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ORIGINAL RESEARCH

Comparative Encapsulation Efficiency of Lutein in

Micelles Synthesized via Batch and High
Throughput Methods
This article was published in the following Dove Press journal:
International Journal of Nanomedicine

Lauren E Cosby 1 Purpose: Black raspberries (BRBs) and their anthocyanin-rich hydrophilic fractions (BRB-
Kil Ho Lee 2 H) have exhibited significant chemopreventative activity across aerodigestive cancers.
Thomas J Knobloch 3 Lutein, the primary component of the BRB lipophilic fraction (BRB-L), also demonstrates
Christopher M Weghorst 3 bioactivity potential, but is less well characterized, in part because of its poor, innate
bioavailability. For these lipophilic compounds to be accurately evaluated for anticancer
Jessica O Winter 1,2
efficacy, it is necessary to increase their functional bioavailability using delivery vehicles.
1
Biomedical Engineering, The Ohio State Lutein has been delivered in commercial settings in emulsion form. However, emulsions are
University, Columbus, OH 43210, USA;
2
William G. Lowrie Department of unstable, particularly in the gastrointestinal tract, which limit their use as an oral nutraceu­
Chemical and Biomolecular Engineering, tical. Here, we evaluated lutein encapsulation and cellular uptake for nanoparticle (NP)
The Ohio State University, Columbus,
delivery vehicles composed of three different materials synthesized via two different
OH 43210, USA; 3College of Public
Health, Division of Environmental Health approaches.
Sciences, The Ohio State University, Methods: Specifically, NPs were synthesized via smaller scale batch interfacial instability
Columbus, OH 43210, USA
(II) sonication and semi-continuous high throughput electrohydrodynamic-mediated mixing
nanoprecipitation (EM-NP) methods using polystyrene-polyethylene oxide (PSPEO) or poly­
caprolactone-polyethylene glycol (PCLPEG) block copolymers and PHOSPHOLIPON 90G®
(P90G, Lipoid GmbH) lipids. Size distribution, lutein encapsulation efficiency (EE), and
cellular uptake and delivery were evaluated for each NP formulation.
Results: NPs produced via high throughput EM-NP had higher EEs than NPs produced via
batch II sonication, and P90G had the greatest EE (55%) and elicited faster cellular uptake in
premalignant oral epithelial cells (SCC83) compared to other delivery systems.
Conclusion: These qualities suggest P90G could be a beneficial candidate for future lutein
in vitro delivery research and clinical translation for oral cancer prevention.
Keywords: black raspberries, lutein, oral cancer, scalable nanomanufacturing,
nanoprecipitation, emulsification

Introduction
Lutein is a plant-derived, lipophilic, bioactive compound in the carotenoid phyto­
chemical family, commonly found in a variety of fruits, including black raspberries
and leafy green vegetables.1 Epidemiological studies show a strong correlation
between fruit and vegetable intake, lutein, and positive health outcomes.2–5
Lutein is associated with reducing the risk of cardiovascular disease, age related
macular degeneration, and certain cancers.5 Because it is derived from natural food
Correspondence: Jessica O Winter sources, lutein is a particularly attractive compound for oral cancer prevention and
Tel +1 614 247 7668
Email [email protected] treatment (ie, nutraceutical formulation). Lutein has several anticancer properties. It

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demonstrates antioxidant activity, including the ability to One possible alternative is formulation in polymer/lipid
scavenge harmful free radicals, eliciting a protective effect micelles or nanoparticles, which are commonly employed
against deoxyribonucleic acid (DNA) and cellular for delivery of hydrophobic compounds.24,25 For example,
damage.6 Lutein reduces cell proliferation,7,8 modifies polymer nanoparticles synthesized via the flash nano-pre­
gene expression,6,8 reduces cancer cell viability, and elicits cipitation (FNP) process of Prud’homme26 have been
an apoptotic effect.7 Black raspberries are one possible shown to result in very high encapsulation efficiencies of
source of lutein and have independently exhibited chemo­ hydrophobic cargoes, exceeding those of standard sonica­
preventive properties in a number of aerodigestive malig­ tion approaches. We have achieved similar successes
nancies, including oral cancer.9–11 However, because through electrohydrodynamic mixing-mediated nanopreci­
lutein is hydrophobic in nature, it exhibits poor bioavail­ pitation (EM-NP),27 which is based on FNP processes but
ability and bioaccessibility following oral consumption. To does not require impinging jet flows at high Reynolds
evaluate potential health benefits of black raspberries and numbers common to most FNP approaches.28 EM-NP
lutein as chemopreventives for oral cancers, additional employs a water-miscible organic fluid (nonconductive)
solvating amphiphiles and hydrophobic encapsulates that
strategies are needed to enhance delivery.
is delivered via an insulated, electrified, needle immersed
Lutein consists of a long hydrocarbon chain with eight
double bonds; hexene rings anchor each end of the hydro­ in aqueous fluid (conductive).27 Applying high voltage to
the needle creates an electric potential between the tip of
carbon chain (Figure 1). These double bonds and terminal
the needle and a grounding electrode, inducing electrohy­
hydroxyl groups make lutein highly susceptible to degrada­
drodynamic (EHD) flow. This effect induces the rapid
tion by light, heat, or oxidation.12,13 Oil-in-water emulsions
mixing needed to generate uniform particles via nanopre­
can protect lutein against this degradation,14 and many
cipitation of the hydrophobic cargo that are stabilized by
researchers have examined lutein in emulsion form.14–18
amphiphilic block copolymers (BCPs). Unlike the conven­
Commercially, lutein nutritional supplements are available
tional FNP process, rapid mixing depends on the applied
in capsule form, most typically as an oil emulsion for oral
voltage instead of the flow rates employed. Hence, EM-NP
consumption. Despite their widespread use, there are sev­
is an attractive option for synthesizing vehicles encapsu­
eral disadvantages of lutein delivery via emulsions.
lating high-value therapeutics with the potential for high
Emulsions have poor thermodynamic stability that can
controllability over material processing. However, this
result in coalescence and phase separation;19 temperature, approach has not previously been used to encapsulate
pH, and freeze-thaw cycle sensitivity;15 Ostwald ripening;20 bioactive compounds. Further, there are a wide variety of
and lipid oxidation.21 Additionally, upon reaching the sto­ choices for delivery vehicles and predicting encapsulation
mach, emulsion integrity is compromised by the low pH efficiency based on drug/carrier chemical structures can be
and proteolytic environment; sustained release is unachie­ challenging. Encapsulation efficiencies can thus vary
vable. As a result of these factors, lutein experiences che­ widely based on the process and the compounds
mical instability, short shelf life, and minimized employed.29–32
bioavailability. The main approach to overcome these issues To address some of these concerns, we evaluated the
has been to stabilize emulsions through interfacial barriers effect of synthesis method and delivery vehicle chemical
that prevent coalescence,22 including the use of surfactants composition on NP structure, lutein encapsulation, and
such as Tween 20/80 and sodium dodecyl sulfate,19 resultant cellular uptake. NPs were synthesized using scal­
phospholipids,23 and biopolymers, ranging from proteins able EM-NP and batch sonication methods, which per­
to polysaccharides.22 mitted comparison of chemical compatibility trends

Figure 1 Chemical structure of lutein.

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across two different manufacturing platforms. Batch soni­ study and use of lutein as a preventive agent for oral
cation was performed using the interfacial instability (II) cancer and provides some preliminary guidance on poten­
method of Hayward,33 which has been identified as a tial approaches to predict drug encapsulation efficiency
thermodynamically-limited process, whereas EM-NP pro­ trends based on chemical compatibilities.
vides a kinetically-limited high throughput alternative.27
NP delivery vehicles were synthesized using three materi­ Materials and Methods
als to provide a variety of drug-carrier compatibilities. Materials
Poly (styrene)-poly (ethylene oxide) (PSPEO) and poly Carboxyl-terminated polystyrene-b-polyethylene oxide
(caprolactone)-poly (ethylene oxide) (PCLPEO) block (PS-b-PEO-COOH, 9500-b-18,000 Da) and poly (ε-capro­
copolymers were employed because they have previously lactone)-b-polyethylene oxide (PCL-b-PEO-COOH, 6000-
been evaluated as drug delivery vehicles for hydrophobic b-5000 Da) BCPs were used for micelle synthesis. PS-b-
cargoes in scalable nanoprecipitation processes.34,35 These PEO and PCL-b-PEO were obtained from Polymer Source
materials were chosen for their biocompatibility, estab­ Inc. (cat. no. P5755-SEOCOOH and P3130-EOCL,
lished characterization techniques, enhanced encapsulation respectively). Lipid micelles were formed using a com­
abilities, and sustained release dynamics.29 As a compar­ mercial phospholipid, PHOSHPOLIPON 90G (P90G)
ison, we also analyzed PHOSPHOLIPON 90G (P90G), obtained from Lipoid (PHOSPHOLIPID GmbH, item no.
which has been previously used for oral delivery of poorly SA-L368202). Lutein (xanthophyll, cat. no. X6250),
soluble compounds in preclinical studies,36–38 but which Coumarin 6 (cat. no. 442,631) (a fluorescent dye reporter),
has not previously been employed in scalable processes. polyvinyl alcohol (PVA, cat. no. 363,170), and Tween 20
P90G is an FDA-approved, commercially-available, (T20, P1379) were purchased from Sigma Aldrich.
amphiphilic lipid (unsaturated diacyl-phosphatidylcholine)
derived from soybean fatty acids (Figure 2).39 Because Particle Synthesis
P90G is derived from natural, renewable sources, it is Interfacial Instability (II) Sonication
non-toxic, biodegradable and generally regarded as safe Lipid or polymer in tetrahydrofuran (THF, Sigma Aldrich,
(GRAS); therefore, it is commonly used in pharmaceuti­ cat. no.178810) (200 μL, 1 mg/mL) was combined with 5
cal, oral and cosmetic delivery and formulation. Its polar μL of lutein in THF (1 mg/mL), 4 mL of PVA (5 mg/mL),
head group and hydrocarbon chains mimic lipid structures and 4 mL MilliQ water and sonicated (Branson Sonifior
found in the cellular membrane, allowing for enhanced 450) for 5 minutes at a constant duty cycle. In the inter­
cell incorporation, which also aids in gastrointestinal facial instability process, PVA serves to alter the surface
tract uptake and reduces gastric irritation commonly tension of emulsion droplets, promoting droplet fission.33
found with excipients.40 For each carrier, we evaluated After sonication, the solution was transferred to an alumi­
the effect of synthesis technique on size and distribution, num dish and placed on a rocker for three hours to allow
lutein encapsulation, and uptake in premalignant oral can­ solvent to evaporate. The solution was then purified via
cer cells. Thus, this work sets the stage for potential future centrifugal filtration using a 15 mL 100 kDa molecular

Figure 2 General structure of phosphatidylcholine, a constituent of P90G.

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weight cut-off (MWCO) centrifuge filter (Thermo Fisher, purified as described above via centrifugal filtration and
cat.no. UFC910096). Samples were washed three times lyophilization.
with 3 mL MilliQ water for 15 minutes at 4000 rpm.
Samples were then dried overnight via lyophilization for Particle Size and Morphology
storage before further characterization. To characterize the size and shape of resultant NP
micelles, negative stain consisting of 1% uranyl acetate
Electrohydrodynamic-Nanoprecipitation (EM-NP) (20 μL) was applied for 5 minutes to samples mounted
Polymer (200 μL, 1 mg/mL), lutein (5 μL, 1 mg/mL), and on carbon square copper 400 mesh grids (Fisher
Tween 20 (20μL, 1 mg/mL) in THF (400 μL) were pre­ Scientific cat no. 5,024,891). Samples were then imaged
pared prior to EM-NP (Figure 3) as the organic phase.27 using transmission electron microscopy (TEM, FEI
An insulated needle delivering the organic phase via syr­ Tecnai G2 Bio Twin). Images were analyzed using
inge pump was then inserted into the aqueous phase (ie, ImageJ software (NIH) to determine the Feret length,
distilled deionized water). Then, a voltage was applied the measure of the longest axis, which, for spherical
across the grounding electrode and the insulated needle, particles, is the diameter. Data were aggregated into
resulting in rapid electrohydrodynamic mixing of the aqu­ bins ranging from 0 to 100 nm, with widths of 10 nm,
eous phase, which led to atomization of the organic. normalized, and plotted as histograms to evaluate parti­
Because the two phases are miscible, micelles formed cle size distribution for each sample. Histogram plots
nearly instantaneously via this approach. In this process, were fitted using a three parameter logarithmic equation
200 μL of organic was electrosprayed at 12.7 mL/hr into via SigmaPlot 14.0, as lognormal distribution is
10 mL MilliQ water. After spraying, sample solutions expected for drug-loaded micelles.41 Particle size
were placed in aluminum dishes on a rocker for three mean, standard deviation, and polydispersity were deter­
hours to permit solvent evaporation. Samples were then mined using the following equations:

Figure 3 Schematic of EM-NP process.

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� hln x i2 �
0:5
ðx0 Þ Cellular Uptake
A b
yðxÞ ¼ e (1) To enable visualization, coumarin-6 fluorescent reporter
x
(10 μL, 1 mg/mL) was co-encapsulated with lutein (5
1 μL, 1 mg/mL) in delivery vehicles. Cellular uptake of
A ¼ pffiffiffiffiffi (2) co-loaded coumarin-6 and lutein P90G and PCL-PEO
2πb
micelles was evaluated in the premalignant oral cell phe­
� ��
b2 notype, SCC83 cells. SCC83 cells are a patient-derived
Number mean <x � e lnðxo Þþ 2 (3)
cell line that was subsequently shown to have pre-malig­
Geometric standard deviationðσÞ ¼ eb (4) nant characteristics.44,45 The Ohio State University (OSU)
Institutional Biosafety Committee (IBC) is responsible for
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi reviewing research and providing guidance regarding the
ð dev Þðþdev Þ
PDeff ¼ (5) proper acquisition, handling, transfer, and disposal of
hxi
potentially hazardous or regulated biological materials,
where xo and b are the median and the shape parameter, including mammalian cell lines. The current studies were
respectively, and PDeff are the effective polydispersity, approved by the IBC under protocol 2016R00000011-
and standard deviation on the – and + side of the AM2: Models of Multistep Carcinogenesis, Prevention,
distribution. and Therapeutics.
PS-PEO was not evaluated because of its low encapsu­
Encapsulation Efficiency lation efficiency. Cells were cultured in 24 well plates (in
To measure encapsulation efficiency, lyophilized micelle triplicate) to ~6080% confluency and treated with 500 µL
samples were re-suspended in 300 μL THF and evaluated micelles (post-lyophilization) re-suspended in culture
using UV-Vis (in a quartz cuvette) spectroscopy. Lutein media (Dulbecco’s Minimum Essential Media, DMEM,
has a signature peak at 450 nm in THF.42 Thus, a standard 5% FBS, L-Glutamine, antibiotic-antimycotic). At desig­
curve was generated and utilized to determine the final nated time points (0, 5, 15, 30, and 90 minutes), cells were
concentration of lutein in each sample (µg/mL and total rinsed with 1x phosphate buffered saline (PBS) and
mass). The following equation was used to determine the stained with Hoechst 33,342 to observe nuclei (Thermo
percent encapsulation (ie, encapsulation efficiency, EE) for Fisher, cat. no. H3570). Confocal images were taken at the
each particle system: same intensity at 20x magnification for each time point.
DAPI and FITC channels were assigned to Hoescht and
Final Mass Lutein ðμgÞ
EE ¼ � 100 (6) coumarin-6 reporters (respectively) and combined in
Initial Mass Lutein ðμgÞ
ImageJ to visualize micelle cellular uptake over time.
These results were compared to the Hansen solubility
parameters for each system to estimate lutein and polymer
Statistics
interactions and to interpret trends in EE data. The group
JMP Pro 14 software (SAS Institute Inc., Cary, NC, USA)
contribution method developed by Hoftyzer and Van
was used to conduct statistical analysis. Because of the
Krevelen was used to determine three solubility para­
logarithmic nature of the particle size distribution data, the
meters: δd, δp, and δh, representing polymer dispersion,
non-parametric Wilcoxon-Rank Sum test was used. The
polar, and hydrogen bond interactions in the system,
Student’s t-test was used to evaluate the statistical signifi­
respectively.43
cance of encapsulation efficiency between the two differ­
δTOTAL 2 ¼ δ2D þ δ2P þ δ2H (7) ent synthesis processes. p-values <0.05 were considered
statistically significant.
Tables 7.3 and 7.1043 were used to calculate δd, δp, δh
based on drug, lipid, and polymer major chemical structure
groups. It should be noted that structural information for Results and Discussion
P90G is proprietary and its parameters were determined Micelle Particle Size and Morphology
from the standard phosphatidylcholine structure discussed The primary goal of this work was to compare two differ­
above. Thus, true P90G parameters may be different from ent manufacturing processes: batch II sonication, which is
those calculated. thermodynamically controlled,33 and EM-NP, which is

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kinetically controlled, to each other across a range of TEM data were then fitted to a log normal three parameter
delivery vehicle designs. Lutein-encapsulating micelles distribution, where µ and σ were extracted as the mean
were formed via probe sonication or via EM-NP for size and polydispersity (PD), respectively (Table 1).
P90G, PS-b-PEO, and PCL-b-PEO amphiphiles. To iden­ Particle size and distribution are critically linked to bio­
tify effects of synthesis method on particle size and mor­ distribution and ability to cross cellular membranes.46,47
phology, loaded and empty micelles were evaluated using For these reasons, we desired spherical micelles <100 nm
TEM (Figures 4 and 5). Size histograms generated from in diameter with narrow size distributions. Generally, all

Figure 4 Particle size distribution and TEM imaging of unloaded (A–C) and lutein-loaded (D–F) micelles via II sonication. Scale bar = 500 nm.

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Figure 5 Particle size distribution and TEM imaging of unloaded (A–C) and lutein-loaded (D–F) micelles via EM-NP. Scale bar = 500 nm.

systems yielded particles, but with varying morphology. size among different systems and encapsulation efficiency,
Additionally, all vehicles experienced a slight increase in as final carrier size is dictated by amphiphile geometry and
diameter with lutein addition, which suggests lutein size.
encapsulation.41 Particle diameter increase is characteristic In addition, statistical analysis identified a significant
of drug addition to the nanoparticle and is commonly difference in size between the two methods of synthesis.
associated with evidence of encapsulation. However, Comparing II sonication to the scalable EM-NP processes,
there is no expected correlation between loaded carrier particles produced via II sonication were generally the

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Table 1 Particle Size and Polydispersity of Unloaded and Lutein-Loaded P90G, PCLPEO and PSPEO Micelles Synthesized via II
Sonication and EM-NP
Particle Diameter (nm) via Interfacial Instability (II) Sonication

P90G P90G-Lut PCLPEO PCLPEO-Lut PSPEO PSPEO-Lut

Peak 1 Peak 2 Peak 1 Peak 2

Mean 21.6 64.6 22.6 45.6 24.8 25.8 30.0 32.1

+deva +3.5 +25.4 +4.4 +15.5 +6.7 +7.1 +8.5 +8.3
-devb −3.0 −18.2 −3.7 −11.6 −5.3 −5.6 −6.6 −6.6
PDeffc 0.151 0.333 0.178 0.294 0.239 0.244 0.249 0.231

Particle Diameter (nm) via EM-NP

P90G P90G-Lut PCLPEO PCLPEO-Lut PSPEO PSPEO-Lut

Peak 1 Peak 2 Peak 1 Peak 2

Mean 25.8 - 23.0 - 24.5 24.7 19.7 26.3

+deva +7.68 - +5.2 - +9.0 +8.6 +4.8 +8.8
-devb −6.0 - −4.2 - −6.6 −6.4 −3.9 −6.6
PDeffc 0.263 - 0.204 - 0.315 0.300 0.220 0.290
a, b c
Notes: Standard deviations of the positive and negative sides of the distribution, respectively. Effective polydispersity.
Abbreviations: P90G, PHOSPHOLIPON 90G®; P90G-Lut, lutein-loaded P90G carriers; PCLPEO, polycaprolactone-poly (ethylene oxide) carriers; PCLPEO-Lut, lutein-
loaded PCLPEO carriers; PSPEO, polystyrene-poly (ethylene oxide) carriers; PSPEO-Lut, lutein-loaded PSPEO carriers.

same size or larger than those produced via EM-NP and arrangement is dictated by rapid mixing and change in
less uniform in morphology. PS-b-PEO NPs showed a organic solvent concentration; promoting compact
greater size increase than PCL-b-PEO NPs. However, the arrangement and reduced swelling that may explain the
most striking difference occurred for the P90G samples. smaller diameters observed for some EM-NP particles
Both empty and lutein-loaded P90G micelles formed via II versus II sonication.27,49 Particle uniformity is increased
sonication were granular, aggregated, and characterized by by rapid kinetics, which reduces particle growth time,
a non-uniform, biphasic size distribution (determined by particularly the ability of aggregates of either polymer or
two major peaks for particle diameter mode). (Figure 4A drug to form.
and D). Also, unlike polymer micelle systems, P90G II
sonication was the only system that exhibited a significant Encapsulation Efficiency
number of particles with diameters >100 nm. In contrast to Noting the low aqueous affinity of hydrophobic encapsu­
II sonication, similar morphology and size distribution was lants, drug release was not measured. Previously, we have
observed for all systems made via EM-NP (Figure 5). observed virtually no release in PBS over 7 days and that
P90G particles synthesized via EM-NP (Figure 5A and release is “smart”, occurring primarily as a result of endoso­
D) presented no particles >100nm. These morphological mal rupture.41 Thus, this study focused on the effect of
differences may be attributed to differences in aggregation synthesis method on lutein encapsulation across the three
kinetics between P90G, which has two hydrocarbon tail delivery vehicles. II sonication processes uniformly resulted
chains, versus the single-chain structures of PSPEO and in lower encapsulation efficiencies than scalable EM-NP
PCLPEO polymers. processes (Table 2), suggesting that the kinetically driven
Regarding differences between the two processes, II nature of EM-NP may allow for greater drug loading.27
sonication is a thermodynamically driven process by This is similar to results reported for other scalable NP
which particle formation is dictated by the lowest energy processes, such as flash nanoprecipitation.50 It has been
state between polymer and drug. Alternatively, EM-NP is suggested that the fast kinetics of nanoprecipitation processes
a kinetically-driven process for which particle size and can result in aggregation of drugs followed by polymer
formation are dictated by nucleation and growth time.48 stabilization, which inherently increases encapsulation in
Further EM-NP has a much lower organic/aqueous solvent the core compared to processes in which thermodynamic
ratio than traditional sonication approaches. Chain equilibrium is attained.

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Table 2 Encapsulation Efficiency (EE) of Lutein in Each System Synthesized via II Sonication and EM-NP
% Encapsulation of Lutein

II Sonicationa EMNPb

P90G PCL-PEO PS-PEO P90G* PCL-PEO PS-PEO

Average ± SE 17.4 ± 3.77 9.86 ± 1.24 2.09 ± 0.49 55.6 ± 3.25 14.08 ± 5.07 4.46 ± 0.94
Notes: aInterfacial Instability Sonication, bElectrohydrodynamic Mixing- Nanoprecipitation, SE = Standard Error, *EE significantly different between manufacturing
systems (p = 0.0024).
Abbreviations: P90G, PHOSPHOLIPON 90G®; P90G-Lut, lutein-loaded P90G carriers; PCLPEO, polycaprolactone-poly (ethylene oxide) carriers; PCLPEO-Lut, lutein-
loaded PCLPEO carriers; PSPEO, polystyrene-poly (ethylene oxide) carriers; PSPEO-Lut, lutein-loaded PSPEO carriers.

Further, lutein is susceptible to damage upon exposure important factor in this analysis because cohesive energy
to heat, light, and oxidizing agents.13,16,51 One main obser­ is largely dependent on polar groups and hydrogen
vation noted in samples formed via II sonication was bonding.52 Non-covalent interactions between hydrogen
increased temperature, which introduces the possibility of bonds and hydrophobic drug groups within the copolymer
heat damage to lutein during nanoparticle synthesis. enhance hydrophobicity, creating stronger interaction
Additionally, the presence of PVA may have influenced between drug and polymer.52 A major weakness of this
lutein encapsulation. After resuspension of lyophilized II approach is that δ cannot be determined directly, therefore
sonication samples in THF (to perform encapsulation effi­ there is large variability in parameter values calculated and
ciency measurements), samples often contained orange- there are a number of methods to determine solubility
tinted PVA that did not dissolve, assumed to be PVA- parameters. Here, we use the group contribution method
trapped lutein. In contrast, the EM-NP utilized Tween 20 established by Hoftyzer and Van Krevelen.43 This method
as a stabilization agent, which may have been more com­ is considered more rigorous compared to others as it
patible with lutein encapsulants. maintains a mean accuracy of roughly 10%.
Comparing across polymer and lipid delivery vehicles, Solubility parameter analysis supports encapsulation
encapsulation of lutein was highest in P90G (55.6%), results observed in both polymer micelle systems (Tables
followed by PCLPEO (14.08%) and PSPEO (4.46%) 3 and 4). δh values of lutein, PS, and PCL are 8.47, 0.00,
(Table 2). To understand these trends, Hansen solubility and 7.25, respectively. The wide contrast between lutein
parameters (HSPs) (δ)43 were calculated for these systems. and PS δh values correlates with the PS polymer system
These parameters represent the cohesive energy of mole­ having the lowest encapsulation efficiency observed in the
cules present in the compound of interest (based on dis­ experimental data. The difference between lutein and PCL
persion (δd), polar groups (δp) and hydrogen bonds (δh)) δh values is considerably lower (Δδh = 1.22) suggesting
and are commonly used to determine polymer-solvent better solubility between the drug and polymer, but only a
compatibility. Additionally, these parameters can be uti­ modest increase in EE (ie, 9.86% and 14.08% for II
lized to evaluate drug–polymer interactions, useful for sonication and EM-NP, respectively) is observed over
describing experimental encapsulation data.43 δh is an that of PS systems (ie, 2.09% and 4.46% for II sonication

Table 3 Hansen Solubility Parameters (HSPs (MJ/m3)1/2) of Lutein and Corresponding Hydrophobic and Hydrophilic Block Copolymer
Segments, P90G, and Water; Calculated Using the Group Contribution Method
Compound δd δp δh δTotal (MJ/m3)1/2

Lutein 15.24 1.80 8.47 17.53

P90G 17.35 9.83 7.30 21.97
PCL 18.30 12.30 7.25 23.21
PS 20.58 1.27 0.00 20.62
PEO 21.46 29.50 8.75 37.51
Water 18.50 16.00 42.30 47.81
®
Abbreviations: d, dispersive; p, polarizing; and h, hydrogen bonding contributions; P90G, PHOSPHOLIPON 90G ; PCL, polycaprolactone; PS, polystyrene; PEO, poly
(ethylene oxide).

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Table 4 Change in δ Values Between Lutein and P90G or Hydrophobic Copolymer Blocks
Polymer Δδd Δδp Δδh

P90G PCL PS P90G PCL PS P90G PCL PS

Lutein −2.10 −3.05 −5.34 −8.04 −10.51 0.52 1.18 1.22 8.47
®
Abbreviations: d, dispersive; p, polarizing; and h, hydrogen bonding contributions; P90G, PHOSPHOLIPON 90G ; PCL, polycaprolactone; PS, polystyrene; PEO, poly
(ethylene oxide).

p
and EM-NP, respectively). The P90G δh value is only Ra ¼ ð4½ðδD1 δD2 Þ�2 þ ½ðδP1 δP2 Þ�2 þ ½ðδH1 δH2 Þ�2
modestly higher than that of PCL (7.30 vs 7.25 for a Δδh (8)
= 1.18), yet encapsulation efficiencies of as much as 55%
Smaller Ra values (the distance between molecular solu­
were observed. Thus, the P90G system had the greatest
bility spheres) equate to greater compatibility. Based on
drug – polymer compatibility.
calculated Ra values (Table 5), compatibility trends reflect
Although the general EE trend predicted by HSPs is
predictions of HSPs and experimental observations: lutein
observed, the dramatic increase for P90G was unexpected.
is most compatible with P90G followed by PCL then PS,
It should be noted that P90G HSP values were based on
PEO, and being least soluble in water. This data is analo­
the hydrophilic (choline head) block of the BCP, since the
gous to EE results and makes a strong case for lutein’s EE
hydrophobic portion is proprietary, which almost certainly
and affinity in P90G. PCL and PS Ra values were similar
contributes to results. Further, the influence of the PEO
(12.22 and 13.64, respectively), and unlike HSP Δδh
block in PSPEO and PCLPEO BCPs should be considered.
values, more closely predicted observed experimental EE
δh values of lutein and PEO are comparable (8.47 and
trends. To further analysis of lutein–polymer interactions,
8.75, respectively). Even though PEO is the hydrophilic
the Flory-Higgins parameter (Χsp) was also evaluated.
block of these systems, it is possible that drug associated
This parameter has been successful in predicting drug
with the hydrophilic block only to be rapidly desorbed
solubility by assessing drug molecule interactions with
during washing and purification processes prior to EE
polymer chains,55 and effectively adds a molar volume
quantification. Thus, although systems have widespread
correction to HSP and Ra equations.
use in the nanoprecipitation and polymer micelle
community,33,53 their suitability for drug encapsulation Ra2 vs
Xsp ¼ (9)
depends heavily on chemical compatibility with the spe­ RT
cific drug to be encapsulated. Based on the high EE of where υs is the molar volume of the drug, R is the gas
lutein in P90G, it is likely that the P90G hydrophobic core constant, and T is temperature in Kelvin. Thus, lowχΧsp
and the choline hydrophobic corona work in a distinctly reflects better polymer-molecule compatibility. From cal­
unique way to provide an ideal soluble environment for culated Χsp values, lutein is more compatible with P90G,
lutein. followed by PCL, PS, PEO, and then water (Table 6). Χsp
Whereas HSPs provide some basis for predicting and results better reflect EE differences seen between the PCL
understanding drug – polymer interactions, the Hansen and PS BCP systems with an 8 point difference in inter­
solubility parameter distance (Ra) may provide a better action values. PEO and water Χsp values (207.2 and 311.9)
measure of interactions. Ra considers molecular interac­ also suggest low lutein EE in PEO blocks and low solubi­
tions in three-dimensional space54 and is calculated by the lity in the aqueous phase. Thus, of these parameter sets,
following equation: Flory-Huggins and Hansen interaction spheres (Ra) were

Table 5 Hansen Solubility Parameter Distance (Ra (MJ/m3)1/2) Values Between Lutein and BCPs and Water
Ra (MJ/m3)1/2

PS PCL P90G PEO Water

Lutein 13.64 12.22 9.15 30.37 37.26

®
Abbreviations: P90G, PHOSPHOLIPON 90G ; PCL, polycaprolactone; PS, polystyrene; PEO, poly (ethylene oxide).

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Dovepress Cosby et al

Table 6 Flory-Higgins Interaction Parameter (Χsp) Values of Lutein – Polymer and Lutein – Water Solutions
Χsp

PS PCL P90G PEO Water

Lutein 41.78 33.53 18.81 207.20 311.96

®;
Abbreviations: P90G, PHOSPHOLIPON 90G PCL, polycaprolactone; PS, polystyrene; PEO, poly (ethylene oxide).

more predictive of EE than differences in Hansen δh coumarin-only micelles using this approach, showing that
values. they initially exhibit punctate signal, indicating individual
micelles, that becomes diffuse cytoplasmic signal over
Cellular Uptake time, most likely resulting from micelles rupturing in the
As a precursor to in vitro analysis of lutein as an oral endosomal environment.41 For P90G micelles, coumarin-6
cancer chemopreventative, we observed cellular uptake of signal could be detected along the cell membrane at 15
lutein–P90G and lutein-PCLPEO micelles in premalignant minutes (Figure 6C). Gradual uptake was observed by 30
oral squamous epithelial cells (SCC83).44,45 [Because of minutes, as the signal intensified. Micelles exhibited a
low encapsulation efficiency, PSPEO micelles were not sustained presence in cells up to 90 minutes after applica­
further analyzed.] To permit observation, lutein was co- tion, eventually filling the cytoplasm surrounding the cell
encapsulated with a fluorescent dye, coumarin-6, and nucleus (Figure 6D and E). This suggests endocytosis as a
imaged over 90 minutes via time-lapse confocal micro­ mechanism by which P90G micelles are internalized and
scopy (Figure 6). Previously, we reported trafficking of trafficked through the lysosomal cycle. Upon reaching the

Figure 6 Cellular uptake of co-loaded coumarin-6 and lutein P90G micelles (A–E) and PCLPEO micelles (F–J) in SCC83 human oral epithelial cells. Scale bar = 500 micron.

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Cosby et al Dovepress

lysosome, reduced pH, proteolysis or both, reduces micelle interaction parameters or Hansen spheres. These data sup­
integrity, resulting in drug/coumarin release throughout the port previous observations61 that scalable nanoprecipita­
cytoplasm. tion processes offer significant advantages in nanoparticle
In contrast, coumarin-6 signal was poorly detected for processing for drug delivery applications. The kinetically
PCLPEO micelles throughout the time course observed, driven nature of EM-NP synthesis may allow for higher
with only faint detection at 90 minutes (Figure 6FJ). The drug loading while maintaining small particle size and
amount of lutein and coumarin-6 used during synthesis narrow distribution. Further, this technique could enable
were kept the same for each particle system; thus, the transition to commercial production and scale-up. The
data suggest that lutein-P90G micelles more successfully primary goal of this work was not to identify a specific
deliver their contents to SCC83 cells than lutein-PCLPEO optimal carrier, but rather to compare systems of carriers
micelles. These observations may result from two factors: in different manufacturing schemes. However, among the
varying EE for coumarin-6 in PCLPEO micelles or differ­ three delivery vehicles, P90G had the highest lutein encap­
ing physicochemical properties between the two vehicle sulation efficiency and delivered lutein to target cells
materials. Size, shape, surface charge, and vehicle within 15 minutes of application. These outcomes most
stiffness56 are among the factors that impact cellular likely result from high drug–lipid solubility and the bio­
uptake. These features dictate cell membrane interactions mimetic structure of P90G lipids. Additional studies
by altering membrane energy dynamics to favor or disrupt should focus on continued characterization of this system,
particle internalization.57 Since vehicle size and shape are including its toxicity, biodistribution, and cell cycle and
similar, particle chemical properties are strong possible apoptosis effects. In particular, animal work could provide
contributors in varying cellular uptake. In studies evaluat­ additional information on ability of these carriers to cross
ing particle transport in normal and carcinogenic cells, oral mucosal barriers or gastrointestinal trafficking that
PCLPEO micelles initially experience slow uptake and would be crucial for oral formulations. P90G is an attrac­
do not exhibit a distinct presence in cells until 2–3 hours tive candidate for lutein delivery in the food industry
post-incubation.58,59 Additionally, whereas the total net because of its plant-derived nature.39 These qualities sug­
charge of P90G head groups is neutral, lipid fusion and gest P90G as a beneficial candidate for future lutein in
membrane association, known as “lipid-raft effect”60 elicit vitro delivery research and clinical translation for oral
a possible endocytosis pathway for rapid cellular uptake. cancer prevention.
Additionally, we recognize that the addition of coumarin-6
may alter nanoparticle structure and lutein EE by influen­
cing drug-drug and drug-polymer interactions, which Acknowledgments
could influence these results. The authors gratefully acknowledge funding from NSF
CMMI-1344567 (KHL, JOW), T32DE014320 (P.I. John
Conclusion F. Sheridan) Comprehensive Training In Oral And
In this study, we synthesized three micelle delivery sys­ Craniofacial Sciences, NIH NIDCR, Pre-doctoral
tems encapsulating lutein using two methods. Size distri­ Fellowship (LEC), NIH grants: U01CA188250,
bution, particle morphology, encapsulation efficiency, and Interactive Omics: Black Raspberry Metabolites and the
cellular uptake in premalignant oral cells (SCC83) were Oral Microbiome in Smokers, PI: Christopher M.
evaluated for each system and method. Whereas a differ­ Weghorst, The Ohio State University and P30CA016058,
ence in particle morphology was not observed for polymer Cancer Center Support Grant, PI: Raphael E. Pollock, The
micelle systems (ie, PSPEO and PCLPEO) between II Ohio State University, Comprehensive Cancer Center. This
sonication and EM-NP synthesis methods, EM-NP gener­ work was supported in part by The Ohio State University
ally provided more uniform morphology and tighter size Institute for Materials Research, the OSUCCC Genomics
distribution for lipid micelle samples (eg, P90G) compared Shared Resource, OSUCCC Analytical Cytometry Shared
to standard sonication processes. Further, EM-NP proces­ Resource, and OSUCCC Nutrient and Phytochemical
sing yielded higher lutein encapsulation efficiency for all Analytics Shared Resource. We would like to thank
three systems compared to II sonication, and generally Yixiao Cui for her assistance with cell imaging, Prof.
produced smaller or similarly sized particles. These results Barbara E. Wyslouzil for helpful discussions on the EM-
were best predicted by the use of Flory-Huggins NP process, and Drs. Jennifer Ahn-Jarvis and Chureeporn

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Dovepress Cosby et al

Chitchumroonchokchai for guidance on black raspberry- 15. Amar I, Aserin A, Garti N. Solubilization patterns of lutein and lutein
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Disclosure oxidants on physical and chemical stability. Food Chem.
Thomas J. Knobloch reports grants from The Ohio State 2018;242:395–403. doi:10.1016/j.foodchem.2017.09.060
17. Mora-Gutierrez A, Attaie R, Nunez de Gonzalez MT, Jung Y,
University College of Dentistry, during the conduct of the Woldesenbet S, Marquez SA. Complexes of lutein with bovine and
study. The authors report no other potential conflicts of caprine caseins and their impact on lutein chemical stability in
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transport of lutein by oral administration of a solid dispersion and a
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