles,
the relationship between EE and hydration solution composition seems to be purely due to
vesicles physical aspects (loading volume capacity). DSC curves showed a change in
bilayer structure with the use of glycerol and polyethylene glycol 400, that may interact
with surfactants and create a less organized structure.
Encapsulation efficiency obtained using dialysis method were higher than obtained for
gel filtration for the same samples. In addition, when dialysis was used, encapsulation
efficiency strongly depended of the hydration media while no differences were observed
when gel permeation was used as purification method. It seems that gel filtration is not an
appropriate purification method for suspensions of large vesicles, especially for viscous
solutions. Mechanical stress of particles during separation could lead to unsatisfactory
results in terms of particle integrity and subsequent EE values.
217
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9. Selected tetraspanins functionalized
niosomes as potential standards for exosomes
detection based on immunoassays
Capítulo/Chapter 9
Selected tetraspanins functionalized niosomes
as potential standards for exosomes detection
based on immunoassays
9.1. Introduction
Extracellular vesicles have emerged as a novel mechanism of intercellular

communication over the last years, playing an important role in both biological and
pathological processes. 1 Exosomes are a subtype of extracellular vesicles (EVs), released by
membrane fusion of multivesicular bodies (MVB) with the plasma membrane. 2 Exosomes
are vesicular subcellular particles (30-150 nm) with an average size around 100-150 nm,
characterized by a particular protein profile which offer information about the original cell
line from which are released, the target cell population, and valuable information about
health status and possible (patho)physiological roles. 3,4
Their possibilities as biomarkers for diagnosis 5 and treatment-response monitoring 6

promote their determination in biological fluids and cell culture media as a routine
practice in cell biology labs, but also in the clinical research. However, as the information
about EVs is constantly growing and evolving, routine practices with possibilities to be
incorporated in hospital facilities must be addressed and validated, 7 which represent a
technological challenge were reference material (RM) play an essential role. Up to date,
several strategies for EV isolation, detection and quantification have been developed, as
reviewed elsewhere. 8-10
EVs can be quantified directly or based on the quantification of biomolecules present in

the vesicles, in the majority of the cases a molecule present in the membrane.8 The specific
recognition of these molecules can be performed by the use of antibodies, aptamers or
other type of molecules with selective interactions, such as proteins with binding
capabilities over lipids. Several strategies have been developed based on this principle,
and optical 11,12 or electrochemical 13,14 transduction are the most popular principles for
223
biosensing of EVs. Regarding the molecules, tetraspanins CD9, CD63 or CD81 are
commonly used as membrane markers, since EVs are enriched in these transmembrane
proteins. 15
During the development and validation of a new analytical tool or method, the use of
standards is essential for the development stage, but also to characterized their
performance, possibilities and potential limitations. Traditionally, for EVs quantification,
suspensions enriched in exosomes isolated from body fluids or cell cultures media have
been used for these purpose. However, the absence of a well characterized and validated
strategy for EVs isolation makes difficult the existence of a robust RM for the evaluation
and performance of methods inter-comparison studies. The co-isolation of other biological
entities (mainly protein aggregates), and the isolation of heterogeneneous EV fractions in
terms of size (i.e., exosomes or microvesicles) or sub-types (i.e., exosome populations from
different cell lines) are some of the most commonly limitations of isolation procedures for
the establishment of EVs-based RM.8
Scientists have focused the attention on mimetic particles and their used in EVs-related
research. 16,17 Synthetic vesicular systems, such as liposomes or niosomes (prepared from
lipids and non-ionic surfactants, respectively) have been postulated as powerful tools due
to their similarities in terms of morphology and chemical behavior to natural EVs. Some
works have explored the applications of synthetic vesicles for the study of EVs biology. 18
On the other hand, some studies have applied these particles for exosomes modifications
for new intended purposes, mainly for diagnostic and therapy. 19
Also, these vesicles have been used in methods comparison studies. For example, Lane
et al. 20 compared different exosome purification methods using a model liposome system,
and they concluded that the studied purification methods (ultracentrifugation, two
sedimentation reagents, a density gradient method, and the ExoSpin exosome purification
system) possessed different efficiencies, but keep constant vesicles size and size
distribution from real sample. Maas et al. 21 used liposomes to compare methods based on
single-particle analysis (nanoparticle tracking analysis or NTA, tunable resistive pulse
sensing or tRPS and high-resolution flow cytometry or hFC) for EVs quantification, and
found absolute quantification differences between techniques and between synthetic
224
counterparts and natural EVs. Interestingly, also some differences were observed for
liposomes with different size. However, the use of this synthetic vesicles for
methodological comparisons is limited to their physical properties and not to functional
characteristics such as the presence of specific molecular markers. This is important, since
some physical properties such as size and monodispersity can influence the outcome of
the analysis by limiting the sensitivity for smaller particle detection and a bias into the
quantification. The introduction of molecular recognition coupled to a proper size, could
create a robust RM that allows to expand the range of techniques to be tested, but also
introduce new possibilities of information acquisition for deep inter-comparison studies.
During the last years, several technologies have raised in order to overcome the
limitations for the use of natural exosomes in biomedical applications, and the so called
artificial exosomes have emerged with a full range of possibilities and capabilities for
diagnosis and therapy. 22,23 However, their use as true standards for analytical purposes
have remained unexplored, and only some probes of concept have been developed. 24,25
The aim of the present work was the design, development and functional
characterization of a potential standard of EVs (RM), more precisely exosomes, for their
application in immunoassay-based methods. Figure 9.1 capture the molecular composition
and structure of our proposal. Recombinant constructions (large extracellular loops, LEL)
of tetraspanins CD63 and CD9 were bioconjugated to the external surface of niosomes
prepared with a size distribution similar to natural exosomes. Mono- (CD9 or CD63) and
double (CD9 and CD63) functionalized particles have been prepared and tested in an
ELISA assay. Their potential as RM for EVs bioanalytics is evaluated, and a versatile
strategy for their customized production is presented.
225
Figure 9.1. Schematic fully artificial exosome produced by bottom-up bio-nanotechnological methods based on
supramolecular chemistry. The different molecular components are detailed with their functions.
9.2. Material and methods
Sorbitan monostearate or Span® 60 (Sp60), Cholesterol hemisuccinate (Cho-suc), and

Phosphate Buffer Saline or PBS (10 mM, pH 7.4) prepared from tablets according with
manufacture instructions, were acquired from Sigma Aldrich (San Luis, MO, USA).
Cholesterol from lamb wool (Cho) was from Across Organics (Geel, Belgium), and
streptavidin (Str) was from G Biosciences (Geno Technology Inc., USA). Ultrapure water
was used for buffer preparation.
Other biochemical have been: Biotinylated-HRP (Life Technologies, Thermo Fisher,

Waltham, MA, USA) TMB, and nitrocellulose membrane (GE Healthcare Life Sciences,
USA).
HiTrap columns packed with Sephadex G-25 (5 mL bed volume) and Sepharose CL-
2B/CL-4B gel filtration media were acquired from GE Healthcare Life Sciences
(Pittsburgh, USA).
226
Biotinylated and purified monoclonal antibodies against CD9 (VJ1/20) and CD63
(Tea3/18) were acquired from Immunostep (Salamanca, Spain). Polyclonal antibody
against CD9 was from Santa Cruz Biotechnology (Dalas, TX, USA) and for CD63 was
acquired from Sigma Aldrich. Secondary antibodies HRP-conjugated, and streptavidin-
HRP were acquired from Thermo Scientific.
9.2.1. Niosomes preparation and size measurement
Nanovesicles formulated with Sp60:Cho:Cho-suc (1:0.5:0.01 molar ratio) were prepared

following a method previously described. 26 Briefly, 20 mL of a 6 mM ethanolic solution
containing bilayer precursors at the mentioned molar ratio were injected (130 mL/h) into
50 mL of ultrapure water at 60 °C and constant stirring (500 rpm). Injection was performed
with a syringe Pump (KDS Instruments, Beijing, China) on a beaker glass over a
heating/stirring plate (IKA, Staufen, Germany). Residual ethanol was removed by
evaporation under vacuum (50 °C, 90 Bar and 35 rpm) (Bütchi Labortechnik AG, Flawil,
Switzerland), and aqueous volume was reduced to 25 mL by water evaporation by
reducing the vacuum down to 50 Bar for approximately 45 min.
Produced vesicles were characterized in terms of size and size distribution by

measuring 3 undiluted independent samples by Dynamic Light Scattering in a ZetaSizer
NANO ZS instrument (Malvern Instruments, Malvern, UK) at 25 °C and 3 runs per
measurement using forward scatter (173°) detector. Low disposable plastic cuvettes from
equipment manufacturer were used for that purpose.
9.2.2. Streptavidin conjugation to niosomes surface
Protein (recombinant streptavidin) conjugation to niosomal surface was carried out

following the carbodiimide method. 27 1-Ethyl-3-(3-dim((ethylaminopropyl)carbodiimide
(EDC) and sulfo N-hydroxysulfosuccinimide (sulfo-NHS) were added to the selected
volume of niosomes suspension to reach 4.3 mM and 9.2 mM, respectively; carboxylic
groups were activated for 30 min at RT with gently shaking. Excess of conjugation
reagents were removed by gel filtration with HiTrap desalting columns packed with
Sephadex G-25. Elution was performed with PBS 10 mM, pH 7.4, a suitable condition for
conjugation to the primary amine-containing molecule. Then, streptavidin was added, and
227
a total sample volume of 2.5 mL was reached by addition of ultrapure water. The solution
was kept at constant mechanical agitation in a vortex for 2 h. To quench possible activated
NHS esters, 1 mg of glycine was added to the suspension.
Removal of unconjugated protein was carried out by gravity elution gel filtration in a
PD-10 empty column packed with Sepharose CL-4B (8.6 mL, bed volume) conditioned
with PBS. A total elution volume of 3.5 mL was recovered in a flow cytometer-grade tube
with sealing cap (BD Plastipak, Vaud, Switzerland), and 0.1% sodium azide in PBS was
used as eluent solution. This concentration was checked to keep vesicles without
modification in colloidal state. A protein quantification kit (based on bicinchoninic acid
assay or BCA, according to manufacturer instruction) was used to determine the elution
profile of a solution of streptavidin to check the suitability of chromatography separation
for purification.
The efficiency of streptavidin conjugation was checked by dot blot, using biotinilated-
HRP enzyme (B-HRP) as protein detection probe and insoluble TMB (suitable for
membranes) as substrate. Briefly, 1 µL of samples (fractions from SEC column) and protein
standards were applied over nitrocellulose membranes (GE Healthcare Life Sciences,
Pittsburgh, USA) and air dried at RT. Membranes were blocked in 5% BSA in PBS-0.05%
Tween® 20 (PBS-T) and then incubated in a 4 µg/mL solution of B-HRP in 0.1% BSA in
PBS-T for 45 min. Membranes were washed and incubated with TMB at variable times,
monitoring the signal from the highest concentration standard to avoid signal saturation.
9.2.3. Tetraspanins (CD9/63) large extracellular loops (LELs)

production
Production of tetraspanin LELs has been performed as previously described.25
9.2.4. Vesicles functionalization with tetraspanins LELs constructions
For mono-functionalization of niosomes with LEL_CD9 or LEL_CD63, 700 µL of

selected LEL stock was added to 1.5 mL of vesicles suspension and incubated overnight at
4ºC with gently shaking. Excess of biotin was used to saturate possible free binding sites of
228
streptavidin in order to avoid possible unspecific signal from biotinylated antibodies used
in ELISA assays.
In the case of double functionalized vesicles, 300 µL of LEL_CD63 was added, while
the amount of LEL_CD9 was reduced to 150 µL according to a previous report showing
that their production yield is approximately the double of CD63.25 This was intended to
keep the ratio of LEL types to 1:1 molar ratio.
In order to remove unbound LEL, sepharose CL-2B columns (10 mL bed volume) were
prepared in plastic syringes (BD Plastipak) with a nylon filter to retain the gel into the
column. A 3 way stopcock (BD Plastipak) was attached to column outlet to control the
elution flow. After equilibration of the column with filtered PBS, the total amount of
vesicles suspension plus LEL was added and a total of 20 fractions (0.5 mL) were collected
into glass vials, and stored at 4 °C.
To check the effectiveness of LEL coupling to streptavidin-coated niosomes, all the

fractions were checked by dot blot analysis with specific monoclonal antibodies against
CD9 and CD63 as primary antibodies, and anti-mouse-HRP as secondary antibody. Blots
were developed with the ECL detecton system (Supersignal® West Femto maximum
sensitivity substrate, Thermo Scientific) in a LAS4000 mini Image System analyzer from
Fujifilm and software ImageQuant-TL (GE Healthcare).
9.2.5. Immunoassays for artificial EVs detection
ELISA assays were performed in 96-well plates (Corning, Corning, NY, USA).
Microplate wells were coated at 4 °C overnight with monoclonal antibodies (10 µg/mL, in
borate buffer saline or BBS buffer, 10 mM and pH 8.2), and blocked with BSA 2% in PBS
for 2h at 37 °C. Samples (100 µL/well) were incubated at 4 °C overnight. Detection was
performed using biotinylated monoclonal antibodies (12.5 µg/mL in PBS) and polyclonal
antibodies (1:250 and 1:500 for anti-CD9 and anti-CD63, respectively) incubated for 1h at
37 °C. Streptavidin-HRP (1:2000) and anti-rabbit IgG-HRP (1:3000) was used as secondary
detection probes. The reaction was developed with o-phenylenediamine dihidrochloride
(OPD, Sigma Aldrich) substrate for colorimetric detection, and signal intensity was
measured at 492 nm in a microplate reader (Tecan Genios, Tecan Trading AG,
229
Switzerland) after addition of stop solution. Washing steps were performed with PBS-T
between incubation steps, and PBS prior to the addition of OPD.
The strategies for the development of artificial exosomas have been reviewed in a
previous publication of our group.23 Their biochemical composition should provide them
with similar physical, optical and functional characteristics to natural EVs, providing a
new range of RM for different isolation and detections strategies. In this work, we have
followed a strategy based on bio-nanotechnology and supramolecular chemistry to create
a synthetic bilayer made of non-ionic surfactants and additives (niosomes) that was then
functionalized with proteins typical of exosomes, against specific receptors are directed in
assays based of molecular recognition such as immunoassays.
9.3.1. Streptavidin-coated niosomes development as generic scaffold

for artificial EVs production
In a previous work,26 the influence of ethanol injection method (EIM) preparation

variables over particle size and monodispersity of the niosomal formulation Span®
60:Cholesterol (1:0.5 molar ratio) was deeply studied. It was found that organic/aqueous
phase volume ratio, the (final aqueous-phase) surfactant-cholesterol concentration, and the
sonication amplitude were the parameters with stronger effects. In order to improve the
results, we decided to use all the information provided by the models, with the aim of
obtaining smaller niosomes with an acceptable size distribution which represented values
in agreement with those observed for natural exosomes.
In a deep study (data not published) we observed that higher sonication amplitude
needed for size reduction really decrease the life time of the probe, and TiNPs could be
observed by TEM in the prepared suspensions. Also, the ageing of the probe introduce not
controlled alterations in the process, and becomes not reproducible as sonication probe
ages. So, it was decided to avoid the use of sonication, and his influence was compensated
by reasonably changing the value of the other two factors. Concentration of membrane
components in the organic phase was reduced to the half, while its volume was kept at 20
230
mL. In order to compensate the dilution of the prepared nanovesicles solution, we used
evaporation under vacuum (50 °C, 45 bar) to reduce the final aqueous suspension down to
25 mL instead of 50 mL.
With these modifications of initial conditions, niosomes with 150 ± 3 nm (PDI 0.060)
were obtained. For bioconjugation purposes, cholesterol-hemisuccinate was added. This
additive not only introduces surface available carboxylic groups as anchor elements, but it
also provides negative charges that enhance the stability of bared niosomes during
storage. The addition of cholesterol-hemisuccinate did not modify the average size of the
vesicles in comparison with formulations lacking cholesterol-hemisuccinate.
Once we got the optimal suspension of niosomes in terms of size, monodispersity and
particle concentration, protein (streptavidin) could be conjugated with the niosomes at the
external surface to create a generic platform for the development of different types of
artificial EVs. The carbodiimide-based bioconjugation strategy is a two-step procedure to
permanently link two biomolecules, or a molecule with a surface or a nanomaterial [ 28].
This method creates a covalent bond between an amine and an activated carboxylic group
by the use of EDC and NHS. In this particular case, between amine groups from basic
aminoacids in the streptavidin molecule and the carboxylic group from cholesterol
hemisuccinate introduce into niosomal formulation as additive.
This strategy has been previously followed for the conjugation of biomolecules with
nanovesicles, and some examples can be found in the literature. 29,30 Interestingly,
carbodiimide method has been applied in the development of artificial exosome for
therapy. 31 The process is carried out in two steps in order to avoid protein aggregation
induced by cross-linked molecules, since proteins exhibit both functional groups, the
amine and the carboxylic. The pH value of each step (acid for the first, neutral to basic for
the second) was carefully adjusted in order to maximize the coupling process. Two
different amounts of protein were tested in order to check variable dependence over
bioconjugation yield. However, no differences were observed, and the lower amount was
selected in order to keep the process cost-effective.
The elution profile from the SEC column of a solution of streptavidin is shown in
Figure 9.2.A. The elution is enough delayed from the death volume, where nanovesicles
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eluted, checked by passing through the column a suspension of NVs with a loaded red
dye for visual purposes (Figure 9.2.A, detail).
As shown in Figure 9.2.B, five different batches (L1 – L5) of streptavidin-

functionalized niosomes (Nio_Str) were analyzed by an adapted dot blot to check the
presence of the protein and the effectiveness of the functionalization method. As shown,
reproducibility is acceptable, with values around 63 µg/ml.
Figure 9.2. Figure 2. (A) Elution profile of a solution of streptavidin from a Sepharose CL-4B SEC gravity
elution column. Signal quantification of each 0.5Ml fraction was measured by BCA total protein assay
according with manufacturer instruction. Graph insight shows (left) the first 3.5 mL collected of a suspension
of red dye loaded niosomes to allow their visualization, after their elution from the SEC column; (right) the
SEC column after the elution of the 3.5 mL of dyed niosomes. Both elements, niosomes and free protein, eluted
from the column enough separate to allow their separation based on Sepharose CL-4B gravity elution
coloumns. (B) Dot-Blot assay for checking the effectively of streptavidin bioconjugation to niosomes through
carbodiimide method (EDC/NHS). Standards of different concentrations allows the semiquantification of the
process by comparison of spot intensity. The result shows the 5 different batches. Biotinylated-HRP (4µg/mL)
was used as detecting agent. (C) Size distribution by DLS of bared niosomes (or NIO) and streptavidin-
conjugated niosomes or (NIO_Str). The average size increment (2nm) shown as peak displacement
demonstrate the effective conjugation of the protein. (D) Size distribution by DLS of the 5 different batches of
NIO_Str (152 ± 3 nm), to demonstrate the reproducibility of the process.
As expected, an increase (2 nm) in hydrodynamic radius of the particles was observed

after bioconjugation of streptavidin over niosomes surface (Figure 9.2.C), and this
increment can be monitored by DLS as routine technique to check the process efficacy. 32
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All the batches were characterized in terms particle size and size distribution (Figure
9.2.D), showing a good reproducibility, with an average value of 152 ± 2 nm.
9.3.2. Artificial EVs production using Nio_Str functionalized with

tetraspanin LEL
In order to develop a functional RM based on artificial exosomes, Nio_Str particles

were incubated with tetraspanin CD9 and/or CD63 large extracellular loops (LEL), to
create mono- or double functionalized niosomes named Nio_LEL (-LEL9, -LEL63, or
LEL9/63). Biotinylated recombinant tetraspanin LELs production has been previously
described.25 Briefly, each construction is biotinylated at the N- and C- terminal by the
introduction of 15 aminoacids of the AviTag peptide, which allows site-specific
biotinylation by the biotin ligase A (BirA) from Escherichia coli. This double biotinylation
will allow LELs to bind them to streptavidin molecules, and this binding process will help
LELs to acquire the proper spatial conformation for antibodies specific recognition during
molecular recognition assays performance, such as dot blot and ELISA assays.
LELs coupling to Nio_Str was performed by co-incubation at constant stirring

overnight at 4 °C in a tube rotator. In the special case of Nio_LEL9/63, niosomes were
incubated with both type of LEL at a molar ratio 1:1, in order to obtained particles with
equal amount of surface tetraspanins.
After incubation, excess of LELs was removed by gel filtration (Sepharose CL-2B), and
several fractions were collected into glass vials. The fractions were analyzed by dot blot for
immunodetection of the LEL. Figure 9.3.A shows the detection of LEL_CD9 and
LEL_CD63 constructions in both mono- and double-functionalized niosomes, and
confirms the single and co-functionalization with LELs. The first five fractions correspond
to the void volume of the column and no signal was detected. A progressive increment in
the signal was observed in fractions 5/6 to 9/10, which correspond to those that showed
the characteristics pale white color of the vesicles in suspension. Then, a reduction in
signal is observed prior to another increment in the signal corresponding to the elution of
free LEL that is used in excess.
233
Both characteristics (color ad signal) were taken into consideration, and the 5 fractions
showing higher signals were pooled, so the final volume achieved of Nio_LEL was 2.5 mL
for each type of modified niosomes. Signal differences between CD63 and CD9 for double
functionalized vesicles are due to differences in exposure time. It was needed longer
exposures for CD63 detection. Nio_LEL particles were produced in the range of 5.2 x 1011 –
1.0 x 1012 particles/mL, measured by NTA.
Nio_LEL were also characterized by NTA to check particle average size and size
distribution, and a sample of natural exosomes was also measured for comparative
purposes. Besides differences in terms of peaks intensities across the distributions (Figure
9.3.B), average size of Nio_LEL9, Nio_LEL63, Nio_LEL9/63 and natural exosomas are
similar (185, 160, 159 and 162 nm respectively), and the size distribution limits are similar
for all the particles. However, artificial vesicles are more homogeneous (as expected) since
they are lab-made products, while natural exosomas are more heterogeneous regarding
their natural origin. Mono- and double-functionalized particles remains similar between
types of particles.
The next step was to test the recognition of the Nio_LEL by their specific anti-
tetraspanin antibody and the possible unspecific signal from the other anti-tetraspanin.
Negative controls were introduced (bared niosomes, and niosomes functionalized with
Streptavidin with/without biotin saturation), and a sample of natural exosomes
(mesenchymal triple-negative breast cancer cell line SUM159) was used as positive control.
Both polyclonal and monoclonal antibodies (biotinylated or purified) were tested.
Effective molecular recognition was carried out by dot blot assay as performed to check
niosomes-LEL functionalization. This rapid and simple technique is suitable for screening
of antibodies. 33
234
Figure 9.3. (A) Dot-blot assays for revelation of LEL, CD9 or CD63, positive fractions collected from a
Sepharose CL-2B gravity elution SEC column. Mon- and double-functionalized Nio_Str have been produced.
(B) Size distribution measured by NTA (Nanosight, Malvern Instruments) of previous described fully
artificial exosomas (NIO_LEL). A sample of natural exosomas isolated from mesenchymal triple-negative
breast cancer cell line SUM159 was also measured for comparison purposes.
The results of the different assays carried out showed that the best detection was
obtained with the polyclonal antibodies (Figure 9.4), in terms of specificity (referred to
Nio_LEL recognition and discrimination). Those antibodies exhibit some unspecific
recognition for the other tetraspanin, but less intense than the observed for biotinylated
monoclonal antibodies. In addition, the signal from negative controls was more intense for
235
both types of monoclonal antibodies. However, between them, purified ones offered better
results. Based on these observations, capture by purified monoclonal antibodies and
detection using polyclonal antibodies were selected as configuration for a sandwich-based
ELISA experiments to detect artificial, according to a classical configuration for this type of
immunoassays. 34
Figure 9.4. Dot-blot assay for the screening and selection of α-tetraspanin antibodies for their future use in
ELISAs for the detection of fully artificial exosomes (NIO_LEL). Secondary antibodies labelled with HRP
were appropriate selected. Different negative controls were also introduced (bared niosomes, niosomes
functionalized with streptavidin with and without biotin saturation), as the use of isolated exosomas from
mesenchymal triple-negative breast cancer cell line SUM159 as positive control. Values are uniplicate assays.
(PU) monoclonal purified antibody; (biot) Biotynilated monoclonal antibody; (poli) polyclonal antibody.
9.3.3. Development of ELISA assays using artificial exosomas
9.3.3.1. Single tetraspanin functional particles
To test the potential use of monofunctionalized Nio_LEL as RM, ELISA assays were
carried out using different combinations of capture/detection antibodies for both types of
vesicles (Nio_LEL9 and Nio_LEL63). This proposed ELISA assay with colorimetric
detection used monoclonal antibodies for capture and polyclonal antibodies for primary
detection, with the use of adequate HRP-labeled secondary. The combination of the proper
236
detection and capture antibodies is crucial in the development of ELISA assays, especially
in terms of signal-to-noise ratio and specificity. Multiple antibodies combinations were
tested, and their unspecific signal were also studied.
From negative controls (Figure 9.5.A), unspecific recognition (capture or detection-

based) can be measured. In this particular case, both types of mono-functionalized
niosomes are recognized by the anti-tetraspanin against the non-carrying protein. Cross
detection of these particles confirms something previously observed by dot-blot assay, that
this unspecific recognition is higher for CD63 compared to CD9. The signal observed for
capture/detection of NIO_LEL63 using anti-CD9 antibodies is higher than that observed
when using anti-CD63. This result was obtained with both batches, and differences in
signal intensity between both batches are probably related to particle concentration (6.9 x
1011 and 8.2 x 1011 particles/mL for batch 1 and 2 respectively in the case of NIO_LEL9 and
1.0 x 1012 and 7.4 x 1011 for NIO_LEL63).
Then, monofunctionalized niosomes were tested under the different antibodies

combination, to test their selectivity (how efficiently Nio_LEL are discriminated) and
sensibility (how intense is the specific signal). The same controls as for dot blot assay were
used, and Nio_LEL particles were tested in the produced concentration without dilution.
As seen in Figure 9.5.B, a better signal was observed for all the cases were anti-CD9
was used for detection, even when the assayed particles were Nio_LEL63, or in the case of
Nio_LEL9 captured by anti-CD63. These two conditions offer then the unspecific signal for
detection, clearly lower than the specific signal, and in accordance with dot-blot results.
In the case of detection based on anti-CD63, when capture by anti-CD9, this detection
can discern (p˂0.005) between types of Nio_LEL with a really low sensibility, and coupled
to capture also by anti-CD63 is the only combination that cannot differentiate Nio_LEL
type.
237
Figure 9.5. (A,B) Cross-reactive responses for mono-functionalized LEL-tetraspanin niosomes (fully articial
exosomas) for different configuration of capture (c) and detection (d) anti-tetraspanin antibodies. (C) ELISA
assays for the detection of NIO_LEL9 (N9) and NIO_LEL63 (N63) fully artificial exosomes using different
combination of capture (c) and detection (d) antibodies. Monoclonal α-CD9 or α-CD63 antibodies were used
for capture, whereas polyclonal α-CD9 or α-CD63 antibodies were used for detection. (D) ELISA assays for
the detection of NIO_LEL9/63 using different combinations of capture (c) and detection (d) antibodies. The
graphs shows the mean ± SD of 3 independent experiments. *** p˂0.005, Student’s t-test.
The large difference between specific capture-detection of Nio_LEL9 y –LEL63 could

be explained by differences in antibodies affinity, since differences in particles
concentration are no so evident (6.9 x 1011 vs 1.0 x 1012 for Nio_LEL9 and Nio_LEL63
respectively). Based on these results, capture and detection by anti-CD9 seems to offer the
better sensibility with capabilities to discern the type of vesicles.
238
9.3.3.2. Double tetraspanin functional particles
However, single molecule detection is often used, but the simultaneous detection of
two different tetraspanins (at least one of them must be CD63, being the others CD9 or
CD81) is accepted as a molecular identification of the endosomal origin of the exosomes,
and can be used as molecular markers in exosomes detection based on immunoassays. 35
For this purpose, double functionalized niosomes (Nio_LEL9/63) were produced and
tested by the same antibodies combination in order to identify which one offers better
sensibility.
Nio_LEL9/63 are recognized by all the combinations of capture/detection antibodies

(Figure 9.5.D), however a clear higher detection is observed when using anti-CD9 as
capture and detection (p˂0.005). The next combination with acceptable sensibility is the
one that uses capture by anti-CD63 and detection by anti-CD9. The other two possibilities
(c9-d63 and c63-d63) offer a really low sensibility. These observations are in accordance
with those described for mono-functionalized particles, and clearly confirm that
antibodies for CD9 offer better possibilities probably due to a higher affinity constant.
In a previous work of our group, 36 an ELISA procedure was developed for the
detection of exosomes based on the simultaneous recognition of two tetraspanin (one used
for capture, and the other for detection). For this purpose, melanoma cell line (Ma-Mel-
86c) derived exosomes isolated by ultracentrifugation were used, and different
combination of antibodies recognizing CD9, CD63, and CD81 were tested. Results
revealed that capture by CD81 and detection carried out by CD9 presented the best signal-
to-noise ratio. However, the same exosomes were better detected in a Lateral Flow
Immunoassay (LFIA) using CD9 for capture and CD63 for detection, besides this
combination offered a higher background signal in ELISA compared to CD81/CD9 for
capture/detection respectively.
In this type of analytical platforms, only Nio_LEL9/63 could be tested, since mono-
fucntionalized particles are not suitable due to how it works a LFIA test. However, the
best capture-detection pair described for this systems is in our case the less sensible one, so
our proposal of RM based on artificial exomes is adequate for ELISA and could not be for
LFIA.
239
Finally, only in the combinations where anti-CD9 is used as detection, the sensibility
was enough to allow the visualization of dose-response signals (Figure 9.6). This result is
the manifestation of those observed in figure 34.D, where it can be seen that detection
based on CD63 is not a good option. In all the cases, a linear correlation is observed. When
the signal intensity proportioned by a specific antibody configuration was enough to allow
visualization of dose-response, this response was fitted to a linear equation, demonstrating
that working condition where into the linear range of the typical sigmoidal response
related to a sandwich assay, which confirms that our RM proposal is suitable.
Figure 9.6. Dose-response graphs for different types of fully artificial exosomas detected by ELISA assay
using monoclonal and polyclonal antibodies α-tetraspanins CD9 y CD63 for capture and detection
respectively. As secondary appropriate α-IgG-HRP was used. N9 and N9/63 are NIO_LEL9 and
NIO_LEL9/63 respectively, which are mono- and double-functionalized LEL-tetraspanin niosomes.
9.3.4. Commercial potential of our artificial exosome model
The work of Lane et al.20 has highlighted the physical similarities between synthetic
vesicles and EVs, and those particles have been used as reference materials for
methodological comparisons (NTA, tRPS and hFC). However, all those methods are
classified as unspecific concentration determination methods 37 since they rely on general
physical characteristics and not in a specific molecular marker which allows also
phenotyping possibilities. In this scientific challenge we propose fully artificial exosomes22
as a new potential tool to help into the development and validation of new analytical
methods and platforms, as demonstrated with our results. In our opinion, our proposed
RM could be competitive to those in the market up to date.
240
Tabla 9.1. Some commercial available kits based on ELISA for exosomes quantification in biological samples.
Most of them are based on colorimetric signal quantification based on HRP substrates, with a typical format
of 96 well microtiter plate.
Product Manufacturer Biomarkers Assay format Standard

used for
calibration
plots
ExoELISA SBI System CD9/CD63/CD81 Exosomes are immobilized Lyophilized
Biosciences for detection directly into the well Exosomes
ExoTestTM HansaBioMed CD9 for detection Sandwich assay using CD9 for Exosome
detection. Capture not lyophilized
specified by the manufacturer
ExoQuant Centaur CD9 for detection Sandwich assay using pan- Lyophilized
Genprice Exosome biomarkers (data not Exosomes
specify by the manufacturer
ExoEL- BioVision CD9 for detection Sandwich assay using pan- Exosome
CD81A1 Exosome biomarkers (data not lyophilized
specify by the manufacturer
PS Fujufilm Wako CD63 for Exosomes are captured by a Lyophilized

CaptureTM Pure Chemical detection phosphatidylserine binding Exosomes
Exosome Corporation protein immobilized in the
ELISA KIT wells
CD9/CD63 Cosmo BIO CD63 for Sandwich assay using CD9 for CD9/63 Fusion
Exosome CO. Ltd detection capture protein
ELISA Kit
ExoAssayTM CD Creative Not specified by Sandwich assay using CD9 for Lyophilized
®
Diagnostics the manufacturer capture Exosomes
Details extracted from products data sheets or proportionated by the manufacturer
Commercially available kits based on ELISA assays are marketed with different
configurations (Tabla 9.1). Some of them are based on direct capture of exosomes into
plate wells (such as ExoELISA-(Ultra), SBI System Biosciences), while other relies on
exosome-capture mediated by antibodies-coated weels (such as ExoTESTTM,
HansaBioMed). Majority of them, use lyophilized exosomes as standards, and some
241
manufactures let clear that signal can be different depending on the amount of protein per
vesicle between different types of exosomes, with potential bias of extrapolated
concentration. This fact hinders the quantification of these types of analytes, since the
same intensity could be related to a higher concentration of analytes with a reduced
expression level of detection antigen, or a low concentration of analytes carrying a higher
number of detection epitopes. This is a key point to take into account for analytes such as
exosomas, where the expression levels of CD9 and CD63 differs between cell lines, 38 or
even exosomas from cells with different heath status. This is something that clearly
difficult the development of a universal standard for EVs quantification, especially for
exosomes, as mentioned previously.
In this diverse market our proposal can be compatible, since both types of Nio_LEL
(mono- and double-functionalize) could be potentially applied. On the other hand, our
platform is really versatile, since antigen density can be tuned by changing the density of
streptavidin over their surface, and any biotinylated peptide could be used to
functionalize, giving lots of options to create specific types of artificial exosomas.
Complementary studies about measurements of effective concentration of LEL in the NVs
and their stoichiometry in the case of multiple protein functionalization) will re-enforce
the results presented here. Also, the application of calibration curves obtained with this
innovative RM to different real sample will help to consolidate our model.
9.5. Conclusions and future work
The results demonstrate the potential use of this new biomaterial as analytical standard
for molecular recognition based assays, such as immunoassays or aptamer-based assays.
The development of artificial exosomes based on tetraspanin (recombinant LEL) surfaced
functionalized niosomes have been described. The production of mono- and double-
functionalized vesicles with CD9 and/or CD63 is possible, and these particles can be
detected and discerned by sandwich ELISAs, using a classical format based on capture
through monoclonal antibodies and detection based on polyclonal antibodies with
secondary enzyme-labeled antibodies. Dose-response of this particles has been checked,
since a linear fitted response is essential for their use as standard for obtaining calibration
plots used for quantification purposes.
242
The methodology proposed in this study allows the preparation with a tunable
functionalization based on changes in density surface functionalization (by variation in
cholesterol hemisuccinate molar ratio) or changes in the stoichiometry of proteins (by
variation in molar ratio of proteins during coupling to streptavidin-coated niosomes). The
exploration of this variables could be an interesting starting point for future works. Also,
further validation studies must be performed, in order to test their usability for different
cell-line derived exosome quantification. Additional proteins, different than classical
tetraspanins, could be used for the development of pathology-specific standards.
Another interesting field to be explored is the exploration of other methods for

niosomes production, such as microfluidics, that allows an exceptional control of size
control while the chemical consumption is really low. This is essential when formulation
use really expensive compounds, or scarce material such as highly purified proteins are
needed. Furthermore, microfluidics may be a potential platform for the assembly or
reconstitution of proteins into synthetic bilayers while those are being self-assembled.
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Chapter 23, in Immunoassay, edited by Christopoulos T.K. and Diamandis E.P, Academic Press
Inc., San Diego, California, USA.
[34] Christopoulos T.K. and Diamandis E.P., Immunoassay configurations, Chapter 10, in
Immunoassay, edited by Christopoulos T.K. and Diamandis E.P, Academic Press Inc., San Diego,
California, USA.
[35] Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J. P., Primdal-Bengtson, B., Florent
Dingli, F., Loew, D., Tkach, M., Théry, C. (2016). Proteomic comparison defines novel markers to
characterize heterogeneous populations of extracellular vesicle subtypes. Proceedings of the
National Academy of Sciences, 113(8), E968-E977.
[36] Oliveira-Rodríguez, M., López-Cobo, S., Reyburn, H. T., Costa-García, A., López-Martín, S.,
Yáñez-Mó, M., Cernuda-Morollón, E., Paschen, A., Valés-Gómez, M., Blanco-López, M. C. (2016).
Development of a rapid lateral flow immunoassay test for detection of exosomes previously
enriched from cell culture medium and body fluids. Journal of extracellular vesicles, 5(1), 31803.
[37] Rupert, D. L., Claudio, V., Lässer, C., & Bally, M. (2017). Methods for the physical
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Biophysica Acta (BBA)-General Subjects, 1861(1), 3164-3179.
247
10. Conclusiones/Conclusions
Capítulo 10. Conclusiones
La conclusión general que se extrae de los resultados obtenidos a lo largo de la

presente Tesis Doctoral es la probada posibilidad de realizar un correcto control sobre
la producción de nanovesículas con tamaño controlado y monodispersas. Por otra
parte, se mejora la encapsulación de compuestos hidrofílicos y estudia su relación con
el tamaño de partículas. Además, se demuestra su potencial para el desarrollo de
modelos bio-miméticos de exosomas, un tipo natural de vesículas extracelulares con
elevado interés clínico y biológico, y su empleo a modo de estándar analítico. Esta
conclusión es el resumen de los siguientes puntos:
• Se planteó el estudio del efecto de las variables de un método clásico, como es

la inyección de etanol, sobre las propiedades finales de las partículas
(liposomas y niosomas), concretamente el tamaño y la monodispersidad. El
adecuado planteamiento de los diseños de experimentos ha permitido reducir
de diez variables metodológicas iniciales con posible efecto sobre las
respuestas a sólo tres (concentración final de compuestos de membrana en la
fase acuosa, relación de volumen entre fase acuosa y orgánica, y amplitud de
sonicación), que son las de mayor efecto. Se han establecido modelos
matemáticos en forma de ecuaciones polinómicas que permiten predecir el
tamaño y la dispersión del mismo (PDI) en función de los valores de los
factores metodológicos.
Adicionalmente, estudios de estabilidad coloidal llevados a cabo mediante

medidas de potencial Zeta (ζ) y de perfiles de retro-dispersión de luz, así como
de eficacia de encapsulación de compuestos bioactivos, han demostrado su
potencial uso en aplicaciones biotecnológicas diversas, como la distribución y
liberación controlada de compuestos bioactivos, sistemas de amplificación de
señal analítica, y desarrollo de elementos biomiméticos.
• Se ha evaluado la impresión 3D tanto convencional como de alta resolución a

la hora de la fabricación de un dispositivo de microfluídica y su módulo
termostatizado. Esta técnica, ha permitido la fabricación de dispositivos
funcionales y de bajo coste, aunque la morfología y dimensiones de los
microcanales del chip difieren de aquellos planteados en el diseño por
251
ordenador. Estas diferencias podrían atribuirse a parámetros operacionales de
las impresoras, y deben ser estudiados más a fondo en futuros trabajos.
Los resultados de este estudio han mostrado la idoneidad de este tipo de

dispositivos a la hora de preparar nanovesículas. El desarrollo de un sistema
de control de temperatura adaptado al dispositivo microfluídico, ha permitido
el uso de tensioactivos no iónicos con elevada temperatura de transición (Tm),
los cuales se ha demostrado que son los más adecuados en la formulación de
niosomas.
Por un lado, el diseño del dispositivo y su acoplamiento a un microscopio

invertido ha permitido obtener información de procesos clave en la formación
de estas partículas como es la difusión molecular en flujo paralelo. También se
ha observado el papel de la temperatura sobre dicha difusión y sobre el
principio en el que se basa el dispositivo microfluídico: el enfoque
hidrodinámico de fluidos. De forma análoga al estudio anterior, se ha podido
establecer la importancia de los parámetros metodológicos, siendo la relación
de flujos orgánico:acuoso el parámetro fundamental para controlar la
eficiencia del mezclado y las propiedades morfológicas de las partículas.
Por otra parte, la concentración de los componentes de membrana en la fase

orgánica se ha demostrado que condiciona los tamaños finales y los valores de
PDI. Sin embargo, el flujo volumétrico total posee un escaso efecto, y sin
embargo influye de forma importante sobre la eficacia del mezclado de fases,
así como sobre el tiempo de producción. La temperatura puede llegar a
mejorar las propiedades de las partículas, como reducir el tamaño promedio y
los valores de PDI.
• La encapsulación de compuestos hidrófilos en sistemas vesiculares representa

un reto, y no es adecuada de llevar a cabo mediante métodos de inyección de
solventes. Por ello se llevó a cabo un estudio de la influencia de la
composición del medio de hidratación durante el método denominado
hidratación de película delgada. Se ha podido comprobar como la eficacia de
encapsulación (EE) se puede mejorar con la composición del método de
hidratación, y además se observó una clara influencia del mismo sobre el
tamaño de partícula final.
252
Por otra parte, se han descrito efectos particulares del peso molecular de los
compuestos a encapsular tanto sobre el tamaño de partícula como sobre la EE
de dichos compuestos en las vesículas formuladas.
Mediante técnicas calorimétricas, se ha podido comprobar como la

composición del medio de hidratación altera el orden y empaquetado de las
bicapas, pudiendo ser el responsable de los cambios encontrados en la
distribución de tamaño y la EE.
Además, se ha podido comprobarla idoneidad de la diálisis y la filtración en

gel para la purificación de estos sistemas vesiculares formulados en medios
viscosos. Se ha observado una discrepancia entre ellos para este tipo de
suspensiones, y se ha concluido que medios muy viscosos impiden una
correcta separación e introducen un sesgo de tamaño en la población de
vesículas.
• Por último, la información que se ha obtenido de los modelos obtenidos para

la inyección de etanol ha sido empleada para la mejora de las características de
los niosomas formulados con Span® 60: Colesterol (relación molar 1:0,5). En
concreto, la relación de fases orgánica:acuosa se ha mantenido elevada
mientras que se ha reducido la concentración de los componentes de
membrana. Esto ha permitido obtener niosomas de un tamaño similar al de
exosomas naturales, con valores de PDI adecuados, y todo ello evitando
procesos de sonicación, ya que se ha observado que el envejecimiento de la
sonda introduce variabilidad en el proceso.
Estas partículas han sido funcionalizadas en la superficie con moléculas de

estreptavidina tras una optimización del proceso, y han permitido la posterior
funcionalización con tetraspaninas recombinantes biotiniladas, en concreto
CD9 y CD63. Se han podido obtener partículas mono- y bi-funcionalizas con
tamaños similares a exosomas naturales, aunque con distribuciones de tamaño
ligeramente diferentes, probablemente debido al proceso de purificación por
cromatografía de exclusión de tamaños empleado en etapas finales para su
preparación. Estos exosomas totalmente artificiales y producidos mediante
técnicas bio-nanotecnológicas basadas en química supramolecular, han sido
reconocidos y diferenciados en función de la tetraspanina en superficie
253
mediante immunoensayos sándwich de tipo ELISA. A su vez, la señal
analítica responde a la concentración de dichas partículas, lo que en conjunto
las hace candidatas idóneas a ser empleadas como estándares analíticos en
este tipo de métodos de detección basados en reconocimiento molecular.
Sin embargo, el diseño de estas partículas condiciona la selección de

anticuerpos que pueden usarse para su detección, debiendo evaluarse si
dichas configuraciones son adecuadas para muestras biológicas. Además,
estos exosomas deber ser caracterizados en mayor profundidad para conocer
la densidad superficial de proteínas, así como la relación molar real entre ellas
cuando son doblemente funcionalizados. Este parámetro es importante para
explicar la diferencia en concentración de exosomas naturales en una muestra
que se ha obtenido al emplear un calibrado obtenido con exosomas artificiales
y el valor obtenido mediante NTA (Nanoparticle Tracking Analysis). Esta
información ayudará a la mejora del diseño de este tipo de biomaterial con
fines analíticos, y a su adecuación al tipo de exosomas naturales que se desee
mimetizar, ya que es conocida la enorme diversidad fenotípica que existe.
254
Chapter 10. Conclusions
The general conclusion that can be drawn from the results obtained throughout the
work carried out in this Ph.D. Thesis is the possibility to carry out a correct control
over the production of nanovesicles with controlled size and monodisperse size
distribution. On the other hand, the encapsulation of hydrophilic compounds and their
relation to particle size is improved. In addition, it is demonstrated their potential for
the development of biomimetic models of exosomes, a natural type of extracellular
vesicles with high clinical and biological interest, and its use as an analytical standard.
This conclusion is based on the following points:
• The study of the effect of the variables of a classical method, such as Ethanol
Injection Method or EIM, on the final properties of the particles (liposomes
and niosomes), specifically size and monodispersity was proposed. The
adequate approach of the DoE has allowed reducing from ten initial
methodological variables with potential effect on the responses to only three
(organic/aqueous phase volume ratio, membrane precursors final
concentration in the aqueous phase, and sonication amplitude), which are
those with a higher effect. Mathematical models base don polynomic
equations have been setted for the prediction of particle size and
monodispersity (PDI) according with the methodological parameters values.
Additionally, studies of colloidal stability carried out by mmeassuring Zeta

potential (ζ) and backscattering profiles, as well as encapsulation efficiency of
bioactive compounds, have demonstrated their potential use for
biotechnological applications, such as the distribution and controlled release
of bioactive compounds, analytical signal amplification systems, and
development of biomimetic elements.
• Conventional and high-resolution 3D printing have been evaluated for

manufacturing the thermostatic and microfluidic device respectively. This
technique has allowed the production of functional and low-cost devices,
although the morphology and dimensions of the chip microchannels differed
from those proposed in the computer-assisted design. These differences could
255
be attributed to the operational parameters of the printers and should be
further studied in future works.
The results of this study have demonstrated the suitability of these devices for
the preparation of nanovesicles. The development of a temperature control
system adapted to the microfluidic device has allowed the use of non-ionic
surfactants with a high transition temperature (Tm). Those have been
demonstrated as suitables for the formulation of niosomes.
On the other hand, the device design and its coupling to an inverted
microscope have allowed obtaining information on key processes in the
formation of these particles such as molecular diffusion in parallel flow. The
role of temperature on this diffusion and on the principle on which the
microfluidic device is based, the hydrodynamic flow focusing, has also been
reported. In a similar way to the previous study, it has been possible to
establish the importance of the methodological parameters, being the flow rate
ratio of organic:aqueous streams the fundamental parameter to control the
mixing efficiency and the morphological properties of the particles.
On the other hand, the concentration of the membrane components in the

organic phase determined the final sizes and the PDI values. However, the
total volumetric rate had reduced effect, but it has an important influence on
the extent of mixing, as well as on the production rate. The temperature can
improve the properties of the particles, such as reduce size and PDI values.
• The encapsulation of hydrophilic compounds in vesicular systems represents

a challenge, and it is not adequate to be carried out by means of solvent
injection methods. Therefore, a study of the influence of the composition of
the hydration solution was carried out during the method called thin film
hydration. It has been possible to verify how the encapsulation efficiency (EE)
can be improved with the composition of this solution, and also it was
observed a significant influence over particle size.
On the other hand, particular effects of the molecular weight of the

compounds to be encapsulated have been described over the size and EE
values for the vesicles.
256
Using calorimetric techniques such as DSC, it has been possible to verify how
the composition of the hydration medium alters the order and packaging of
the bilayers, which may be responsible for the observations on size and EE in
the study.
Aditional, it has been possible to verify the suitability of dialysis and gel
filtration for the purification of these vesicular systems formulated in viscous
media. A discrepancy between them has been observed for this type of
suspensions, and it has been concluded that very viscous media prevent a
correct chromatographic separation and introduce a size bias in the
population of vesicles.
• Finally, the information obtained from the models for the ethanol injection
method has been used to improve the characteristics of the niosomes
formulated with Span® 60: Cholesterol (1: 0.5 molar ratio). Specifically, the
organic:aqueous phase volume ratio has set high while the concentration of
the membrane components has been reduced. This has allowed obtaining
niosomes with a similar size to that of natural exosomes, with acceptable PDI
values, and all these avoiding sonication processes, since it has been observed
that the aging of the probe introduces uncontrollable variability in the process.
These particles have been surface-functionalized with streptavidin molecules

after optimization of the process, and have allowed subsequent
functionalization with biotinylated recombinant tetraspanins, specifically CD9
and CD63. Mono- and double-functionalized particles have been obtained
with sizes similar to natural exosomes, although with slightly different size
distributions, probably due to the purification process by size exclusion
chromatography used in final steps for its preparation. These fully artificial
exosomes produced by bio-nanotechnological techniques based on
supramolecular chemistry have been recognized and differentiated based on
surface tetraspanins by sandwich ELISA type immunoassays. In fact, the
analytical signal is concentration-dependant, which makes them suitable
candidates to be used as analytical standards in this type of detection methods
based on molecular recognition.
257
However, the design of these particles determines the selection of antibodies
that can be used for their detection and makes necessary the evaluation of
whether such configurations are suitable for biological samples. In addition,
these exosomes must be characterized in depth to know the surface density of
proteins, as well as the real molar ratio between them when they are doubly
functionalized. This parameter is important to explain the difference in
concentration of natural exosomes in a sample that has been obtained by using
a calibration obtained with artificial exosomes and the value obtained by NTA
(Nanoparticle Tracking Analysis). This information will help to improve the
design of this type of biomaterial for analytical purposes, and its adaptation to
the type of natural exosomes that want to mimic since it is known the huge
phenotypic diversity that exists.
258
ANEXO I. Publicaciones derivadas de la Tesis
Doctoral
Article
pubs.acs.org/IECR
Using Factorial Experimental Design To Prepare Size-Tuned

Nanovesicles
Pablo García-Manrique,†,‡ María Matos,† Gemma Gutiérrez,† Oscar R. Estupiñań ,†,‡
María Carmen Blanco-López,‡ and Carmen Pazos*,†
†
Department of Chemical and Environmental Engineering and ‡Department of Physical and Analytical Chemistry, University of
Oviedo, Julián Clavería 8, 33006 Oviedo, Spain
*
S Supporting Information
ABSTRACT: The aim of this work was to prepare size-tuned

nanovesicles using a modified ethanol injection method (EIM)
by applying factorial experimental design. Stable size-tuned
nanovesicles (liposomes and niosomes) with controlled sizes
and high EE values for hydrophobic compounds (Sudan Red
7B and vitamin D3) were achieved. Equations that were able to
predict the mean particle sizes, in the ranges of 55−156 nm for
liposomes and 224−362 nm for niosomes with PDI values
between 0.032 and 0.378, were obtained. These customized
soft nanoparticles could be suitable in food, cosmetic, pharmaceutical, or medical applications, such as diagnosis or therapy.
1. INTRODUCTION cholesterol) produced larger sizes and increased EEs. It is useful

Controlled preparation of nanoparticles has attracted great to deliver efficient amounts of a selected drug into superficial skin
interest in recent years.1 Nanovesicles are an important family of layers without systemic absorption.10 On this basis, the goal of
organic nanoparticles, produced by bottom-up nanotechnology, our work was to set up a bulk method for producing nanovesicles
with relevant applications in biomedicine,2 food science,3 of controlled size that could be subsequently modified for specific
analytical chemistry,4,5 and biosensors.6 They are considered applications.
soft nanoparticles because interactions among their molecular Vesicles are colloidal particles in which a concentric bilayer
components are similar to those arising from biological systems.7 made up of amphiphilic molecules surrounds an aqueous
Most of the work describing their preparation for specific uses compartment. These vesicles are commonly used to encapsulate
has focused on the optimization of their composition with the both hydrophilic and lipophilic compounds, for food, cosmetic,
aim of maximizing encapsulation efficiency, delivery, or delivery pharmaceutical, or medical applications, such as diagnosis or
control. therapy.11 Hydrophilic compounds are entrapped into the
However, size is one of the most critical properties (together aqueous compartments between bilayers, whereas lipophilic
with shape and surface chemistry) for understanding cell-uptake compounds are preferentially located inside the bilayers.12,13 The
processes and, therefore, bioavailability and targetability.7 Several most common types of vesicles are liposomes and niosomes.
studies have focused on the optimization of the drug Liposomes were first described by Bangham et al. in 1965,14
encapsulation efficiency while considering size as just a property and they are basically spherical bilayer vesicles formed by the self-
for controlling administration parameters, such as penetration assembly of phospholipids. This self-assembly process is based
kinetics in topical formulations. For example, Padamwar et al.8 on the interactions occurring between phospholipids and water
studied the encapsulation of vitamin E in liposomes and found molecules, where the polar head groups of phospholipids are
that the amount of lipids yielded a positive correlation with size, exposed to the aqueous phases (inner and outer) and the
which was, in turn, negatively correlated with penetration hydrophobic hydrocarbon tails are forced to face each other in a
efficiency into the skin. Sometimes, size has been found to bilayer.15 Because of the presence of both lipid and aqueous
increase with higher amounts of membrane components, such as phases in liposome structures, they can be used for the
cholesterol, whereas it decreased with higher amounts of encapsulation, delivery, and controlled release of hydrophilic,
surfactants (e.g., Tween 80). Simultaneously, cholesterol or lipophilic, and amphiphilic compounds.15,16
surfactants can affect encapsulation efficiency (EE). Optimal On the other hand, niosomes are vesicles formed by the self-
situations can be reached as a compromise at intermediate levels assembly of nonionic surfactants in aqueous media resulting in
of both factors. In that case, Taha9 also reported an interaction
between membrane-component concentration and size reduc- Received: April 21, 2016
tion by ultrasound, making factor optimization an essential task. Revised: July 28, 2016
In other cases, an opposite effect was observed, and higher Accepted: August 10, 2016
concentrations of membrane components (such as Span 60 and Published: August 10, 2016
© 2016 American Chemical Society 9164 DOI: 10.1021/acs.iecr.6b01552

Ind. Eng. Chem. Res. 2016, 55, 9164−9175
Industrial & Engineering Chemistry Research Article
Table 1. Plackett−Burman Fractional Factorial Design: Responses, Levels, and Factors

response code meaning
Y1 Z-average size of PC liposomes
Y2 PDI of PC liposomes
factors
formulation injection evaporation sonication
level O/A (X1) C (X2) (g/L) I (X3) (mM) QV (X4) (mL/h) TI (X5) (°C) NS (X6) (rpm) TE (X7) (°C) NR (X8) (rpm) A (X9) (%) t (X10) (min)
−1 5:50 2.5 10 50 30 350 35 30 25 15
1 20:50 6.0 150 215 60 900 60 120 42 30
batch X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 Y1 Y2
PB1 1 1 −1 −1 −1 −1 1 −1 1 1 90 0.254
PB2 1 −1 −1 1 −1 1 1 1 1 −1 93 0.129
PB3 −1 −1 −1 −1 −1 −1 1 1 −1 −1 97 0.152
PB4 1 −1 1 −1 1 0 1 −1 1 −1 72 0.205
PB5 −1 1 −1 1 −1 −1 1 −1 −1 1 258 0.413
PB6 1 1 1 1 −1 −1 −1 1 −1 −1 102 0.176
PB7 −1 −1 1 1 −1 −1 −1 −1 1 1 84 0.218
PB8 1 −1 −1 1 1 1 −1 1 −1 1 106 0.240
PB9 −1 −1 −1 −1 1 1 −1 1 1 1 71 0.229
PB10 1 −1 1 −1 −1 −1 −1 −1 −1 1 81 0.141
PB11 −1 1 1 −1 1 −1 1 1 −1 1 152 0.316
PB12 1 1 1 1 1 1 1 1 1 1 65 0.260
PB13 −1 −1 1 1 1 1 1 −1 −1 −1 87 0.189
PB14 −1 1 1 −1 −1 1 −1 1 1 −1 115 0.273
PB15 1 1 −1 −1 1 1 −1 −1 −1 −1 113 0.199
PB16 −1 1 −1 1 1 −1 −1 −1 1 −1 74 0.271
closed bilayer structures.13,17,18 As liposomes, their formation However, a large number of variables are involved in this
process is a consequence of unfavorable interactions between modified EIM, and selection of the most important of them
surfactants and water molecules, and they can also entrap (screening) is a crucial step in rationally preparing vesicles by this
hydrophilic, lipophilic, and amphiphilic compounds.19,20 versatile method. In this work, the Z-average size and
Niosomes exhibit a number of advantages over liposomes, polydispersity index (PDI) were selected as the dependent
such as higher stability, easy access to raw materials, lower variables. They are considered to be of great importance in
toxicity, high compatibility with biological systems, non- nanovesicle design because most of the final applications of these
immunogenicity, and versatility for surface modification.20 vesicular systems are directly related to these two parameters.
Cholesterol is commonly used as a membrane additive for Factorial experimental design and the analysis of variance
nanovesicle preparation to improve vesicle stability, entrapment (ANOVA) methodology are appropriate and efficient statistical
efficiency, and release under storage.20 It increases vesicle size tools that permit the effects of several factors that influence
and rigidity, improving encapsulation efficiency, but at high responses to be studied by varying the factors simultaneously in a
concentrations, it can adversely affect the encapsulation rate.21,22 limited number of experiments.
Cholesterol also plays a fundamental role in niosome formation In the recent past, design of experiments (DoE) has been
when hydrophilic surfactants are used (hydrophilic/lipophilic extensively used for the study and optimization of vesicles and
balance of ∼10).20 other similar organic materials. Different designs can be applied
More than 20 different methods have been identified for to reduce the number of factors involved in the preparation
nanovesicle preparation, and these methods were recently techniques28 and, therefore, to minimize the number of
reviewed.23,24 In this work, a modified ethanol injection method experiments without losing valuable information. Plackett−
(EIM) is used, because it offers some advantages over other Burman design is a type of fractional design involving relatively
methods, such as simplicity, absence of potentially harmful few runs,29 commonly used for the screening of variables.
chemicals, and suitability for scaleup.25 Another important role of DoE is in the optimization of
The conventional EIM was first described in 1973.26 In this nanovesicle composition for the enhancement of intended
technique, lipids/surfactants and additives are first dissolved in purposes. For instance, it has been applied to the formulation of
an organic solvent, such as diethyl ether or ethanol, and then liposomes (phospholipid and cholesterol ratio) for the topical
injected slowly through a syringe into an aqueous phase delivery of vitamin E,8 hybrid liposomes (with both low- and
containing the compound of interest. Then, the organic solvent high-transition-temperature phospholipids) to improve the
is removed using a vacuum rotary evaporator. When ethanol is encapsulation and delivery of silymarin,30 and niosomes for
used as the organic solvent, the spontaneous formation of topical delivery applications.10,31 DoE has also been used to
vesicles occurs as soon as the organic solution is in contact with enhance the transdermal flux of raloxifene hydrochloride32 and
the aqueous phase,27 but vigorous agitation is needed to obtain diclofenac diethylamine33 loaded transfersomes and of other
narrow size distributions. For this purpose, a final sonication polymeric nanoparticles encapsulating an anticancer drug.34
stage was applied in this study after organic-phase removal by Moreover, the interactions between vesicles and proteins, such as
vacuum evaporation. pectin, to improve drug-delivery properties has been studied by
9165 DOI: 10.1021/acs.iecr.6b01552
Ind. Eng. Chem. Res. 2016, 55, 9164−9175
DoE.35 Nanostructured lipid carriers (NLCs) loaded with Table 2. Full Factorial Design (23) with Center-Point
flurbiprofen were also produced under optimal conditions Repetitions (n = 5): Factors, Levels, and Responses
using full factorial design.36
response code meaning
In this work, an initial fractional factorial design with two levels
(Plackett−Burman) was used to screen the most important Y1 Z-average size of PC liposomes
factors in vesicle preparation by the EIM. Then, a 23 two-level full Y2 PDI of PC liposomes
factorial design using center-point replicates was applied to study Y3 Z-average size of S60:Cho niosomes
the influence of the main factors and their interactions on the Z- Y4 PDI of S60:Cho niosomes
average size and PDI. Once the appropriate operating conditions factors
were determined, vesicle stability was studied by using multiple level O/A (X1) C (X2) (g/L) A (X3) (%)
light scattering technology and by measuring the encapsulation −1 (low) 5:50 2 30.0
efficiencies (EEs) of different compounds. 0 (medium) 12.5:50 5 42.5
1 (high) 20:50 8 55.0
2. MATERIALS AND METHODS
2.1. Materials. Phosphatidylcholine (PC) (predominant batch X1 X2 X3 Y1 Y2 Y3 Y4
species C42H80NO8P, MW = 775.04 g/mol) from soybean FF1 1 −1 1 65 0.299 305 0.075
(Phospholipon 90G) was a kind gift from Lipoid (Ludwigshafen, FF2 1 1 −1 97 0.249 362 0.136
Germany). Sorbitan monostearate (Span 60, S60) (C24H46O6, FF3 −1 1 −1 149 0.296 294 0.206
MW = 430.62 g/mol) and cholesterol (Cho) (C27H46O, MW = FF4 −1 1 1 88 0.307 262 0.291
386.65 g/mol) were purchased from Sigma-Aldrich (St. Louis, FF5 −1 −1 1 64 0.342 242 0.120
MO). All membrane components were dissolved in absolute FF6 1 1 −1 100 0.257 360 0.143
ethanol (Sigma-Aldrich, St. Louis, MO). FF7 1 1 1 64 0.272 241 0.182
Methanol, acetonitrile, 2-propanol, and acetic acid of high- FF8 −1 −1 −1 90 0.196 235 0.078
performance liquid chromatography (HPLC) grade were FF9 0 0 0 82 0.219 301 0.195
supplied by Sigma-Aldrich (St. Louis, MO). FF10 1 −1 −1 84 0.205 253 0.032
A phosphate buffer (PB) solution (10 mM, pH 7.4) was used FF11 −1 1 1 107 0.297 276 0.235
in all experiments as the aqueous phase. The buffer solution was FF12 −1 1 −1 156 0.308 275 0.145
prepared in Milli-Q water by dissolving proper amounts of FF13 1 −1 1 65 0.378 248 0.066
sodium dihydrogen phosphate and sodium hydrogen phosphate, FF14 1 −1 −1 97 0.246 268 0.045
supplied by Panreac (Barcelona, Spain). Sodium chloride from FF15 −1 −1 −1 84 0.173 239 0.094
Panreac (Barcelona, Spain) was added to increase the ionic FF16 0 0 0 75 0.224 305 0.253
strength when required according to the experiments listed in FF17 0 0 0 84 0.250 317 0.118
Table 1. For the encapsulation experiments, Fat Red Bluish or FF18 1 1 1 55 0.307 224 0.203
Sudan Red 7B dye (C24H21N5, MW = 379.46 g/mol) and FF19 0 0 −1 77 0.242 308 0.241
cholecalciferol or vitamin D3 (C27H44O, MW = 384.64 g/mol) FF20 0 0 0 84 0.251 337 0.171
were purchased from Sigma-Aldrich (St. Louis, MO). FF21 −1 −1 1 69 0.343 233 0.124
2.2. Factorial Design of Experiments. Factors that could
potentially affect the size of vesicles produced by the EIM were
classified in four groups, according to the different steps involved (version 17) was used for all data analysis. Analysis of variance
in this preparation method: formulation (organic/aqueous phase (ANOVA) was used for this purpose.
volume ratio, phospholipid concentration, and ionic strength), Once the models were obtained taking into account significant
injection (injection flow rate, temperature, and stirring speed), factors and interactions, a set of selected size-tuned vesicles were
evaporation (temperature and rotation speed), and sonication prepared and characterized.
(amplitude and time of sonication). 2.3. Vesicle Preparation. For liposome preparation,
To identify the relative effects of variables on the response, a appropriate weighed amounts of PC were dissolved in different
two-level fractional factorial design was used. A Plackett− volumes of absolute ethanol (5−20 mL range). The same
Burman (P−B) resolution III design with n = 16 runs was procedure was applied to niosome preparation by weighing and
proposed for screening of the initial factors. Two levels were dissolving S60 and Cho in a 1:0.5 weight ratio. Then, the organic
selected for each variable. solution was injected, with a syringe pump (KD Scientific,
Table 1 lists the factors and levels involved in the P−B Holliston, MA) at a flow rate of 120 mL/h, into Milli-Q water
fractional factorial design used, where O/A is the organic/ that was kept at 60 °C and stirred at 500 rpm. Once vesicles
aqueous phase volume ratio, C is the concentration of formed, ethanol was removed at 40 °C under reduced pressure
phospholipid, I is the ionic strength, QV is the injection flow (90 kPa) in a rotary evaporator. The resulting vesicular systems
rate, TI is the injection temperature, NS is the stirring speed were further sonicated for 15 min (CY-500 sonicator, Optic
during injection, TE is the evaporation temperature, NE is the Ivymen System, Biotech SL, Barcelona, Spain), using and
evaporator rotation speed, A is the sonication amplitude, and t is amplitude of 30−55%, a power of 500 W, and a frequency of 20
the sonication time. kHz. The sonication probe was placed in a 100 mL glass beaker at
In a second step, a 23 full factorial design with center-point a constant depth, 1.5 cm above the container bottom.
repetitions (n = 5) was carried out to study the main effects and 2.4. Vesicle Characterization. 2.4.1. Vesicle Size. The Z-
interactions between factors previously selected by the screening average size and PDI of vesicles were determined by dynamic
design (Table 2). All other factors were fixed at certain values. light scattering (DSL) using a Zetasizer Nano ZS system
In both designs, mean diameter (Z-average size) and PDI were (Malvern Instruments Ltd., Malvern, U.K.). Three independent
selected as response variables. Minitab statistical software samples were taken from each formulation, and measurements
9166 DOI: 10.1021/acs.iecr.6b01552
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Figure 1. Pareto chart of the standardized effects of independent variables (factors) on the (A) Z-average size and (B) PDI of PC liposomes for the
Plackett−Burman fractional factorial design.
were performed three times at room temperature without rupture and to extract the encapsulated compound. EE was
dilution. calculated according to the equation
2.4.2. Vesicle Morphology. Morphological analysis of vesicles
was carried out by negative staining transmission electron peak area of filtered sample
EE (%) = × 100
microscopy (NS-TEM), using a JEOL-2000 Ex II transmission peak area of unfiltered sample (1)
electron microscope (Tokyo, Japan). A sample drop was placed
on a carbon-coated copper grid, and excess sample was removed The RP-HPLC system was equipped with an HP G1315A UV/
with filter paper. Then, a drop of 2% (w/v) phosphotungstic acid vis absorbance detector (Agilent Technologies, Palo Alto, CA).
(PTA) solution was applied to the carbon grid and allowed to The column was a Zorbax Eclipse Plus C18 column with a
stand for 1 min. Once the excess staining agent had been particle size of 5 μm, 4.6 mm × 150 mm (Agilent Technologies,
removed with filter paper, the sample was air-dried, and the thin Palo Alto, CA). The mobile phase consisted of a mixture of (A)
film of stained and fixed vesicles was observed with the 100% Milli-Q-water and (B) 100% methanol with gradient
transmission electron microscope. elution at 0.8 mL/min. The step gradient started with a mobile
2.4.3. Vesicle Stability. Vesicle stability was determined by phase of 80% A, running 100% mobile phase B starting in minute
measuring backscattering (BS) profiles in a Turbiscan Lab Expert 5 for 10 min. Mobile phase B was fed for 2 min after each
apparatus (Formulaction, L’Union, France) provided with an injection to prepare the column for the next sample. The
aging station (Formulaction, L’Union, France). separation was carried out at 30 °C. Different wavelengths were
Samples were placed in cylindrical glass test cells, and used for the UV/vis detector, namely, 533 nm for Sudan Red 7B
backscattered light was monitored at 30 °C as a function of and 270 nm for vitamin D3.
time and cell height every 2 h for 7 days.
The optical reading head scans the sample in the cell, 3. RESULTS AND DISCUSSION
providing BS data every 40 μm in percentages relative to 3.1. Effects of Variables on Morphological Character-
standards as a function of the sample height (in millimeters). istics. The responses (Z-average size and PDI) of each batch
These profiles build up a macroscopic fingerprint of the sample at from P−B design were measured by dynamic light scattering
a given time, providing useful information about changes in the (DLS). The relative importance of the main effects on the Z-
size distribution or appearance of a creaming layer or a average size and PDI of PC liposomes are shown in the Pareto
clarification front with time.3,37,38 chart given in Figure 1.
2.4.4. Encapsulation Efficiency (EE). EE also provides useful Researchers must be aware of the confusion of main effects
information related to the stability of the vesicle membrane. with two-factor interactions in this type of design (resolution III),
Hydrophilic compounds are entrapped in aqueous compart- where the alias structure is too complex. However, we decided to
ments between bilayers, whereas lipophilic compounds are use the initial Plackett−Burman design only for screening
preferentially located within the surfactant or lipid bilayer.39 purposes and selection of the main factors from the Pareto chart,
Substances such as drugs, bioactive compounds, dyes, and as is usually accepted. Effects were selected by applying the
nanomaterials incorporated into vesicles can also affect the hierarchical ordering principle, known sometimes as the sparsity-
morphology and stability of the final dispersion. of-effects principle, where higher-order effects (three- or four-
For the purpose of determining EEs, Sudan Red 7B and way interactions) are sacrificed to study lower-order effects
vitamin D3 (hydrophobic compounds) were encapsulated in the (main effects first and two-way interactions next). This principle
two different formulations. suggests that priority should be given to the estimation of lower-
Each compound was analyzed by reverse-phase high-perform- order effects, especially when resources (time and money) are
ance liquid chromatography (RP-HPLC) (HP series 1100 scarce. This postulate is an empirical principle whose validity has
chromatograph, Hewlett-Packard, Palo Alto, CA). Before RP- been confirmed by the analysis of many real experiments.
HPLC analysis could be performed, the nonencapsulated According to these data, the most important variables for both
compound had to be removed by passing the sample through a responses are the organic/aqueous phase volume ratio, the (final
Sephadex G-25 column (GE Healthcare Life Sciences, aqueous-phase) phospholipid concentration, and the sonication
Wauwatosa, WI). Then, both filtered and nonfiltered samples amplitude. These results are in good agreement with previous
were diluted 1:10 (v/v) with methanol to facilitate vesicle studies carried out by Kremer et al.,40 who evaluated the effects of
9167 DOI: 10.1021/acs.iecr.6b01552
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Figure 2. Pareto chart of the standardized effects of independent variables (factors) on the (A,C) Z-average size and (B,D) PDI of (A,B) PC liposomes
and (C,D) S60:Cho niosomes (1:0.5, w/w) for the 23 full factorial design.
some preparation variables on the size and polydispersity of liposomes (with standard deviations ranging from 0.304 to 4.40
liposomes made from two different natural phosphatidylcholines. nm for size and from 0.003 to 0.053 for PDI). Similar size ranges
Their experimental results showed that the most important were also obtained using the EIM in other previously reported
factor in the final size of the liposomes was the lipid studies.22,27,41,43,44
concentration in the alcohol injected into the buffer solution. The normality, variance homogeneity, and randomness
This factor corresponds to the interaction of the lipid amount assumptions were tested with a normal probability plot,
and the flow rate of organic solvent injected, two factors present frequency histogram, and residuals versus fits and residuals
in the Pareto chart in Figure 1. The same explanation was versus order plots, respectively (Supporting Information, Figure
postulated by other authors,8,41,42 confirming that the lipid S2).
concentration clearly affects the liposome size. This factor was No clear aberrant tendencies were observed, because the
found to be the most relevant one for controlling morphological residuals tended to form a line, no typical cornet pattern was
characteristics of phosphatidylcholine liposomes. Szoka43 found observed, and no time-based pattern was detected. Only some
that stirring, ionic strength, and temperature of the aqueous outlier values were detected (Cook’s distance and DFITS values
phase could also contribute to the final size, but the effects of are given in Table S3 of the Supporting Information).
these factors were smaller than those observed for lipid The ANOVA results allowed for an analysis of the
concentration, organic/aqueous phase ratio, and chemical nature contributions of the effects of the independent variables on the
of the organic solvent (a parameter not included in our study).
response function (mean size of PC liposomes). In this case,
Therefore, the experimental results in Figure 1 confirm the
significant two-way interactions were identified: (O/A) × C and
previously reported observations.43
C × A (see Figure 3). Larger sizes are reached when the organic
The ethanol injection method is usually chosen because it
avoids the sonication step, which is needed in several other solution has a higher lipid concentration (more than 20 g/L). On
methods of liposome preparation, such as the thin-film hydration the other hand, the C × A interaction reveals that the degree of
method. Preliminary experiments (data not shown) indicated size reduction upon application of a higher amplitude depends
that sonication is a crucial step for reducing the size of both on the total lipid concentration present in the medium (referred
liposomes and niosomes. Alternatively, small vesicles can be to the final volume of the dispersion).
produced without sonication by using low concentrations of All of the main effects are significant (p < 0.05), with a positive
lipids/surfactants, but with low yield. This is why we decided to effect on the mean size (a higher response value with an increase
include this step as a factor in the present study. in the factor level) for the total lipid concentration and a negative
3.2. PC Liposomes. The first three main effects from the effect (a lower response value with a decrease in factor level) for
Pareto chart obtained for the P−B design were selected for the 23 the organic/aqueous phase volume ratio and the sonication
full factorial design. The ANOVA results for Z-average size and amplitude.
PDI values are listed in Tables S1 and S2 (Supporting These effects can be explained according to a previously
Information), respectively, whereas the corresponding Pareto reported vesicle formation model.45−47 This model relies on the
chart is shown in Figure 2. Mean sizes in the range of 55−156 nm formation of vesicles through intermediate structures, such as
with PDI values between 0.173 and 0.378 were obtained for PC phospholipid bilayer fragments and sheet-like micelles. These
9168 DOI: 10.1021/acs.iecr.6b01552
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Figure 3. Three-dimensional (3D) response surface plots for the factors O/A (organic/aqueous phase volume ratio), C (lipid or surfactant/stabilizer
concentration, g/L), and A (sonication amplitude, %) for the (A,C) Z-average size and (B,D) PDI of (A,B) PC liposomes and (C,D) S60:Cho niosomes
(1:0.5, w/w).
intermediates are the result of amphiphilic self-assembly because applied to vesicles during the sonication process as a result of the
of their characteristic physicochemical properties.48 effect of induced cavitation.49,50 The final effect of ultrasounds
During the injection of ethanol droplets into the aqueous can be controlled by varying the input power, ultrasound
phase, lipid reorganization inside these dispersed droplets to frequency, sonication time, and probe depth into the container.
form bilayers is favored by the fact that lipids energetically prefer As frequency increases, liposomes of smaller size are produced as
a parallel molecular arrangement.45 These planar structures give a result of stronger acoustic cavitation events. This assumption
rise to closed vesicles when their size induces enough surface was confirmed by our results, in accordance with previous
tension to close the structure and minimize the bending energy. studies.49,50 It is important to point out that, to minimize the
The sizes of these intermediates depend directly on the effects of variations in the probe depth, this factor was kept
number of lipid molecules (concentration) and the dispersion constant at 1.5 cm above the container bottom.
degree (solubilization) in the organic phase. It is obvious from Another aspect to be taken into consideration is the effect of
the previous assessment that higher concentrations of lipids in sonication time. It was reported by Silva et al.49 that sonication
the droplets will form higher membrane fragments, as our time plays an important role in decreasing vesicle size, although
experimental results and previous observations confirm.8,40−42 they observed that this effect reached a plateau at about 21 min.
It is also important to know how easily lipid droplets are Our P−B design revealed a positive effect of sonication time on
dispersed, as well as their size and homogeneity. Lipids of higher the Z-average size (from 15 to 30 min), although it was weaker
solubility will then form smaller lipid droplets and, consequently, than the effects of the other variables selected for the 23 full
shorter membrane fragments (and ultimately tiny vesicles).40 factorial design (especially sonication amplitude). A similar
This explains, in a simplified way, why higher organic/aqueous influence was observed for the PDI response, but with a stronger
phase ratios yield smaller liposomes. effect. We preferred to select sonication amplitude instead of
The negative effect of the sonication amplitude is explained by sonication time because one of the goals of controlling factors is
vesicle rupture, which takes place when an excess of energy is to obtain a narrow size distribution.
9169 DOI: 10.1021/acs.iecr.6b01552
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Table 3. Estimated Coded Coefficients for the Considered Effects on the Z-Average Size and PDI of PC Liposomes and S60:Cho
Niosomes (1:0.5, w/w)
coefficientsa
response constant X1 X2 X3 X1X2 X1X3 X2X3 X1X2X3 R2
Z-Average Size
liposome (Y1) 89.68 −11.14 12.40 −17.50 −11.75 − −5.97 − 96.69
niosome (Y3) 269.82 12.72 16.87 −15.94 − −12.15 −19.92 −16.04 91.27
PDI
liposome (Y2) 0.280 − − 0.038 −0.012 − −0.029 − 89.35
niosome (Y4) 0.136 −0.026 0.057 0.026 − − − − 84.73
a
X1, organic/aqueous phase volume ratio; X2, PC or S60:Cho concentration (g/L); X3, sonication amplitude (%).
As the design included a center point with several repetitions are shown in Figures 2 and 3, respectively. Mean sizes in the
(n = 5), the presence of curvature in the response variables could range of 224−362 nm with PDI values between 0.032 and 0.291
be tested (Figure 3). Because curvature seemed to be significant were obtained for S60:Cho niosomes (with standard deviations
(p < 0.05), a term involving center point (Ct Pt) was included in ranging from 1.05 to 7.28 nm for size and from 0.009 to 0.052 for
the equations for its estimation. PDI). Similar size and PDI ranges were reported for niosomes
With all of this information about the effects and their prepared by the EIM using Span 60 as the membrane
estimated coefficients, the following equation (R2 = 96.69%) for component.17
the Z-average size value of PC liposomes (Y1) was generated Two-way interactions (O/A × A, C × A) and a three-way
interaction (O/A × C × A) were detected, with sonication
Y1 = 62.8 + 2.55(O/A) + 0.449C − 0.185A amplitude (A) as the common factor in these interactions (see
− 0.0185(O/A) × C − 0.00555C × A − 9.26(Ct Pt)
Figure 2C). Therefore, it can be postulated that sonication
amplitude is the key factor in the niosome size response. The
(2)
response depends on both the O/A and C factor levels, with a
Different behavior was observed regarding PDI, which was higher interaction between the sonication amplitude and the
strongly affected by the sonication amplitude as the only total amount of membrane components. Differences in the
significant main effect and its interaction with the total lipid magnitude of the coefficient of this factor between liposomes and
amount. The O/A × C interaction was also detected, but with a niosomes can be attributed to the initial size before sonication
lower effect on the PDI response. (smaller for liposomes) and vesicle stability.50
To understand the C × A interaction, it is important to take The three main effects are significant, but in contrast to the
into account the effect of the sonication amplitude as the main case for liposomes, the organic/aqueous phase volume ratio (O/
effect. An increase in this factor leads to a less monodisperse size A) shows a positive effect on niosome size. This behavior could
distribution, that is, higher PDI values. However, according to be due to different molecular features of the surfactant and
the interaction, this response depends highly on the total amount stabilizer that result in different interactions with the organic
of lipids present in the sample. At a low level of the lipid amount, phase and, therefore, poor or insufficient solubility.
the reduction in size is more effective (as previously mentioned), The other two variables (C, A) have effects similar to those
but the size distribution is large. However, at a high level of the described above for liposomes. Therefore, the same explanation
lipid amount, this enlargement of the size distribution is regarding surfactant concentration and sonication amplitude can
significantly lower. be applied here to justify their effects on niosome size. In this
Curvature in the response was also tested, again revealing a case, the stronger effect of C is explained by the influence of
significant presence (p < 0.05). For the PDI response (Y2), the cholesterol on the final size of vesicles, as reported by Padamwar
following equation with an R2 value of 89.35% was obtained and Pokharkar.8
Once again, curvature was detected for the Z-average size
Y2 = −0.160 + 0.00939A − 0.0000420(O/A) × C response. The following equation was obtained to model this
− 0.0000250C × A − 0.0425(Ct Pt) case, with an adjusted correlation coefficient (R2) of 91.27%
(3)
These equations were formulated with uncoded coefficients, Y3 = 236.9 − 4.31(O/A) − 0.012C − 0.56A
making it easier to use them to predict selected target size and
PDI values. + 0.0461(O/A) × C + 0.00363C × A
3.3. S60:Cho Niosomes. To investigate whether the − 0.00114(O/A) × C × A + 44.00(Ct Pt) (4)
selected factors in the P−B design for PC liposomes (a reference
model for vesicular systems) produced similar effects with other On the other hand, a completely different behavior was observed
different formulations, the same 23 full factorial design using regarding the PDI response. Only the three main effects (O/A, C,
center-point replicates was performed for a typical niosome A) were found to be significant, and no interactions were found.
formulation, in this particular case, S60:Cho niosomes (1:0.5, w/ Two positive effects on the niosome PDI were detected:
w). The main variables were the organic/aqueous phase volume surfactant/stabilizer concentration and sonication amplitude. In
ratio (O/A), the total concentration of surfactant and stabilizer this case, the total concentration of membrane components
(C), and the sonication amplitude (A). seemed to have an important role in the vesicle size distribution,
The ANOVA results for Z-average size and PDI values are as can be seen in the correspondent Pareto chart (Figure 2). This
listed in Table S1 (Supporting Information), and the observation once again can be attributed to the solubilization of
corresponding Pareto chart and three-dimensional surface plot membrane components in the organic phase. Higher concen-
9170 DOI: 10.1021/acs.iecr.6b01552
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Figure 4. Optimization plot and values of individual (d) and composite (D) desirability provided by the response optimizer (Minitab, version 17) for an
example of size-tuned PC liposome (desired size = 70 nm, with a minimum PDI value).
trations of these components require better solubilization in The estimated coded coefficients for the considered effects on
dispersed droplets to reach small membrane fragments. the Z-average sizes and PDIs of PC liposomes and S60:Cho
It is important to note that some combinations of factors niosomes are listed in Table 3, as a summary of the factors’
yielded narrow size distributions, namely, PDI ≤ 0.100, a value influence. Coded coefficients were used to maintain the
frequently obtained by other preparation methods, such as orthogonality of the designs and to allow for a direct comparison
microfluidic hydrodynamic focusing51 also using S60:Cho as the between coefficients.
formulation. 3.4. Vesicle Characterization. Size-tuned vesicles were
A negative effect was detected for the organic/aqueous phase prepared under selected operating conditions by applying the
volume ratio (O/A). As the final concentration of ethanol models obtained from the experimental design (eqs 2−5) and
increased during the injection process, a smaller size distribution the assistance of the response optimizer and response predictor
was obtained. As previously mentioned, no interaction between in Minitab statistical software (version 17). These tools can be
this factor and the total concentration of membrane components
applied to the simultaneous optimization of several responses
was observed.
only when the same set of factors are studied separately, because
The following equation with an R2 value of 84.73% was
obtained for the niosome PDI model response (Y4) a common experimental region is needed.
The operating conditions were selected to prepare PC
Y4 = 0.053 − 0.00392(O/A) + 0.000039C + 0.00067A liposomes with a mean size of 70 nm and the minimum PDI
value (predicted values of Y1 = 67 ± 4 and Y2 = 0.317 ± 0.013)
+ 0.0597(Ct Pt) (5) and S60-Cho niosomes with a mean size of 240 nm and the
9171 DOI: 10.1021/acs.iecr.6b01552
Ind. Eng. Chem. Res. 2016, 55, 9164−9175
Figure 5. Optimization plot and values of individual (d) and composite (D) desirability provided by the response optimizer (Minitab, version 17) for an
example of size-tuned S60:Cho niosome (1:0.5 w/w) (desired size = 240 nm, with a minimum PDI).
minimum PDI value (predicted values of Y3 = 239 ± 11 and Y4 = vesicles, as a result of the interactions of the electron beam with
0.120 ± 0.025). These sizes and PDI values were selected only as PTA, which produces a selective deposit of metal ions that
an example. The factor output values were O/A = 5:50, C = 2 g/ enhances morphological details. The micrographs show spherical
L, and A = 55% for the liposomes and O/A = 5.9:50, C = 2 g/L, structures of approximately 80 nm for the liposomes (Figure 6C)
and A = 55% for the niosomes. Figures 4 and 5 show optimization and about 250 nm for the niosomes (Figure 6D). These values
plots and values of individual and composite desirability for size- agree with the DLS measurements.
tuned liposomes and size-tuned niosomes, respectively. Figure 6D shows clusters of niosomes that are all similar in
The experimental results showed that the models obtained
size. Aggregation arose during the drying step prior to TEM
with the experimental design were accurate, because mean sizes
of 69 ± 0.5 nm (PDI = 0.245 ± 0.005) and 233 ± 3 nm (PDI = measurements, because no flocculation phenomena were
0.112 ± 0.004) were obtained for the PC liposomes and S60:Cho monitored with the Turbiscan apparatus.
niosomes, respectively. The relative error was low for the Slight differences were noticed in the zeta potential measure-
experimental results regarding mean size (3% for Y1 and Y3) but ments, exhibiting low values for both types of vesicles. Niosomes
higher for the size distributions (22% for Y2 and 7% for Y4). had values of about −16.8 ± 0.7 mV, whereas the liposomes had
The sizes and morphologies of the vesicles were investigated values of −6.9 ± 0.3 mV. This small value for the liposomes could
by TEM, using a negative contrast. Figure 6 shows black-stained be due to neutralization of the negative charge from the
9172 DOI: 10.1021/acs.iecr.6b01552
Ind. Eng. Chem. Res. 2016, 55, 9164−9175
Figure 6. (A,B) BS profiles and (C,D) TEM micrographs of empty vesicles designed with a controlled size and PDI values by applying the models
obtained from experimental design: (A,C) PC liposomes and (B,D) S60:Cho niosomes (1:0.5, w/w).
phosphate groups by sodium cations present in the medium High EE values were obtained for both Sudan Red 7B and
(from sodium chloride in the PB solution). vitamin D3, as expected taking into account their hydrophobic
The formulated vesicles exhibited a high stability after 1 week character. EE values up to 90.1% and 88.0% were obtained for
of monitoring time. BS profiles obtained for the PC liposomes Sudan Red 7B encapsulated in PC liposomes and S60:Cho
are given in Figure 6, where a variation of 4.5% in the middle part niosomes, respectively. Experiments carried out with vitamin D3
of the cell (from 10 to 30 mm) is noticed. A simultaneous slight led to EE values of 99.2% for PC liposomes and 73.9% for
clarification process was observed in the middle and top parts of S60:Cho niosomes. These results are in good agreement with
the cell in the corresponding transmission profile (results not those of previous studies, where compounds with similar
shown). This was promoted by some movement of the PC chemical properties were encapsulated.12,13,27
liposomes toward the bottom of the cell, resulting in a slight
increase in BS (sedimentation). However, this was a reversible 4. CONCLUSIONS
process, caused by differences in concentration, with the sample In this work, an adequate approximation using DoE was applied
remaining stable and maintaining its initial properties (size and to study the influence of experimental factors of the EIM on the
PDI). The vesicles were again characterized after gentle agitation mean size and size distribution of PC liposomes and S60:Cho
of the cell at the end of the monitoring time with analogous niosomes (1:0.5, w/w).
results. An initial screening design enabled a reduction of the number
For the S60:Cho niosomes (Figure 6B), the BS profile of variables. This was a necessary step before carrying out a full
factorial design. Finally, response models were applied to prepare
remained nearly constant (variations of approximately 0.5%)
selected size-tuned nanovesicles, which were characterized from
with time, showing high stability. Some variation was also
a stability point of view.
observed in the transmission profile all along the cell height, This was achieved with a low number of experiments (58
because the sample was not translucent. runs). This methodology enabled two different formulations
3.4.1. Encapsulation Efficiency (EE). Vesicles containing (liposomes and niosomes, the most common types of nano-
Sudan Red 7B and vitamin D3 as model compounds (both vesicles) to be studied in a comparative way. Stable liposomes
lipophilic) were also prepared and characterized. No differences and niosomes of the targeted sizes were successfully prepared
were observed regarding mean size and PDI values or TEM, zeta with the model equations obtained, with encapsulation
potential, or Turbiscan measurements, meaning that the efficiencies higher than 73.9% in all cases for selected
entrapped compounds did not affect the vesicle’s behavior. hydrophobic compounds.
9173 DOI: 10.1021/acs.iecr.6b01552
Ind. Eng. Chem. Res. 2016, 55, 9164−9175
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■
Maccioni, A. M.; Sinico, C.; Fadda, A. M. Development of novel
AUTHOR INFORMATION diolein−niosomes for cutaneous delivery of tretinoin: Influence of
formulation and in vitro assessment. Int. J. Pharm. 2014, 477, 176−186.
Corresponding Author (19) Mahale, N. B.; Thakkar, P. D.; Mali, R. G.; Walunj, D. R.;
*Tel.: +34 985103509. E-mail: [email protected]. Chaudhari, S. R. Niosomes: Novel sustained release nonionic stable
Notes vesicular systems  An overview. Adv. Colloid Interface Sci. 2012, 183−
The authors declare no competing financial interest. 184, 46−54.
■
(20) Moghassemi, S.; Hadjizadeh, A. Nano-niosomes as nanoscale
drug delivery systems: An illustrated review. J. Controlled Release 2014,
ACKNOWLEDGMENTS 185, 22−36.
This work was supported by the Ministerio de Economiá y (21) Mali, N.; Darandale, S.; Vavia, P. Niosomes as a vesicular carrier
Competitividad (MINECO, Spain), under Grant MINECO-13- for topical administration of minoxidil: formulation and in vitro
CTQ2013-47396-R. This study was also cofinanced by the assessment. Drug Delivery Transl. Res. 2013, 3, 587−592.
Consejeriá de Educación y Ciencia del Principado de Asturias (22) Fan, M.; Xu, S.; Xia, S.; Zhang, X. Preparation of salidroside nano-
(ref FC-04-COF-50-MEC, PCTI Asturias 2006-2009, ref liposomes by ethanol injection method and in vitro release study. Eur.
Food Res. Technol. 2008, 227, 167−174.
EQP06-024, and FC15-GRUPIN14-022). We especially thank
(23) Marianecci, C.; Di Marzio, L.; Rinaldi, F.; Celia, C.; Paolino, D.;
Prof. Antonia Salas (University of Oviedo) for her advice with Alhaique, F.; Esposito, S.; Carafa, M. Niosomes from 80s to present: The
the statistical work.
■
state of the art. Adv. Colloid Interface Sci. 2014, 205, 187−206.
(24) Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S. W.;
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Supporting Information
Using factorial experimental design to prepare size-tuned

nanovesicles
Pablo García-Manrique1,2, María Matos1, Gemma Gutiérrez1, Oscar R.

Estupiñán1,2, María Carmen Blanco-Lopez2 and Carmen Pazos1*
1
Department of Chemical and Environmental Engineering, University of Oviedo,
Julián Clavería 8, 33006 Oviedo, Spain.
2
Department of Physical and Analytical Chemistry, University of Oviedo, Julián
Clavería 8, 33006 Oviedo, Spain.
*Corresponding author. Tel: +34 985103509

E-mail address: [email protected] (Carmen Pazos)
- S1-
Table S1. ANOVA results (coded units) for Z-average size of PC-liposomes for
the 23 full factorial design; results for S60:Cho niosomes are also given (cursive
numbers)
Source DF Seq SS Adj SS Adj MS F P
Main effects 3 9349.7 9349.7 3116.6 86.7 0.000

11209.8 11209.8 3736.6 14.9 0.000
2-Way
3 2805.3 2805.3 935.1 26.0 0.000
Interactions
8828.3 8828.3 2942.8 11.7 0.001
3-Way
1 113.2 113.2 113.2 3.2 0.101
Interactions
4116.8 4116.8 4116.8 16.4 0.002
Curvature 1 326.9 326.9 326.9 9.1 0.011
7370.0 7370.0 7370.0 29.3 0.000
Residual Error 12 431.2 431.2 35.9
3015.0 3015.0 251.3
Pure Error 12 431.2 431.2 35.9
3015.0 3015.0 251.3
Total 20 13026.3
34539.9
ANOVA indicates analysis of variance; DF, degrees of freedom; SS, sum of squares; MS,
mean of squares; F, Fischer’s ratio; P, p-value.
- S2-
Table S2. ANOVA results (coded units) for PDI of PC-liposomes for the 23 full
factorial design; results for S60:Cho niosomes are also given (cursive numbers)
Source DF Seq SS Adj SS Adj MS F P
Main effects 3 0.0256 0.0256 0.0082 16.72 0.0000

0.0728 0.0728 0.0243 18.30 0.0000
2-Way
3
Interactions 0.0162 0.0162 0.0054 11.02 0.0010
0.0017 0.0017 0.0006 0.43 0.7370
3-Way
1
Interactions 0.0017 0.0017 0.0017 3.39 0.0900
0.0002 0.0002 0.0002 0.18 0.6830
Curvature 1 0.0069 0.0069 0.0069 14.05 0.0030

0.0136 0.0136 0.0136 10.22 0.0080
Residual Error 12 0.0059 0.0059 0.0005

0.0159 0.0159 0.0013
Pure Error 12 0.0059 0.0059 0.0005

0.0159 0.0159 0.0013
Total 20 0.0550
0.1042
ANOVA indicates analysis of variance; DF, degrees of freedom; SS, sum of squares; MS,
mean of squares; F, Fischer’s ratio; P, p-value.
- S3-
Table S3. COOK's distance and DFITS values obtained for each response in the
full factorial designs
BATCH COOK Y1 DFIT Y1 COOK Y2 DFIT Y2 COOK Y3 DFIT Y3 COOK Y4 DFIT Y4

FF1 0.000 -0.019 0.708 -3.530 0.706 3.518 0.003 0.168
FF2 0.015 -0.358 0.007 -0.245 0.001 0.073 0.002 -0.130
FF3 0.089 -0.889 0.016 -0.369 0.077 0.823 0.156 1.207
FF4 0.530 -2.693 0.011 0.307 0.046 -0.626 0.131 1.096
FF5 0.028 -0.486 0.000 -0.031 0.020 0.413 0.001 -0.074
FF6 0.015 0.358 0.007 0.245 0.001 -0.073 0.002 0.130
FF7 0.116 1.024 0.139 -1.131 0.063 0.740 0.018 -0.393
FF8 0.058 0.705 0.060 0.720 0.004 -0.173 0.011 -0.299
FF9 0.001 0.103 0.023 -0.457 0.022 -0.440 0.000 -0.009
FF10 0.255 -1.614 0.191 -1.355 0.046 -0.626 0.007 -0.242
FF11 0.530 2.693 0.011 -0.307 0.046 0.626 0.131 -1.096
FF12 0.089 0.889 0.016 0.369 0.077 -0.823 0.156 -1.207
FF13 0.000 0.019 0.708 3.530 0.706 -3.518 0.003 -0.168
FF14 0.255 1.614 0.191 1.355 0.046 0.626 0.007 0.242
FF15 0.058 -0.705 0.060 -0.720 0.004 0.173 0.011 0.299
FF16 0.033 -0.554 0.012 -0.325 0.010 -0.291 0.086 0.980
FF17 0.016 0.368 0.012 0.315 0.002 0.122 0.158 -1.571
FF18 0.116 -1.024 0.139 1.131 0.063 -0.740 0.018 0.393
FF19 0.010 -0.294 0.002 0.116 0.005 -0.208 0.054 0.729
FF20 0.015 0.361 0.014 0.341 0.077 0.909 0.016 -0.370
FF21 0.028 0.486 0.000 0.031 0.020 -0.413 0.001 0.074
- S4-
Figure S1. Contour Plot for the factors O:A (organic:aqueous phase volume
ratio), C (lipid or surfactant/stabilizer concentration, g/L) and A (sonication
amplitude, %) on Z-average size, nm (A) and PDI (C) of PC-liposomes, and Z-
average size, nm (B) and PDI (D) of S60:Cho niosomes (1:0.5, w/w).
- S5-
Figure S2. Testing for normality, variance homogeneity and randomness assumptions of ANOVA for the full
factorial design of PC-liposomes and S60:Cho niosomes (1:0.5 w/w).
- S6-
9. Olle, M. and Virsile, A. (2013) The effects of light-emitting
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extensively dependent on narrow[83_TD$IF]-band- Blackwell cacy evaluation. Their molecular
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brane and cargo, includes lipids, proteins,
and nucleic acids, providing information
Acknowledgements
This work was supported by UEF spearhead project
Forum about their cells of origin and their status.
Our ability to now acquire this valuable
CABI (JKH, RJ-T) and the European Social Fund (ESF)
grant no. S12530 (JKH, MK).
Fully Artificial cellular information and the fact that it also
Exosomes: Towards comes from hard-to reach tissues, has
resulted in the term ‘liquid biopsy’, a
1
Department of Environmental and Biological Sciences,
University of Eastern Finland, P.O. Box 1627, FI-70211
Kuopio, Finland
New Theranostic new frontier in the clinical field.
2
Department of Environmental and Biological Sciences,
University of Eastern Finland, P.O. Box 111, [82_TD$IF]FI-80101,
Biomaterials In addition, exosomes combine the
Joensuu, Finland
1,2 advantages of nanocarriers (i.e., particles
Pablo García-Manrique,
used to efficiently deliver molecules) and
*Correspondence:
jarmo.holopainen@uef.fi (J.K. Holopainen).
Gemma Gutiérrez,1 and
therapeutic agents. Exosomes are cur-
http://dx.doi.org/10.1016/j.tibtech.2017.08.009 Maria Carmen Blanco-López2,* rently considered among the most prom-
ising drug delivery systems, especially for
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10 Trends in Biotechnology, January 2018, Vol. 36, No. 1

Top-down methods Boom-up methods
Fully arficial exosomes

Nanoporous membranes Microfluidics A key to future Supramolecular chemistry
• Mass producon of NVs nanomedicine? • Producon of a high pharmaceucal
• NVs with natural target properes, specificity grade product
tuneable by precursor cell line selecon • Ideal plaorm for basic studies of
• Expression of receptors and co-receptors independent elements
allowing true cell interacons • Known composion, able to be adapted
• Naturally immunotolerated to specific applicaons
• No selecvity during cargo sorng • No scaled-up funconalizaon
• Need for purificaon steps and trained • Challenging incorporaon of mulple
personnel, me-consuming surface elements
Figure 1. Advantages (Green) and Disadvantages (Red) of Two Approaches for the Bionanotechnological Development of Fully Artificial Exosomes
as Theranostic Agents. Abbreviation: NV, nanovesicles.
Despite the great expansion of techni- membrane fragments that will be used proliferation enhancement [8]; and (ii) spe-
ques for developing semisynthetic exo- to form the vesicles. Different methodolo- cific microfluidic devices. Simple pressur-
somes, the main drawbacks concerning gies based on this approach have been ization over a device based on an array of
their clinical application include produc- developed, mainly for drug delivery, but parallel hydrophilic microchannels has
tion, isolation, modification, and purifica- also for enhancing cell proliferation and been described for the production of
tion on a large scale and at a suitable for generating exosome-mimetic models NVs for the delivery of endogenous
clinical grade. However, these drawbacks for biodistribution analyses (i.e., monitor- RNA to targeted cell cultures [9]. The
have resulted in the design and manufac- ing exosomes after exogenous adminis- fabrication of microchannels on micro-
ture of fully synthetic exosome-mimetic tration) (Table 1). Two of the most relevant blades (fabricated in silicon nitride)
particles using bionanotechnology. Here, strategies involve: (i) extrusion over poly- resulted in a device able to slice living cells
we provide an overview of these techni- carbonate membrane filters. This is a as they flowed through the channels [10].
ques, organized according to two trends common way to reduce the mean size The incorporation of exogenous material
in nanofabrication: top-down and bot- and homogenize the size distribution of in the cell suspension enhanced their
tom-up approaches (Figure 1). colloidal systems. By applying this tech- encapsulation by plasma membrane frag-
nique to cultured cells, NVs have been ments during reassembly and suggested
Top-Down Methodologies: produced with a simple commercial lipo- a method for in vitro exogenous material
Bioengineering Cells as some extruder and diminishing pore size delivery.
Membrane Fragment Precursors filters [5,6]; they have been used to treat
Top-down methodologies rely on the pro- tumors by targeted encapsulated chemo- Both of these methods are suitable for the
duction of nanosized materials by break- therapeutics. A scaled-up version was production of larger amounts (by a factor
ing bigger and more complex units into also developed with a device designed of more than 250) of biologically active
smaller parts. In this case, the production to be used with conventional laboratory particles compared with natural exosome
of artificial exosomes begins with cultured centrifuges [7]. This device was used to release yields. Sizing and biochemical
cells that are then used to produce mass-produce NVs in a study of cell profiling showed that these NVs were
Trends in Biotechnology, January 2018, Vol. 36, No. 1 11

Table 1. [145_TD$IF]Fully Artificial Exosomes based on Top-Down and Bottom-Up Bionanotechnology[146_TD$IF].a
Mechanism of membrane Precursor cell lines Type and example of material Application Refs
fragmentation incorporated
Studies based on top-down approaches
Manual extrusion over Human monocytes (U937) and Exogenous, chemotherapeutic drugs Targeted delivery of [5]
polycarbonate membrane murine mouse macrophages (doxorubicin, 5-FU, gemcitabine, and chemotherapeutics to an in vitro model
filters with a device for (Raw 264.7) carboplatin) (TNFa-treated HUVECs) and in vivo-
liposome preparation induced malignant tumors (CT26
mouse colon adenocarcinoma cells)
Murine mouse macrophages Exogenous, radiolabeling agent 99mTc- In vivo biodistribution of exosomes and [6]
(Raw 264.7) HMPAO artificial counterparts
Centrifugal-induced extrusion Murine mouse embryonic Endogenous, precursor cell Gene delivery to NIH-3T3 fibroblast [7]
over membrane filters in a stem cell line (D3) characteristic RNA (mOct 3/4 and cells
device designed to be used in mNanog)
lab centrifuges
No intention to encapsulate any Enhanced in vitro cell proliferation for [8]
specific compounds regenerative medicine (murine skin
fibroblasts)
Pressurization over hydrophilic Murine mouse embryonic Endogenous, precursor cell Gene delivery to NIH-3T3 fibroblast [9]
microchannels array on a stem cell line (D3) characteristic RNA (mOct 3/4 and cells
microfluidic device mNanog)
Living cells sliced with silicon Murine mouse embryonic Exogenous, polystyrene beads as Material delivery to mouse embryonic [10]
nitride blades in a microfluidic stem cell line (D3) representative exogenous material fibroblasts
device
Type of formulation NV preparation strategy Proteins for NV functionalization Application Refs
Studies based on bottom-up approaches
Classical liposome, PC:Chol Thin film hydration method MHC class I peptide complexes and Ex vivo and in vivo T cell expansion for [12]
(TFHM) and maleimide-based FAB regions against T cell receptors for immunotherapies
bioconjugation strategy adhesion, early and late activation, and
survival
Mimicking exosome lipid TFHM and Ni2+/His-Tag APO2L/TRIAL-His10 recombinant Downregulation of T cell activation in an [13,14]
composition, PC:Chol:SM protein coordination as proteins autoimmune disease animal model
bioconjugation strategy (antigen-induced arthritis);
immunotherapy for apoptosis induction
in hematological tumors
Innovative liposomes, PC: Microemulsion and micelle- Monoclonal antibody against DEC205 Proof of concept of artificial antigen [15]
CpEL:DOPE assembling method for vesicle dendritic cell antigen presentation to dendritic cells
formation
a
Abbreviations: 99mTc-HMPAO, 99mTc-hexamethylpropyleneamineoxime; Chol, cholesterol; CpEL, chemopor EL; DOPE, dioleoyl-phosphoethanolamine; HUVECs,
human umbilical vein endothelial cells; MHC, major histocompatibility complex; PC, phosphatidylcholine; SM, sphingomyelin; TFHM, thin film hydration method.
substantially similar to natural exosomes issue regarding the production of these include those used in the cosmetic and
[4–9]. Their ability to exhibit receptors and mimetic particles is the need for final puri- drug-delivery industries for the prepara-
co-receptors, which is essential to target fication steps identical to those used for tion of liposomes, which are particles
and effectively interact with receptor cell exosome isolation, which require trained formed by a lipid bilayer that resembles
populations, is of special relevance. In personnel and are time-consuming. the plasma membrane. These materials
addition, their natural origin from cells are a promising starting point to design
Bottom-Up Techniques:
renders them immunotolerant [4–9]. and manufacture artificial exosomes. The
Mimicking the Plasma Membrane self-assembled synthetic bilayer can be
However, these methods have some by Preparing Artificial Bilayers then functionalized with selected proteins
drawbacks. Cargo sorting lacks selectiv- By contrast, bottom-up techniques cre- to mimic the desired exosomal functions.
ity due to the passive encapsulation of the ate complex structures from molecular With this purpose in mind, special lipids
surrounding medium during membrane building blocks by using their physical can be incorporated into the liposome
fragment self-assembly. Another relevant and chemical properties. These methods formulation. These can be modified with

chemical groups (such as carboxylic, to be functionalized with proteins). The platform for particle production and drug
amine, or metal chelating agents) with attachment of multiple molecules to encapsulation. This strategy can rapidly
the capacity to create covalent bonds NVs is also challenging because conjuga- test multiple formulations thanks to the
with the selected biomolecules through tion procedures require stable and spe- fast product generation and also con-
known bioconjugation techniques [11]. cific conditions [14]. sumes a significantly smaller amount of
chemicals.
Among the many methods for preparing [149_TD$IF]On the other side, another advantage of
liposomes, one of the most often used to this method is that it can be adapted to
In terms of the composition of NVs,
develop mimetic NVs is the thin film the encapsulation of nucleic acids, which
alternatives to lipids are being explored.
hydration method. This is a two-step pro- is challenging due to the unstable nature
Current research is testing the possibili-
cess where a dried film of lipids is of the molecules. The best encapsulation
ties of niosomes (vesicles formulated
hydrated by an aqueous medium contain- efficiencies are obtained with cationic lip-
with non-ionic surfactants) as a sustain-
ing the compounds to be encapsulated. ids, which are more immunogenic than able alternative to lipid-based particles
Artificial exosomes have been produced their uncharged counterparts [15]. in the development of artificial exo-
with a classical liposome formulation [12] somes. The main advantages of these
and a lipid formulation simulating the Concluding Remarks and Future vesicles are their wide array of starting
composition of exosomes [13]. Using Perspectives compounds, lower price, better physical
chemical bioconjugation procedures, cer- New therapies with fully artificial exo- and chemical stability, and higher
tain protein and/or peptide complexes somes could be a basis for future person- biocompatibility.
and ligands involved in T cell receptor alized nanomedicine. However, whereas
interactions and activation have been clinical trials of natural EVs have just As new production routes are improved,
attached to vesicles for immunotherapy begun, biomimetic materials produced novel NVs closer to real exosomes will
(ex vivo and in vivo cell expansion, by the synthetic routes discussed here become available, making personal nano-
Table 1). In addition, the incorporation have not yet reached clinical translation. medicine and theranostic agents adapted
of certain ligands was reported to induce Current challenges include the establish- to particular needs possible.
apoptosis and downregulate T cell acti- ment of regulatory protocols, mode of
vation in autoimmune diseases, such as actions, and clarification of safety [143_TD$IF]Acknowledgments
antigen-induced arthritis, [13]. These NVs aspects. Commercialization also requires This work was financed by the Consejería de
have also been evaluated for the treat- scaling-up procedures. Automatization of Economía y Empleo del Principado de Asturias (Plan
ment of hematological tumor cells [14]. purification steps would also be highly de Ciencia, Tecnología e Innovación 2013-2017),
desirable. A multidisciplinary approach, under the Grant GRUPIN14-022. Support from the
European Regional Development Fund (ERDF) is
More recently, a method based on with contributions from molecular biology,
gratefully acknowledged.
microemulsification and micelle assembly engineering, biotechnology, and chemis-
was described for encapsulating BSA as try, will be essential to overcome these 1Department of Chemical and Environmental Engineering,
an artificial exosome mimicking antigen limitations. Research to improve the University of Oviedo, Julián Clavería 8, 33006 Oviedo,
Spain
presentation to dendritic cells (DCs) manufacturing methods could include 2Department of Physical and Analytical Chemistry,
[15]. To specifically target DCs, a mono- the combination of both approaches: University of Oviedo, Julián Clavería 8, 33006 Oviedo,
clonal antibody against a highly semi- and fully synthetic artificial Spain
expressed receptor on the surface of exosomes. *Correspondence:
DCs that facilitate endocytosis was [email protected] (M.C. Blanco-López).
selected. Techniques for modifying cells before http://dx.doi.org/10.1016/j.tibtech.2017.10.005
exosome isolation could be easily cou-

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121–132

Journal of Extracellular Vesicles
ISSN: (Print) 2001-3078 (Online) Journal homepage: https://www.tandfonline.com/loi/zjev20
Therapeutic biomaterials based on extracellular

vesicles: classification of bio-engineering and
mimetic preparation routes
Pablo García-Manrique, María Matos, Gemma Gutiérrez, Carmen Pazos &

María Carmen Blanco-López
To cite this article: Pablo García-Manrique, María Matos, Gemma Gutiérrez, Carmen Pazos &
María Carmen Blanco-López (2018) Therapeutic biomaterials based on extracellular vesicles:
classification of bio-engineering and mimetic preparation routes, Journal of Extracellular Vesicles,
7:1, 1422676, DOI: 10.1080/20013078.2017.1422676
To link to this article: https://doi.org/10.1080/20013078.2017.1422676
© 2018 The Author(s). Published by Informa Published online: 17 Jan 2018.

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https://www.tandfonline.com/action/journalInformation?journalCode=zjev20
JOURNAL OF EXTRACELLULAR VESICLES
2018, VOL. 7, 1422676
https://doi.org/10.1080/20013078.2017.1422676
REVIEW ARTICLE
Therapeutic biomaterials based on extracellular vesicles: classification of

bio-engineering and mimetic preparation routes
Pablo García-Manrique a,b, María Matos b
, Gemma Gutiérrez b
, Carmen Pazos b
and María Carmen Blanco-López a

a
Department of Physical and Analytical Chemistry, University of Oviedo, Oviedo, Spain; bDepartment of Chemical and Environmental
Engineering, University of Oviedo, Oviedo, Spain
ABSTRACT ARTICLE HISTORY

Extracellular vesicles (EVs) are emerging as novel theranostic tools. Limitations related to clinical Received 24 July 2017
uses are leading to a new research area on design and manufacture of artificial EVs. Several Accepted 22 December 2017
strategies have been reported in order to produce artificial EVs, but there has not yet been a clear KEYWORDS
criterion by which to differentiate these novel biomaterials. In this paper, we suggest for the first artificial extracellular
time a systematic classification of the terms used to build up the artificial EV landscape, based on vesicles; biomimetic
the preparation method. This could be useful to guide the derivation to clinical trial routes and to materials; nanomedicine;
clarify the literature. According to our classification, we have reviewed the main strategies drug-delivery nanocarrier
reported to date for their preparation, including key points such as: cargo loading, surface
targeting strategies, purification steps, generation of membrane fragments for the construction
of biomimetic materials, preparation of synthetic membranes inspired in EV composition and
subsequent surface decoration.
Extracellular vesicles in nanomedicine: macromolecules (proteins and nucleic acids) combined

possibilities and limitations with a minimum cytoskeleton that allows deformability
while keeping the whole integrity of the vesicle. (3) size
Extracellular vesicles (EVs) represent an important por-
distribution; (4) great stability in the blood due to the
tion of the secretome. An overview of their functions in
evasion of the innate immune system; (5) adaptative
physiological conditions of EVs was compiled by a
responses that cause clearance from the blood, with the
recent position paper from the International Society of
corresponding decrease in bioavailability.
Extracellular Vesicles (ISEV) [1]. Some of the described
EVs can be used as a therapeutic agent by themselves or
properties can be used for therapeutic uses, and their
as delivery systems. The great potential of EVs as drug-
testing has been transformed sometimes into several
delivery vehicles has been acknowledged in the literature
registered clinical trials [2]. Exosomes are being applied
[7–12]. In most cases, the encapsulated drug acts in colla-
in antitumour immunotherapy [3], as therapeutic agents
boration with elements naturally present in the EVs, creat-
against infectious diseases [4], unmodified exosomes for
ing a synergetic effect. In other cases, EVs serve only as
immune-modulatory [5] and regenerative therapies [6],
vehicles to reach a specific target population, sometimes
and modified ones for targeted drug delivery [7], espe-
highly protected from conventional administration routes.
cially in gene therapy [8]. Although some of the
Nevertheless, limitations of EVs as therapeutic agents
mechanisms behind their properties remain unde-
have also been reported, including the absence of a good
scribed, some general characteristics of EVs make
definition from a pharmaceutical point of view [13], an
them advantageous over other therapeutic strategies.
incomplete understanding of their role in the develop-
Both the structure of the membrane and the forma-
ment and spread of pathology, the absence of methods
tion route are the origin of the following advantageous
for the isolation of homogeneous populations and sub-
aspects: (1) high selective targetability and minimum
populations of EVs, and cost-ineffective technology for
off-target effect, thanks to a set of molecules involved
the availability of sufficient quantities for clinical trials
in targeting, signalling and receptor-mediated uptake,
with constant characteristics. Moreover, it has been
complete with all the co-receptors needed for the inter-
acknowledged that there is little understanding on how
nalization process; (2) capacity of extravasation due to
biological barriers are crossed by EVs, and a need for
a gel-state core derived from the presence of hydrated
CONTACT María Carmen Blanco-López [email protected]

© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 P. GARCÍA-MANRIQUE ET AL.
loading methods with scalable properties in clinical In order to provide a systematic classification to
translation has been identified [14]. move around in this new emerging field, we suggest
the nomenclature given in Figure 1. This artificial EVs
landscape is based on the concept behind the term. In
Bio-engineered and mimetic EVs for
this way, “artificial EVs” will be used as a general
nanomedicine: classification of artificial EVs
concept to designate all vesicles, modified or manufac-
EVs have been modified in the search for broader tured (from natural or synthetic sources), with the aim
therapeutic capability. Sometimes, this included the to mimic EVs (mainly exosomes) for therapeutic uses.
incorporation of new elements for targeted puporses, Behind this term, two categories of artificial EVs can be
in vitro or in vivo traceability, or the material to be discerned: “semi-synthetic EVs” and “fully synthetic”
delivered. In other cases, modification was aimed at the or “EVs mimetic vesicles”, corresponding to modified
enhancement of colloidal stability, or change in surface natural EVs (pre- or post-isolation) and artificial struc-
charge to increase their uptake rate. These new tures, lab-made or generated from cultured cells.
approaches have generated new terms such as bio- The former generate semi-synthetic products, as
engineered exosomes, artificial exosomes [15], exo- they start from a natural substrate, which can be sub-
some-mimetic nanovesicles [16], exosome-like nanove- sequently modified before or after their isolation. The
sicles [17,18] and exosome-based semi-synthetic modification affects the structures of the outer surface
vesicles [19]. These expressions have been used with of the vesicles, the membrane or the cargo that travels
different meanings in the literature, but to date, there within, and could also include hydrophobic molecules
has not yet been a clear criterion for their classification. at the membrane.
One example is the term “exosome-like nanovesicles”. The term fully synthetic, on the other hand, stresses
In some works, this concept is used to name artificial the artificiality of the product. We have recently briefly
vesicles made from cells through different techniques commented their potential in therapeutics [23]. These
to mimic exosomes [17,18]. However, cell-derived vesi- techniques can be classified on the basis of their man-
cles with morphological and biochemical characteris- ufacturing route: those starting from larger substrates
tics similar to exosomes were also named exosome-like (cells) that are reduced to units for the creation of
nanovesicles by other authors [19,20]. Other authors small size vesicles (top-down nanotechnology) or
working with non-animal research models used this those taking individual molecules (lipids, proteins,
term to refer to vesicles with size and flotation density etc.) that self-assemble in higher-order structures with
values similar to those of exosomes. For example, tunable composition (bottom-up nanotechnology).
Regente et al. [21] described the presence of exo- Top-down products differ from natural EVs in terms
some-like vesicles in sunflower plant fluids, and of micro-structure and biochemical composition, since
Prado et al. [22] showed evidence of vesicles quite they are formed from cell fragments: the characteristic
similar to exosomes during pollen germination. membrane microdomains [24] (lipid rafts) and
Figure 1. Artificial EV landscape: explored routes to date for the preparation of artificial EVs for specific purposes. *EBSSNs [19].
JOURNAL OF EXTRACELLULAR VESICLES 3
associated pools of surface markers (especially tetraspa- scientific community. The combination of both sources
nins) are absent, and the minimal cytoskeleton is not of information would be the perfect scenario for the
present. design of future artificial EV-based therapies.
In any case, both types of artificial EVs should meet
product specifications related to “purity, identity,
Impact of the artificial EV classification in the
quantity, potency and sterility” in concordance with
design of new therapeutic agents based on EVs
the pharmaceutical market regulations [28]. Once
The preparation route chosen is important for the final more, there are several important differences in the
purpose of artificial EVs, but it could be critical for the definition of these parameters depending on the semi-
clinical trials and subsequent commercialization. The or fully synthetic character of EVs. These key points
extensive manipulation of EVs during the bio-engi- will be considered and discussed in the following
neering methods is the reason for their classification sections.
as advanced therapy medicinal products (ATMPs) [2],
with a particular regulatory framewok. Following the
Semi-synthetic exosomes: biotechnological
same criteria, fully artificial EVs produced from cul-
modification of naturally released exosomes
tured cells (top-down) should also be incorporated into
this category. The simplest idea to manufacture EVs would be to use
On the other hand, bottom-up artificial mimetic the natural mechanisms for the formation of vesicles,
EVs are more difficult to assign to one or another that is, the cellular machinery itself. It is known that
category. To date, a set of proteins have been fixed the composition of the EVs at all levels responds to a
on lipidic vesicles with an undefined purpose. While high degree of control at very selective cellular
there is some evidence that the EV membrane is mechanisms [29]. Therefore, the composition of the
important for the uptake process [1], the role of the EVs could be controlled by intentional alteration of
artificial membranes is not yet clear. Comparative stu- the cellular environment.
dies of the effects over target cell lines with conven- This method would lead to the creation of EVs with
tional liposomes and exosome-mimetic nanovesicles a composition profile adapted to a specific purpose.
would be very useful to clarify this. The work in this The technological methods used for bioengineering
field is reviewed in the section “Bottom-up methodol- EVs are explained in the following sections. Two key
ogies: artificial membranes decorated with functional aspects are the selection of producer cells (and their in
proteins to mimic EVs functions.” vitro harvesting conditions) and the EV isolation/
A critical evaluation of the fully synthetic EVs con- enrichment procedures. Both choices will condition
cept would imply providing an answer to questions subsequent uses.
such as: “What we are trying to mimic from EVs?”;
“Is the biochemical composition, the morphology or
Selection of EV cellular origin
the whole entity?”; “Is it worth mimicking a specific
function?” This is still a challenge in the field, since Cell lines could potentially release EVs vesicles, but
some of these questions are being answered at the same there are great differences in release rate and biochem-
time for natural EVs. The best approach would involve ical composition and their susceptibility to modifica-
an extensive biochemical characterization of natural tions [30]. It is also accepted that before translation to
EVs and a detailed description of their functions. clinical use of EVs, limitations regarding biocompat-
Regarding functionality, other populations and not ibility, economic viability, harvesting methods and
only the target cell lines should be assayed. This immunotolerance must be overcome. A summary of
could provide information about possible side effects. cell lines used for the production of EVs for clinical
In the same way, multiple parameters should be regis- purposes, especially drug delivery, can be found in the
tered as output factors, not just the process targetted by literature [31]. Dendritic cells and cancer cell lines,
the EV-based treatment. Proteins, nucleic acids and such as melanoma, are the most commonly used lines
lipid composition from specific types of exosomes are for EV production.
registered in specialized databases such as ExoCarta Mesenchymal stem cells (MSCs) are one of the most
[25], EVpedia [26] and Vesiclepedia [27]. But this is promising sources of EVs, especially exosomes [32].
not enough: a database with assays performed with EVs Yeo et al. [33]. defended their use for mass production
reporting treated cell lines, EV type as therapeutical of exosomes with future therapeutic purposes based on
agents, type of assay (in vitro or in vivo) and experi- some important facts related to their advantages over
mental conditions could be of great interest to the other cell lines. Mainly, MSCs are easy to obtain from
all human tissues (even those considered as medical specific cell lines. Evidence about immunotolerance
waste), and they have a high ex vivo expansion capacity should also be provided in order to avoid any inter-
compatible with immortalization without compromis- ference in the results.
ing their therapeutic efficacy. These two facts are essen- In any case, studies involving the encapsulation of
tial to establish a scalable and long-term source of well- the same drug into different cell lines-derived EVs
characterized EVs, particularly exosomes [14,28]. In would be desirable in order to clarify whether the
addition, their immunomodulatory effect gives them beneficial effect is due to the drug or the combination
and their derived EVs important features in autologous of drug/type of carrier.
and allogenic therapeutic applications.
Dendritic cells (DCs) have important roles in immu-
Obtaining a good substrate for modification:
nity (both innate and adaptative). Some authors have
isolation procedures
paid attention to this cell line in order to enhance the
production of clinical grade DC-derived exosomes for Since the final destination of artificial EVs would be the
immunotherapy [33]. Properties of DCs have even administration for therapeutic purposes, the highest
been enhanced with different nanoparticles [34]. level of standards would be required in order to pre-
Exosomes from DCs modified to express indoleamine serve patient safety [27,43]. A key point in artificial
2,3-dioxygenase (IDO), a tryptophan-degrading EVs development is the enrichment from different
enzyme that is important for immune regulation and biological samples, from cell-culture supernatants to
tolerance maintenance, have been used in the treat- several body fluids. There are different reviews [43–
ment of rheumatoid arthritis [35]. In another study, 45] and technique-comparative papers [46–49] about
DC-derived exosomes modified to express FasL on the isolation procedures. They involve ultracentrifugation,
surface (ligand involved in apoptosis induction) were filtration, immunoaffinity isolation, polymeric precipi-
tested as inflammatory and autoimmune therapy [36]. tation and microfluidics techniques, with different
Other recent works are related to the role of tumour- degrees of purity for the final product [50]. In this
released exosomes to load antigens into DCs for the review, we have focussed on scalability, reproducibility
therapy of malignant mesothelioma [37]. and synthetic EV potential damage or physical
To overcome the problem of low release rate, some modification.
authors lowered the micro-environmental pH, mimick- Since the efficient function of EVs depends on their
ing the natural cancer mechanism [38]. By culturing size distribution, aggregation and size changes after
HEK293 cell lines at different pHs, these authors found isolation are important. Lane et al. [47] studied these
that low pH values were best to isolate high amounts of parameters in four isolation methods: two aggregation
exosomes. In spite of the impact of these results, it kits, a density-based method and ultracentrifugation.
would be necessary to test similar effects in non-can- These authors found that some methods kept a con-
cerous cell lines and to determine how the change in stant vesicle size, but large differences were observed
the harvesting conditions affects EV composition regarding yield of isolation (the two sedimentation
(membrane components and cargo). methods gave recovery values two orders of magnitude
Not only human cells have been explored as a source higher than the other methods). Another reported
of EVs: exosomes isolated from bovine milk and loaded obstacle is the co-purification of material with similar
with different drugs were a promising strategy for mass physical characteristics to EVs [42,51,52].
production of therapeutic EVs [39]. They can be easily Scalability is also important, since the batch size is
isolated by differential centrifugation. The biocompat- correlated with the homogeneity of the final product;
ibility of milk-derived exosomes was checked by clin- sometimes small batches are more prone to being sus-
ical biochemical analysis in an animal model by oral ceptible to bias during the process, but on other occa-
gavage during 6 h (short-term toxicity) and 15 days sions, higher batches yield more heterogeneous
(medium-term toxicity). populations due to microenvironments during
In recent years, some attention has been drawn to procedures.
non-animal (especially plant) EVs and their potential The scale-up step with ultracentrifugation (UC) is
use in therapy [40]. In particular, fruit-derived exo- limited by the number of rotor positions and the max-
somes (lemon [41] and grapes [42]) have been isolated, imum sample volume. On the other hand, methods
characterized and tested as beneficial products. Perhaps such as size exclusion chromatography (SEC) are easy
this new source of EVs could be used in the near future to scale up by using large columns, but with the asso-
for the development of EBSSNs following modifica- ciated longer separation time. Pressure application to
tions to express the desired targeting molecules against reduce processing time can disturb EVs [43]. Other
methods, such as immunoaffinity isolations, are only Table 1. Classification of techniques for the production of
used for small amounts of original sample because of artificial EVs, mainly exosomes, according to type of final pro-
the high price of the reagents required. Finally, micro- duct (semi- or fully synthetic) and the principle of the obten-
tion mechanism.
fluidic methods [45,53] are promising, with the possi-
Semi-synthetic exosome production: modification of vesicles naturally
bility of being coupled to online analysis [54]. produced by cells
Reproducibility is crucial when the product is going Pre-isolation modification
to be used in the clinical field. Comparative results of Class I Co-localization of cargo and exosomal carrier
moiety thanks to the natural tropism of
UC are difficult to obtain due to the high number of the second
models available in the market, and this could affect Class II Use of sequences (i.e. nucleic acid-based
sequences) for the exosomal biogenesis
the quality of the isolated product [43]. The use of a pathway signalling
fixed instrument would keep low variability between Class III Take advantage of passive loading via
increments of their presence, by genetic
batches [47]. overexpression or active loading of
Welton et al. [55] reported that ready-to-use SEC producer cells
Post-isolation modification
columns could overcome some problems related to Class IV or passive Methods that use passive adsorption of
homemade poured columns [52], thereby avoiding var- methods molecules into external surface of EVs,
iations from column to column, and facilitating robust owing to their hydrophobicity nature
Class V or active methods V.a (Physical methods), based on the creation
protocols to be used routinely. The main problem asso- of transitional alteration in the integrity of
ciated with this method is dilution of EVs in the final EVs that allows cargo to enter the vesicles
by concentration gradient or by passive
sample and the subsequent need for concentration using incorporation during subsequent restoring
precipitating agents or UC. This increases retention of initial status post-stimuli
V.b (Chemical methods), based on induced
time and the possibilities of co-precipitation of other chemical reactions between EVs and cargo
molecules with the same size and physical properties. with or without previous introduction of
functionalization agents into vesicles
Considerable effort has been made in the field of EV Creation of artificial mimetic structures of the natural exosomes
isolation methods, which are still limiting the expan- Type I or top-down bio- Starting from larger substrates (cells) that are
sion of this field. There is not yet a perfect and uni- nanotechnology reduced to units for the creation of
vesicles with reduced size
versal method, and it is also accepted that selection of Type II or bottom-up bio- Starting with individual molecules (lipids,
the isolation method could impact downstream nanotechnology proteins, etc.) that are assembled in a
controlled way for generating complex
steps [56]. structures of higher order
Strategies for biochemical modification In this case, the fusion between the gene of a protein to
Pre-isolation modification using own cellular be incorporated and the gene of an exosomal-localized
machinery protein can be used for the expression of the former on
The advantages and disadvantages of the methods the outer surface of exosomes. This has been referred
applied to incorporate proteins of nucleic acids are to in the literature as Exosome Display technology [63],
shown in Tables 2 and 3. Based on the study of exosome and it enables the manipulation of the protein content
biogenesis-related mechanisms, several methods have of exosomes and the subsequent tailoring of activities.
been developed [50,57–62]. The following criteria have The potential of the method was successfully demon-
been identified for their classification (see Table 1): strated by the production of specific antibodies against
human leukocyte antigen (HLA), a low immunogenic
(I) location of exosomal functional entities, such as antigen [64].
transmembrane proteins and the use of their Lactadherin C1C2 domain was also used for simi-
natural tropism to co-localized the exogenous lar purposes by Zeelemberg et al. [65] and Hartman
element; et al. [66] to induce expression of chicken egg oval-
(II) strategies using molecular mechanisms for the bumin (OVA) peptide and the human epidermal
introduction of exogenous molecules into EVs growth factor receptor 2 antigen (HER2), respec-
for cytosolic delivery; tively. Álvarez-Erviti et al. [67] described a method
(III) increasing the amount of molecules into the cel- of inducing surface expression of the central nervous
lular plasma to be encapsulated by passive system-specific rabies viral glycoprotein (RVG) pep-
mechanism during MVB formation. tide on exosomes isolated from immature dendritic
cells derived from mice. Complementarily, these
Class I methods (Table 2) involve the design of chi- brain-targeted exosomes were loaded with siRNA
meric constructions of proteins by genetic engineering. for the first time by electroporation. The delivery of
Table 2. Pre-isolation methods for cargo incorporation into EVs.

Cargo incorporation previous to the release of exosomes
Category of
EV modification
modified (according
Method component with Table 1) Advantages Disadvantages Molecules incorporated
Genetic fusion of Surface Class I Efficient exposure of targeting Only successfully explored with Peptides, small proteins
cargo gene moiety on the surface of EVs exosomal membrane proteins such as GE11 peptide
with an By selecting the EVs protein to [69], HLA [63], OVA [65],
exosomal be fusioned with, different HER2 [66], and RVG [67]
protein gene expression rate can be
achieved
Exosome surface Surface Class I Suitable to induce expression of Not tested with high-molecular- Fluorescent proteins [62]
display protein in both, extravesicular weight proteins such as GFP and RFP
technology and intravesicular sides at the
same time
RNA zipcodes Cargo Class II Alternative to electroporation of Applicable only to mRNA and miRNA mRNA [70] and miRNA [71]
miRNA Influence of mRNA size not tested modified with zipcodes
EXPLORE Cargo Class II Excellent platform to load Limited loading capacity due to the mCherry, Bax, SrIκB and Cre
platform proteins to be delivered to the presence of fluorescence reporter recombinase proteins [61]
cytosol of the target cell proteins in future work, this
Expected better results than protein can be omitted
commercial solutions available
TAMEL platform Cargo Class II By selection of one component Highly cost-effective method RNA [59] with variable
of the platform, the EV- Required highly experimented length
enriched protein loading personal
efficiency can be controlled EE values depending on RNA
molecule size
Overexpression of Cargo Class III Used in all types of exosomes Nonspecific loading mechanism RNA and proteins by
RNA cargo into Applicable to all types of RNA Low efficiency, especially for mRNA expression of RNA into
producing cells cell producer cytosol
Fusion with Surface Class III High efficiency Cellular uptake rate can be decreased Hydrophobic and
liposomes and/or Both hydrophilic and Better efficiencies for hydrophobic hydrophilic compounds,
cargo hydrophobic compounds can molecules such as DiI and calcein
be loaded respectively [72]
Photosensitizer drugs, such
as ZnPc [72]
GAPDH-siRNA specifically to neurons, microglia moiety was selected by the elevated expression of his
and oligodendrocytes in the brain resulted in a spe- receptor (EGFR) in tumours of epithelial origin.
cific gene knockdown. This was considered the first More recently, Stickney et al. [62] developed another
example of EV-based genetic therapy. One of the genetic engineering method for surface expression of pro-
most important facts of this work was the successful teins in human cells, called surface display technology. In
treatment of a highly protected tissue, the brain, in this case, tetraspanin CD63 was used as a scaffold for the
spite of the existence of brain–blood-barrier presentation in both extravesicular and intravesicular
selectivity. orientations.
Tian et al. [68] used lysosome-associated membrane Class II methods include a heterogeneous group of
glycoprotein 2b to target electroporated doxorubicin- strategies that have in common the use of specific
loaded exosomes (20% of loading efficacy) produced in molecular interactions between two elements and can
dendritic cells. Ohno et al. [69] used platelet-derived be used to transport the complex into the exosomes.
growth factor receptor transmembrane domain to anchor One example of this strategy is the interaction between
GE11 peptide, a ligand of the epidermal growth factor specific sequences in RNA molecules and proteins that are
receptor (EGFR). This construction was transfected to present in the route of exosomes formation [16]. Highly
Human Embryonic Kidney cell line 293 (HEK293) using observed sequence motifs into RNA types studied in EVs,
pDisplay vector and FuGENE HD transfection reagent. called EXOmotifs, were found in mRNA [70] and miRNA
As a model cargo, siRNA let-7 was selected for its ability to [71]. Exosomes for Protein Loading via Optically
alter cell-cycle progression and reduce cell division in Reversible protein–protein interactions (EXPLORs) and
cancer cells. This siRNA was introduced into EV producer Targeted and Molecular EV Loading (TAMEL) are tech-
cells by the lipofection method. Modified exosomes (15– nologies based on the molecular interaction between cer-
21% of total released exosomes) were isolated by centri- tain types of proteins or between RNA special structures
fugation and intravenously injected into an animal model and specific proteins. Proteins of interest can also be loaded
with induced breast cancer. GE11 peptide as the targeting into the inner compartment of exosomes for their direct
Table 3. Post-isolation methods for cargo incorporation into EVs.

Cargo incorporation after the release of exosomes
Category of
modification
EVs Modified (according
Method component with Table 1) Advantages Disadvantages Molecules incorporated
Co-incubation Cargo Class IV The simplest method More suitable for Low- and medium-molecular-weight
with Inexpensive hydrophobic molecules hydrophobic molecules such as
exosomes Compatible with the addition curcumin [74], placlitaxel [95],
of a small amount of organic cucurbitacin I [75], celastrol [76]
solvent for the enhancement and different porphyrins [58]
of hydrophobic drug Enzymes, such as catalase [80]
dissolution
Electroporation Cargo Class Va Used in all types of exosomes Applicable only for RNA, especially siRNA [67]
Able to incorporate large hydrophilic compounds Different drugs such as placlitaxel
compounds, such as 5 nm RNA type-dependence [60], porphyrins [58]
NPs effectiveness SPIONs [79]
Slight differences in EE
depending on the
cellular origin (cell line)
of the exosomes
Induce aggregation of
siRNA, with valuable
reduction in EE.
Exosome aggregation trend
during electroporation
process
Extrusion Cargo Class Va Simple method Induces changes in EVs Small molecules such as Porphyrins
which reduce delivery with different hydrophobicity [58]
efficiency Enzymes, such as catalase [80]
Saponin- Cargo Class Va Similar loading efficiency to Low efficiency for some
assisted electroporation, but without large molecules, but
loading the associated problems better than simple
Saponin can enhance in some incubation
Hypotonic Cargo Class Va cases the efficiency of co- Not tested with large
dialysis incubation molecules
Sonication Cargo Class Va Enhance simple incubation Not tested with hydrophilic Small molecules such as placlitaxel
through decreasing bilayer molecules [95] or Doxorubicin
rigidity Not tested with different EV Enzymes, such as Catalase [80]
populations
Click chemistry Surface Class Vb Keep constant morphology or A two-step procedure with Fluorescent dyes such as azide-Flour
functionality of EV properties subsequent purification 545 [81]
Applicable to any molecule steps to remove Potentiality to any type of molecule
previously modified unbound molecules and susceptible to being modified by
activate agents azide groups
Fusion with Surface and/or Lipids with different chemical nature
liposomes membrane [85] (zwitterionic, cationic,
anionic, PEGliated, etc.)
delivery to the cytosol of the target cell as an alternative for between an EV-enriched protein and an RNA-binding
therapeutic target locations. EXPLORs [61] has recently domain. This construction is transfected into EV pro-
presented for that purpose. The system integrates two ele- ducer cells to make his function. Different loading
ments: one is produced by the genetic fusion of the photo- degrees can be obtained by selecting the EV-enriched
receptor cryptochrome 2 (CRY2) to the protein to be protein. This is related to the natural expression of
loaded, and the other is made by the fusion of a truncated different proteins into EVs.
version of the CRY-interacting basic-helix-loop-helix 1 Class III methods includes the simplest method:
(CIBN) to tetraspanin CD9. Both elements can be transi- passive loading into vesicles through their biogen-
torily attached by exposure to blue light, which induces the esis. There can be two different approximations to
interaction between CRY and CIBN, and the interaction this objective. First, the overexpression of RNA
can be stopped once the blue light is not present. cargo in the producer cells. The major disadvantage
TAMEL is another genetic engineering tool that of this method is the lack of selectivity in the load-
has recently been published [59] for the active cargo ing process, since it is gradient-driven (the higher
of RNA. This platform is a fusion between an engi- the concentration in the cytoplasm, the higher its
neered EV-loading protein and the RNA to be loaded. possibility of being trapped into exosomes during
Engineered EV-loading protein is also a fused product invagination of MVB formation). On the other
hand, the great advantage of the method is that by Physical and chemical post-isolation modifications
translation of mRNA into receptor cell cytoplasm, These are the methods that require an external force
codified proteins can also be passively loaded (chemical or physical) to incorporate new molecules on
into EVs. previously isolated exosomes (Table 3).
The second approximation explores the active load- Passive methods. Incubation of EVs and cargo. The
ing of the producer cells, i.e. by nanocarriers such as simplest way to incorporate any cargo into cell culture
fusogenic liposomes. This strategy is based on phy- or body fluid isolated EVs is the co-incubation of both
sico-chemical properties that govern the type of elements. This strategy was explored by Sun et al. [74],
mechanism (the fusiogenic properties of the two ele- who found that curcumin exosome-loaded exhibited a
ments that take place in the method, cells and lipo- better stability and higher bioavailability in serum in an
somes). Second, they modify the whole cell and not animal model. For these therapeutic-modified exo-
only the exosomes. somes, an improvement in in vivo anti-inflammatory
As an alternative method for the incorporation of and septic shock was observed.
exogenous molecules (specially designed for hydro- In another study [75], two anti-inflammatory com-
phobic compound) into EVs, Lee et al. [72] pre- pounds were loaded into exosomes and microparticles,
sented the use of membrane fusogenic liposomes and they were administrated intranasally, opening up
(MFLs). By the treatment of cells with MFLs loaded new therapeutic possibilities. The effects of solvents
with a hydrophobic compound (DiI) and a hydro- and drug release kinetics from loaded exosomes by
philic molecule (calcein), these authors obtained dialysis have been studied [39,76].
EVs modified with both molecules. Only slight dif- Active methods. Physical methods: electroporation and
ferences in the efficiency of incorporation into EVs other temporary membrane disruptive methods. The
were found, since a hydrophobic compound would most commonly used method for cargo incorporation
remain in the plasma membrane after liposome into EVs after their release is electroporation [77]. This
fusion and in the subsequent formation of EVs technique involves the temporary permeabilization of
membrane through their routes of biogenesis. In membranes through the creation of pores due to the
contrast, a hydrophilic molecule would be released application of high-voltage electricity. Some authors
into the cytosol. Intercellular transport of both have pointed out that this method is not suitable for
molecules mediated by EVs was successfully siRNA cargo into EVs due to technical problems, and
observed in vitro in a multicellular tumour spheroid an overestimated encapsulation into EVs could be
model. observed. It has been reported that electroporation
A similar method was used to modify the composi- induces siRNA aggregation and co-pelletization with
tion of EVs, with a special focus on the modification of EVs during purification by ultracentrifugation, without
the properties of the EV membrane [57]. Dyes, fluor- any dependence on electroporation buffer composition
escent lipids with different lengths and saturation grade [78]. They also postulated that slight differences could
of acyl chains, and chemotherapeutics were loaded into be found between different EVs regarding their cellular
cells by means of EVs. origin (e.g. primary cells).
These authors also carried out a membrane sur- Another relevant problem concerning electropora-
face modification, with the possibility of conjugation is exosome aggregation and subsequent decrease
tion with molecules for targeting purposes. They in functionality. To avoid these problems, Hood et al.
first prepared MFLs containing azide-modified [79] electroporated exosomes from mouse B16-F10
lipids which were fused with cells. By simple incu- melanoma cells by incorporating 5 nm superparamag-
bation with dibenzocyclooctyne (DBCO)-modified netic iron oxide nanoparticles (SPIONs) as model exo-
peptides, a covalent bond was created due to the genous cargo. Other authors compared the loading
fast and selective reaction between DBCO and the efficiency of different porphyrins with different hydro-
azide group [73]. This strategy will allow new pos- phobicities into EVs with various origins by passive
sibilities of surface ligand decoration on EVs for loading (co-incubation), and by active loading, such
targeting purposes (such as peptides, aptens or anti- as electroporation, extrusion, saponin-assisted drug
bodies) or for the introduction of molecules with loading and hypotonic dialysis [58]. The best results
therapeutic properties via interaction with selective were obtained for hydrophobic compounds and for
receptors. By the combination of a different head- electroporation. Interestingly, zeta potential (ζ) related
group modified lipids, several different ligands to the chemical composition of EV membranes seems
could be incorporated into EV outer membranes, to play a role in loading efficiency, since higher ζ values
including receptors and co-receptors. led to higher EE. The chemical properties of cargo are
also relevant, since their charge will condition the final and allows the incorporation of exogenous molecules to the
outcome. Electroporation did not induce drug surface of EVs.
precipitation. Finally, there is another type of cargo modification
In contrast, extrusion over polycarbonate mem- that has been applied into artificial vesicular systems
branes altered the morphology of vesicles and, sub- (liposomes) with potential applicability to EVs. This
sequently, their delivery efficacy. Other authors used method is based on the ability of some peptides to be
the sonication of EVs in the presence of drug solu- incorporated into lipidic membranes causing disrup-
tions [60].The loading efficacy was found to follow tion [83,84]. By fusion of the peptide D1-7 to an adhe-
the order: incubation at RT < electroporation ≪ sion molecule expressed in cells, targeted lipidic
mild sonication. A similar trend was observed for carriers with therapeutic cargo were produced and
size changes after the loading procedure. On the successfully tested in vitro and in vivo. Another inter-
other hand, surface charge and protein profile were esting application of this strategy is its ability to insert
similar after loading, evidencing no alteration in peptidic cargo into live cell membranes, giving possi-
exosome stability. These authors explained the bilities of imaging live cells and modifying cell surfaces.
results concerning sonication as a decrease in bilayer This last property could be explored for the modifica-
rigidity after sonication, which allowed a better tion of plasma membrane in EV producer cells.
incorporation of the hydrophobic drug. Therefore, The modification of EV membranes results in
mild sonication should be considered as an enhance- changes in surface charge, fusiogenic properties,
ment of co-incubation. Additionally, loaded exo- immunogenicity decrease and colloidal stability [85].
somes were stable over large periods of time at Engineered hybrid exosomes were prepared by mem-
different temperatures. brane fusion with liposomes formulated with different
A similar comparative study was carried out with types of lipids (i.e. zwitterionic, anionic, cationic and
the enzyme catalase [80]. For the preparation of PEGlilated). Fusion properties with cell-culture-derived
exosomes modified with this oxidative stress-protect- exosomes were studied according to the chemical nat-
ing agent used for the treatment of Parkinson’s dis- ure of liposome lipids [86]. It was found that zwitter-
ease, these authors selected four methods: incubation ioninc and anionic lipids did not alter the uptake rate,
at RT in the presence/absence of saponin, freeze/ while the introduction of cationic lipid greatly
thaw, sonication and extrusion. Sonication yielded decreased the phenomena, and PEGilated lipid
the higher EE (26.1%) and the more stable product. increased it by twofold. Therefore, it can be concluded
In contrast, this method also produced the higher that functional properties could be tuned by modifying
increment in size, from 105 nm naïve exosomes to the membrane composition.
183.7 nm in catalase-loaded exosomes. Associated
with size increment, AFM observation also revealed
a change in morphology, with a final non-spherical Top-down and bottom-up methods for the
shape. development of full synthetic EVs
Despite these promising results regarding the encap- Production of artificial EVs by generation of
sulation of different molecules into exosomes, standar- plasma membrane fragments: a top-down-inspired
dization in systematic conditions followed by the study methodology
of several cell lines is still necessary to strongly support
the use of these methods as routine practice in the Different approaches based on top-down nanotechnol-
clinical field. ogy have been developed for the production of EVs
Chemical methods: click chemistry mediated functionaliza- mimetic nanovesicles using cells as precursors of plasma
tion and other targeted drug-delivery strategies. The che- membrane fragments. Those strategies rely on the prin-
mical copper-catalysed reaction between an alkyne and an ciple of self-assembly of lipids and lipid membranes into
azide that forms a triazole linkage (click chemistry) has been spherical structures and the encapsulation of surround-
used for the surface functionalization of exosomes [81]. ing material into the aqueous cavity of generated nano-
These were first chemically modified by the incorporation vesicles. Current methods include extrusion over
of alkyne groups into amine groups from proteins by membrane filters [16,17,87], hydrophilic microchannels
carbodiimide chemistry [82]. These authors conjugated [88] or cell slicing by SixNy blades [18] (see Table 4).
azide-Fluor 545 (a fluorescent compound) to activated
EVs. Since no differences in morphological and functional Extrusion over polycarbonate membrane filters
properties were found, it was concluded that modification Jang et al. [16] used a serial extrusion through poly-
by click chemistry does not alter exosome characteristics carbonate membrane filters with decreasing pore sizes
Table 4. Summary of the published work about the generation of mimetic EVs nanovesicles by top-down bio-nanotechnology (cell
source and type of cargo are encapsulated, and main characteristics are given).
Type of material
Generation technique Precursor cell type encapsulated Nanovesicles characteristics Reference
Manual extrusion over Monocytes and Exogenous, Mean size and distribution similar to that of exosomes [16]
polycarbonate membrane macrophages chemotherapeutic Exosomal protein profile similar to that of natural exosomes
filters drugs EE of chemotherapeutics dependent on the original amount
used during extrusion
High rate of drug release
100 times more that of nanovesicles than exosomes from the
same number of cells
Results reproducible with different cell types
Pressurization and extrusion Murine embryonic Endogenous, proteins Average size in the exosome range [88]
over hydrophilic parallel stem cells and RNA Similar intracellular and membrane protein and total RNA profile
microchannels in a to the original cells and exosomes
microfluidic device Same ability to deliver RNA content as exosomes
Centrifugal force and Murine embryonic Endogenous, proteins NVs size and morphology similar to exosomes [87]
extrusion over a filter with stem cells and RNA Cargo of RNA, intracellular proteins and plasma membrane
micro-size pores into a proteins similar in types to exosomes
polycarbonate holder Small RNA profile differs in quantity, especially in miRNA with
structure respect to exosomes
Intravesicle contain twice the concentration of natural exosomes
250 times more vesicles than naturally secreted exosomes
Slicing living cell membrane Murine embryonic Exogenous, Generated NVs in the size range of exosomes [18]
with silicon nitride blades stem cells polystyrene latex Nanovesicle production 100 times more productive than natural
in a microfluidic device beads exosome
30% of EE for 22 nm nanoparticles as model of exogenous
material encapsulation
NVs can deliver exogenous encapsulated material
(10 μm, 5 μm and 1 μm) in a mini-extruder, similar to murine embryonic stem cells for the treatment of mice
those commonly used for the preparation of liposomes. isolated skin fibroblasts. These authors wanted to
Human monocytes were chosen as precursors for explore the potential of mimetic exosomes to induce
membrane fragments. The yield production of NVs proliferation and recovery after injury in an in vitro
was 100-fold in comparison with the production of model. Genomic and proteical profiles similar to origi-
exosomes by using the same number of cells. nal cells were assessed by PCR and Western blotting of
Morphological studies of these NVs by crio-TEM and specific markers, and it was confirmed that successful
NTA showed many similarities with the exosomes, delivery of genetic material by NVs was reached.
round shape and a peak diameter around 130 nm.
Even the exosomal protein marker profile (CD63, Pressurization, extrusion and slicing over hydrophilic
Tsg101, moesin and beta-actin) checked by Western parallel microchannels in a microfluidic device
blot was identical for the NVs and exosomes. The Jo et al. [87] produced exosome-mimetic nanovesicles
chemotherapeutic drugs, doxorubicin, 5-FU, gemcita- by extruding cells over hydrophilic microchannels,
bine and carboplatin were added to the buffer where with the aim of delivering endogenous RNA across
cells were resuspended. The encapsulation efficiency in the plasma membrane with high efficiency and low
the final purified NVs was found to be dependent on toxicity. These authors developed a microfluidic device
the initial amount of added drug. made of PDMS by soft lithography. This device had an
Looking for a scaled-up process using extrusion as array of 37 parallel microchannels, with a common
the generation procedure of NVs, Jo et al. [87] devel- inlet and outlet connection for the pumping with a
oped a device that uses centrifugal force to extrude cells syringe pump and the collection of NVs, respectively.
over polycarbonate filters (10 μm and 5 μm pore sizes). The higher amount of nanovesicles generated by extru-
The device has a central piece where filters are located sion over hydrophilic channels with a similar size to
and connected to two syringes where the sample is exosomes was obtained with a length of 200 μm and a
dispensed by the centrifugal force. Uniformly sized width of 5 μm. These results showed that an appro-
100 nm NVs with a yield 250-fold higher than that of priate total shear force induced by the channel has to
exosomes from the same number of cells was achieved. be reached in order to produce NVs with acceptable
Analysis of the filters by TEM showed that many cells results. This force is responsible for NV generation due
remained trapped in the structure. to elongation of the plasma membrane on the micro-
The same device previously cited was employed by channel surface. When the elongated membrane
Jeon et al. [17] to produce exosome-mimetic NVs from reaches a certain value, it breaks into small portions
that directly form nanovesicles, thanks to the self- individual molecules (lipids, proteins and cargo) into
assembly property of lipids in aqueous media. complex structures, such as a bilayer structure resem-
With the appropriate channel morphology, these bling EV membranes functionalized with proteins for
authors produced 100 nm nanovesicles similar in com- mimicking EV functions. This could be achieved by
position (proteinical and nucleic acid profile) to natu- assuming that not all components in natural exosomes
rally produced exosomes. The analysis of these NVs are essential for specific and efficient delivery [13],
[88] revealed that the formation of exosome-like NVs including the transport of a message through direct
through hydrophilic channels produced a delivery sys- contact with target cell receptors. Another assumption
tem of endogenous material with identical results to that encourages researchers to explore this route is
those with exosomes. that, from a structural and biochemical point of view,
More recently, Yoon et al. [18] reported the produc- exosomes are liposomes with attached proteins.
tion of exosome-mimetic nanovesicles by the slicing of Therefore, this type of vesicular system could be an
cells through SixNy blades aligned to the flow direction ideal substrate to develop exosome-mimetic structures.
over hydrophilic microchannels. These authors com- The main functional components of exosomes to be
bined the induced shear stress formation on NVs with incorporated in mimetic materials have been reviewed
the fragmentation of plasma membrane by the blades [13]. The three main components of exosomes
to obtain fragments for the generation of exosome-like reported were lipids, membrane proteins and therapeu-
nanovesicles and the co-encapsulation of exogenous tic cargo.
material (polystyrene latex beads as a model sub- One of the main advantages of fully artificial EVs
stance). It was found that NV diameter increased as over previous strategies is the production of pure and
the width channel increased. This is because channel well-defined biomaterial. In addition, production stra-
morphology is proportional to the Reynolds number tegies of artificial EVs based on liposomes are more
(Re). In this particular case, Re is proportional to the sustainble and easier to scale up [11,89]. This fact is
hydraulic diameter and, therefore, to the inertial force, quite important for preclinical and clinical studies and
which directly increases with the channel width. In in order to manufacture products ready to be sent to
other words, NVs travelling through wide channels the market [2].
have a higher inertial force when they reach the blades, Liposome preparation techniques have been exten-
generating larger sliced fragments that produce larger sively reviewed [89–101], but not all the methods yield
NVs. These have a similar composition to that of vesicles suitable for becoming an artificial exosome. It
parental cells and naturally released exosomes by could be considered that only small unilamellar vesicles
those cell lines. (SUVs) are ideal precursors due to their similarities to
One of the most interesting works in the literature natural exosomes (size range and membrane
[18] describes the encapsulation of 22 nm fluorescent disposition).
polystyrene latex beads as an exogenous simulated Methodologies for SUV preparation (Table 5) can
material, adding these nanoparticles to the media be classified according to different criteria [92]. For
where cells were diluted before slicing. With a final example, number of steps to reach SUV. Another
corrected EE of 30%, these NVs containing exogenous classification is based on the physical principle
material were given to fibroblasts in an in vitro experi- applied to prepare vesicles: mechanical processes,
ment. After a period of time, red dots corresponding to organic solvent replacement, detergent removal and
fluorescent beads were detected in the cytoplasm of other techniques as microfluidic-based methods.
fibroblast by confocal microscopy. The delivery effi- Reverse-phase evaporation, ethanol injection method,
ciency of encapsulated beads into exosome-like NVs ether injection method (EtIM), thin-film hydration
was higher than that of bead-aggregated NVs, revealing method (TFH), homogenization techniques, French
that exogenous material delivery with these NVs was press cell extrusion, microfluidization, extrusion over
possible, but the efficiency was still lower than that membranes, ultrasound and membrane contactors are
achieved with parental cell-component generated NVs. some of the techniques developed for SUV
preparation.
All these techniques rely on the self-assembly of
Bottom-up methodologies: artificial membranes
amphiphilic molecules, such as lipids, in ordered struc-
decorated with functional proteins to mimic EV
tures due to their physicochemical behaviour in aqu-
functions
eous media [93,94]. This principle is the basis of
The third option for obtaining artificial EVs is their bottom-up nanotechnology. Vesicles with a size range
construction in a fully synthetic way by assembling similar to that of natural EVs could be obtained [95]
Table 5. Advantages and disadvantage of most frequently used methods for small unilamellar vesicles (SUVs) preparation.
One-step method for Physical method
Method SUVs preparation applied for preparation Advantages Disadvantages
Ether injection Yes Organic solvent Scale-up adapted Not suitable for thermosensitive
method replacement High hydrophobic compound compounds
encapsulation Solvent not suitable for some
No mechanical degradation of biocompounds
compounds
Ethanol Yes Organic solvent Scale-up adapted Ultrasounds are needed when
injection replacement Non-dangerous substances are handled concentrated samples are produced
method High hydrophobic compound Low encapsulation efficacies of low-
encapsulation molecular-weight hydrophilic
No mechanical degradation of compounds
compounds Not suitable for thermosensitive
compounds
Reverse-phase No Emulsification/organic Widely used Frequently used solvents are not suitable
evaporation solvent replacement Suitable for mass production for some biocompounds
Thin-film No Mechanical processes Applied for any type of amphiphilic Difficult to scale up production
hydration molecules Timely and cost-ineffective due to
method High encapsulation of Hydrophilic necessary downsizing techniques
compounds compared to other
methods
Downsizing / Mechanical processes Good reproducibility Product loss associated with clogging of
Techniques Adapted to scale-up requirements membrane by concentrated samples
French press cell
extrusion
Microfluidization
Extrusion over
membranes
Ultrasounds Yes Mechanical processes Simple methodology Degradation of biological compounds
Possibility of being scaled up Scale-up unadapted
when operational variables were oprtimized by design authors have used classical separation methods, such
of experiments. In addition, a wide spectrum of mole- as ultracentrifugation [102] or gel filtration [15,103]
cules with biological activity, independently of their (SEC) with high exclusion limit resins (Sepharose CL-
physicochemical nature (hydrophilic or hydrophobic, 2B, 4B mainly). Dialysis, however, is not used due to
low molecular weight or macromolecules), can be the high molecular weight of biomolecules selected for
incorporated into liposomes, during or after their for- mimicking exosomes.
mation [96]. Undecorated liposomes have also been used in the
Functionalization of liposomes with biomolecules is EV research field as EV models for comparing isolation
possible, owing to the different headgroup-modified efficacy and physical integrity [47], detection by flow
lipids that are available [97]. Headgroup modification cytometry [104] and EV refractive index study [105].
usually includes a molecule of polyethylene glycol as a However, their use as a scaffold for artificial EV devel-
spacer between the functional group and the polar opment could offer new possibilities in basic research
region of the lipid. This avoids the sterical hindrance about EVs and theranostic applications. To date, there
caused by the proximity of biomolecules and liposome have been few examples of this approximation for the
surface. The chemical modification includes the intro- development of mimetic exosomes, and no compara-
duction of different types of functional groups, such as tive results are available owing to the great differences
biotin, amine, maleimide, carboxylic acid, folate, cya- between the methods used. A summary of the main
nur, DBCO, azide and succinyl groups. These groups experimental work on mimicking exosomes by bottom-
determine the crosslinking strategy [98–101] which up nanotechnology is given in Table 6 and briefly
should not compromise the biological function. commented on below.
Bioconjugation should ideally be carried out under The most frequent preparation technique for SUVs
mild conditions, aqueous media and chemoselectivity, as templates for EVs mimicking is the TFH method
and with a high yield. combined with extrusion over polycarbonate mem-
Successful conjugation of peptides/proteins with branes and with [102] or without [15] previous
liposomes can be checked using conventional molecu- freeze–thaw cycles. Martínez-Lostao et al. [102] had a
lar biology techniques such as dot-blot [15], SDS- formulation that included lipids and stoichiometry
PAGE [102] or even flow cytometry [102]. A prelimin- inspired in natural exosomes. The introduction of
ary purification step is required in order to remove only 5% (w/w) of an iminodiacetic acid derivative or
unconjugated biomolecules. For this purpose, the DOGS-NTA allowed the binding of APO2L/TRAIL-
Table 6. Summary of published work about the development of mimetic EVs nanovesicles by bottom-up bio-nanotechnology,
showing formulation of the vesicles, molecules for the surface functionalization and main physical characteristic (size).
Formulation Preparation method Conjugation strategy Size Protein for functionalization Reference
PC:SM:Cho:DOGS-NTA Thin-film hydration Ni2+-NTA headgroup 150–200 nm APO2L/TRAIL-His10 [102]
(55:30:10:5) weight ratio method (KCl 100 mM, functionalized lipid +
For fluorescent labelling, 0.25% HEPES 10 mM pH 7.0, histidine-tagged
mole/mole DSPE-RhodB EDTA 0.1 mM; KHE recombinant
buffer) peptides
Filtered and degassed + 37°C, 30 min
extrusion over 200 nm
membranes
PC:Cho:DSPE-PEG:DSPE-PEG-MAL Thin-film hydration Maleimide headgroup 100 nm MHC class I peptide complexes and [15]
(1:0.5:0.04:0.01) Molar ratio method (Hepes 25mM, functionalized lipid + FAB regions against T-cell
For fluorescent labelling, 1% of NaCl 140mM; pH 7.4) Traut’s reagent protein receptors (adhesion, early and
PC amount of DSPE-RhodB Filtered and degassed + activation late activation and survival)
extrusion over 100 nm 1h RT 20/1 ratio
membranes
Micro-emulsion phase Micro-emulsion and Carboxilic group from 82 nm Monoclonal antibody against [103]
PC:CpEL micelle combining ChoS and amine DEC205 antigen expressed on
(7:3, w/w) method + sonication group from protein dendritic cells
Micelle phase step for 3min EDC/NHS
In 10:1 v/v DE:A 4°C for 12 h
DOPE:DC-Cho
(4:1, w/w)
In 1:2 v/v EtOH:DW
His10 to liposomes in a single step. Its bioactivity was a Box–Behnken experimental design to optimize (max-
higher activity than that of the soluble ligand. imize) the EE by adjusting some formulation para-
Moreover, a treatment based on these synthetic exo- meters. The final optimized formulation is
somes achieved 60% of disease improvement in a rheu- summarized in Table 6. In this particular case,
matoid arthritis-induced animal model. In another researchers selected mimetic exosomes for the potential
study, liposome-bound Apo2L/TRAIL overcame the transmission of antigen to DCs by a controlled target
resistance to the soluble ligand exhibited by chemorre- delivery using a conjugated monoclonal antibody anti-
sistive tumour cell mutants [106]. The mechanism of DEC205, a highly expressed receptor on the surface of
action of LUV-TRAIL in haematologic cells [107] was DCs. The introduction of cholesterol succinate in the
also studied using mimetic structures of exosomes. outer layer of the liposomes allowed the bioconjugation
Another approximation to artificial EVs (exosomes) of the Ab by EDC/NHS chemistry.
for therapeutic purposes was carried out by De la Peña Despite the promising results and the advantages of
et al. [15] using a reported formulation [108,109]. The these methods for the development of liposome-based
main components were phosphatidylcholine and cho- artificial EVs, there are several limiting aspects that
lesterol, and headgroup-modified lipids such as DSPE- hinder the transfer to the clinical field. While techno-
PEG DSPE-PEG-MAL. In order to make traceable logical progress has allowed the design, development
NVs, both in vivo and in vitro, a fluorescent lipid was and production of nanomedicines with high pharma-
included in the formulation, and magnetic nanoparti- ceutical grade, their clinical impact has been smaller
cles (SPIONs) were encapsulated during a thin-film than expected due to a lack of sufficient information
hydration step. After optimization of chemical-acti- about in vivo interactions and fates inside the human
vated ligands binding, mimetic SNVs simulating DCs body [11]. Specific challenges [13] are related to the
derived exosomes were successfully tested as new tools functionalization of vesicle surface with a combina-
in basic and clinical immunology. A T-cell expansion tion of functional proteins at the same time because
rate higher than that with previously reported experi- actual methodologies are time-consuming and
ments using conventional methods was achieved. because of the incorporation of nucleic acids with
An innovative methodology for the production of acceptable efficiencies. The dependence of vesicle for-
protein encapsulated nanoliposomes was also reported mulation on parameters such as fusion properties and
[103]. This produced 82 ± 4 nm antibody-coated lipo- stability could be another limit of special relevance to
somes with approximately 93% EE of BSA. The pre- immunotolerance. Finally, the knowledge about key
paration route that combined a micro-emulsion components in exosomes is not yet complete, since
contained the protein to be encapsulated, with micelles, they may vary from one cell line to another. They
in order to create a lipid bilayer formed through layer- could even be health-state-dependent and sensitive
by-layer assembly. In this work, the authors selected a to harvesting conditions.
Despite attempts to mimic the exosome natural lipid pathological conditions, novel opportunities for the
composition, there is a need for actually checking development of enhanced therapeutic biomaterials have
whether the formulation is active and involved in the arisen. These observations could help in the production
expected functions or is just a passive element involved of new materials inspired by natural vesicles, without the
only in the scaffolding of true functional elements. classical inconveniences associated with up-to-date syn-
Comparison of the efficiency of the intended integrated thetic alternatives (liposomes, polymersomes, inorganic
component in differently formulated vesicles could be nanoparticles, etc.). EV-based therapies include tissue
an interesting experiment to elucidate the role of mem- regeneration or immunomodulation, but drug delivery
brane components. Parameters of uptake route and is one of the most promising applications. Production,
incorporation efficiency could also be measured with isolation, modification and purification at a large-scale
different cell lines, in order to assess the role of the clinical grade are the main limitations of EVs becoming a
target cell. Other compounds as an alternative to lipids, true clinically settled therapeutic agent.
with a high grade of biocompatibility, could also be These limitations have promoted the development
used for the formulation of artificial exosome bilayers. of mimetic materials inspired by EVs, the so-called
One option is to use non-ionic surfactants [110] for the artificial EVs. In this article, we have introduced a
preparation of niosome-based artificial exosomes. systematic classification of the types of artificial EVs
These compounds offer several advantages [111] over according to their preparation routes. Two well-defined
lipids, such as price, versatility and sustainability. On strategies have been developed: semi-synthetic or fully
the other hand, their chemical structure offers synthetic products. The first strategy uses natural exo-
enhanced stability from both a chemical and physical somes as precursors that are modified at the moment
point of view. Niosomes with a size range close to that of their biogenesis (pre-isolation modifications),
of EVs can also be produced [95]. whereas the second strategy modifies the vesicles after
In recent years, microfluidics has been playing an their release by cells and their isolation from cultured
important role in the development of enhanced vesi- media or biological fluids. Genetic engineering-based
cular systems, enabling robust and highly controlled modifications, active loading platforms, specific signal-
preparation routes of vesicles [112] and allowing ling sequences for selective sorting or precursor cell
rapid characterization of products [113]. Another modifications with nanomaterials are some of the
important aspect in the development of exosome- methods developed for exosome-based semi-synthetic
based therapy, regarding any preparation route, is the nanovesicle production.
creation of reduced systems for the study of traffic and Fully synthetic vesicles with EVs mimetic properties
delivery into in vivo microenvironments [19]. Again, can be produced by bio-nanotechnology. Top-down
microfluidic-based systems are opening up new possi- techniques that produce vesicles made of membrane
bilities by the development of organ-on-a-chip plat- fragments obtained from the extrusion or slicing of
forms that enable the study of these processes in an cells, or bottom-up techniques that take advantage of
innovative and highly efficient way [114]. supra-molecular chemistry (mainly self-assembly) to
It is expected that microfluidic synthesis of nanove- produce vesicles from individual molecules, represent
sicles will open the path for new artificial EV routes, the technology developed for that purpose.
with the required control of size and EE, and minimal Despite the great potential of artificial EVs, some
consumption of reagents. limitations to their development as therapeutic tools
Other recently explored drug-delivery systems have have been identified. There is no perfect technique,
developed platelet-mimetic nanoparticles by also using and, depending on the final purpose of artificial EVs,
bottom-up technology. These authors have produced combinations of procedures could offer new insights in
unilamellar polymeric nanoparticles functionalized the field. Systematic studies with different cellular ori-
with immunomodulatory and adhesion antigens, and gins and target cell lines would expand and consolidate
they have tested them as another approach to disease- the applications of artificial exosomes. Comparative
targeted delivery [115]. work including the encapsulation into different artifi-
cial vesicles would be interesting in order to identify
effects due to the carrier.
Conclusions and future perspectives
Multidisciplinary teams with complementary actions
Knowledge about all the biological aspects related with in the fields of applied biology, pharmacology, chemi-
EVs, especially exosomes, has opened up new frontiers in cal engineering, material sciences and medicine would
the clinical field. After an explosion of publications in allow the definitive consolidation of these therapeutic
recent years about the role of EVs in physiological and biomaterials in clinical routines.
Highlights [4] Aline F, Bout D, Amigorena S, et al. Toxoplasma

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Competitividad (MINECO, Spain), under the Grant Microvesicles and exosomes: opportunities for cell-
CTQ2013-47396-R. This study was also financed by the derived membrane vesicles in drug delivery. J Control
Consejería de Economía y Empleo del Principado de Asturias Release. 2012;161:635–644.
(Plan de Ciencia, Tecnología e Innovación 2013-2017), under [10] Ha D, Yang N, Nadithe V. Exosomes as therapeutic
the Grant GRUPIN14-022. Support from the European drug carriers and delivery vehicles across biological
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Disclosure statement from the liposome field. J Control Release.
No potential conflict of interest was reported by the authors. 2014;195:72–85.
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Colloids and Surfaces B: Biointerfaces 182 (2019) 110378
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb
Continuous flow production of size-controllable niosomes using a T

thermostatic microreactor
Pablo García-Manriquea,b, Gemma Gutiérrezb, María Matosb, Andrea Cristaldic, Ali Mosayyebic,
⁎ ⁎
Dario Carugod, Xunli Zhangc, , María Carmen Blanco-Lópeza,
a
Department of Physical and Analytical Chemistry, University of Oviedo, Spain
b
Department of Chemical Engineering and Environmental Technology, University of Oviedo, Spain
c
Bioengineering Sciences Group, School of Engineering, Institute for Life Sciences (IfLS), University of Southampton, United Kingdom
d
Mechanotronics and Bioengineering Sciences Research Groups, School of Engineering, Institute for Life Sciences (IfLS), University of Southampton, United Kingdom
A R T I C LE I N FO A B S T R A C T
Keywords: The new roles of vesicular systems in advanced biomedical, analytical and food science applications demand
Organic colloids novel preparation processes designed to reach the new standards. Particle size and monodispersity have become
Niosomes essential properties to control. In this work, key parameters, involved in a microfluidic reactor with hydro-
Size control dynamic flow focusing, were investigated in order to quantify their effects on niosomes morphology. Particular
Hydrodynamic flow-focussing
attention was given to temperature, which is both a requirement to handle non-ionic surfactants with phase
Microreactor
transition temperature above RT, and a tailoring variable for size and monodispersity control. With this aim,
3D-printing
niosomes with two different sorbitan esters and cholesterol as stabilizer were formulated. High resolution and
conventional 3D-printing technologies were employed for the fabrication of microfluidic reactor and thermo-
static systems, since this additive technology has been essential for microfluidics development in terms of cost-
effective and rapid prototyping. A customised device to control temperature and facilitate visualization of the
process was developed, which can be easily coupled with commercial inverted microscopes. The results de-
monstrated the capability of microfluidic production of niosomes within the full range of non-ionic surfactants
and membrane stabilizers.
1. Introduction groups for stability enhancement or bioconjugation, (iii) wide range of

surfactant types available (with single or double acyl chain, with dif-
A precise control over local environment during production of col- ferent length or saturation), (iv) high immunological tolerance, and (v)
loids is essential to minimise perturbations in chemical characteristics cost effectiveness. Firstly introduced in the cosmetic industry by L’Oreal
that could lead to heterogeneous populations, and then, differences in [8] for dermal bioactive compounds delivery, over the last 15 years
particle properties. To achieve such homogeneity and uniform proper- their applications have expanded to many fields. Food fortification [9],
ties, a strict control of particle size is necessary [1,2]. diagnostic agents [10], analytical chemistry [11], nanomaterial synth-
Nanovesicles (organic colloids) are particles formed by self-as- esis [12], and drug delivery [13] are just some of examples. For all of
sembled amphiphilic molecules into closed bilayered structures with an these applications, a product with specific characteristics, homogeneity
inner aqueous core. Depending on the chemical nature of bilayer con- and reproducibility is desired, and in particular, controlled size and
stituents, these particles are categorised into liposomes (lipids), nio- monodispersity are essential.
somes (non-ionic surfactants) or polymersomes (block copolymers), as Effort has been made to the production of niosomes by traditional
most frequently found in the literature [3–5]. methods with tight control over size and size distribution [14] for some
Niosomes exhibit unique advantages over the other types of vesi- specific applications [15]. For example, in our previous work [16], we
cular systems due to their inherent characteristics of non-ionic surfac- have used experimental design to study the influence of variables in the
tants [6,7]. These advantages include; (i) better chemical and physical ethanol injection process, in order to improve particle size tunability.
stability of suspensions due to the absence of oxidation-related de- One of the most popular chemical families for niosome production
gradation, (ii) easy derivatization to introduce different functional involves sorbitan esters (commercially available as Span®). Span family
⁎
Corresponding authors.
E-mail addresses: [email protected] (X. Zhang), [email protected] (M.C. Blanco-López).
https://doi.org/10.1016/j.colsurfb.2019.110378
Received 14 March 2019; Received in revised form 16 June 2019; Accepted 17 July 2019
Available online 18 July 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.
P. García-Manrique, et al. Colloids and Surfaces B: Biointerfaces 182 (2019) 110378
Fig. 1. Schematic diagram of a continuous

flow microreactor based on hydrodynamic
flow focusing for vesicular systems production.
The reduction of focused stream width under
laminar flow conditions makes possible the
mixing of chemical species by molecular dif-
fusion, since time for mixing decreases with
the square root of distance. By changing flow
rates, the kinetics and extension of mixing can
be modified, and then, the size of particles.
Amphiphilic molecules are self-assembled into
bilayers once critical concentration of solvent
is reached, and molecules acquired an ordered
state to minimize the interaction with water
molecules. At a certain size bending modulus
induce planar bilayer to be closed into vesicles.
members differ in terms of acyl chain length and saturation, with a big facilities [27]. Among the techniques explored, additive manufacturing,
range of hydrophilic-lipophilic balance values (HLB), where HLB is an especially 3D-printing, has emerged as a promising method for micro-
important parameter with implications in drug encapsulation efficiency fluidic device manufacturing [28]. The rapid development of 3D-
and morphological characteristics of particles. This parameter is also printing technology and the commercialization of desk printers have
related to the physical state at room temperature (RT), and influences enabled researchers to explore its utility in microfluidic prototyping
the minimum temperature (together with gel-to-liquid transition tem- and manufacturing [29–31], that generally use low cost raw materials
perature, or Tc) that is required at the very stage of the particle for- and can print objects with desired resolution.
mation. On the other hand, some of the compounds used in formula- The aim of the present work was to develop a thermostatic micro-
tions with great loading capacity, low release rate and stability in reactor platform for the continuous flow production of niosomes in a
solution are solid at RT. For these reasons, a higher and controlled size-controllable manner. The microfluidic reactor was designed with a
temperature level is mandatory for this process. hydrodynamic flow focusing configuration, and fabricated in order to
Microfluidics technology is very promising for precise control over allow visualization of the dynamic process including molecular diffu-
input variables when mixing chemical species [17]. Other advantages sion, with the aid of an inverted microscope and a digital image ac-
include low consumption of chemicals (relevant in formulation opti- quisition system. 3D-printing technology was used for fabricating the
mization), scale-up possibilities for industrial production, on-line cou- microfluidic device (positive mould) and thermostatic system. The ef-
pling to other processes (such as purification steps), and efficient con- fect of operational parameters was investigated on the final morpho-
trol over temperature if required [18]. Jahn et al. [19] reported for the logical characteristic of niosomes. Niosomes were formulated with non-
first time the hydrodynamic flow focussing (HFF) technique (Fig. 1) for ionic surfactants with different transition temperatures (Tm) with con-
liposomes production. Following that, other researchers have used this trolled temperature as a tailoring parameter to tune the size and
method to examine various liposomes formulations and for en- homogeneity of particles.
capsulating either hydrophobic or hydrophilic molecules [20,21].
Under laminar flow conditions within the HFF configuration, a stream 2. Materials and methods
of lipids in organic phase is focussed between two aqueous streams in
microchannels, allowing the mixing of chemical species by molecular 2.1. Materials
diffusion. At the two organic/aqueous interfaces, bilayers can be
formed and self-assembled into liposomes once a critical concentration Sorbitan monostearate or Span® 60 (Sigma-Aldrich), sorbitan
is reached. By controlling the flow, the extension of mixing and hence monolaureate or Span® 20 (Sigma-Aldrich), cholesterol from lamb wool
the size of liposomes, could also be controlled. However, the production (Akros Organics), Phosphate Buffer Saline (10 mM, pH 7.4) prepared
of niosomes through microfluidic routes remains less explored, and from tablets according to manufacture instructions (Sigma-Aldrich),
limited attention has been paid to using HFF technique [22–24]. Bromoxylenol blue (Sigma-Aldrich), and technical grade solvents such
At present, the high temperature required for the preparation of as ethanol absolute, 2-propanol (or isopropyl alcohol, IPA), and acetone
niosomes has not been well taken into account in microfluidics routes. (all from J.T. Baker, Avantor, USA) were used in this work. Ultrapure
For example, the previous work that firstly explored microfluidics as- water was used for all experiments. Poly(dimethylsiloxane) monomer
sembly of niosomes faced such temperature related challenge, thus only Sylgard® 184 or PDMS was purchased from Dow Corning Corporation
included Span® 20 and Span® 80 (Tm =25 °C and -30 °C, respectively) in (Auburn, AL, USA). Other materials used for devices fabrication are
the study. [22] specified in the following respective sections.
Along with the wide application of continuous flow microreactors
for organic colloids preparation [18] is the development of micro-
reactor itself, including design and manufacturing of such micro- 2.2. Thermostatic system fabrication
devices, with simpler and more affordable production methods [25]. As
a result, some traditional fabrications methods which stem from the Thermostatic chamber was design in Autodesk® Inventor® and 3D-
photo-electronics field, such as photolithography [26], are being sub- printed with PLA filaments using a special printer for fused deposition
stituted by new processes that require less expensive equipment and modelling (Ultimaker 2 + 3D printer, Ultimaker B.V., The
can be performed in common labs with no need for clean rooms Netherlands). Main chamber and cap of the device were produced se-
parately. A microscope glass slide of 50 x 70 mm (Corning® microscope
2
slides, Sigma-Aldrich, Gillingham, UK) was sealed to the chamber with 2.4. Niosomes production and morphological characterization
a 2-phase adhesive glue special for plastic materials, bought in a local
store. A transparent piece of plastic was glued to the cap aperture with Working solutions of 5 and 20 mM of Span® 60:cholesterol and
the same adhesive used with the other piece. Teflon tape was used to Span® 20:cholesterol (1:0.5 molar ratio) were prepared by dilution from
enhance the closure of both elements in a removable way. Holes for the a 50 mM stock solution. Ethanol absolute was used as organic solvent,
inlets and outlet pipes of the microfluidic device were manually pre- since it is miscible in aqueous buffer (PBS, 10 mM pH 7.4). Aqueous and
pared with a sharp tool. organic phases were pumped into microfluidic device once appropriate
The previously described chamber was connected to a temperature- temperature was reached. Three different total flow rates (QT) were
controllable recirculation system (F12-MC, Julabo GmbH, Germany) studied (50, 100 and 200 μL/min), and aqueous:organic flow rates were
through the inlet, and a peristaltic pump (MasterFlex®, Cole-Parmer adjusted to five different flow rates ratios (FRR) (5, 15, 25, 35 and 50).
Instruments Company, USA) through the outlet. The plastic pipes were Span® 20:cholesterol formulation was injected at 30, 40, 50 and 60 °C;
those from the recirculator, and connections to the chamber were made while Span® 60: cholesterol was only injected at 50 °C. All the combi-
with common plastic adapters (see supplementary material). nation of membrane components concentration, QT, FRR, and tem-
External supply of the recirculator was set approximately at a flow perature was conducted by duplicate.
rate of 55% of the total volume, while peristaltic pump revolution rate A total volume of 2.5 mL was collected from the outlet of the device
was adjusted to remove water from the chamber at a rate that allowed a for each experimental condition in a glass vial. Size (z-average or peak
continuous and constant flow through it. Temperature inside the value, depending on the number of peaks in the size distribution) and
chamber was monitored with a digital temperature probe (Testo 110, homogeneity (PDI) of particles were measured by Dynamic Light
Testo SE & Co., Germany). The sensor probe was introduced into the Scattering (DLS) in a Zetasizer NANO-ZS equipment (Malvern
chamber through a hole placed in one side of plastic window of the cap Instruments Ltd, Malvern, UK). Samples were measured undiluted by
(see Fig. S2). triplicate, with the 173° backscatter detector in disposable low volume
cuvettes (Malvern Instruments Ltd, Malvern, UK).
2.3. Microfluidic devices manufacturing and channel characterization
2.5. Mixing efficiency visualization
Master mould of devices was designed in Solidworks® CAD 2016
software and 3D-printed onto VeroClear™ resin with the HR-3D printer Solvent and no-solvent diffusion by hydrodynamic flow focusing
Objet350 Connex™ (Stratasys Ltd., USA). A post-printing process was was monitored by an adaption of a previous published methodology
also needed. First, mould was flushed with (I) IPA, (II) deionized water, [32]. Briefly, a change in colour of a pH indicator dye (bromoxylenol
(III) acetone, and finally compressed air. Then, it was cured overnight blue) was used, since this dye exhibits a strong yellowish colour at pH
at 60 °C, and on the following day a treatment of the inner surface was below 6.0 and blue at pH above 7.6. A saturated solution of dye in
carried out with Aquapeel® (to avoid interference of the resin with absolute ethanol acidified with acetic acid was focused by PBS adjusted
PDMS curing process). Three individual moulds were printed. to pH 10.0 with 2 M NaOH solution. Once focused, a change in colour of
Once the positive mould was ready, a mixture of degassed PDMS the stream from yellow to blue indicated a molar fraction of aqueous
curing agent (1:10 w/w) was poured into it, and left overnight in an phase close to one, and then, completes mixing by diffusion.
oven at 40 °C. For degassing the PDMS mixture, a bench centrifuge was
used at 3000 rpm for 10 min. It should be noted that pouring into 3. Results and discussion
master mould must be done slowly to minimise bubble formation. On
the following day, the replica of the mould was carefully peeled off With the microreactor platform developed, systematic character-
from the mould, and inlets and outlet holes were prepared with a isation and operation were conducted in terms of 3D printing outcomes
1.5 mm biopsy punch with plunger (Miltex®, Fischer Scientific, UK). and nanoproduction, as detailed below. (The performance and opti-
Oxygen plasma (PVA-TePla 300 plasma cleaner, Wettenberg, mization of the thermostatic system are described in the Supplementary
Germany) treatment was applied to bond a microscope glass slide material, Fig. S1).
(50 x 70 mm; Corning® microscope slides, Sigma-Aldrich, Gillingham,
UK) to the PDMS replica to complete the microfluidic channel. Four 3.1. High resolution 3D-printing of master moulds for microfluidic devices
pieces of thermic resistant plastic (Ø 8 mm and 3 mm height) were fabrication
glued in each corner at the bottom of the glass slide, to elevate the
device allowing a flow of hot water under the channels. As a key element of the device, mixing channel morphology was
Polytetrafluoroethylene (PFTE, 0.5 mm I.D.) pipes (Cole-Parmer, characterized by mechanical profilometry onto 3D-printed positive
UK) were inserted into the holes, and the other end was attached to a moulds. A considerable difference in nominal dimensions between
syringe needle to create a connection for introduction of the fluids from Computer Aided Design (CAD) and printed object was observed (Table
syringe pumps (NE-300, NEW ERA Pump Systems Inc., USA). Luer lock S1). With an original squared cross sectional geometry of 100 μm width
syringes (Becton, Dickinson and Company, UK) of 1, 10 or 20 mL were and 100 μm height, printed features onto VeroClear® resin showed a
used depending on the selected Flow Rate Ratio (FRR), i.e. volumetric curved morphology five times wider and approximately half of the
flow rate of total aqueous phase/volumetric flow rate of organic phase. height. At the same time, variations in width and height of the mixing
The mixing channel (23 mm long) on the 3D-printed positive mould channel were found between the three 3D-printed positive moulds (see
was characterized in terms of morphology, accuracy and reproduci- Table S1) even following the same fabrication procedure. However,
bility by mechanical profilometry (Talysurf-120 L, Taylor-Hobson, these dimensions were reasonably constant along the mixing channel
United Kingdom). Three equidistance measurements were taken (2 mm length, especially for channel height.
across the channel, perpendicular to it), and data were processed with A possible explanation for these variations in channel dimensions
OriginPro 18 (OriginLab Corporation, USA) software. could be related to printer operational parameters. Objet350 Connex3
The whole setup (microfluidic device inside the thermostatic printer used Polyjet™ inkjet-head patented technology for a layer-by-
chamber with respective inlets and outlets) was placed over the stage of layer process based on Stereolithography [33]. The jetting head dis-
an inverted microscope (IN200TAB series, AmScope, USA) with a di- pensed a proper amount of a photopolymer resin onto a build tray and
gital imaging system to capture images (5 M.P USB CCD camera, instantly cured them with UV light. The process took place in XY-axes
AmScope, USA) supported with the software supplied by the camera to create a 2D sheet (down to 16 microns thickness), and by lowering
manufacturer. The entire experimental setup is illustrated in Fig. S2. the build tray, another layer was created over the previous one. The
3
cycle was repeated until the whole design was completed. With a re- preparation routes [18], and with important advantages such as better
solution of 600 × 600 × 1600 dpi (X-Y-Z-axes respectively) and an control over particle preparation and the subsequent final character-
accuracy of 20–85 microns for features below 50 mm (up to 200 mi- istics (size and monodispersity, i.e.). This is important for biomedical
crons for full model size), the final features depended on geometry [1], food [35] and analytical chemistry [2] applications. In this regard,
(proximity between elements), build parameters (exposure time, the influence of operational conditions over particles physical proper-
printing speed) and model orientation [29]. Comina et al. [29] reported ties was tested by analysing the results of 3 total flow rates (QT), two
the successful printing of positive moulds for microfluidics devices with different concentrations of bilayer components, for 5 different FRR.
elements from 50 μm to 2 mm, however, some artefacts were described Particle size (nm) and size distribution (PDI) were measured by DLS as
between close elements with 50 μm in dimension differences, though output variables. All the combinations were conducted at 50 °C, a
working with optimized parameters. Unfortunately, no details about temperature over surfactant Tm.
cross section geometry were given for these channels. Some other au- In general terms, smaller particles were produced as the FRR in-
thors [34] have reported differences between CAD and printed designs creased (Fig. 3A and B) for both concentrations (5 and 20 mM), and for
with efforts in resin formulation optimization. all the QT levels. At a concentration of 5 mM (Fig. 3A), the particle size
In our recent work [31], we found that 3D printed channels with the decreased from 278, 298 and 358 nm (when FRR = 5) to 155, 129 and
Objet350 Connex3 printer were smoother than channels printed with a 143 nm (when FRR = 50), where QT = 50, 100 and 200 μL/min, re-
conventional desk 3D printer (Ultimaker 2+). However, for the same spectively. At a concentration of 20 mM (Fig. 3B), similarly, the particle
dimensions and aspect ratio, accuracy in cross sectional shape was size reduced from 342, 361 and 386 nm (when FRR = 5) to 164, 147
lower for the HR-3D printer even at large dimensions (1 mm squared and 151 nm (when FRR = 50) at the three QT levels of 50, 100 and
channels). It suggested that further studies are needed to understand 200 μL/min, respectively. Size reduction was rapidly reached with an
this effect with the scale and for different materials in order to inform increment in FRR from 5 to 15, and this reduction became less pro-
printing parameters optimization in terms of element dimensions, nounced from FRR 15 to 50. It is important to take into account that
geometry, and printing materials. Apart from the difference between when FRR increased the total amount of bilayer components decreased,
CAD and 3D-PMs, the cross sectional area of Mould 3 was similar to that not only producing vesicle with smaller size, since particle concentra-
previously used by Lo et al. [22], on which the selected operational tion was also reduced.
parameters of the present work were based. No significant effect of different QT was observed, while only some
differences were noticed in some particular combinations of parameters
at low surfactant concentration (5 mM), as seen in Fig. 3A. These ob-
3.2. Production of nanoparticles with temperature control for formulations servations were in accordance with previous studies [22] carried out
with high Tm non-ionic surfactants with identical chip configuration for the production of niosomes for-
mulated with other sorbitan esters (Span® 20 and 80), and also for the
The use of non-ionic surfactants for the formulation of organic production of liposomes [19,20,36,37]. At lower QT, also the linear
colloids, especially for NVs preparation, exhibits numerous advantages velocity was lower (hence larger residence time) what can counteract
[4,6]. However, a strict control of the temperature is necessary if Span® the effect of the bilayer components concentration.
60 (Tm = 45 °C), one of the most commonly used surfactant in niosome The increase in FRR, and the subsequent decrease in initial focused
formulation) is involved. Fig. 2 shows its precipitation at RT in mi- width (Wf), reduced the time needed for a complete mixing between
crochannels once reaching the focusing region, highlighting the sig- solvent and no-solvent (tmix), thus the critical concentration to induce
nificance of temperature effect. molecules self-assembly was reached faster. This led to smaller vesicles
In Fig. 2 surfactant precipitation was observed at the focussing re- since the total amount of bilayer components was reduced [38]. On the
gion and persists along the channel length when Span® 60 is used at other hand, the reduction of solvent introduced in the mixing channel
25 °C. However, at 50 °C a complete mix of both phases were produced also decreased the possibility of particle fusion into bigger unities by
without the presence of any surfactant precipitation. Moreover, the Ostwald-ripening phenomena [20,39]. A reduced tmix also led to com-
production of niosomes at this temperature conditions were observed plete mixing in limited length channels. In other cases, no diffused
using Transmission Electron Microspcopy (TEM) and negative staining solvent containing amphiphilic molecules self-assembled out of the
protocol. channel under entirely different conditions (outlet pipes, with no
This technique has been less explored than traditional bulk
Fig. 2. Precipitation of Span® 60 (Tm = 45 °C) at room temperature (upper arrow) at the focusing region (left), 0.5 and 1.0 cm downstream (centre and right). At a
temperature above surfactant Tm, focusing is complete and vesicles formation could be checked by negative staining (Phosphotungstic acid 2%) and Transmission
Electron Microscopy (TEM).
4
Fig. 3. Size (nm) and size distribution (PDI, a.u.) measured by DLS in undiluted samples from niosomes formulated with Span® 60:Cholesterol (1:0.5 molar ratio) at
5 mM (A,C) and 20 mM (B,D) in a continuous flow microreactor based on hydrodynamic flow focusing at controlled temperature (50 °C). Each condition was tested
twice, and each batch was measured by triplicate.
laminar flow characteristics). stream exhibited a “beating pulse” like effect that was likely produced
Regarding size distribution of particles (Fig. 3C and D), PDI value by the syringe pump due to its own pumping mechanism. These pulses
reduced as FRR increased from 5 to 15, (for 5 mM: from 0.119, 0.141 created really short increments in the width of the focused fluid that
and 0.163 at FRR = 5 to 0.092, 0.071 and 0.093 at FRR = 15; for introduced alteration in solvent exchange kinetics and the subsequent
20 mM: from 0.080, 0.131 and 0.118 at FRR = 5 to 0.061, 0.057 and changes in the local concentration of bilayer precursors and solvent
0.071 at FRR = 15; for both concentration values are indicated for concentration.
QT = 50, 100 and 200 μL/min respectively). and remained without Surprisingly, lower PDI values were obtained at 20 mM for all FRRs
significant changes at higher FRR for both concentrations. Some au- at the three different QT. Indeed, these differences were higher at
thors [19,20,32] reported a significantly increase in PDI with the in- 50 μL/min. At low concentration, those mentioned instabilities can in-
crement of FRR for an identical chip configuration, but for liposomes duce more pronounced local changes in bilayer precursor’s abundances,
production instead. However, our observation was in line with that of with the corresponding effect in particle monodispersity. To gain in-
Bottaro et al. [32] in a “Y”-shaped device, while Joshi et al. [21] de- sights into these observations further studies are needed.
scribed also a reduction in PDI as FRR increase during liposome for- On the other hand, larger particles were obtained when a higher
mation. No significant differences on PDI were observed for all QT le- concentrated ethanolic solution of bilayer components was used
vels applied. (20 mM vs. 5 mM). This was observed at all QT and FRR levels (see
The use of microreactors with different channel configurations, and Supplementary material, Fig. S4). The same observation was also re-
the use of static mixing enhancers [40], could be the reason of different ported by other authors when producing liposome using microchannels
results among published works. Some of them have highlighted the [38], and in agreement with the mechanism of vesicle formation under
influence of channel dimensions and configurations over mixing effi- microfluidic flow dynamic mixing.
ciency and particle properties [19,22,38]. The preparation of solvent The efficiency of mixing under the assayed working conditions was
mixture containing bilayer precursors can also influence the extension studied following a published methodology [32]. With this method,
and homogeneity of solubilisation, and in consequence, nanoprecipi- mixing efficiency was measured through the change in colour of a pH
tation process. In the present work, ethanol was used as solvent for indicator dye (bromoxylenol blue), that changed from yellow (acidic
microfluidic-based preparation of niosomes for the first time, and this ethanolic solution containing bilayer precursors) to blue (basic aqueous
limited the possibility for comparison with other studies. phase, PBS pH = 10). A shift in focused fluid colour from yellow to blue
We have noticed that at high FRRs some transitory perturbations of indicated that molar fraction of water into the stream was close to 1 and
the focused fluid were recorded, especially at 50 μL/min. The focused the subsequent molar fraction of EtOH became close to 0, evidencing a
5
of Wf with QT was observed at lower FRR values (Fig. 5A). It was also
observed that a lower QT generated wider focused streams probably due
to the lower pressure exercised by the lateral aqueous flows to the
middle solvent flow, but these differences became less pronounced at
higher FRRs. A similar trend was observed by Bottaro et al. [32] in an
identic channels configuration, but contrary to Jahn et al. [19] who
reported a non-variation in Wf with modifications in QT.
Moreover, an intense inverse correlation (potential) between FRR
and Wf was observed at all the QT levels (Table S2). However, a strong
negative correlation (linear) between particle size and Wf was observed
at the two different concentrations studied. These correlations reflect
that particle size is governed by focusing parameters. It is clear that
niosomes size can be tuned with the selection of the appropriate FRR
and QT values, which are key parameters for Wf and residence time.
3.3. Production of niosomes at different temperatures to study potential

tailoring effect over particle morphology
In this part of work QT of 100 μL/min and 5 mM of components

concentration were selected, since these have been the best operating
conditions in terms of smaller particles with narrower size distributions.
The effect of temperature was examined in a rage of 30 °C and 60 °C
as another operating parameter on size-tuned niosomes formation
through flow-focused based microfluidics. For this purpose, a non-ionic
surfactant with low Tm was needed that allowed to test a wide range of
working temperatures. Sorbitan monolaureate or Span® 20 (Tm =25 °C
and HLB 8.6) was selected, another common surfactant used for nio-
somal formulations [42], and surfactant:cholesterol molar ratio was
kept in 1:0.5 as for Span® 60 in order to allow formulations compar-
isons.
Fig. 6 depicts the results of particle size at the same FRRs previously
used for Span® 60 niosomes at different working temperatures: 30, 40,
50 and 60 °C. At 30 °C, a reduction in particle size from FRR = 5 to
FRR = 15 was observed. As FRR increased size became also larger
(even higher that those particles produced at FRR = 5). This phenom-
enon could be related to the observation of cholesterol precipitates
inside the mixing channel that were formed immediately after focusing
Fig. 4. Images (4X) of focusing region and end of the mixing channel eviden- region. The low solubility of cholesterol in water at nearly room tem-
cing hydrodynamic flow focussing of a central ethanol stream at different FRR perature induced its precipitation as crystals. Those precipitates mod-
values for QT = 100 μL/min and 50 °C. Yellow colour indicates acid pH (pure ified the flow properties and introduced turbulences that induced micro
EtOH, no mixing), while blue colour indicates basic pH (complete mixing by co- domains in the fluid with different concentrations of bilayer compo-
diffusion of solvent and no solvent). Bromoxylenol blue dye was dissolved in nents, and particles with different morphologies. Also the depletion of
EtOH (acidified with acetic acid), and PBS was adjusted with NaOH to pH 10 cholesterol could generate different particles than those produced in
(For interpretation of the references to colour in this figure legend, the reader is their presence. These perturbations were magnified at higher FRR, since
referred to the web version of this article). as seen in Fig. 5B the width of focused fluid became smaller with the
increment of FRR, and this stream was relatively smaller than the
complete mixing by solvent and aqueous effluents. This change in formed crystals (around 100 μm structures).
colour was easily detected in the inverted microscope, and recorded For the rest of temperatures, a similar behaviour as for Span® 60
with the digital camera. As an example, results for QT = 100 μl/min at niosomes was observed. Particles size became smaller with an inverse
several FRRs are shown in Fig. 4. correlation with FRR. At higher temperatures, focused ethanol stream
Complete mixing was only reached at high values of FRR (35 and was wider, and these differences were reduced with the increment in
50) for QT = 50 μl/min and QT = 100 μl/min, and only at the high FRR FRR. Only slight differences in particle size could be detected (Fig. 6A).
(50) for QT = 200 μl/min. As QT increased, residence time of the fluid Regarding temperature effect some authors reported an increase in
inside mixing channel reduced (from 0.5 s at 50 μl/min to 0.13 s at particle size as temperature increased [24] which were attributed to the
200 μl/min), preventing to stay the necessary time to reach complete bilayer expansion at higher temperature [43]. In our case, such increase
mixing. Only at high FRR value, tmix was short enough to be compatible in particle size was not observed. It is know that collapse pressure and
with low values of residence time for our channel dimension surface compressional moduli decrease with temperature for all sur-
(tmix = 17 ms and 8 ms for FRR = 35 and 50, respectively) predicted factants, and this implies that Span monolayers are more expanded with
according to a theoretical model (Eq. (1), [41]). In this model Wf re- increments in temperature. However, as temperature increases planar
presents the width of focused stream, where w is the channel width and bilayer precursors are less rigid, which could be easily bended to closed
D is solvent diffusion coefficient. structures, and this effect could lead then to smaller particles [39].
Regarding size distribution and temperature, it was observed that
Wf 2 w2 the increment of temperature yielded more monodisperse particles,
t mix ∼ ≈
4D 9D (1+FRR)2 (1) especially at 50 °C. Complete mixing can be reached at FRR = 35 and
FRR = 50 at any temperature. Only at 50 and 60 °C PDI values re-
As seen in Fig. 4, Wf decreased as FRR increased, and a dependence mained nearly constant (after a first reduction from FRR = 5 to 15)
6
Fig. 5. Values of ethanol focused stream (Wf, μm) as flow rate ratio (FRR) increased for different values of volumetric rates (QT) at constant temperature (50 °C), and
different temperatures at constant QT (100 μL/min). Values represent the average of two independent measurements, taken at approx. at 100 μm from the end of
focussed region.
with the increment in FRR. 4. Conclusions
Novel prototyping and additive manufacturing techniques such as

3.4. Effect of surfactant acyl chain length over particles size and (HR)3D-printing have been applied for the fabrication of a microfluidic
monodispersity continuous flow reactor for hydrodynamic flow focusing at controlled
temperature compatible with commercial inverted microscopes.
Another interesting finding resulted from the comparison between Despite some alteration in cross sectional dimensions and morphology
niosomes formulated with different non-ionic surfactants under iden- accuracy with respect to the original CAD design, high resolution 3D-
tical preparation conditions. In this work niosomes with sorbitan esters printed positive moulds allow us to create functional microreactors for
with different saturated acyl chain lengths (C12 and C18 for Span® 20 organic colloid production under different working conditions, and to
and 60 respectively) were prepared. As seen in Fig. 7, shorter chains study their effect on aqueous/solvent mixing efficiency through mole-
generally yielded larger niosomes. That was contrary to what would be cular diffusion, and its relationship with particles morphology.
expected; it is generally understood that shorter chains increase the This work shows that temperature is an essential parameter that
curvature radius of the bilayer, according to the critical packing para- must be taken into consideration when formulating niosomes with
meter (cpp) of the molecules [6], allowing smaller particles. However, surfactants with Tm over RT. Also it can be used to modify the prop-
if was taken into account the higher hydrophilic character of Span® 20 erties of particles (size and dispersity) produced with non-ionic sur-
compared to Span® 60 (higher HLB value) the higher hydrophilicity factants with Tm above RT.
could enhance water soak into the inner core of the vesicle, resulting in We have found that flow focussing at controlled temperature fol-
larger vesicles size. Similar results were reported by Gutierrez et al. lows the same patterns as for RT, with the ratio between aqueous and
[44] when niosomes were prepared by mechanical agitation. Regarding solvent streams being the main parameter to control focused stream
niosome size distributions, no differences between both types of sur- width and hence, mixing efficiency and kinetics. However, total flow
factants were observed. rate only has insignificant effect when FRRs are set to low values, whilst
it can influence residence time, and subsequently, mixing efficiency. In
Fig. 6. Size (nm) (left) and size distribution (PDI, a.u.) (right) measured by DLS in undiluted samples from niosomes formulated with Span® 20:Cholesterol (1:0.5
molar ratio) at 5 mM in a continuous flow microreactor based on hydrodynamic flow focusing at different controlled temperatures (30, 40, 50, and 60 °C). Each
condition was tested twice, and each batch was measured by triplicate.
7
Fig. 7. Influence of acyl chain length (C12 and C18 for Span® 20 and Span® 60 respectively) of two different sorbitan sters used in niosomes formulation (surfac-
tant:cholesterol 1:0.5 molar ratio, 5 mM), and produced under different conditions by hydrodynamic flow focusing at controlled temperature (50 °C) and a flow rate
QT = 100 μl/min.
general terms, an increase in FRR yields a focused stream being nar- Appendix A. Supplementary data
rower, and then, smaller particles due to the reduction in residual
solvent and the introduction of less amount of bilayer components. This Supplementary material related to this article can be found, in the
reduction allows complete mixing, even at high total flow rate, re- online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110378.
sulting in the size distribution of generated particles being more
homogeneous. The counterpart is that production yield is reduced, References
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9
Supplementary Material-
Controlled size niosome production using a thermostatic

microfluidic device
Pablo García-Manrique1,2, Gemma Gutiérrez2, María Matos2, Andrea Cristaldi3, Ali Mosayyebi3,
Dario Carugo4, Xunli Zhang3*, Mª Carmen Blanco1*
1 Department of Physical and Analytical Chemistry, University of Oviedo; Spain
2 Department of Chemical Engineering and Environmental Technology, University of Oviedo; Spain
3Bioengineering Sciences group, Faculty of Engineering and the Environment, Institute for Life Sciences (IfLS),
University of Southampton; United Kingdom

4Mechanotronics and Bioengineering sciences research groups, Faculty of Engineering and the Environment,
Institute for Life Sciences (IfLS), University of Southampton; United Kingdom
*Corresponding authors; E-mail address: [email protected], [email protected]
1
Table S1. Morphological characteristics of mixing channel for original Solidworks® CAD 2016
design and 3D-Printed positive moulds (3D-PM) onto VeroClearTM resin with the 3D printer
Objet350 ConnexTM (Stratsys). Average and standard deviation values are given for the
parameters.
Mould Width (µm) Height (µm) Cross sectional area (µm)
CAD 100 100 10000
3D-PM1 535 ± 40 47 ± 2 13090 ± 485
3D-PM2 423 ± 14 51 ± 1 11605 ± 262
3D-PM3 507 ± 28 68 ± 3 18007 ± 1092
2
Figure S1. Calibration plot (A) and temperature stability (B) of the in-house designed
thermostatic chamber for microfluidics chips, fabricated by 3D-printing technology with PLA
filaments. Values represented are the average of three independents measurements.
3
Figure S2. Pictures composition showing the whole setup (central) and detailed components
(sides) used in this work for niosomes production by Hydrodynamic Flow Focusing with controlled
temperature.
4
Table S2. Correlation factor between flow focusing parameters (FRR and Wf) and particle size
at different concentration of bilayer components and variable QT (A), and at fixed
concentration and QT for different working temperatures (B).
(A)
R2 (T = 50 °C) 50 µl/min 100 µl/min 200 µl/min
(*)
FRR vs Wf 0.99 0.97 0.92
(**)
Wf vs Particle size 0.86 (5 mM) 0.98 (5 mM) 0.97 (5 mM)
0.98 (20 mM) 0.99 (20 mM) 0.93 (20 mM)

FRR, Flow Rate Ratio; QT, Total volumetric rate; Wf, initial width of focused stream; (*) potential correlation; (**)
linear correlation
(B)
R2 (QT = 100 µl/min) 30 °C 40 °C 50 °C 60 °C
(*)
FRR vs Wf 1.00 0.99 0.97 0.98
(**)
FRR vs Particle size 0.24 0.56 0.91 0.65
FRR, Flow Rate Ratio; QT, Total volumetric rate; Wf, initial width of focused stream; (*) potential correlation; (**)
linear correlation
5
Figure S3. Effect of bilayer components concentration for niosomes formulated with Span®
60:Cholesterol (1:0.5 molar ratio) and produced under the same flow conditions (QT and FRR)
for size (upper row) and size distribution (lower row).
6
ANEXO II. Publicaciones relacionadas con la
Tesis Doctoral
New BIOTECHNOLOGY 47 (2018) 1–7
Contents lists available at ScienceDirect
New BIOTECHNOLOGY
journal homepage: www.elsevier.com/locate/nbt
Easy-to-perform and cost-effective fabrication of continuous-flow reactors

and their application for nanomaterials synthesis
Domenico Andrea Cristaldia,c, Fatih Yanara, Ali Mosayyebia, Pablo García-Manriqueb,
⁎⁎ ⁎ ⁎⁎
Eugen Stulzc, , Dario Carugod, , Xunli Zhanga,
a
Bioengineering Group, Faculty of Engineering and the Environment, Institute for Life Sciences (IfLS), University of Southampton, UK
b
Departments of Physical and Analytical Chemistry, and Chemical Engineering and Environmental Technology, University of Oviedo, Spain
c
School of Chemistry & Institute for Life Sciences, University of Southampton, Highfield, Southampton, UK
d
Mechatronics and Bioengineering Science Research Groups, Faculty of Engineering and the Environment, Institute for Life Sciences (IfLS), University of Southampton, UK
A R T I C L E I N F O A B S T R A C T
Keywords: The translation of continuous-flow microreactor technology to the industrial environment has been limited by
3D printing cost and complexity of the fabrication procedures and the requirement for specialised infrastructure. In the
Lab-on-a-chip present study, we have developed a significantly more cost-effective and easy-to-perform fabrication method for
Soft lithography the generation of optically transparent, continuous-flow reactors. The method combines 3D printing of master
Nanoparticles
moulds with sealing of the PDMS channels’ replica using a pressure-sensitive adhesive tape. Morphological
Liposomes
Continuous-flow reactors.
characterisation of the 3D printed moulds was performed and reactors were fabricated with an approximately
square-shaped cross-section of 1 mm2. Notably, they were tested for operation over a wide range of volumetric
flow rates, up to 20 ml/min. Moreover, the fabrication time (i.e., from design to the finished product) was <
1 day, at an average material cost of ∼£5. The flow reactors have been applied to the production of both
inorganic nanoparticles (silver nanospheres) and organic vesicular systems (liposomes), and their performance
compared with reactors produced using more laborious fabrication methods. Numerical simulations were per-
formed to characterise the transport of fluids and chemical species within the devices. The developed fabrication
method is suitable for scaled-up fabrication of continuous-flow reactors, with potential for application in bio-
technology and nanomedicine.
Introduction polymethyl methacrylate, PMMA). This procedure does not require the
fabrication of photomasks via photolithography, which is typically
Photolithography has been widely used to manufacture continuous- performed in a cleanroom environment. However, the method requires
flow reactors at high spatial resolution, in terms of both size and shape the use of micromilling machines, as well as oxygen plasma bonding for
of the channels’ architecture [1]. However, this process involves nu- the sealing of the polydimethylsiloxane (PDMS) channels onto a glass
merous steps and generally requires specialised cleanroom facilities, layer. Alternatively, positive moulds can be fabricated in a single step
and expensive materials or instrumentation. In addition, the whole using high-resolution 3D printing (with channel width down to 50 μm),
process (i.e., from the design of the device architecture to the end as demonstrated by Comina et al. [4]. Similarly, three-dimensional (3D)
product) is highly time-consuming. The combination of these factors PDMS microfluidic reactors can be fabricated via UV-activated 3D
has hindered the widespread adoption of this technology by industries printing, as described by Chan et al. [5]. Although 3D printing of resin
and researchers, particularly in the non-specialised or less resourced moulds may be less complex to perform compared to μMi-REM, it re-
laboratories. quires specific treatments of the moulds prior to PDMS casting, due to
In the last decade, efforts have been made to develop more cost- the inhibition effect of the resin on the PDMS cross-linker [6,4]. In
effective and user friendly manufacturing approaches [2]. For instance, addition, both methods rely on the use of oxygen plasma during the
the micromilling-replica moulding (μMi-REM) technique recently de- bonding process. On the other hand, microfluidic devices could be
veloped by Carugo et al. [3] involves the fabrication of positive epoxy entirely 3D printed, as demonstrated by Kitson et al. [7]. This method is
masters obtained from negative micromilled moulds (made of cost-effective and easy-to-perform; however, devices are not optically
⁎
Corresponding author.
⁎⁎
Co-corresponding authors.
E-mail addresses: [email protected] (E. Stulz), [email protected] (D. Carugo), [email protected] (X. Zhang).
https://doi.org/10.1016/j.nbt.2018.02.002
Received 25 July 2017; Received in revised form 2 February 2018; Accepted 4 February 2018
Available online 06 February 2018
1871-6784/ © 2018 Elsevier B.V. All rights reserved.
D.A. Cristaldi et al. New BIOTECHNOLOGY 47 (2018) 1–7
transparent, thus limiting the ability to optically monitor flow and moulds. The following printing settings were adopted: bottom/top
mixing processes. The optical transparency of 3D printed channels was thickness = 0.5 mm, fill density = 100%, print speed = 50 mm/s, and
recently improved by Gaal et al. [8], using a custom built 3D printer to nozzle size = 0.4 mm. PDMS replicas were prepared by pouring a 10.2/
create polylactic acid/polydimethylsiloxane (PLA/PDMS) architectures. 0.8 (w/w) ratio of degassed PDMS precursor and curing agent mixture
However, careful adjustments of the 3D printer settings were needed (Sylgard® 184, Dow Corning Corporation, Michigan, USA) over the
during the fabrication process, and the cross-section of the produced mould (Scheme 1, Step 2). Degassing was carried out by prior cen-
channels differed significantly from the original computer-aided design trifugation at 3000 rpm for 15 min, using the Eppendorf Centrifuge
(CAD). 5804 and Corning Centristar™ tubes (50 ml). After pouring, the liquid
Developments have also been made in terms of the microchannel PDMS was left at room temperature for 4 h and any formed gas bubble
dimensions obtainable using 3D printing, with recent studies re- removed using a sharp tool (every 1 h). This step could be significantly
searching novel resin formulations for stereolithgraphy (SL) and 3D accelerated by placing the mould under vacuum. PDMS curing was
printing with Digital Light Processing (DLP) [9]. With this technique, performed in the oven at 40 °C overnight. The cured PDMS replica
Gong et al. demonstrated the generation of remarkably small microchannel was peeled off from the master mould, and a 1.5 mm diameter
channels (i.e., with cross section as small as 18 × 20 μm) [10]. Sig- biopsy punch with plunger (Miltex®, Fischer Scientific, UK) was used for
nificant efforts have also been devoted to development of simple creating inlets/outlets. Sealing of the PDMS layer was performed on
bonding techniques for microfluidic devices. For example, Serra et al. ThermalSeal RT™ tape purchased from Excel Scientific (USA) (Scheme
[11] recently demonstrated the use of a commercially available sealing 1, Step 3). To characterise the morphology of the 3D printed moulds, a
tape (Thermalseal RTS™) for bonding of various substrates. The PDMS non-contact Alicona Infinite Focus 3D optical profilometer was em-
channel architectures in this study were fabricated from micromilled ployed (5 × magnification lens, vertical resolution: 410 nm, lateral re-
brass masters. solution: 6.59 μm). In order to evaluate the method’s repeatability,
In the present study, we developed a fast, cost-effective and facile three identical devices were printed and two-dimensional (2D) images
manufacturing process to fabricate optically transparent flow reactors, (at 2.5 × magnification) were acquired to measure (i) the width of the
with milli- or sub-millimetre scale flow channels (i.e., with 0.5–1.0 mm mixing channel at five equidistant locations along the channel (se-
channel width). A combination of techniques was employed including paration distance between measurements = 2 mm) starting from the
3D printing of positive moulds followed by PDMS casting (3D printed junction, and (ii) the radius of curvature of both inlet channels. Mea-
mould casting, 3DPM-C), and direct sealing of the PDMS layer using a surements were performed using Image-J software (NIH, USA).
pressure-sensitive adhesive tape. The 3D printed moulds were char- The manufacture of μMi-REM devices was performed following a
acterized in terms of surface roughness and cross-sectional shape, and protocol previously reported [3]. The PVA-TePla 300 plasma cleaner
reactors were tested over a wide range of operational conditions. To was employed to assist the bonding of the PDMS layer with a
demonstrate the usability of the developed fabrication technique, re- 50 × 70 mm glass sheet (Corning® microscope slides, Sigma Aldrich,
actors were applied to the production of silver nanospheres (SNSs) and Gillingham, UK).
liposomes, as examples of inorganic and organic synthesis, respectively. The two fabrication methods employed in this study are herein
Silver nanospheres (SNSs) have been employed in many research defined as 3DPM-C/Tape and μMi-REM*Glass, where ‘/’ and ‘*’ indicate
fields ranging from photocatalysis [12] to optoelectronics [13], as well adhesive tape and plasma bonding procedures, respectively.
as for biological applications due to their antibacterial properties [14].
SNSs synthesis typically needs a carefully balanced stoichiometry, in Synthesis of inorganic and organic nanoparticles
order to obtain the desired particle size and/or shape [15]. Recently,
Barber et al. demonstrated a coaxial glass reactor for continuous-flow The same experimental set-up was used for the production of both
production of SNSs [16], with superior control over the fluidic en- silver nanospheres and liposomes. Syringe pumps (AL-1010) were
vironment and properties of the produced SNSs, when compared to purchased from World Precision Instruments (UK). Luer Lock syringes
bulk methods. Liposomes, spherical vesicles comprising an aqueous (20 ml) (BD Plastipak) were purchased from BD (Becton, Dickinson and
core surrounded by a lipid bilayer, are employed as vehicles for Company, UK). Polytetrafluoroethylene (PTFE) tubing and connectors
transporting and administering pharmaceutical actives [17]. Produc- (Cole-Palmer, UK) were employed for interfacing the device inlets and
tion of liposomes via solvent exchange mechanism in continuous-flow outlet with syringes and collection vials, respectively. The length of the
reactors has emerged as a promising technique, offering a higher degree tube from the outlet to the collection vial was 26.70 cm.
of control over the physical and dimensional properties of the end Silver nanoparticles were synthesized using silver nitrate 99.9999%
product, compared to batch methods. (AgNO3), tri sodium citrate dihydrate ≥ 99.0% (TSCD), poly-
In this study, we demonstrate continuous-flow production of both vinylpyrrolidone (PVP), and sodium borohydride 99% (NaBH4), which
types of nanoscale particles using cost-effective and easy-to-operate were purchased from Sigma Aldrich UK (Gillingham, UK). Propan-2-ol
reactors. (or isopropyl alcohol, IPA) laboratory reagent grade was purchased
from Fisher Chemical (UK). Milli-Q water was collected using the Q-
Materials and methods Gard purification filter, connected to the Milli-Q Gradient A10 system
(Merck Millipore, USA).
Design, fabrication and morphological characterization of flow reactors Scheme 2 shows the experimental set-up employed for the synthesis
of both silver nanoparticles and liposomes. Syringes were spatially ar-
Fabrication of the 3D printed mould casting (3DPM-C) channels is ranged in a way that allowed performing the same experiment for both
illustrated in Scheme 1. Solidworks® CAD 2016 software was used for 3DPM-C/Tape and μMi-REM*Glass devices, by simply changing the
designing the master mould. The flow reactor architecture has two reactor.
semi-circular inlet channels of 0.50 mm × 1.00 mm (width × height) For SNSs production, a 20 ml syringe was primed with a Milli-Q
and 1.50 mm radius. Inlets converge in to a straight channel of water solution containing AgNO3 (1.02 mM), TSCD (15.02 mM), and
1.00 mm × 1.00 mm × 60.00 mm (width × height × length). The PVP (0.45 M). The second syringe was primed with 15 ml of an IPA/
channel architecture was positioned at the bottom surface of a box Milli-Q solution (9:1 v/v) of NaBH4 (5.28 mM). The total flow rate
structure having a 7.00 mm high edge, which acted as a container for (TFR = FRA + FRB) was kept at the constant value of 1 ml/min,
uncured PDMS (see Step 1 in Scheme 1). The Ultimaker 2+ 3D printer, whereas the flow rate ratio (FRR = FRA/FRB) was varied (5, 7, 9 and
loaded with PLA filaments, was employed as a representative fused 11). Each sample was separately collected in a 1.5 ml Eppendorf tube,
deposition modelling (FDM) tool for the production of the master and 1.5 ml were collected in a waste vial in between each experimental
2
Scheme 1. Graphical representation of the manufacturing steps for the 3DPM-C/Tape reactor: 1) Low-cost 3D printing of the positive mould; 2) PDMS casting; and 3) sealing of the cured
PDMS layer onto adhesive tape.
run. In order to evaluate the robustness of the reaction, experiments measure the mean diameter (z-average), the polydispersity index (PDI),
were repeated in triplicate at selected TFR and FRR values. and zeta potential of all liposomal formulations, produced with both
For the synthesis of liposomes, Phospholipon® 90G (lipids) and 3DPM-C/Tape and μMi-REM*Glass reactors. Liposome dimensional
purified phosphatidylcholine from soybean lecithin, were kindly pro- stability was assessed by measuring the mean diameter of samples
vided by Lipoid GmbH (Germany). Pure ethanol (99.9%) was purchased stored at both 4 °C and 25 °C, every 5 days for a total 30 days. All
from Sigma-Aldrich Company Ltd. (UK). A 100 mM lipid solution in measurements were performed using the Zetasizer Nano ZS Malvern,
ethanol was prepared, and the lipid concentration selected to produce UK.
liposomes of a clinically relevant size and mass [18–20]. Milli-Q water
was injected into one inlet (Syringe A) and ethanol-containing lipids
was injected into a second inlet (Syringe B), for each reactor (Scheme Results and discussion
2).
Experiments were carried out maintaining a constant FRR of 25, at Characterisation of the 3D printed moulds and PDMS channels
varying TFRs of 1, 3 and 6 ml/min to demonstrate devices’ usability for
producing liposomes at high-throughput. Three samples were collected, The morphology and roughness of the flat base of the 3D printed
at each TFR and for each device tested. master are important characteristics affecting spatial uniformity and
durability of the sealing. From the morphological examination, diag-
onal grooves can be observed due to the oblique motion of the nozzle in
Characterisation of nanoparticles the x-y plane during printing (see Fig. 1a). The average roughness (Ra)
value of the master mould is 6.56 μm (Fig. 1a), and a maximum peak-to-
The UV–vis characterization of silver nanospheres (SNSs) was car- valley (Rz) value of 85.13 μm is detected in proximity to the diagonal
ried out using a Varian Cary300Bio UV–vis Spectrophotometer. All features over the base surface (representative cross sectional profiles
measurements were performed in the 200–800 nm range, with an in- are shown in Figure SI-1a). Notably, we observed that grooves could
crement step of 0.5 nm. SNS analytical samples (1 ml) were collected promote fluid leakage at flow rates greater than 5 ml/min.
from the flow reactors, and diluted to 3 ml with Milli-Q water into a In order to overcome this limitation, the PDMS mixture (monomer/
quartz cuvette for the spectrophotometric characterization. The base- curing agent) was prepared using 8% curing agent (instead of the
line was subtracted from each experimental condition (i.e., considering commonly used 10% by mass), to reduce PDMS stiffness. Notably, a
the specific Milli-Q/IPA volume ratio). softer PDMS could be deformed more easily by applying external
Transmission electron microscopy (TEM) characterization of SNSs pressure during sealing. Manual compression using a plastic spatula
was also performed. Images were acquired using the TEM Hitachi was initially performed (at an estimated pressure of 1 bar), followed by
HT7700. Silver nanoparticles were prepared by drop-casting of the compression at ∼0.5 bar for 1 h. In this way, no leakages were observed
colloidal synthesis solution (5 μl), on carbon and Formvar coated Cu/Pd even at a total flow rate of 20 ml/min (see Supporting information
200 mesh grids, and left to dry under atmospheric conditions at room video, at https://youtu.be/EpmnLZDXtBo), confirming the effective-
temperature. ness of the sealing procedure for continuous-flow synthesis at high-
A dynamic light scattering (DLS) technique was used instead to throughput.
Scheme 2. Schematic of the experimental set-up and list of chemicals injected through syringes A and B for the production of SNSs and liposomes, respectively.
3
Fig. 1. Morphology and cross-sectional shape of the 3D printed moulds. a) Morphological characterisation of the 3D printed base, including average roughness (Ra) and maximum peak-
to-valley value (Rz). b) Cross section of a representative channel design of 0.60 × 0.80 mm (width × height), obtained from optical profilometry.
Accurate reproduction of the designed (i.e., nominal) channel size Fig. 2a). The radius of curvature of the inlet channels was also very
and shape should ideally be achieved by the 3D printing process. In this comparable between different moulds (mean value = 1.770 ±
respect, the high resolution (HR) resin 3D printer (Object Connex 350) 0.012 mm), and only slightly differed from the nominal value of
was tested against the Ultimaker 2+ by printing channels of different 1.75 mm (see Fig. 2b).
cross-sectional dimension, as illustrated in Figure SI-1b. The HR 3D
printer was able to create smaller and smoother channels; however, Synthesis of silver nanospheres and liposomes via 3DPM-C/Tape & μMi-
reproduction of the cross sectional shape was less accurate, even for the REM*Glass reactors
relatively large channel dimension of 1.00 × 1.00 mm (see cross section
in Figure SI-1c obtained from profilometry). Thus, the Ultimaker 2+ Nanoparticle synthesis via 3DPM-C/Tape reactor was performed in
was selected to generate the master moulds in the remaining experi- parallel with the already established μMi-REM*Glass technique. In
ments. order to compare the performance of the two fabrication methods, the
Two additional test channels were printed having different size but physical properties of the produced nanomaterials were measured and
the same aspect ratio of 1.33, using the Ultimaker 2+. The mean ex- evaluated at varying fluid dynamic boundary conditions.
perimental values obtained with the optical profilometer (re-
presentative test Channel 1 in Fig. 1b) are compared with the nominal
Synthesis of silver nanospheres
dimensions of both channels in Table 1. The channel height is accu-
rately reproduced, whereas the width is affected by a 0.09 mm differ-
Silver nanospheres (SNSs) were synthetized using the chemical re-
ence for both test channels 1 and 2 (a 3D cross section of test Channel 2
duction method [15], which was adapted for usage in a continuous-flow
is shown in Figure SI-1c). This is due to the orientation angle (90°) of
reactor. Specifically, NaBH4 solution was prepared initially by dissol-
the side walls of the channels with respect to the light source of the
ving the solid in 1.5 ml of Milli-Q water, followed by dilution to 15 ml
profilometer. This effect is clearly visible in Fig. 1b in which the side
with IPA. The use of IPA was to minimise the generation of H2, pro-
walls of the channel are represented as the projection of the edge of the
duced from the degradation of NaBH4 in water [21]. This drastically
channel roof towards the base, causing overestimation of the measured
reduces the formation of gas bubbles within the channels, which may
width.
significantly alter the flow field or cause clogging. This effect may
Nonetheless, in order to assess repeatability of the reactor’s fabri-
however be less problematic in millimetre-scale channels. An alter-
cation method, three replicas of the 3D printed moulds were manu-
native approach may involve the generation of a strong basic condition
factured using the Ultimaker 2+ (Fig. 2). The width of the mixing
(NaOH, 14 M) in water, as described by Barber et al. [16]. However,
channel (measured at five separate and equidistant locations along the
with the protocol described in this study, we were able to produce SNSs
channel) and the radius of curvature of the inlet channels were mea-
at all FRRs investigated, obtaining the maximum absorbance (Amax) of
sured, and results are shown in Fig. 2a and b respectively. Insets il-
2.9 ± 0.2 nm after 1:3 dilution with Milli-Q water. Nevertheless, the
lustrate the positions at which measurements were taken.
stoichiometry played a critical role in SNSs synthesis, and although the
The width of the mixing channel is comparable between the three
numerical results (see Figure SI-2b) show a marginal increase in mixing
different moulds, as illustrated in Fig. 2a. The maximum difference is at
efficiency at FRR = 11, the SNSs production performance was more
6 mm from the junction, between mould n.1 and n.3, and is equal to
effective at FRR = 7.
0.0011 mm only. All moulds had a slightly larger channel width in
Fig. 3a reports the UV–vis spectra of SNSs prepared using the 3DPM-
proximity to the junction, which gradually reduced along the channel
C/Tape and the μMi-REM*Glass reactors, applying the same operational
and reached a plateau value at about 8–10 mm from the junction (see
conditions in both devices (TFR = 1 ml/min; FRR = 7). In order to
assess the reproducibility of the reaction, samples were produced in
Table 1 triplicate under the same conditions for both devices.
Size comparison between the CAD design and the 3D printed channels. The typical absorption spectra, due to SNSs surface plasmon re-
sonance (SPR) [22], are observed. Notably, the SNSs spectra obtained
Design-Morphology Width × Height (mm) Width × Height (mm)
using the two different fabrication methods are almost overlapping
Comparisons Test Channel 1 Test Channel 2
across the whole wavelength spectrum. The mean value of maximum
CAD Design 0.60 × 0.80 0.80 × 1.33 absorbance (Amax) is equal to 3.252 ± 0.050 and 3.236 ± 0.068 for
the Tape and Glass reactors, respectively (see Fig. 3a and inset). More
3D Printed mould 0.69 × 0.80 0.89 × 1.33
importantly, in both types of reactor the maximum absorption value of
profile (mean
values) 398.0 nm is obtained, further indicating a comparable performance
between them. The absorbance value is also related to the particle size
4
Fig. 2. a) Width of the mixing channel at 2, 4, 6, 8, and 10 mm from the junction between inlets, for three different 3D printed moulds. An image of a representative channel (at
2.5 × magnification) showing the measurement lines (red lines) is reported in the inset. b) Radius of curvature of both right (R) and left (L) inlet channels, for three different 3D printed
moulds. A graphical representation of the measurement method is reported in the inset, for the right inlet channel.
[23], which ranged from 10 to 20 nm as shown in the TEM images min and FRR = 25, have a diameter of 188.61 ± 1.62 nm and
(Fig. 3b). 191.37 ± 3.19 nm, respectively. Moreover, by increasing the TFR from
1 to 6 ml/min caused only a slight increase in liposome diameter in
Synthesis of liposomes both devices, which is consistent with the numerical simulations
showing a relatively small difference in the mixing index at the dif-
Having demonstrated that flow reactors fabricated with different ferent flow regimes investigated (see Figure SI-2b). Fig. 4b shows that
methods have comparable performance when employed to produce liposomes have a relatively small dispersity, and that PDI slightly in-
silver nanoparticles, they were also tested for continuous-flow synthesis creased with increasing the TFR from 1 to 6 ml/min.
of liposomes. Devices were operated at total flow rates which were Liposomes produced by the 3DPM-C/Tape reactor had a larger
significantly higher than those typically used in microfluidic reactors mean dispersity at the higher flow rates investigated, which could be
[20], to demonstrate their potential utility for scaled-up synthesis. potentially attributed to the surface roughness of the PDMS channels.
Fig. 4 shows the size (z-average) and polydispersity index (PDI) of Further investigations will be performed to better understand the effect
liposomes produced at varying TFR values (1, 3, 6 ml/min) and a fixed of surface properties on the transport of fluids and chemical species in
FRR of 25. A representative intensity-based liposome size distribution these devices.
(at TFR = 1 ml/min and FRR = 25) is also shown in Figure SI-3a, for In addition, the diameter of liposomes produced by 3DPM-C/Tape
the μMi-REM*Glass reactor. Notably, liposome size and dispersity are reactor (at FRR of 25 and TFR of 1 ml/min) was measured up to 30 days
not significantly different across different devices, for all the hydro- from production, at two different storage temperatures (4 °C and 25 °C),
dynamic conditions investigated. For instance, liposomes produced as shown in Fig. 4c. The size of liposomes stored at 4 °C increased over
with the 3DPM-C/Tape and μMi-REM*Glass reactors at TFR = 1 ml/ time, and reached a maximum% increase of approximately 15% after
Fig. 3. a) UV–vis characterization of SNSs prepared using the 3DPM-C/Tape (black) and the μMi-REM*Glass (red) reactors. Spectra are shown as the mean of triplicate samples prepared
using both types of reactor, at the same operating conditions (TFR = 1 ml/min; FRR = 7) (individual spectra are shown in the inset). b) Representative TEM image of the SNSs prepared
with the 3DPM-C/Tape (TFR = 1 ml/min; FRR = 7); with a magnified view shown in the inset.
5
Fig. 4. Comparison of liposome size and dispersity. Size (z-average) (a) and dispersity (PDI) (b) of liposomal formulations produced by μMi-REM*Glass and 3DPM-C/Tape reactors. Each
experiment was performed at TFR of 1 ml/min, 3 ml/min and 6 ml/min, at a fixed FRR of 25. Data are reported as the mean of three independent samples, with the corresponding
standard deviation. c) Size stability of liposomal formulations produced by 3DPM-C/Tape reactor, at FRR of 25 and TFR of 1 ml/min. Liposome size was measured every 5 days and up to
30 days, at storage temperatures of both 4 °C and 25 °C.
30 days. These results are comparable with the ones previously reported (liposomes). They exhibited comparable performance to reactors fab-
in the literature [24], although they refer to different Phospholi- ricated using more laborious and expensive fabrication methods. The
pon90G-based liposomal formulations. The increase in vesicle diameter manufacturing technique presented in this study is therefore potentially
could be attributed to sterical hindrance of bilayer stability [25] or suitable for scaling-up, as devices can be fabricated at low-cost, and
aggregation, which may result in liposome coalescence [26]. Con- without resorting to sophisticated instrumentation and time-consuming
versely, the size of liposomes stored at 25 °C was almost unchanged multistep procedures. Moreover, devices produced with this technique
over time. Moreover, the zeta potential of liposomes produced using the can be operated at relatively high total flow rates ( > 20 ml/min),
3DPM-C/Tape rector (at FRR of 25 and TFR of 1 ml/min) was which is a desirable characteristic for application in continuous-flow
−15.06 mV, which is coherent with the literature [27]. chemical synthesis.
Conclusions Acknowledgment
We have developed an easy-to-perform and cost-effective method Authors would like to thank the University of Southampton for
for the fabrication of PDMS based continuous-flow reactors, through 3D supporting the research through a PhD studentship.
printed mould casting (3DPM-C). In this method, the positive mould
was “printed” using a desktop 3D printer, followed by PDMS casting to Appendix A. Supplementary data
produce replica channels of millimetre or sub-millimetre width and
height. Channels on the PDMS replica were then sealed using a com- Supplementary data associated with this article can be found, in the
mercially available pressure-sensitive adhesive tape. It was also de- online version, at https://doi.org/10.1016/j.nbt.2018.02.002.
monstrated that the whole fabrication process can be completed within
24 h from the CAD design of the channel architecture, at an average References
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Supporting information
SI-1. Morphological characterisation of the 3D printed moulds
Figure SI-1a: Cross sectional profile of the 3D printed mould base, showing the presence of diagonal grooves.
Figure SI-1b: Comparison between the Ultimaker 2+ 3D printer (white PLA material) and the High Resolution
3D printer Object Connex 350 (Veroclean® resin material). Although in this test the 0.4 mm channel was not
printed by the Ultimaker 2+, channels of 0.5 mm could be generated (these correspond to the inlet channels of
the reactor). The HR can reach smaller dimensions but the designed (i.e. nominal) cross sectional shape could
not be accurately reproduced (see SI-1c).
Figure SI-1c: a) Ultimaker 2+ mould test using the Alicona microscope. b) 3D morphological reproduction of
the designed 0.80 mm × 1.33 mm (width × height) channel. c) Object Connex 350 mould test under the Taylor-
Hobson profilometer. d) Profile graph of the actual channel (solid black line) compared with the designed
(nominal) 1.00 mm × 1.00 mm (width × height) channel (dotted line). The reproduction error for the HR 3D
printed channels is greater for smaller channel sizes.
SI-2. Computational fluid dynamic (CFD) simulations
SI-2a. Simulation methods
The transport of fluids and chemical species within the reactors was characterised
numerically via computational fluid dynamic (CFD) simulations. The process comprised the
following steps: (i) design of the flow reactor geometry using Inventor Pro 2016 (Autodesk
Inc., San Rafael, CA, USA); (ii) meshing of the fluidic domain in ICEM CFD 17.0 (Ansys
Inc., Concord, MA, USA) using finite volumes of tetrahedral shape. The mesh element edge
length was equal to 0.05 mm, and was identified from a mesh dependence study as the
optimal compromise between solution accuracy and computational cost (Figure SI-2a).
Figure SI-2a: mesh dependence study performed at mesh volume sizes of 0.5, 0.1, 0.07, 0.05 and 0.03 mm. a)
velocity magnitude (in m/s) along the channel midline and b) inset of velocity (in m/s) for the mesh sizes of 0.1,
0.07, 0.05 and 0.03 mm. 48-64 processors were used for running the simulations at the TFR of 1 ml/min and
FRR of 11. Simulations at the mesh size of 0.05 mm took 3 hours and about 3500 iteration to converge, whereas
simulations at the mesh size of 0.03 mm took about 12 hours and 10,000 iteration to converge. Therefore, a
mesh size of 0.05 mm was selected as the best compromise between solution accuracy and computational cost.
The total number of mesh volumes was equal to 8’123’298; (iii) solving for steady state mass
and momentum conservation equations (i.e., Navier-Stokes equations at laminar flow regime,
see Equations 1 & 2 below), and species transport (i.e., advection-diffusion) equations (see
Equations 3 & 4 below), using Ansys® Fluent 17.0 (Ansys Inc., Concord, MA, USA).
∇ ∙ (𝒗) = 0 (Eq. 1)
𝜌𝒗 ∙ ∇𝒗 = −∇𝑃 + 𝜇∇2 𝒗 (Eq. 2)
∇ ∙ (𝜌𝒗𝑀𝑓,𝑖 ) = −∇ ∙ 𝐽 (Eq. 3)
𝐽 = −𝜌𝐷𝑖 ∇𝑀𝑓,𝑖 (Eq. 4)
Where v, ρ, µ and P represent fluid velocity, density, dynamic viscosity and pressure,
respectively. Mf,i is the mass fraction of species i. J is the diffusion flux, and Di is the mass
diffusion coefficient for species i.
To reproduce the experimental conditions, the following boundary conditions were imposed:
(i) mass flow boundary condition at the inlets, (ii) atmospheric pressure at the outlet, and (iii)
no-slip at the channel walls. The experimental values of TFR and FRR were replicated
numerically.
Fluids were assumed incompressible and Newtonian, and the IPA/ethanol-water diffusion
coefficient was set to 1 × 10−9 m2/s [1,2]. The effect of solvents’ mixing on fluid density and
viscosity was taken into consideration in the simulations. The mass fraction of IPA or ethanol
was calculated in a cross sectional plane located in the vicinity of the outlet, and the mixing
index in this specific location was determined for each TFR and FRR investigated.
SI-2b. Numerical analysis of fluids and species transport
CFD simulations were performed in order to gain insights into the flow field and the transport
of chemical species within the reactor (Figure SI-2b(a)), to aid the interpretation of the
experimental findings.
Figure SI-2b: Mixing index calculated over a cross sectional plane in the vicinity of the outlet surface, as shown
in a). Results refer to b) IPA-water at TFR of 1 ml/min and FRRs of 5, 7, 9 and 11; and c) ethanol-water at
TFRs of 1, 3 and 6 ml/min and FRR of 25. Contours of IPA or ethanol mass fraction are reported next to each
data point.
The extent of mixing was quantified via the mixing index [3], which is defined as a measure
of the homogeneity of chemical species (in this case water and ethanol/IPA), over a selected
surface in the reactor. Figure SI-2b shows the mixing index calculated at different fluidic
conditions, which correspond to those employed in the production of SNSs and liposomes, (b
& c respectively). Figure SI-2b(b) demonstrates that increasing the FRR from 5 to 11, while
keeping the TFR constant at 1 ml/min, resulted in increased mixing efficiency between water
and IPA. However, complete mixing was not achieved within the reactor, suggesting that the
production of SNSs continued within the collection tube at these flow regimes. Stratification
of different chemical species due to differences in density could also be observed.
Figure SI-2b(c) shows that increasing the TFR from 1 ml/min to 6 ml/min, at a fixed FRR of
25, resulted in a reduction of the mixing efficiency between ethanol and water. This could be
attributed to interfacial instabilities or vortical flow occurring at the higher flow rates, and
merits further investigations. It should however be noted that variations in the mixing index
are within a 10% range, for the different flow rates investigated.
SI-3. Liposome size distribution
Figure SI-3a: Representative liposome size distribution obtained by DLS, at TFR of 1 ml/min and FRR of 25. All
liposomes have mean hydrodynamic radius between 63 and 615 nm. The mean diameter is of 190 nm, with a
PDI of 0.180.
Bibliography
1. Bottaro, E., Mosayyebi, A., Carugo, D. & Nastruzzi, C. Analysis of the diffusion process by pH
indicator in microfluidic chips for liposome production. Micromachines 8, 1–16 (2017).
2. Hills, E. E., Abraham, M. H., Hersey, A. & Bevan, C. D. Diffusion coefficients in ethanol and in water
at 298K: Linear free energy relationships. Fluid Phase Equilib. 303, 45–55 (2011).
3. Hashmi, A. & Xu, J. On the quantification of mixing in microfluidics. J. Lab. Autom. 19, 488–91
(2014).

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UNIVERSIDAD DE OVIEDO

Departamento de Química Física y Analítica

Programa de Doctorado de Análisis Químico, Bioquímico y Estructural y

NANOVESÍCULAS DE TAMAÑO CONTROLADO PARA

PABLO GARCÍA MANRIQUE

Departament of Physical and Analytical Chemistry

Ph.D. program: Chemical, Biochemical and Structural Analysis and

SIZE CONTROLLED NANOVESICLES FOR

PABLO GARCÍA MANRIQUE

1.- Título de la Tesis

Programa de Doctorado: Análisis químico, bioquímico y estructural, y modelización

RESUMEN (en español)

Las nanovesículas, un tipo particular de coloides orgánicos, está generando un interés

El sexto capítulo presenta el desarrollo de un modelo matemático multivariante de Diseño

En el séptimo capítulo se ha abordado la producción de niosomas de tamaño controlado y

En el octavo capítulo se estudia la mejora de la encapsulación de compuestos hidrófilos

Finalmente, en el noveno capítulo se parte de los modelos obtenidos para niosomas en

RESUMEN (en Inglés)

The sixth chapter includes de development of a mathematical model of Design of

In the eighth chapter the enhacement of hydrophilic compounds encapsulation is studied

SR. PRESIDENTE DE LA COMISIÓN ACADÉMICA DEL PROGRAMA DE DOCTORADO EN

Índice de Figuras ................................................................................................................. 11

Índice de Tablas ................................................................................................................... 19

Lista de Abreviaturas ......................................................................................................... 21

3. Consideraciones básicas ..................................................................................................... 43

3.1. Sistemas vesiculares: generalidades .......................................................................... 45

3.1.1. Moléculas anfifílicas.............................................................................................. 45

3.1.2 Clasificación de los sistemas vesiculares ............................................................ 52

3.1.3. Tipos morfológicos de vesículas ......................................................................... 59

3.2. Métodos de preparación de vesículas ....................................................................... 60

3.2.1. Métodos que implican cambio de polaridad en el medio ............................... 61

3.2.2. Métodos directos ................................................................................................... 62

3.2.3. Métodos de nueva generación............................................................................. 63

3.3. Procesos asociados al uno biotecnológicoy bioanalítico de los sistemas

3.3.1. Encapsulación ........................................................................................................ 67

3.3.3. Purificación ............................................................................................................ 70

3.4.1. Exosomas como modelo de inspiración de vesículas mejoradas ................... 75

3.4.2. Retos tecnológicos asociados a los exosomas y soluciones biomiméticas..... 75

3.5. Referencias bibliográficas............................................................................................ 77

4.1. Dynamic Light Scattering (DLS): tamaño y potencial ζ .......................................... 95

4.2. Microscopía Electrónica de Trasmisión (TEM): morfología................................... 97

4.4. HPLC fase reversa: eficacia de encapsulación ......................................................... 99

4.5. Lector de placas .......................................................................................................... 100

4.6. Analizador para la medida de Western Blot .......................................................... 101

4.7. Equipo de Differential Scanning Calorimetry ........................................................ 101

5. Therapeutic biomaterials based on extracellular vesicles: classification of bio-

5.1. Extracellular vesicles in nanomedicine: possibilities and limitations ................. 105

5.2. Bio-engineered and mimetic EVs for nanomedicine: classification of artificial

5.4. Semi-synthetic exosomes: biotechnological modification of naturally released

5.4.1 Selection of the EVs cellular origin .................................................................... 109

5.4.3 Strategies for biochemical modification ............................................................ 113

5.5.1. Production of artificial EVs by generation of plasma membrane fragments: a

5.5.2 Bottom-Up methodologies: artificial membranes decorated with functional

5.6. Conclusions and future perspectives ...................................................................... 131

5.7. Referencias bibliográficas .......................................................................................... 133

6. Using factorial experimental design to prepare size-tuned nanovesicles ................. 142

6.1. Introduction ................................................................................................................ 143

6.2. Materials and methods .............................................................................................. 146

6.2.1 Materials ................................................................................................................ 146