Journal Pre-proof

Recent advancements in liposome technology

Nina Filipczak, Jiayi Pan, Satya Siva Kishan Yalamarty, Vladimir

P. Torchilin

PII: S0169-409X(20)30068-5
DOI: https://doi.org/10.1016/j.addr.2020.06.022
Reference: ADR 13589

To appear in: Advanced Drug Delivery Reviews

Received date: 11 April 2020

Revised date: 16 June 2020
Accepted date: 21 June 2020

Please cite this article as: N. Filipczak, J. Pan, S.S.K. Yalamarty, et al., Recent
advancements in liposome technology, Advanced Drug Delivery Reviews (2020),
https://doi.org/10.1016/j.addr.2020.06.022

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Recent advancements in liposome technology

Nina Filipczak1,a, Jiayi Pan1,a, Satya Siva Kishan Yalamarty1,a, Vladimir P. Torchilin1,2,*
1
Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston,
MA 02115, USA
2
Department of Oncology, Radiotherapy and Plastic Surgery, I.M. Sechenov First Moscow State
Medical University (Sechenov University), Moscow, Russia
a
-equal contribution of the authors
*-Corresponding author- E-mail address: [email protected]
Postal address: 360 Huntington Ave., 140 The Fenway Building, Room 214, Boston, MA 02115,
USA

Abstract:

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The liposomes have continued to be well-recognized as an important nano-sized drug delivery
system with attractive properties, such a characteristic bilayer structure assembling the cellular

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membrane, easy-to-prepare and high bio-compatibility. Extensive effort has been devoted to the
development of liposome-based drug delivery systems during the past few decades. Many drug
candidates have been encapsulated in liposomes and investigated for reduced toxicity and
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extended duration of therapeutic effect. The liposomal encapsulation of hydrophilic and
hydrophobic small molecule therapeutics as well as other large molecule biologics have been
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established among different academic and industrial research groups. To date, there has been an
increasing number of FDA-approved liposomal-based therapeutics together with more and more
undergoing clinical trials, which involve a wide range of applications in anticancer, antibacterial,
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and antiviral therapies. In order to meet the continuing demand for new drugs in clinics, more
recent advancements have been investigated for optimizing liposomal-based drug delivery
system with more reproducible preparation technique and a broadened application to novel
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modalities, including nucleic acid therapies, CRISPR/Cas9 therapies and immunotherapies. This
review focuses on the recent liposome’ preparation techniques, the excipients of liposomal
formulations used in various novel studies and the routes of administration used to deliver
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liposomes to targeted areas of disease. It aims to update the research in liposomal delivery and
highlights future nanotechnological approaches.
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Keywords: liposomes; recent advancement; cancer immunotherapy; microfluidics; nucleic acid

therapies; routes of administration.
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1. Introduction

The liposome is an ideal and safe form for drug delivery with characteristic lipid bilayers that
resemble the plasma membrane of the cell. They can incorporate a variety of drug candidates
within their hydrophilic and hydrophobic compartments. It is clear that even gasses such as nitric
oxide can be encapsulated into the liposomes {US patent US007976743B2(2011)} to treat
pulmonary hypertension. Many large-scale techniques have been described in the recent past.
They include the age old extrusion method [1, 2], electroformation [3, 4], freeze-drying [5, 6],
hydration or swelling [7, 8] double emulsions (water-in-oil- in-water, or W/O/W) [9, 10] and
bubbling [11]. However, these methods offer limited process control and reproducibility. Thus,
they often result in inconsistency and inefficient use of the materials in clinical translations.

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Figure 1. Liposomal formulations present in clinical trials [12].

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The continuing development of liposome technology has now allowed use of many liposome-
based preparations today and many products to undergo various clinical trials (Figure 1). Since

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the first liposome product Doxil® developed by Gabizon and Barenholz [13], many other anti-
cancer formulations have been successfully formulated including DaunoXome® [14], Depocyt®
[15], Myocet® [16], Mepact® [17], Marqibo® [18] or OnivydeT M [19]. Additionally, liposomal
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application is not limited to anti-cancer therapies. They are also useful tools for the delivery of
anti- fungal agents (Abelcet® [20], Ambisome® [21], Amphotec® [22]), pain relief agents
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(DepoDurT M [23], Exparel® [24]) and oral anti-viral agents (Epaxal® [25], Inflexal® V [26]).
The range of potential applications of liposomes are constantly being expanded. This is
evidenced by the number of liposomal formulations present in the different phases of clinical
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trials. The liposomal formulations present today in clinical trials should continue to bring
measurable benefits to a wide range of patients.
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The properties of liposomes can be controlled via the type of lipids used in the ir preparation. A
recent study used porphyrin-phospholipids in a liposome preparation that can release the cargo
upon near infrared irradiation [27]. Fatty acid elastin like polypeptide was used to prepare a
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temperature triggered liposome, where temperature is used as an external stimulus [28].

Clinically temperature sensitive liposomes have advantages such as widening intravenous
administration of the drug, increased drug accumulation due to their in vivo stability and
improved drug release from the liposomes [28].

2. Recent liposome production approaches

Techniques such as Electroformation and hydration and Extrusion are well established bulk
methods and used by many research groups for different applications. Electroformation and
hydration involves formation of a lipid layer on an electrode Later, an electric field is applied in
the presence of an aqueous solution to peel off the lipids for self-assembly into vesicles [3]. An
electric field is a potential drawback in this method that may degrade proteins sensitive to
electric fields. Another method that is similar to electroformation, hydration, involves formation
of the vesicles without the application of an electric field. Furthermore, extrusion methods
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involve passage of the prepared vesicle solution through a polycarbonate membrane to control
the size and lamellarity of the vesicles [2, 29]. Other recent methods will be discussed in the
upcoming sections.

Bulk methods result vesicles that are not uniform both in their lamellarity and size due to poorly
controlled mechanical and chemical conditions during vesicle formation. The advantages of
these novel production approaches include controlling of both size and lamellarity of the vesicles
formed and also the reproducibility of these novel microfluidic methods are more than the
conventional bulk methods. Furthermore, the one big advantage of the microfluidic systems over
the bulk methods would be their ability to remove the organic phase from the final vesicles
unlike in bulk methods where there is a high possibility of the organic layer being entrapped in
the lipid bilayer. The disadvantages of these methods in comparison to the bulk methods will be
its low volume of the manufacturing process and some of the methods are cumbersome to set it

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up. Usually, these microfluidic approaches are robust however, they work with very low
quantities of solutions.

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2.1. Liposomes on chips

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Microfluidic technologies offer a high degree of control over the process and the manufacture
and overcome problems of reproducibility. Microfluidics refers to the precise control of fluids in
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a geometrically constrained volume, microliter or picolitre amounts. This is usually in sub
millimeter lengths with a low Reynolds number. This reduction of reaction volumes can decrease
the cost of the reagents and thereby increase throughput and analytical performance [30].
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Mechanical and physical characteristics of the membrane could also be altered by the presence of
residual solvents in the bilayer [31-35]. Another significant aspect of the membrane is its
lamellarity or the number of bilayers present. The manufacturing techniques also play a role in
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the size and size distribution of the prepared liposomes. Most applications require the vesicle
population to be monodisperse [36]. Stability, encapsulation efficiency, membrane curvature and
rates of transport across the membrane all depend on size distribution. Any variability in size
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leads to variability of these parameters [36-39]. The key advantage of the method is its ability to
produce monodisperse vesicles. In addition to membrane composition and size, the stability of
the vesicles also depends on factors such as osmolarity, salinity, pH and temperature [40-42].
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Instability of vesicles can cause budding, coalescence, aggregation, or lysis [43]. Tan et al,
reported unilamellar vesicles that are stable for more than 26 days when formed by the emulsion
transfer process [44]. To prepare durable vesicles, a higher amount of process control is needed
which can be achieved with microfluidic systems Factors such as vesicle size, temperature,
osmolarity, pH, salinity and fluid mechanical forces all can be controlled very precisely using the
microfluid systems.

Usability is another factor to be considered when comparing different microfluidic technologies.

Since setting up and operation of the microfluidic tools can be difficult and cumbersome, the
ease of its use often decides what is used. Bulk methods that can be used as microfluidic
approaches in liposome preparation include double emulsion templating [45], pulsed jetting [46],
transient membrane ejection [9] ice droplet hydration [47], droplet emulsion transfer [48], and
hydrodynamic focusing [49]. A hydrodynamic pinch off mechanism a microfluidic method with
excellent encapsulation efficiency with monodisperse unilamellar liposomes were reported [50].
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2.2. Flow focusing

This technique demonstrated by Jahn et al.,[51] consists of a central stream of solution

containing phospholipids in alcohol encompassed by aqueous solution on either side. When the
three flows are merged into a microchannel, where the alcohol diffuses into the aqueous layer,
thus diluting it past a critical concentration by the lipids self-assemble to form liposomes. The
vesicles formed using this method are monodisperse and around 50 – 150 nanometers (nm) in
diameter depending of the flow rates. Lin et al.,[52] described microfluidic tweezing which is
comparable to flow focusing, used to develop membrane tubes adapted for the formation of
vesicles from tubes [53]. The vesicles resulting from this method are polydisperse. The method
of flow focusing can used in a continuous process to prepare vesicles [54, 55]. It allows for the
preparation of organic and aqueous solutions stocked and connected to a microfluidic device to

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allow for continuous manufacturing of vesicles. Although, encapsulation efficiency is not
discussed much by Jahn et al., it should be noted that since the formation of vesicles is dependent

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on diffusive process from organic layer into aqueous layer, it is possible that some of the
material escapes into its surroundings before the vesicles are formed. Also, there is a chance that
liposomes formed from this method could have alcohol entrapped in them by partitioning into

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the membrane and leading to stability issues [56].
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2.3. Pulsed jetting

This method is an interesting technique similar to blowing soap bubbles through a loop. Using a
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micro- nozzle, aqueous solution is shot from small jets into a planar lipid membrane. The
momentum of the aqueous solution carries them further and pinches the lipid membrane thus
forming a vesicle [57]. It was first described by Funakoshi et al [58]. The encapsulation
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efficiency is high with this method. One of the limitations is with encapsulation of large
molecules such as proteins. It is unclear whether they can withstand the shear stress. Another
major problem with this technique is residual solvent left over in the membrane from the
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phospholipid solution [58]. Using Raman spectroscopy Kirchner et al., found that decane used in
the membrane formation can be found in the final vesicles at up to tens of nanometers thick [59].
Two more shortcomings of this method are it is cumbersome to set it up and is very sensitive to
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operating conditions and materials. It is very challenging to properly position the micro-nozzle or
micro-pipette. In this bilayer formation step, the manual pipetting the micro-nozzle had to be
repositioned every time the instrument was used. The parameters such as viscosity, temperature
and membrane composition determine how the membrane is deformed by the fluid jet and are
very critical. Solutions might often have to be changed every time the apparatus is used.

2.4. Double emulsion template

This method involves the evaporation of the oil phase from lipid stabilized water-in-oil- in-water
emulsion using a mixture of organic solvents such as toluene and chloroform [60, 61]. As the oil
phase is removed the external and internal oil- water phases come together to form lipid bilayer
and thus forming a liposome. The high throughput technique was first illustrated by Shum et al
[60]. It is a method that produces vesicles with high encapsulation efficiencies and vesicles that
are monodisperse. The major problem that is encountered in this method is the incomplete
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evaporation of the solvent in the evaporation step. Lorenceau et al., showed that it is feasible to
remove the oil phase without using organic solvents and used this method to produce
polymerosomes that resemble vesicles with membranes consisting of deblock copolymers [62].
Furthermore, Tan et al., used a similar method but used a microfluidic device and oleic acid to
produce the lipid stabilized water-in-oil emulsion and later transferred to an aqueous solution
containing ethanol and water [63]. The oleic acid used dissolves in ethanol and forces the
phospholipids to self-assemble into a lipid bilayer and thus forming unilamellar and
monodisperse vesicles. The parameters such as size a nd stability can be easily controllable. The
vesicles formed using this simple method is shown to last for over 26 days. The same group later
performed the entire process in a microfluidic device [64]. Moreover, the use of ethanol to
remove oleic acid makes it more biocompatible than the previous methods for double emulsion.
Furthermore Deshpande et al., described a technique which is similar to double emulsion
technique known as Octanol assisted liposome assembly (OLA) which forms liposomes that are

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unilamellar, monodisperse and cell sized (Figure 2). It uses 1-octanol as a lipid carrying phase
which is biocompatible which leads to easy solvent extraction process. A double emulsion

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droplet is quickly transformed into intermediate of aqueous volume encircled by lipid bilayer
with side attached solvent pocket. As shown in figure 1 the lipid bilayer completely zips along
entire interface separating the 1-octanol pocket from the assembled liposome. The formation of

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droplet emulsion in a single step using 1-octanol is an efficient and user friendly technique over
other methods [65].
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Figure 2: (a) A schematic representation of octanol-assisted liposome assembly. (b)
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Corresponding fluorescence images of the steps involved. (c) Separation of 1-octanol from the
liposomes using temporal-resolution sequences [65].
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2.5.Droplet emulsion transfer

Pautot et al., described this technique on a macroscale. A water-in-oil emulsion was stabilized by
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phospholipids and transferred the droplets to an aqueous medium. The droplets gather a second
lipid layer as they pass the interface of oil and aqueous phases. The vesicle formed using this
method are unilamellar bilayered [10]. Norieaux et al. used the droplet emulsion method to
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prepare vesicle encapsulated for gene expression [66]. The emulsification process was carried
out by vortexing, resulting in vesicles that are not monodisperse. The emulsification method was
significantly improved with a microfluidic device to form a droplet emulsion. This was then
transferred into a bulk pre-formed oil-water interface [67-69]. Polydispersity issues were
eliminated by using a well-accepted microfluidic droplet formation technique. Although the
emulsion transfer method overcomes several other liposome manufacturing challenges, it was
still not able to eliminate oil matter residues despite using different implementations [70].

2.6.Hydrodynamic focusing

Unlike the other previously mentioned methods that result in large vesicular systems or
microtubules. This hydrodynamic focusing method results in a nanoscale vesicular system with
the potential for clinical application. The methods mentioned earlier cannot be strictly considered
as a microfluid system. Microfluidic hydrodynamic focusing (MHF) consists of a typical
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microfluidic system with a low Reynolds number and diffusion-dominated mass transfer.
Microfluid devices with a 3D angular coaxial geometry or with a cross flow geometry were used
for MHF [71]. Typically, the central channel of the device flowe s through an alcohol solution
containing lipids which are sheathed by coaxial flows of an aqueous phase of either water or
buffers. The lipid solution is hydrodynamically focused into narrow sheets with cross-flow
geometry or a circular cross-section in the case of 3D annular coaxial chips. Parameters such as
the volumetric flow rate ratio (FRR) and total flow rate (TFR) can be used to control the size of
the focused stream [72]. This MHF technique produced monodispersed liposomes, and the
parameters such as size could be controlled by either FRR or TFR. Jahn et al, hypothesized that a
micrometer length scale of sample stream allows for controllable and reproducible che mical and
mechanical conditions across the stream width in comparison to the traditional bulk-phase
preparation techniques [73]. M. Guimarães Sá Correia et al., investigated different FFR and TFR
on the size of the liposomes and observed that lower FFR and TFR caused an increase in the

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particle size. In their study, the particles formed at 20 ml min -1 were smaller than the ones
formed at 6 ml min-1 [74]. This system provides an opportunity to easily scale up to an industrial

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level and can be used to manufacture clinically relevant batches [56].

2.7. Other methods

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Transient membrane ejection was established by Ota et al., using microfluidic technology [48].
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It involves the formation of a lipid bilayer and its disruption to form vesicles. A laser is used to
heat up the aqueous solution to the one side of the membrane. This causes inflation of the
solution resulting in a bubble that breaks off into a liposome. The major advantages of this
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method are complete integration with the microfluidic platform, resulting in monodisperse
unilamellar vesicles. Since it is fully integrated with the microfluidic platform, the size of the
vesicles is tunable. Shortcomings of this method include the depletion of the membrane used to
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form vesicles. This causes a problem where the user needs to pay attention to possible
intermittent oil phases separating batches of liposome samples. Since both the internal and
external aqueous phases are kept separate by the lipid bilayer membrane the encapsulation
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efficiencies are high. Another method described by Sugiura et al [47] was Ice droplet hydration.
It involves stabilization of a monodisperse water- in-oil emulsion by span-80 and stearylamine.
The encapsulation efficiencies are high with this method because both the internal and external
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aqueous phases are separated by the lipid bilayer. While the water droplets are still frozen the oil
phase is evaporated and the aqueous phase is added. This method results in low encapsulation
efficiencies and unilamellar vesicles after extrusion [75] which makes the method cumbersome.

Unlike the bulk methods that require post-processing steps, MHF has the ability to produce
liposomes that are uniform with an exceptional control over size and lamellarity [76].
Continuous production coupled with scaling up by microreactor parallelization is offered by this
microfluidic technology, and is a significant improvement over traditional bulk methods along
with in situ monitoring of the liposomes [77]. Another high-throughput method was mentioned
by E. Kastner et al., which used a staggered herringbone micromixer, which used chaotic
advection as shown in Figure 3. The stretching and folding of fluid streams over the channel
cross sectional area are caused by chaotic advection, effectively, increasing the mass transfer
together with increasing herringbone structure on the channel floor. This method also showed
increased transfection efficiency [78].
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Figure 3: Staggered Herringbone Micromixer (SHM) schematic. Solutions containing lipids and
aqueous buffers are injected separately and chaotic advection is aided by the grooves on the
channel floor [78].

Porphyrin-phospholipid containing liposomes can be used to release the payload upon the
incidence of near- infrared light. Using near- infrared (NIR) light is a form of externally controlled
drug release system where the lipids used in the liposomes consists of devinyl hexyloxyethy-
pyropheophorbide (HPPH)-based monomers that are capable of forming stable lipid bilayers.
Unlike the pyro- lipid monomer used in the earlier studies this HPPH based monomers create a
superior packing assembly due to space filling by its hexyl ether moiety. Carter et al., also made
porphyrin-phospholipid doped liposomes that are thermally stable at even 90 0 C but triggered the

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release of their cargo when irradiated with NIR [27]. These liposomes with porphyrin-
phospholipids show a great promise in the controlled release of cargo based on external stimuli.

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Recently, Wang et al., have used porphyrin-phospholipid liposome for sonodynamic therapy
with low intensity focused ultrasound to release the cargo of the liposome [79]. Figure 4 shows a
schematic of the ultrasound therapy of antineoplastic agent such as doxorubicin via this novel
approach.
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Figure 4: Ultrasound activatable Dox- loaded porphyrin-phospholipid- liposomes for anti- tumor
treatment [79].
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3. Types of payload in liposome-based drug delivery system

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3.1. Liposomes for nucleic acid delivery

Recently, tremendous effort has been made to the development of delivery platforms for
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pharmacological biologics with large molecule weight, especially for nucleic acid molecules.
The pharmacokinetic properties of nucleic acid therapeutics, including plasmid DNA, messenger
RNA, small interfering RNA, microRNA and other types of antisense oligonucleotides, prevents
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efficacious therapeutic outcomes. Their highly negatively charged backbones significantly limit
their cellular interaction and transfection into the target cells. Thus, delivery of nucleic acid
molecules with non- viral vectors has become a well-recognized strategy for successful
administration of nucleic acid therapy (Figure 5). Liposomal nucleic acid delivery systems have
aroused attention to both academic and industrial development efforts.

Figure 5. Schematic illustration of the intracellular trafficking of liposome-based nanoparticles

for delivery of nucleic acid/oligonucleotide molecules to cells. Reproduced with permission from
[80].

Cationic liposomes, consist of cationic lipids, electrostatically bound to anionic nucleic acids,
forming stable lipoplexes that facilitate their uptake into cells through endocytosis. As the core
functional component of the liposome, these cationic lipids neutralize the anionic nucleic acid
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molecules, enabling the delivery across the cellular membrane. The cationic charges also
facilitate the interaction and escape of nucleic acid- loaded liposomes to the cytoplasm. As shown
in Figure 6, diverse novel synthetic cationic lipids have been used in the preparation of
liposomes with improved biocompatibility, nucleic acid loading capacity and transfection
efficiency.

Helper lipids, which are usually neutral in charge, have been routinely used in formulating
cationic liposomes for nucleic acid delivery. One type of these helper lipids is fusogenic lipid,
such as dioleoylphosphatidylethanolamine (DOPE), which has been widely applied in liposomal
formulations to promote the destabilization of the cell bilayer and aid endosomal escape of the
complexed nucleic acid molecules into the cytoplasm. Substitution of DOPE for DOPC
significantly hampered the transfection efficiency of cationic liposomes [81]. Another crucial
helper lipid is cholesterol, which when inserted between the phospholipids increases the rigidity

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of the liposomes. In a new approach, cationic cholesterol, such as 3β-{N-(N′,N′-
dimethylaminoethane) carbamoyl} cholesterol (DC-Chol), have been investigated when

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complexed to nucleic acids in liposome delivery systems instead of using cationic phospholipids
[82, 83]. It should be noted in designing the liposomes for nucleic acid delivery that the
application of cationic lipids can destabilize the anionic cellular membrane, resulting in

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undesired cytotoxicity. Thus, incorporation of PEGylated neutral lipid chains has become a
general approach used to shield the exposed cationic charges in liposomal formulations for
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nucleic acid delivery [84].

3.1.1. siRNA
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As one of the most extensively studied strategies in RNA interference (RNAi) technology,
delivery of siRNA using liposomes has gained much attentions over the past several decades.
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Recent studies of siRNA delivery using liposomes focused on understanding the impact of
excipient and surface properties on intracellular delivery efficiency. Cationic lipids, such as 1,2-
dioleoyl-3-trimethylammonium-propane (DOTAP) [85], 1,2-dioleoyl-3-dimethylammonium-
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propane (DODAP) [86], 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) [87], ,2-di-O-

octadecenyl-3-trimethylammonium propane (DOTMA) [88], have been formulated together with
DOPE/Cholesterol/PEG at various mole ratios by various groups for siRNA delivery (Figure 6).
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Another popular liposomal formulation for siRNA delivery was DC-Chol and DOPE at 1:1 mole
ratio. These DC-Chol/DOPE liposomes efficiently delivered siRNA systemically and resulted in
an efficient SK-OV-3 ovarian tumor growth inhibition [89]. However, on other study suggested
that DC-Chol/DOPE liposomes were more toxic than DOTAP/Chol ones at the same mole ratios
[85].

Figure 6. Chemical structures of recently used cationic lipids for liposome-based nucleic acid
delivery.

Anionic and neutral liposomes may still serve as promising siRNA delivery platforms. A current
phase I clinical trial (NCT01591356) aimed to determine the safety and tolerability of EphA2
siRNA delivered via 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based neutral
liposomes by intravenous administration in patients with advanced/recurrent malignancies.
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Combination with a small molecular weight cationic polymer provides another possibility for
anionic liposomes. For example, DOPE/DPPC/Chol liposomes were used to encapsulated 22
kDa polyethylenimine (PEI)-complexed siRNA as a potential siRNA delivery system to ovarian
tumor [90]. Another study also involved delivery of siRNA to both in vitro and in vivo ovarian
tumor models. However, the egg PC/Chol liposomes complexed siRNA through a surface-
conjugated 600 Da PEI [91].

3.1.2. microRNA

Delivery of microRNA (miR), which has a structure similar to siRNA, has become the focus of
recent investigations due to its crucial effect on disease progression and prognosis. The
successful delivery of miR by synthetic cationic lipids-based liposomes can suppress the target
mRNA translation and regulate the levels of other functional genes, thus exhibiting their

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potential as therapeutics [92]. For example, vascular endothelial growth factor (VEGF-A) was
correlated with metastasis and recurrence of thyroid carcinoma [93]. VEGF-A is also regarded as

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one of the most important factors in controlling the angiogenesis in tumors. Delivery of miR-
34b-5p via DOTAP/Chol/DOPE liposomes to thyroid carcinoma suppressed the expression of
VEGF-A both in vivo and in vitro [94]. Intravenous administration of miR-34b-5b liposomes

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resulted in a 2.5-time decreased VEGF-A expression and reduced tumor size in a xenograft
BHT-101 thyroid carcinoma.
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A similar DOTAP-based liposome formulation was applied to deliver microRNA for treatment
of osteoporosis. Modified with eight repeating sequences of aspartate (D-Asp8), which bind
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preferably to bone resorption surface, DOTAP /Chol/DOPE liposomes targeted t he antagomiR-

148a to osteoclasts. Liposomal delivery of antogomiR-148a demonstrated ~80% downregulation
of miR-148a levels, leading to reduced bone resorption and improved bone microarchitecture
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without liver or kidney toxicity in osteoporotic mice [95, 96].

Another interesting study of microRNA delivery was aimed at the regeneration of eye and brain
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(Figure 7) [97]. Cationic lipids with symmetric di-octadecyl tails and asymmetric
ocatdecyl/oleyl tails were synthesized and formulated into respective cationic liposomes or at a
1:1 mole ratio together with cholesterol. All three liposomes efficiently complex with anti- miR-
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124 and deliver them into live schmidtea mediterranea planarians. These cationic liposome
formulations exhibited an efficient fusion with planarian cell membranes and robust
downregulation of the miR-124 functions[97].

Figure 7. (A) Chemical structures of the synthetic symmetric and asymmetric cationic lipids. (B)
Characterization of liposomes consist of various ratios of symmetric and asymmetric cationic
lipids. (C) Membrane fluidity analysis by the diphenylhexatriene (DPH) fluorescence anisotropy
measurement in three types of liposomes. Error bars indicate S.D. (D) Schematic illustration of
self-assembled liposomes and anti-miRs- loaded lipoplex formation.
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Using a nontraditional liposome formulation, a recent report demonstrated that miRNA-124-
loaded bubble liposomes improved blood flow by promoting the angiogenesis in an experimental
hindlimb ischemia model [98]. The bubble liposome was formulated using 1,2-distearoyl-sn-
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glycero-phosphatidylcholine (DSPC), 1,2-distearoyl-3-dimethylammonium-propane (DSDAP)

and PEG2000 and pressurized with perfluropropane gas in a bath sonicator. These cationic lipid-
shelled nanobubbles are potential carriers for extensive delivery into tissues. Delivery of miR-
126-loaded bubble liposomes via intravascular injection led to the angiogenic factors induction
and blood flow improvement.

3.1.3. mRNA

Messenger RNA (mRNA), encoding various therapeutic proteins, has been investigated for
applications in cancer immunotherapy [99], infectious disease vaccines [100] and protein
replacement therapy [101]. Some liposome formulations for siRNA delivery have also been used
mutually for the delivery of mRNA. A very classic formulation, DOTAP:Chol at 1:1 mole ratio
was used to deliver mRNA encoding survivin- T34A in both in vitro and in vivo C26 colon

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cancer models[102]. First incubated for 10 mins with protamine sulfate solution (1:1, w/w) and
followed by another 15-min incubation with liposomes at room temperature, survivin- T34A

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mRNA was loaded into DOTAP liposome in a liposome:protamine:mRNA ratio of 1:1:1
(w/w/w). Both local and systemic administration of survivin-T34A mRNA- loaded nanoparticles
resulted in a superior antitumor effect with low toxicity.

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In another case, di- linoleylmethyl-4-dimethylaminobutyrate (D-Lin-MC3-DMA)-based cationic
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liposomes were designed for the delivery of mRNA encoding the cystic fibrosis transmembrane
conductance regulator (CFTR), which is an anion channel necessary for chloride efflux from
secretory epithelial cells [103]. The nanoparticles were pipetted onto the nostrils for spontaneous
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inhalation into the lungs of female BALB/c mice. Results suggested that exposure to the CFTR
mRNA- loaded liposomes on two consecutive days restored approximately one third of normal
chloride efflux with effects for at least 2 weeks. Such amount of protein restoration was regarded
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as sufficient to ameliorate disease [104].

It should be noted that the chemical and structural properties of mRNA vary from the ones of
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siRNA/microRNA. The increased molecular length and charge density of mRNA require
significant modification on the liposome delivery formulations developed for siRNA/microRNA
delivery. Complexation of highly charged anionic mRNA with cationic liposomes could result in
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uncontrollable aggregation. Thus, microfluidics technique, PEGylation and adjustment on the

lipid/mRNA ratios are often required to prepare mRNA-loaded liposomes with a uniform size
distribution [105] Kauffman et al. optimized the previously used siRNA liposome formulation
for mRNA delivery using an in vivo C57BL/6 mice model [106]. The liposome formulation for
siRNA delivery consisted of C12-200 cationic lipid: DSPC: Cholesterol: PEG at 50:10:38.5:1.5,
whereas the optimal formulation for mRNA delivery is C12-200 cationic lipid: DSPC:
Cholesterol: PEG at 35:16:46.5:2.5. The mole ratio between cationic lipid and mRNA was also
adjusted from 5:1 to 10:1. Changing of the formulations compositions significantly increased the
potency of the liposome in mRNA delivery.

3.1.4. pDNA

Delivery of plasmid DNA (pDNA) shares some similar challenges, such as achieving a high
loading capacity and avoiding aggregation due to the high anionic charge and high molecular
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weight of pDNA. More importantly, unlike mRNA, pDNA has to reach the nucleus rather than
just the cytoplasm for protein translation. The delivery of pDNA is also accompanied by the risk
of integrating into the genome and resulted in mutation [107].

Previous research demonstrated that inclusion of DOPE in cationic liposomes improved in vivo
intracellular trafficking in target cells [108]. Based on that, most cationic liposomes used for
pDNA delivery contain DOPE. In addition, the preparation method for DNA-complexed
liposomes can have a huge impact on the transfection efficiency and cytotoxicity. Cationic DC-
Chol and DOTAP together with DOPC and DOPE at a ratio of 1:1:1:1 were prepared either by
self-assembling method or with microfluidic mixing. Self-assembled liposomes consist of
multilayers, while microfluidic mixing techniques provide liposomes that possess few lipid
bilayers, larger numbers but contain less DNA molecules individually. As a consequence,
delivery of the same amount of pDNA, liposomes prepared by microfluidic mixing led to a

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higher transfection events but lower transfection intensity per cell [109].

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A similar effect of microfluidic preparation was also reported by another group [110], who
compared the performance of a combination library of cationic and helper lipids in transfection
both in vitro and in vivo. Substituting 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-(1,3)-dioxolane

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(DLin-KC2-DMA) for heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate
(DLin-MC3-DMA) in liposomes resulted in a higher transfection efficiency. In particular, the
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transfection potency order was DLin-KC2-DMA > DLin-MC3-DMA > 1,2-dilinoleyloxy-3-
dimethylaminopropane (DLinDMA) > 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP).
Replacement of saturated PC by unsaturated PC and PE in the liposome formulation of pDNA
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could also enhance the transfection efficiency. The necessity of helper lipids was supported by
the fact that little or no transfection was observed for formulations in the absence of helper lipids.
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Versatile membrane-tethered DNA has also been examined. DNA molecules modified with a
cholesterol anchor through a flexible tetra (ethylene glycol) (TEG) linker at the strands’ 5’
terminus was conjugated [111]. The cholesterol moieties anchored the DNA into the bilayers of
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liposomes made up of DOPE and DOPC. Quantitative analysis showed that cholesterol DNA
packed tightly on the membranes with a maximum density of one strand per 0.02 to 0.04 nm2 ,
depending on the lipid composition. A higher affinity on the liposomal membrane was
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demonstrated in double strand DNA than single strand DNA of the same length, despite doubling
the negative charge. The study is expected to facilitate creation of biomimetic DNA versions of
natural membrane nanopores and cytoskeleton for nanobiotechnology research [111].
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Table 1. Examples of liposomal formulations used in delivery of nucleic acid for various
diseases.

3.2. Liposomes for CRISPR/Cas9 delivery

The clustered regularly- interspaced short palindromic repeats (CRISPR) technology has
revolutionized the field of molecular biology as a novel genomic editing tool [112]. The
CRISPR/Cas9 therapeutics designated to treat a variety of genetic diseases and cancers by
targeting and downregulating genes of interest. However, CRISPR/Cas9 therapeutics have
usually been administered in the form of Cas9 protein with sgRNA, or Ca9 mRNA with sgRNA,
or CRISPR/Cas9 plasmid [113]. Each of them suffered from their poor stability and were
vulnerable to the enzymatic digestion in the serum. The negatively charged plasmid molecules

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have a poor cellular uptake because of the electrostatic repulsion by negatively charged cell
membranes. Nevertheless, liposome-based delivery systems have shown potential in overcoming

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these challenges[114].

Because of the high cellular uptake and efficient endosomal escape, cationic liposomes were

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considered as potential delivery platform for CRISPR/Cas9. For example, a recent study reported
DOTAP-based liposome delivery of Cas9 and sgRNA plasmid achieved 39% gene-editing
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efficiency in knocking out a GFP reporter in the HEK293 cell line [115]. Another study used
DOTAP-based liposomes to deliver CRISPR/Cas9 plasmid for treatment of
mucopolysaccharidosis type I (MPS I). The liposomes were complexed with CRISPR/Cas9
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plasmid using microfluidic mixing. Genetic editing performance was evaluated in both an in
vitro human fibroblast model and in an in vivo murine model [116]. In both studies, PEGylated
DOTAP- liposomes provided serum stability, higher endosomal escape and transfection
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efficiency, supporting promise for therapeutic application.

A DOTAP-liposome-templated hydrogel nanoparticle (LHNP) was developed for targeted

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delivery of CRISPR/Cas9 to inhibit the polo- like kinase 1 (PLK1) gene in tumors [117]. The
core of LHNP was a hydrogel formed by crosslinking cyclodextrin- polyethylenimine (PEI, 25
kDa) with adamantine-PEI (25 kDa) to encapsulate Cas9 protein. The shell consisting of DOTAP
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efficiently delivered the sgRNA. The LHNP was further modified with internalizing RGD (Arg-
Gly-Asp) for improved accumulation and tumor growth inhibition of xenograft U87 tumors in
mice. R8-dGR is another cancer targeting moiety that binds to integrin αvβ3 and neuropilin-1.
This multifunctional peptide has been attached on the surface of DOTAP liposome for specific
delivery of CRISPR/Cas9 plasmid targeting on hypoxia- inducible factor-1 alpha (HIF-1α) to
cancer cells [118]. The inhibition of HIF-1α was expected to prolong the survival time by
suppressing metastasis. These R8-dGR- modified cationic liposomes were co- loaded with both
paclitaxel and CRISPR/Cas9 plasmid to achieve a combinational therapeutic efficacy.

A high genome editing efficiency in vitro often cannot be translated into a similar editing
efficiency in vivo [119]. The challenges and obstacles of CRISPR/Cas9- mediated genome editing
via non- viral delivery systems were summarized by Li et al. [113]. First, the characteristic
properties of CRISPR/Cas9 plasmids or proteins that prone to the recognition and clearance by
the reticuloendothelial system (RES) are obstacles in terms of achieving the efficient
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encapsulation and stable blood circulation [120]. The second obstacle is triggering the host
immune response by the elements of CRISPR/Cas9 system, which was initially derived from
bacteria. Though inclusion of PEG can help escape from the RES system and reduce the immune
response, some recent reports indicated that specific antibodies were generated following the
administration of PEGylated liposomes [121, 122]. These formulation-specific antibodies created
uncertainties on the efficacy and safety of PEGylated nanoparticle therapeutics [123]. Another
obstacle is the insufficient accumulation of CRISPR/Cas9- loaded non-viral vectors in the
targeted tissues. Cationic liposomes are well-recognized as triggers for higher cellular
association in vitro and intracellular endosomal escape, which are critical aspects in terms of
CRISPR/Cas9 function. Some reports suggested that the cationic charges could result in poor
tumor penetration, non-specific accumulation and increased macrophage uptake [124]. By
contrast, neutral liposomes lead to a deeper tissue penetration, but sacrifice the cellular uptake.
For example, neutral liposome composed of lecithin, cholesterol and 1,2-dioleoyl-sn-glycerol-3-

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((N-(5-amino-1-carboxylpentyl)iminodiacetic acid)succinyl)-(nickel salt) (DOGS-NTA-Ni) was
used to delivery recombinant Cas9 protein and sgRNA complex [125]. To increase the

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encapsulation efficiency, positively charged PEI 2 kDa was utilized as a fusion material to
compensate for the negative charge of the Cas9-sgRNA complex. The sgRNA sequence
specifically targeted dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-

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like peptide 1 in liver for type II diabetes therapy. Blood analysis showed normal liver and
kidney function probably because of lessened drug dispensation. Overall, both cationic and
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neutral liposomes showed capability as promising delivery platforms, but still require further
development. Anionic liposomes were not preferred formulations as the loss of transfection
efficiency.
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3.3. Gas-filled liposomes

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Nitric oxide (NO) besides its vasodilatory effects has anti-atherogenic effects due to its anti-
inflammatory effects, lowered formation of plaque plus inhibition of platelet aggregation and
adhesion. Systemic delivery of NO may have side effects due to its off- target effects. Huang et
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al., filed a US patent to further explore the possibility of preparing NO-containing liposomes.
Gas containing liposomes were prepared by combining the liposomes with gas under pressure
and later freeze- thawing the gas-liposome dispersion [126]. In some cases, a second gas was
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also encapsulated to regulate the release rate of NO from the liposome. The amount of gas
encapsulated is proportional to the NO pressure. According to Henry’s law gas uptake by the
liposomes is proportional to the amount present in the solution. The freezing step in the
preparation has two purposes: to increase the local concentration of dissolved gas and formation
of small pockets of bulk gas phase. Liposomes that can encapsulate both NO and CO are
described in this invention and are actively targeted due to their diverse bioactivities of both the
gases. Later ultrasound imaging was used to track the particles after in vivo administration.

3.4. Liposome for immunotherapy

Immunotherapy is a potent tool used to fight cancer. It offers a broad spectrum of possibilities.
To make it effective, the appropriate antigen has to be delivered to antigen presenting cells and
released at appropriate locations. Both conditions must be met to induce cross-presentation and
activation of immunocompetent cells. Both conditions must be met to induce cross-presentation
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and make immunocompetent cells. Liposomes are good candidates to use as antigen-presenting
carriers since they are easy to functionalize. [127].

Surface alteration of liposomes with fusogenic or pH-sensitive moieties can deliver antigen to
the cytoplasm, followed by cross-presentation of antigen via a "cytosolic pathway". On the other
hand, antigen escape in early endosomes induces cross-presentation via the "vacuolar pathway",
after recognition of surface receptors in antigen-presenting cells. [128].

Figure 8. Scheme of liposome-based antigen-presenting carriers for immunotherapy of cancers.

[129].

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To achieve better immunity- inducing properties of the liposomes, adjuvants such as synthetic

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cationic lipids, Toll- like receptor agonists, or bioactive polysaccharides are incorporated.
Systems that can reverse immunosuppression in a tumor microenvironment are also useful tools
to increase the antitumor resistance of antigen-presenting delivery systems. Moreover,

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comprehension of the immunity- inducing action and the molecular base of tumor
immunosuppressive environments is a signature characteristic for the successful design of
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liposome-based antigen delivery systems.

3.4.1. Stimulation of interferon genes

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Immune systems have the ability to eliminate cancer cells from the body. The spontaneous
immune response to cancer contributes to the checkout of neoplasm growth in cancer patients
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since the permeation of pre-existing T cells in the tumor microenvironment is effective.

Infiltration of pre-existing T cells into the tumor microenvironment is favorable for patients
undergoing anti-tumor immunotherapy using specific antibodies for T cell-associated protein 4
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(CTLA-4) and programmed cell death protein 1 (PD-1) [130]. Therefore, it seems likely that
launching of endogenous immune responses boosts cancer therapy. Though the host's
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mechanism for initiating spontaneous cancer detection remains largely unknown, there is an
eventual answer to this query. The main mechanism of innate immune detection of cancerous
cells occurs via the interferon gene pathway host simulator (STING) [131]. Researchers from
Harvard University recently showed that liposomal form of STING agonists (cGMP) can boost
anti-tumor resistance. They used positively charged liposomes to prove their anti-tumor power
against lung's metastases of melanoma. Liposomes have been used to transfer STING ligands
and vaccines to the lymph nodes. Those two components joined together showed that
PEGylated liposomes containing cGMP were able to reduce the size of melanoma tumors [132,
133].

STING technology was also utilized as inhalation of aerosolized liposomal-cGAMP to control

metastases in lungs in mice with lung metastases [134].
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Figure 9. Scheme of STING signal path activation. In the first stage, IFN type I and
proinflammatory cytokines are produced, which then activate natural killer cells. Their activation
leads to the penetration of NK cells into cancer cells and, as a result, their death. [132].

STING senses cytosolic, cyclic dinucleotide (2’3’-cGAMP), produced by the cyclic-GMP-AMP

(cGAMP) synthase (cGAS), which leads to further activation of tank binding kinase 1 (TBK1)-
interferon regulator factor 3 (IRF3) pathway. As a result of path activation, type I interferons
(IFNs) are produced by activated antigen presenting cells (APCs). The final result of this
activation is the priming of CD8+ T cells against tumor-specific antigens [130, 131]. Based on
this strategy, the first commercial DNA immune stimulator product, that contains bacterial
plasmid DNA rich in unmethylated CpG motifs enclosed in a cationic lipid coating, ZelNate®
was introduced as the veterinary product in 2015. ZelNate® is used as a support in the therapy of
bovine respiratory disease caused by Mannheimia haemolytica [135]. ZEL product uses cGAS

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and STING to activate the IRF3 pathway. ZEL liposomes consist of cationic DOTIM and
cholesterol, and pMB75.6 plasmid DNA entrapped inside. It is well known that when plasmid

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DNA is complexed with, or when encapsulated in positively charged lipid particles that involve
endocytosis, residence in endosomes, and escape into the cytosol in the endosomal compartment
which leads ZEL to get access to a cytosolic pool of DNA, followed by activation of specific
pathways [136].
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3.4.2. Cancer vaccines
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Therapeutic cancer vaccines are designed to stimulate the body's immune activity to fight cancer
cells. An endogenous anti-tumor immune response is frequently ineffective. In addition, specific
activation of antigen-presenting cells (APCs) (e.g. dendritic cells) by oncological vaccines is
technically difficult. Activated APCs present antigen to effector cells, (e.g. T cells), which
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identify and kill tumor cells. Currently, the only oncological vaccine approved by the US Food
and Drug Administration (FDA) is Sipuleucel-T against prostate cancer [137].
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Table 2. Examples of liposomal formulation used as cancer vaccine delivery system.

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However, that vaccine requires separation of the dendritic cells and their stimulation in vitro
before re-administration. A different method of dendritic cell activation uses nanoparticles with
tumor antigen labeled with specific antibodies for these cells. Promising results of oncological
vaccines from the German Jan Gutenberg University in Mainz were published in the Nature
magazine. They have developed a simple way to activate dendritic cells without having to isolate
them from the body or label nanoparticles with antibodies. Liposomes composed of the broadly
used lipids DOTMA and DOPE of specific particle size (200–400 nm) containing various RNA
fragments encoding the tumor antigen and stimulated the body's immune response was used.
After intravenous administration, these liposomes, due to a slightly negative charge, had a
superior and distinct affinity for the spleen and other lymph nodes in which APC cells are found.
Vaccine anti-tumor efficacy tests were performed on murine models of lung cancer. As a result
of vaccination, the mice developed long- lasting activation of the specific immune response
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against tumor antigens as well as reduction of the tumor volume. These vaccines have also
passed pharmacological safety tests on murine and macaque models [138].

4. Analytical development in liposome

4.1. Characterization methods for size and zeta potential

Characterization of liposomes is important since physical and chemical parameters need to be

monitored to make sure that the preparation of liposomes is reproducible and has the desired
function. Size is a parameter that is very important to monitor since it can affect the functions of
liposomes [139]. The physical stability of the liposomes can be assessed by their size
distribution and polydispersity. A variety of methods including electron microscopy,
fluorescence microscopy, atomic force microscopy, field flow fractionation, dynamic light

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scattering, nanoparticle tracking analysis, flow cytometry, size exclusion chromatography,
scanning ion occlusion sensing, centrifugal sedimentation and differential scanning calorimetry

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can be used for the size analysis of liposomes. Relatively new technologies for the size and zeta
measurement are discussed.

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Scanning electron microscopy is a common technique used for the size measurement of
liposomes. These methods can visualize the liposomes and also determine their lamellarity and
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morphology. A special variant of transmission electron microscopy used is cryo-transmission
electron microscopy (Cryo- TEM) where the liposomes are visualized in a frozen state. The grid
of liposomes is put into liquid nitrogen. This rapid freezing does not cause crystal formation and
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thereby preserves the liposomes in an intact form. Each liposome can be visualized individually
which gives detailed information about the size of the liposomes. However, this method cannot
measure the hydrodynamic radii of the liposomes which is essential for size determination of
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PEGylated liposomes [140]. Moreover, adding to the shortcomings this method can be regarded
as a tedious one. Another method is freeze-fracture transmission electron microscopy [141]. The
sample on the carbon film grid is shock-frozen in melting nitrogen. Later, the sample is inserted
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into a cryo transfer holder for imaging. The advantage of this method is that no structural
disruption of the sample is observed [139]. A drawback of this method is that it needs large
amount of sample [40].
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Field Flow Fractionation (FFF) is a gentle method of separating macromolecules and

nanoparticles such as liposomes [142]. The separation of the sample occurs in a narrow ribbon-
like channel. External electric fields are applied perpendicularly to the carrier flow by which
separation occurs. Carrier flow is from the inlet of the channel to the detector. A parabolic flow
profile is created in the channel. The perpendicular flow affects the liposomes and forces them to
move towards the membrane by their Brownian motion. Due to their higher diffusion coefficient
and greater distance from the wall smaller liposomes elute first. FFF can be used in a sample
with size distribution of highly polydisperse solutions and the retention is predictable. Some of
the drawbacks include, complexity of the channel construction, measurement of spherical
particles and background correction of the UV detector needed [142].

The surface charge of the liposomes is calculated by measuring the zeta potential of the liposome
preparation. The charge on the liposome is either positive, negative or neutral depending on the
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composition of the liposome. This zeta potential makes the liposomal suspension stable by
preventing it from aggregation. Particles carrying charge repel each other, so that liposomes with
a zeta potential of higher than 30 mV and less than -30 mV are considered stable. One of the
techniques besides light scattering methods is the laser doppler velocimetry used for the rapid
detection of the charge on the liposome. In light scattering methods, the electric filed applied
causes movement of the liposomes due to their charge and results in a doppler shift in frequency
detected by the fluctuations in the detected light.

4.2. Quantification of liposomal drug release in plasma

Liposome formulations of a variety of drug candidates have been widely developed for treatment
of various diseases in the clinics. The significantly a ltered drug pharmacokinetics and
biodistribution make liposomal drugs different entities from the non-encapsulated free drugs

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[143]. After intravenous administration into plasma, drug molecules-delivered by liposomes can
be classified into three distinct forms: liposome-entrapped drug, protein-bound drug and free

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drug. Disposition of these three forms is closely related to the pharmacokinetics, biodistribution,
toxicity and therapeutic effects. In 2018, The Food and Drug Administration of United States
(FDA) released the Liposome Drug Products Guidance for Industry, specifically emphasizing

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that pharmacokinetic measures or parameters should include both the free and the total drug in
plasma, as appropriate. Hence, pharmacokinetic stud ies that analyze only total drug plasma
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concentration for a conventional dosage form is not suitable for liposomal drug evaluation. It is
essential for analytical development to track the pharmacokinetic profiles of both liposome-
associated and non-liposomal drugs after administration in plasma [144].
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The isolation of free and liposomal drugs in plasma has been conducted through ultrafiltration
[145], ion-exchange chromatography [146], size-exclusion chromatography [147], solid-phase
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extraction (SPE) [148] and capillary electrophoresis [149]. However, all these methods have
obvious limitations. Ion-exchange chromatography showed poor reproducibility with low
recovery. Gel chromatography involved high sample dilution and slow separation. Ultrafiltration
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was limited by the drug adsorption on devices and SPE had potential drug release problems
during the separation procedures. A satisfactory separation method should be a fast, simple, and
done with high-throughput analysis to avoid the drug leakage during the process and to assure a
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high recovery efficiency. Of note, protein-bound drug should ideally be considered as part of free
drug, but these fractions are even more challenging to separate. Protein binding alters the
properties of the drug, further limiting the application of separation methods, including
ultrafiltration, ion-exchange chromatography and SPE.

Some recent studies have optimized the separation procedures, mainly by using SPE for
separation and LC-MS/MS for detection. Xie et al. developed a modified SPE methods for
determination the concentration of non- liposomal and liposomal doxorubicin concentration in
plasma and compared the result with an ultrafiltration technique [144]. By using Oasis®
hydrophilic- lipophilic-balanced (HLB) reverse-phase sorbents and pre-coating the sorbent with
plasma, they demonstrated effective separation with good specificity and high sensitivity. The
free doxorubicin concentration in plasma was quantified by LC-MS/MS after separation. The
LLOQ of the separation method was 3.13 ng/ml for non- liposomal doxorubicin and 0.156 µg/ml
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for liposomal doxorubicin. This HLB-sorbent-based SPE method was also successfully applied
to characterize the pharmacokinetic profiles of Doxil® after i.v. administration in canine.

In another study, a similar SPE method was followed by LC-MS/MS detection and applied to
analyze the free and liposomal Amphotericin B (AMB) concentration in rat plasma [150]. Free
AMB has a higher lipophilicity than doxorubicin, and thus, leads to a higher protein-binding. In
this SPE method, rat plasma containing liposomal AMB was pretreated with 0.1% NH4 OH to
reduce the drug-protein interaction. Using the HLB reverse phase cartilage, the LLOQ of this
method was reported as 0.2 µg/ml for free AMB and 0.5 µg/ml for liposomal AMB. All
separation efficiency data were accurate within 5% in rat plasma spiked with 0.5-80 µg/ml of
liposomal AMB concentration.

In summary, recent studies developed an efficient and easy-to-use SPE method for separation of

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free drug and liposome-encapsulated drug in plasma. Successful separation requires pretreatment
of column with plasma to minimize the sorbent adsorption. Depending on the lipophilicity and

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protein-binding affinity of target drug molecules, it is necessary to take the amount of protein-
bound drug into consideration. The plasma samples can be incubated with a certain amount of
NH4 OH or formic acid to reduce the percentage of protein-bound drug without interference in

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the LC-MS/MS analysis. Additionally, to overcome the liposomal breakdown during SPE
separation resulting from the low osmotic pressure encountered in the cartridges, it is necessary
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to precondition plasma samples with 5% glucose and pretreat the SPE sorbent with a 5% glucose
solution.
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5. Administration routes of liposome

The actual problem for many treatments is not the lack of an effective drug, but the difficulty of
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delivering it to the pathological target. The first reason is that drug molecules are often unable to
overcome biological barriers, such as the blood-brain barrier, skin, or mucous membranes in the
small intestine, nose and mouth. These barriers are natural obstacles that screen out foreign
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substances before entering the body, that particularly impact most biologics, including
recombinant proteins, antibodies and gene therapies. The second reason is the host-triggered
immune response the body uses to ride itself of therapeutics. Therefore, the most effective
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delivery system assist protecting drug candidates from the immune response and enabling their
penetration across these physical barriers.

5.1. Oral administration

Liposomes have been used for parenteral drug delivery since they were discovered. However,
oral delivery of liposomes still has problems due to their low stability, weak penetration
properties and difficulties in bulk production. Tod ay many of these problems have been solved
by alteration of the bilayer composition and attachment of polymers or ligands to the surface.
This improves both the durability and permeability of liposomes, making them a useful tool for
oral administration [151]. Conventional liposomes are not resistant to the joint detrimental
effects of gastric acid, gall, and lipases in the gastrointestinal tract, which reduce the
concentrations of intact liposomes and promote cargo release. Conventional liposomes are
characterized by poor permeability across enteric epithelia because of their big size and the
presence of diverse barriers. Two main pathways of liposome-based oral drug delivery were
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proposed. In the first pathway, the drug could be released in the gastrointestinal lumen or via
conversion of liposomes into mixed micelles and followed by penetration of drug trough the
intestinal epithelia [151]. The second way is an uptake of liposomes by M cells staying in the
follicle-associated epithelium (FAE) of Peyer's patches [152].

Several strategies have been used to improve liposomes as an oral drug delivery system.
Addition of sterylamine to the bilayer composition to change the charge on liposomes, and so
make the liposomes able to repress the digestion of cargo by trypsin, was shown in an example
with insulin [153]. Another commonly used approach has been to stabilize liposomes by coating
their surface with polysaccharides e.g. pectin [154], chitosan [155], O-palmitoylpullutan [156] or
polymers like Eudragit S 100 [157]. The use of chitosan, it’s derivatives or other polysaccharides
also enhances mucoadhesion of liposomes. This feature prolonged liposomes’ GUT residence,
allowing longer contact of liposomes with intestinal epithelia subsequently enhanced oral

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absorption of liposomes or only the cargo [158]. The glycosylation of lipids and proteins present
in cell membranes of the digestive tract was utilized as a strategy to overbear the low

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permeability of traditional liposomes. This approach uses the lectin’s affinity for glycans. [159].
Lectin modified liposomes can be also up taken by M cells thanks to particular glycosylation
patterns presented in these cells. An advantages of this approach is that M cells are the least of

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all secured cells by mucus, which make them more accessible for liposomes[160]. A novel
approach that combines pH-responsive gold nanoparticles with liposomes has been developed
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for the stomachic delivery of antimicrobial agents. Chitosan- modified gold nanoparticles were
linked with the external surface of anionic liposomes. This modification ensures liposome
stability at gastric pH. However, at inert pH, the gold nanoparticles separate from liposomes and
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as a result the doxycycline-charged liposomes rapidly fuse with bacterial membranes. Compared
to free doxycycline liposomes achieve a more effective antibacterial effect. [161].
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The research focused on liposomal oral delivery has grown, but substantial breakthroughs are
still desired to evolve and commerce these products for clinical exert. The main problem in the
successful formulation of oral liposomes consist of a weak understanding of the mechanisms
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behind liposomes absorption in the gastrointestinal tract.

5.2. Transdermal administration

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The three main barriers to local and transdermal delivery are the skin itself (cuticle and dermis),
skin surface, and hypodermic tissue with its systemic circulation. Disinfectants, insect repellents
and cosmetics are applied mainly to the surface of the skin. Aiming at the different stratum of the
skin remains important when the illness condition is located in the organ itself, e.g. tumors,
inflammatory disorders, and microbial infections. The local way provides direct access to targets
situated within several microns below the skin surface, providing prolonged effects and higher
effectiveness, reducing the therapeutic dose and risk of side effects compared to parenteral
administration. Transdermal administration, in which the subcutaneous tissue or systemic
circulation is the main focus, is an alternative to systemic and oral routes of administration. The
transdermal route offers numerous advantages over the oral route, including avoiding
degradation by a gastrointestinal environment, avoiding first-pass systemic metabolism, and high
patient compliance. However, transdermal delivery of hydrophilic and high molecular weight
drugs has its limits. The first is permeability through the stratum corneum (SC), consisting
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largely of lipids and that represents a penetration barrier for hydrophilic molecules. The second
is the low skin partition coefficient (<< 1), which leads to non-absorption of drugs by the skin
[162, 163]. Nanotechnology has been used to deliver substances through the skin, by facilitating
the penetration of the drug, enabling topical administration, and protecting the drug from
degradation. Liposomes were first used as transdermal carriers since 1980. Traditional liposomes
usually remain on the surface of the skin because of their poor penetration abilities. [164, 165].
Deformable liposomes, also known as elastic or transferosomes, were introduced in 1992 [166].
Their self-adaptability and high flexibility make this type of liposomes able to penetrate the
stratum corneum barrier in comparison to other similar in size nanoparticles.

Elastic liposomes have been used as topical vehicles for zinc phthalocyanine (ZnPc) and the
nitrosyl ruthenium complex {Ru (NH.NHq)(tpy)NO}3+ (RuNO) as a photosensitizer. These
liposomes were made up from the phospholipid, dioleylphosphocholine (DOPC), and Tween 20

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as an edge activator. [167]. Transferosomes have been used to deliver nonsteroidal anti-
inflammatory drugs (NSAIDs) like diclofenac and ketoprofen (Transfenac® and Diractin®,

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respectively) [168]. Other anti- inflammatory drugs delivered by elastic liposomes include
piroxicam [169] and lornoxicam [170]. Ultra-deformable liposomes are also used to deliver acne
treatments e.g. transferosomes composed of phosphatidylcholine and Tween 80 with tretinoin

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[171]. Topical delivery of anti- infective drugs also uses elastic liposomes advantage. Neomycin
sulfate[172] , clotrimazole [173], metronidazole [174] can be efficiently entrapped in this kind of
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liposomes and release in target place. Most of the se drugs can be administered using
transferosomes to increase the efficacy of the drug for localized action e.g. insulin [175],
curcumin [176] , methotrexate [177], loratadine [178]. The usage of elastic liposomes helps to
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solve drug-associated physicochemical and clinical problems such as low aqueous solubility,
skin irritation, gastrointestinal after effects namely vomiting, nausea, and diarrhea, and
accumulation of the drug in bone marrow when orally administered. [179]. Although ultra-
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deformable liposomes can solve many topical drug delivery problems, challenges that need to be
addressed include storage stability of the liposomes, their stability after administration to the
skin, maintenance of their intact structure inside deeper layers of the skin, and the development
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of appropriate lipid compositions to meet the need for both flexibility and stability.

5.3. Delivery across the blood brain barrier

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The brain is a very sensitive and delicate organ, which is why it is separated from the
bloodstream by a dynamic physical and biological barrier that plays a key role in regulating its
internal environment. The blood-brain barrier ( BBB) protects nerve cells against a variety of
harmful factors (viruses, bacteria, toxins), but also limits the transport of most xenobiotics from
the blood to the brain, including anti-cancer drugs, antibiotics and many other products used to
treat neurodegenerative diseases [180]. Due to its specific structure and properties, the barrier
prevents 98% of small and 100% of large particles from reaching the brain [181]. The blood-
brain barrier consists of a single layer of polarized squamous cells lying on the inner surfaces of
the capillaries in the brain built from glycosaminoglycans linked to proteins and cell membrane
lipids. The barrier contains many membrane receptors, enzymes and specific proteins that enable
the transport of glucose as well as amino acids from the blood to the brain. Tight connections
between cells constitute a additional barrier that prevents the diffusion of ions and molecules into
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the intercellular space. However, thanks to the pores, the selective passage of many compounds
is enabled.

Figure 10. The different mechanisms that can be used by the nanoparticles to cross the blood–
brain barrier. (A) transcellular lipophilic pathway. (B) transport proteins. (C) receptor- mediated
transcytosis and (D) adsorptive transcytosis [182]

5.3.1. Glioma treatment with liposomal preparations

Glioblastoma multiforme (GMB), also called an "octopus tumor" because of the way cancer cells
spread to adjacent tissue, is inoperable, resistant to therapy, and always ends with the death of
the patient, usually within 15 months of onset. Each year, glioblastoma multiforme kills around

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15 000 people only inside US. One of the main obstacles to therapy is the blood-brain barrier, the
network of blood vessels that allows nutrients to reach the brain, but blocks access to other

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substances. Thus, there are needs for ways to efficiently transport drugs across this barrier. In
this circumstance, liposomes provide an ideal and safe form of drug-carrier system due to their
lipid bilayer which resembles the plasma membrane of the cell. Besides their characteristic

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bilayer, it is also feasible to incorporate both hydrophilic and hydrophobic substances within
them.
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Facilitation of BBB penetration is possible by injection of layered liposomes containing
epirubicin in the aqueous layer, tamoxifen in the lipid bilayer and transferrin attached to the
polyethylene glycol (PEG) chain. The selection of liposome components was associated with the
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fact that transferrin receptors are located o n the BBB and on the surface of glioma cells, and the
tamoxifen acts on P-glycoproteins belonging to multi-drug resistance proteins. The amount of
cytostatics that penetrated the BBB from this multi-component liposome (52.2%) was twice as
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high compared to free epirubicin. Intravenous administration of layered liposomes with 92%
cytostatics, 34 mg tamoxifen / mmol lipids, 123 mg transferrin / mmol lipids at a dose of 5 mg /
kg induced an increase in median survival up to day 23 in mice model, while for epirubicin free
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it was 15 days [183]. Similar approach to enhance BBB penetration utilizes liposomes with
CB5005 peptide, used as a cell membrane penetration enhancer and NF-κB inhibitor. The PEG-
peptide conjugate was used to make doxorubicin bearing PEGylated liposomes. In vivo research
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proved that these liposomes can cross BBB and reached tumor cells [184].

The use of hyperthermia as a supporting glioma treatment when used concurrently with systemic
chemotherapy is now also a promising strategy. There is evidence of increased antitumor activity
of several chemotherapeutic agents with suitable heat exposure (40–44 °C). Hyperthermia for
clinical purposes can be induced by an alternating magnetic field with a combination of magnetic
nanoparticles, such as thermosensitive magnetoliposomes that contain doxorubicin and iron
oxide in the form of magnetic nanoparticles. Magnetoliposomes can be heated to 43 °C (the
phase transition temperature of the lipid used) within a few minutes. During this process
doxorubicin is sustainably released. In vivo results showed that magnetic drug targeting has a
strong anti- glioma effect associated with complete remission of the tumor. Such a drug delivery
strategy could provide clinical benefits compared to currently available methods [185].

5.3.2. Liposomal drugs to treat the Alzheimer’s disease

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The primary problem in the treatment of Alzheimer's disease is the difficulty of delivering active
substances across the blood-brain barrier. Many strategies have been developed. One is the direct
administration of drugs to the brain. However, only nanotec hnology enabled the intensive
development of techniques for targeted delivery of therapies using carriers - liposomes or
polymer nanoparticles [186].

One therapeutic strategy in Alzheimer's disease has been the reduction of the amount of amyloid
β (Aβ) in peripheral blood. This action prevents the deposition of Aβ in the small arteries and
capillaries of the brain, which prevents the formation of senile plaques. The effect of liposomes
containing phosphatidic acid (phosphatidic acid liposomes, PA-LIP) and cardiolipin (cardiolipin
liposomes, CL-LIP) on the level of β-amyloid in plasma, blood and brain was studied. It has
been found that structures containing PA or CL affect the distribution of Aβ can directly or

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indirectly modify brain metabolism. Both types of liposomes enabled the reduction of β-amyloid
levels in peripheral blood. This was associated with the rapid interaction of phosphatidic acid
with Aβ prior to its removal by liver and spleen macrophages. Both phosphatidic acid and

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released cardiolipin bound to amyloid β. The resulting junction was removed from the brain to
peripheral blood. The obtained therapeutic effect is an important example of the use of liposomes
for therapy of Alzheimer's disease [187].
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Another promising strategy for treating Alzheimer's disease was the development of
multifunctional liposomes (mApoE-PA-LIP) in tests on mice. The study aimed to facilitate
passage through the blood-brain barrier and to impede the formations and increase of breakdown
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of large Aβ aggregates into smaller fragments. The protein receptors (mApoE) present on the
surface of liposomes bound to specific sites on the surface of brain endothelial cells, which
enabled the carriers to cross the blood-brain barrier. MApoE-PA-LIP liposomes reached the
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brain intact and accumulated in the hippocampus. They reduced the of amyloid plaques in the
brain by binding phosphatidic acid to Aβ and prevented its aggregation in the form of senile
plaques. However, treatment with mApoE-PA-LIP did not eliminate the causes of Aβ
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overproduction, but slowed the neurodegeneration process in transgenic mice [188].

BBB prevents the entry of most toxins as well as drug molecules. However, special arrangements
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prevalent in blood-brain barrier can enhance the transport of some necessary substances like
glucose, required for the proper brain functioning. Such substances cross the BBB via a receptor-
mediated way. This feature can be utilized for the successful delivery of the drug into the brain.
Liposomes composed of cholesterol and soybean phospholipid, loaded with coumarin 6 were
modified with glucose by incorporation to the lipid bilayer varied chain lengths of PEG- glucose
conjugates. The results show that developed liposomes with a medium-chain PEG were found in
higher concentrations in the brain compared to peripheral organs. On the other hand, liposomes
prepared with short-chain PEG show higher accumulation in the peripheral organs than the brain
itself. In this case, glucose was not represented on the surface of the liposome, because
PEGchain was not long enough to expose it, and in the consequence made the liposomes unable
to cross the BBB via the glucose transporter. Longer chain PEG shows some elasticity leading to
a fold of PEG on the surface of liposomes and reduces the ability of liposomes to interact with
glucose receptors and consequently to cross the BBB [189]. This showed that not only a proper
choice of receptor is important but also its’ exposure on the surface of the drug carrier. Various
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other attempts targeting BBB have successfully used modified liposomes. One is G-
Technology® a well-established, patented, FDA approved technology proved to enhance brain
targeting. It includes PEGylation of liposomes combined with glutathione that crosses the BBB
via sodium-dependent transporter [190].

The intranasal route of administration offers an alternate way to deliver a drug directly to the
brain bypassing BBB. This strategy, avoids the first-pass metabolism and enzymatic degradation
of the liposomes, has a larger absorption area for the drug and directly delivers the drug to the
nose to the brain via the olfactory pathway. However, some improvements, such as the
absorption of hydrophilic molecules, and an increase in the ability to target drugs are needed.
Liposomes composed of EPC69, DMPC-PEG2000, cholesterol and H102 peptide have been
successfully used to treat Alzheimer's disease. H102 is a new molecule that prevents the
formation of β-amyloid plaque through the breakage of β-sheets responsible for amyloid β

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accumulation. The liposomal formulation of this peptide, administered intranasally, showed
prolonged drug release and higher accumulation in brain tissue in vivo [191].

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5.3.3. Liposomal drugs to treat the Parkinson’s disease

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Parkinson’s disease (PD) is a neurodegenerative disorder that affects 1–2 people per 1000
globally. The major cause of PD is the loss of dopaminergic neurons (DA) located in the
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substantia nigra (SN) of the midbrain. It has been believed that PD occurs due to oxidative stress
leading to the formation of unstable free radicals, that contribute to nerve cell death. Divers
therapeutic strategies are available for the treatment of Parkinson’s disease, but there are
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limitations to their effectiveness. Among these challenges, the most problematic ones include the
delivery of agents across the blood-brain barrier to targeted brain tissue and the aftereffects
associated with long term administration. Many drug delivery systems such as micelles,
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dendrimers, liposomes, niosomes, and other nanoparticles are being intensively investigated for
diagnosis and therapy [192]. One approach to treat Parkinson's disease is to deliver dopamine
HCl encapsulated in the charged liposomes. The advantages of this approach are the effective
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delivery of the cargo to the brain via a passive way and protection of the drug against
degradation in an animal model [193]. Another approach based on liposomal tyrosinase delivery
directly to the tumor utilizes tyrosinase ability to increase the dopamine level in the brain tissue
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by providing L-tyrosine. Liposomes containing glutathione have also been developed for PD
treatment. The goal of this approach was to maintain the proper glutathione level in the
mesencephalic neuronal cells, leading to their protection [194]. L-DOPA, a classic anti-
Parkinson’s drug recently has been encapsulated in chlorotoxin- modified liposomes. Chlorotoxin
was used as targeting moiety in stealth liposomes to efficiently deliver cargo in an animal model
[195].

Glial cell line-derived neurotrophic factor (GDNF) was also used for Parkinson's disease therapy.
The intranasal route of administration of liposome-encapsulated GDNF, produces neurotrophic
effects in the substantia nigra and protects neurons against degradation in the specific animal
model of this disease [196]. Another successful approach for PD treatment is gene therapy based
on the delivery of cDNA and gDNA of the tyrosine hydroxylase gene in combination with
GDFN. Intravenous administration of these Trojan horse liposomes proved to be an efficient
strategy for delivery to the brain, because of the active targeting of rat transferrin receptor [197].
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Similarly, DNA containing, PEGylated liposomes were created for transvacuolar gene therapy of
PD. Additionally, monoclonal antibodies specific to BBB receptors, including insulin and
transferrin receptors were attached to these liposomes to achieve active targeting of brain tissue.
Thanks to that TH activity was restored in rats' brains [198]. The recent advancements in PD
treatment are to combine a few strategies into one treatment. The example of this approach is to
combine ultrasounds and liposomes containing GDNF and nuclear receptor-related factor 1
(Nurr1), with microbubbles. The liposomes-coupled with microbubbles (PLs- GDNF + Nurr1-
MBs) were able to increase the number of dopaminergic neurons in rats' brains. Therefore, the
delivery of PLs-GDNF + Nurr1-MBs into the brain via focused ultrasound looks like a
promising strategy for the treatment of Parkinson's disease [199].

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6. Scaling up of liposome production

Currently, laboratory scale liposome production is not a challenge. However, only a few mass
production techniques are available. Despite their availability, their use is limited by several
restrictions on the entrapment of sensitive molecules due to their exposure to mechanical and/or
chemical stress [200].
The properties of liposomes vary significantly based on the lipid composition used. However,
they can be prepared by the same method regardless of composition. The common steps of the
preparation of the liposomes include lipid film hydration and sizing to obtain a homogeneous
population of vesicles.
Many variations of this method have been developed. However, despite the various
modifications possible consideration has to be taken that lipids associated with a negative or

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positive charge will form smaller vehicles with fewer bilayers and that the characteristic of the
aqueous phase as well as the power and energy input of agitation will influence the final product.

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Most of the liposome preparation methods developed in the 1980s and 1990s, paid very little
attention to the large-scale production of liposomes. Currently, the advancement in a formulation
is primarily focused on mass manufacturing. Nowadays strict control of the product is required

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by pharmaceutical regulations, including features such as sterility, purity, and quality. Until now,
no general, large-scale liposomes production method has been successfully established, mostly
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because of technical difficulties to fine‐tune liposomes with various properties and applications.
The choice of the production method often depends on liposome composition, size, size
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distribution, and the drug release pattern. All these together determine pharmacokinetic
demonstration of adsorption, distribution, metabolism, and elimination (ADME)[200, 201].

So far, scale up methods for liposome production followed the rules of previously established
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methods such as alcohol injection or crossflow technique. All of them use precipitation of
dissolved lipids into anti-solvent (an aqueous solution). This approach utilizes a local solubility
of the lipids which change during this process leading to the spontaneous formation of liposomes.
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API (Active Pharmaceutical Ingredient) can be encapsulated inside the liposome core or in the
lipid bilayer depends on its characteristics. The injection and crossflow methods principles are
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suitable to change them into continuous production methods. The basis that both of these
methods utilize is to maintain all feed streams to produce liposomes, which can be done
continuously. Those kinds of liposome production methods present the most promising approach
to change it into a continuous process, due to ease to maintain sterile conditions. Although they
are the most promising technologies, there are some challenges including formulation
improvement, construction of the system as well as materials that are used to secure maintenance
of sterility, need to be solved before approval of these methods by the pharmaceutical industry.
[201].

7. Summary
Liposomes are attractive drug delivery systems because of their versatile applicability. Feasible
for excipient compositions and chemical modifications, liposomes have been used to deliver
various types of drug modalities and have been administered safely via multiple routes. Since the
discovery of liposomes, the technology of their production has been significantly modified
through the evolution of novel lipid components and preparation procedures. Although there are
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many liposome-based FDA-approved therapeutics on the market with upcoming ones under
active development, the clinical needs of liposome-based therapeutics have not yet been met.
Hurdled to production, include the reproducibility between batches, low entrapment in some
drug candidates, effective sterilization methods, on-shelf stability and, most importantly, scale-
up for clinical use. The production of multifunctional liposomes continues to be a challenge for
the industry. All these factors make medical purposed liposomes expensive. To date, classic
methods such as transmembrane gradients and new methods such as microfluidization and spray
drying have improved the encapsulation efficiency and reproducibility with significantly
effective size control. Recent technological progress provides hope that justifies further
development of liposomes and nanomedicine in general as drug delivery systems.

Acknowledgments:
The authors thank Dr. William C. Hartner for helpful comments during the preparation of the

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manuscript.

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Conflicts of Interest: The authors declare no conflict of interest.

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Table 1:
Nucleic acid Ref
Excipients Formulation composition ratio Application
cargo
siRNA
DOTAP:Chol:DOPE A549 lung
DOTAP splicing [83]
(1:0.75:0.5) carcinoma
factor
DODAP:DSPC:Chol:PEG
DODAP siRNA tetR Liver tumor [110]
(40:10:48:2)
DODMA DODMA:egg PC:Chol:PEG siBcl-2, miR- Liver tumor [85]
 Journal Pre-proof

(45:15:35:5) 122
DOTMA
Cationic lipid:egg PC:PEG siRNA LOR- Acute myeloid
DODMA [86]
(45:54:1) 1284 leukemia
DC-Chol
siRNA
DC-Chol:DOPE:PEG kinesin
DC-Chol Ovarian tumor [87]
(1:1:0.005) spindle
protein
siRNA
DOPE:DPPC:Chol
luciferase
PEI (70:15:15) Ovarian tumor [88]
pCMV-luc,
PEI-nucleic acids
pEGFP-N1
Egg PC:Chol:DOPE-PEI

of
PEI-DOPE siMDR-1 Ovarian tumor [89]
(90:10:0.5)
DOTAP:Chol:DOPE:PEG-C16 Thyroid
DOTAP miRNA-34b [92]

ro
(50:35:5:10) carcinoma
siRNA
DOTAP:Chol:DOPE:PEG-Mal plekhol
DOTAP
(42:15:38:5)
-pmiRNA-33-
5p
Osteoporosis [94]
re
DSDAP:DSPC:PEG Hindlimb
DSDAP miRNA-126 [96]
(30:64:6) ischemia
lP

mRNA
DOTAP DOTAP:Chol (1:1) survivin- Colon Cancer [100]
T34A
DLin- DLin-MC3-
na

mRNA
MC3- DMA:DSPC:Chol:PEG Cystic fibrosis [101]
cmCFTR
DMA (50:10:38.5:1.5)
DLin- DLin-KC2-
ur

KC2- DMA:DOPE:Chol:PEG pDNA Luciferase [108]

DMA (50:10:39:1)
Jo

DOTAP:DOPE (1:1)
DOTAP DOTAP:Chol (1:1) CRISPR/Cas9 HEK293 [111]
DOTAP:DOPE:Chol (1:1:2.1)
DOTAP DOTAP:DOPE:PEG (1:1:0.5) CRISPR/Cas9 MPS I [112]
Pancreatic
DOTAP DOTAP:Chol (1:1) CRISPR/Cas9 [113]
cancer

Table 2:
Formulation Antigen/ Adjuvant Tumor Ref.
[135]
Pegylated PC/cholesterol
α-galactosyl ceramide (α-GC), B16 melanoma lung
liposomes with octa-
lipid antigen metastases
arginine-SA + α-GC
 Journal Pre-proof

neuroblastoma (NB) growth [136]

inhibiting cytosine– HTLA-230
Stealth liposomes
phosphorothioate–guanine neuroblastoma
oligodeoxynucleotide (CpGs)
Cationic TDB/DDA B16-OVATC-1 lung [137]
ovalbumin and HP16 E7 protein
liposomes cancer
[138]
PC/PG/cholesterol
ErbB2 CTL and influenza virus RenCa-lacZ/ErbB2
liposomes with
HATH-peptide epitopes tumors
mannosylated ligands

4 different mRNA drugs [139]

of
Lipo-MERIT encoding melanoma-associated Melanoma
antigens (MAAs)
Lewis lung [140]

ro
DOTAP/DOPE bFGF as angiogenesis stimulator
carcinoma

Graphical abstract:
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