Cholesteryl to improve the cellular uptake of polymersomes within HeLa cells

Article abstract of DOI:10.1016/j.ijpharm.2016.07.036

The need to develop a greater understanding of drug delivery systems has arisen through the development of alternative biological based therapeutics. Drug delivery systems need to adapt and respond to this increasing demand for cellular transportation of highly charged species. Polymersomal drug delivery systems have displayed great potential and versatility for such a task. In this manuscript we present the synthesis, characterisation and biological evaluation of six amphiphilic random co polymers with varying amounts of cholesteryl (0–39%wt) before the subsequent formation into polymersomes. The polymersomes were then analysed for size, zeta potential, encapsulation efficiency, release kinetics and cellular uptake. Results confirmed that the polymersome containing 12%wt cholesteryl polymer displayed a ten-fold increase in cellular uptake of Fitc-CM-dextran when compared to un-encapsulated drug, crossing the cellular membrane via endocytosis. The size of these vehicles ranged between 100 and 500?nm, zeta potential was shown to be neutral at ?0.82?mV ±0.2 with encapsulation efficiencies in the region of 60%. The ease of adaptability and preparation of such systems renders them a viable alternative to liposomal drug delivery systems.

Full text of DOI:10.1016/j.ijpharm.2016.07.036

Accepted Manuscript
Title: Cholesteryl to improve the cellular uptake of
Polymersomes within HeLa cells
Author: Chloe Martin Nino Marino Ciara Curran Anthony P.
McHale John F. Callan Bridgeen Callan
PII:
S0378-5173(16)30666-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijpharm.2016.07.036
IJP 15933
To appear in:
International Journal of Pharmaceutics
Received date:
Revised date:
Accepted date:
13-5-2016
15-7-2016
16-7-2016
Please cite this article as: Martin, Chloe, Marino, Nino, Curran, Ciara, McHale,
Anthony P., Callan, John F., Callan, Bridgeen, Cholesteryl to improve the cellular
uptake of Polymersomes within HeLa cells.International Journal of Pharmaceutics
http://dx.doi.org/10.1016/j.ijpharm.2016.07.036
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Cholesteryl to improve the cellular uptake of Polymersomes within HeLa cells.
a
a
a
a
a
Chloe Martin , Nino Marino , Ciara Curran , Anthony P. McHale , John F. Callan and Bridgeen
Callana*.
aSchool of Pharmacy and Pharmaceutical Sciences, The University of Ulster, Northern Ireland, BT52
1
SA.
*
Corresponding author at: School of Pharmacy and Pharmaceutical Sciences, The University of
Ulster, Northern Ireland, BT52 1SA. Tel: +28 70 123510 Fax: +28 70 123518 b.callan@ulster.ac.uk
Page 1 of 27

GRAPHICAL ABSTRACT
Abstract
The need to develop a greater understanding of drug delivery systems has arisen through the
development of alternative biological based therapeutics. Drug delivery systems need to adapt and
respond to this increasing demand for cellular transportation of highly charged species.
Polymersomal drug delivery systems have displayed great potential and versatility for such a task. In
this manuscript we present the synthesis, characterisation and biological evaluation of six
amphiphilic random co polymers with varying amounts of cholesteryl (0‐39 %wt) before the
subsequent formation into polymersomes. The polymersomes were then analysed for size, zeta
potential, encapsulation efficiency, release kinetics and cellular uptake. Results confirmed that the
polymersome containing 12 %wt cholesteryl polymer displayed a ten‐fold increase in cellular uptake
of Fitc‐CM‐dextran when compared to un‐encapsulated drug, crossing the cellular membrane via
endocytosis. The size of these vehicles ranged between 100 – 500 nm, zeta potential was shown to
be neutral at ‐0.82 mV ±0.2 with encapsulation efficiencies in the region of 60 %. The ease of
Page 2 of 27

adaptability and preparation of such systems renders them a viable alternative to liposomal drug
delivery systems.
Keywords
Drug Delivery System (DDS), Amphiphilic Polymer, Polymersome, Cholesteryl, poly(ethylene) glycol.
1
. Introduction
Recent advances in therapeutics has seen an extensive exploration into personalised medicine; this
has led to the focus shifting from the more common small molecule therapeutics to much larger,
biological based compounds referred to as ‘biologics’(Martin et al 2016) . These naturally occurring,
large, complex molecules (mostly proteins, nucleotides and polypeptides), generally have a specific
therapeutic target for example a certain genotype or protein receptor (Morrow and Felcone 2004).
The introduction of such specificity leads to higher efficacy and fewer unwanted side effects when
compared against general systemic delivery of non‐selective therapeutics. However with these new
favourable developments come new complications, particularly those surrounding the delivery of
such high molecular weight and extensively hydrophilic molecules. Biologics, such as siRNA,
possessing high solubility but low permeability are defined as Class III using the Bio pharmaceutics
Classification System (BCS) (Dahan et al. 2009). Acknowledgement of the absence of an appropriate
drug delivery system (DDS) to overcome these problems has led to extensive research into designing
a suitable amphiphilic nano‐scale vehicle to allow increased solubility, and facilitate permeation
across the cell membrane to enable drugs such as biologics to exhibit their desired therapeutic effect
at the target site (Bartfai et al. 2010; Khan et al. 2010, Lin et al. 2004).
Nano‐carriers such as liposomes, micelles, nanocapsules and polymersomes, offer many advantages
over free drug administration and greatly improve the solubility of encapsulated or adsorbed drug
compounds. They also protect drugs against degradation once administered (Fomina et al. 2012),
allow various routes of administration including oral or inhalation (Gandhi et al. 2014) and most
Page 3 of 27

importantly improve drug targeting and efficacy. This can occur either actively‐ by incorporating
targeting moieties such as RGD‐containing peptides, folic acid and carbohydrates (Khan et al. 2010);
or passively‐ through the enhanced permeability and retention effect (EPR) caused by a combination
of leaky vasculature and insufficient lymphatic drainage (Pasparakis et al. 2014). This beneficially
reduces off site toxicity which is a major limiting factor in chemotherapy, as well as minimising
resistance and allowing the delivery of more than one drug at a time by co‐encapsulation (Dahan et
al. 2009). The widely explored liposomal drug delivery system (DDS) first documented in 1965 by
Bangham (Bangham 1993), successfully allows the delivery of both water soluble and insoluble
drugs. However, being prepared from phospholipids can lead these carriers to possess leaky
membranes resulting in premature release of payload. More recently polymersomes, which
structurally mimic liposomes but are derived from the self‐assembly of amphiphilic polymers, have
become an attractive alternative due to the high loading ability of hydrophilic, hydrophobic or
amphiphilic compounds allowing them to be exceptional in delivering bioactive molecules and
combined loading of multi‐drugs for synergistic therapy (Pasparakis et al. 2014; Bangham 1993).
Their easily adaptable polymeric design, allied to the wide range of available co‐polymers, means a
wide variety of polymersome structures can be prepared with relative ease. By carefully choosing
co‐polymer blocks with specific chemical characteristics as well as altering the hydrophilic to
hydrophobic ratio, a range of properties can be specifically tuned including size (Martin et al. 2015),
encapsulation efficiency, pharmacokinetics including release rates (Alibolandi et al. 2015) ,
degradability and cellular uptake as well as highly stable membranes avoiding drug leakage (Xu et al.
2
014). The use of Poly(ethylene) glycol (PEG) is well known in nanoparticle preparations for its steric
shielding effect, leading to prolonged blood circulation time; its ability to reduce aggregation in
serum; and its immuno‐protection capabilities preventing elimination from the reticuloendothelial
system (RES)(Xu et al. 2011; Kolli et al. 2013; Lee et al. 2001; Lee and Feijen 2012; Magarkar et al.
2
012; Miyata et al. 2011; Wu and McMillan 2009). However, PEGylation has been reported to reduce
cellular transfection and uptake by interference with intracellular trafficking but the dual
Page 4 of 27

incorporation of PEG and lipid molecules such as cholesterol into nanoparticles is thought to provide
a balance between prolonged circulation and essential targeted uptake (Xu et al. 2011). The
importance of cholesterol in nanoparticle formulation is cemented by the FDA, which have only
approved PEGylated liposomal drug delivery systems which incorporate cholesterol as a key
component (Magarkar et al. 2012). Residing in the biological membranes of various organisms, it can
modulate the fluidity of biological membranes involved in many important life events (Zhi‐Yao et al.
2
014). The role cholesterol plays in drug delivery systems is thought to involve packing and stability,
directly affecting drug release rates and by enhancing cellular uptake: as it is recognised as a natural
cell membrane component it is hypothesised that its inclusion will aid cellular uptake and transport
across the cell membrane (Magarkar et al. 2012). Both liposomes and micelles formulated with
cholesterol have shown increased cellular internalization, enhanced colloidal stability, higher
encapsulation volumes and slower release of payload compared to without cholesterol (Magarkar
et al. 2012; Zhi‐Yao et al. 2014). Polymersomes have also been developed with cholesterol for
enhanced purposes. Huang et al. (2013) developed a cholesterol containing polymersome (11.2
%
wt) which was both amphoteric and amphiphilic. The increased electrostatic interactions within
this polymer allow for a facile one step polymersome preparation, whereas the incorporation of
cholesterol is credited with inhibiting binding to proteins and opsonins and enhancing cell uptake.
Yin et al. (2014) dispersed cholesterol within the bilayer of a miktoarm star copolymer based
polymersome and recorded both enhanced stability and cellular uptake of the resulting
polymersome with a 1 %wt loading of cholesterol. Alternatively Jia et al. (2014) created a triggered
release polymersome using an 80 %wt cholesterol containing polymer.
The need for enhanced drug delivery systems with high biocompatibility and cell permeability is of
great interest to this area of pharmaceutics. The amount of cholesterol included within these drug
delivery systems varies significantly between studies. In this work we present an enhanced
polymersomal drug delivery system with cholesteryl incorporated within the architecture (Figure 1)
of the polymer. By varying the amount of cholesteryl incorporated within six amphiphilic co‐
Page 5 of 27

polymers it was possible to identify the optimum concentration of cholesteryl to ensure the greatest
cellular uptake while retaining the required encapsulation efficiency, release kinetics and
hydrodynamic diameter. The comparative study presented in this work allows a better
understanding of the influence of cholesteryl on cellular uptake.
2
. Materials and Methods
Materials and Reagents
Chemicals were purchased from commercial sources at the highest possible purity and used as
received. Poly(ethylene glycol) methacrylate (Mn 500 Da), fluorescein isothiocyanate‐
carboxymethyl‐dextran (Fitc‐CM‐Dextran) (Mn 4KDa), methacrylic acid (99%) and 1‐decanol (99%),
ethylene diamine (99%), cholesteryl chloroformate (95%) and Thiazol Blue Tetrazolium Bromide
(MTT)(98%) CellLight® Reagent BacMam 2.0 GFP were purchased form Sigma‐Aldrich. DOX‐HCl was
purchased from VWR international.
2
2
.1 Synthesis of Monomers
.1.1 Synthesis of Cholesteryl Ethylene‐Diamine Conjugate (3)
Compound
3
(10,13‐dimethyl‐17‐(6‐methylheptan‐2‐yl)‐2,3,4,7,8,9,10,11,12,13,14,15,16,17‐
tetradecahydro‐1H‐cyclopenta[a]phenanthren‐3‐yl(2‐aminoethyl)carbamate) was prepared by a
method similar to that used by Sengupta et al. (2012). Briefly, ethylene diamine (6.26 ml, 93.7 mmol)
(2) was dissolved in 20 ml of anhydrous dichloromethane (DCM) and cooled to 0 °C. Cholesteryl
chloroformate (1) (3.0 g, 6.68mmol) in 20 ml of anhydrous DCM was added to the mixture and the
reaction vigorously stirred for 18h. The contents were then washed with water (50 ml x 3) and brine
(
50 ml x 2). The final DCM layer was dried over anhydrous magnesium sulfate and the solvent
removed under reduced pressure to afford the product as a white powder (2.7g, 85%).
.1.2 Synthesis of Intermediate compound 2,5‐dioxopyrrolidin‐1‐yl methacrylate (6)
2
Page 6 of 27

To a 25 mL round‐bottom flask were added 100 mg of methacrylic acid (4) (1.16 mmol), 160 mg of N‐
hydroxysuccinimide (5) (1.39 mmol), and 598 mg of dicyclohexylcarbodiimide (2.9 mmol). After 8 mL
of dry acetonitrile was injected, the mixture was stirred under nitrogen for about 16‐24 h at 25 °C.
The whole process was monitored by TLC. After reaction, the suspension was filtered and the
solution was evaporated by rotavapor under highly reduced pressure. The residue was purified by
silica column chromatography (Exane/EtOAc, gradient from 4:1 to 3:2). The obtained compound was
recrystallized from EtOAc and produced 200 mg of white powder (6). Yield: 86 %.
2
.1.3. Synthesis of Cholesteryl monomer (7)
(
10,13‐dimethyl‐17‐(6‐methylheptan‐2‐yl)‐2,3,4,7,8,9,10,11,12,13,14,15,16,17‐tetradecahydro‐1H‐
cyclopenta[a]phenanthren‐3‐yl(2methacrylamidoethyl)carbamate) (7). A solution of 6 (100 mg, 0.54
mmol) in dry dichloromethane/DMF (1/1, 15 mL) under nitrogen was treated with 3 (258 mg, 0.54
mmol). The reaction mixture was stirred for 1 h at room temperature and the solvent removed
under reduced pressure. Ethyl ether (60 mL) was added to the residue, washed with water (3 x 10
mL), brine (2 x 10 mL), dried over sodium sulphate and concentrated to dryness. Purification by silica
gel chromatography eluting with 1:1 Exane/EtOAc gave 7 as a white solid (243 mg, 82%).
2
.1.4 Synthesis of Decyl Methacrylate Monomer (9)
Monomer 9 was synthesised following a previously published method (Yildiz et al. 2011). 250 mg of
methacrylic acid 4 (2.9 mmol), were added to 5 ml of 1‐decanol 8 and sulfuric acid in catalytic
amounts. The solution was stirred for 48 h at room temperature. After reaction the mixture was
purified by silica gel chromatography eluting with Exane/EtOAc, gradient from 100% Exane to 95:5 to
give compound 9 in quantitative yield.
2
.2 Synthesis of Amphiphilic Polymers (11 – 16)
Methacrylate monomers, comprising different molar ratios of 7 and 9, were added to a Carius
reaction vessel together with poly (ethylene) glycol methacrylate (10) in anhydrous tetrahydrofuran
according to the molar ratios described in Table 2. Approximately 0.3 molar ratio of free radical
Page 7 of 27

generator 1,1’Azobis (cyclohexanecarbonitrile) (AICN): polymer was also added to the vessel. After
three consecutive freeze‐pump‐thaw cycles the reaction vessel was sealed under vacuum at 80 °C
for72 hours. THF (20mls) and hexane (20mls) were added and the contents centrifuged at 6000rpm
for 5 min, the supernatant was discarded and the resulting pellet re‐dissolved in THF and hexane.
This was repeated 3 times.
The resulting pellet was dried under vacuum to leave the resultant amphiphilic co‐polymers (11 ‐ 16)
as an oil.
2
2
.3 Product Characterisation
.3.1 Characterisation by Nuclear Magnetic Resonance (NMR)
1H Nuclear Magnetic Resonance was carried out on all products synthesised. Samples were prepared
using 10mgs of sample dissolved in 1 ml of deuterated chloroform and analysed using a Varian
5
2
1
00MHz Instrument.
.3.2 Characterisation of monomers by Mass Spectrometry (MS)
mg of sample was added to a glass vial with equal amounts of DCM and methanol. The sample was
diluted with absolute methanol prior to measurement. The measurement occurred in positive
electrospray mode via direct infusion.
2
.4 Preparation of Polymersomes
Six polymersomes were prepared using the polymer ratios described in Table 2. They were prepared
by creating a thin film (1mg) of a particular co‐polymer (11 – 16) in chloroform (CHCl ) (phase 1) then
3
adding 100 µL 2.5mg/ml Fitc‐CM‐Dextran in phosphate buffered saline (PBS). The mixture was
evaporated to dryness, purged with compressed air for 30 sec., before being suspended in 1.5‐2ml
dry chloroform. A further 1mL 2.5mg/ml of the identical polymer used in phase 1 but in PBS (phase
2
) was added and the solution sonicated using a Branson 3510 bath sonicator (230V) at room
Page 8 of 27

temperature for 2‐5 minutes. The organic solvent was removed from the emulsion under reduced
pressure at 40 ⁰C.
2
.5 Evaluation of Polymersomes
2
.5.1 Hydrodynamic radius determined by Dynamic Light Scattering (DLS)
The polymersome solutions were suitably diluted to analyse the particle size distribution and zeta
potential (+/‐ mV) using dynamic light scattering (DLS). The samples were diluted using sterile PBS
and all measurements taken at room temperature on a Malvern Nano‐ZS zetasizer using a
glass/quartz cuvette. The hydrodynamic radius and zeta potential are quoted as size/charge ±
standard error of the mean.
2
.5.2. Scanning electron Microscopy (SEM)
Blank polymersomes were prepared from polymer 13 and added onto aluminium metal stubs in
aqueous solution. Samples were then left covered to air dry before being sputter coated with gold
and palladium. The images were recorded using a FEI Quanta SEM under a high vacuum in secondary
electron mode.
2
.5.3. Determination of Encapsulation Efficiency (%EE)
Encapsulation Efficiency was carried out by preparing polymersomes using the method above and
encapsulating FITC‐CM‐ Dextran. Polymersome solutions were diluted to 5ml with PBS and the
sample placed in pre‐purged Amicon filters (MWCO 30kDa) and centrifuged at 10000 x g for
3
0minutes at 4ºC. The samples were analysed using a Varian Cary Eclipse fluorescence spectrometer
using 1 cm quartz cells with an excitation wavelength of 490 nm, slit width of 5 nm and collecting the
emission between 520 and 620 nm. A standard calibration graph was prepared with the equation of
the line:
ꢀ
ꢁ 21366ꢂ
where y = spectral area between 510 and 700 nm and x = concentration of
fitc‐CM‐dextran (µM)
Page 9 of 27

The % encapsulation efficiency was determined from the following equation:
ꢃꢄꢅꢆꢇꢈꢆꢉꢊꢇꢋ
ꢍ 100ꢎ%
where FD = fluorescence intensity of Fitc‐CM‐Dextran following
ꢃꢌꢊꢆꢇꢈꢆꢉꢊꢇꢋ
centrifugation and F0= fluorescence intensity of Fitc‐CM‐Dextran
added
2
.5.4. Release of FITC CM Dextran from Polymersomes
Polymersomes loaded with FITC‐CM‐Dextran (1.25 mg) were prepared from polymer 13 using the
method described previously in section 2.4. Any un‐encapsulated fluorophore was removed by
centrifugation at 10000 x g using a pre‐purged Amicon filter (MWCO 30 kDa) for 30 min at 4 °C.
Aqueous polymersomes of known FITC concentration (determined by fluorescence) were made up
to a volume of 0.5ml using PBS and placed in dialysis tubing (MWCO 12kDa) in 20mls of PBS at 37 °C
stirring and covered in foil. Measurements of fluorescence were taken at intervals over a 24 h period
under sink conditions and analysed in the method described above using the Cary Eclipse
fluorescence spectrophotometer.
2
2
.6. In‐Vitro Evaluation of Polymersomes
.6.1 Uptake of encapsulated Fitc‐CM‐dextran into HeLa cells
Hela cells were cultured in Hams F‐12 nutrient mixture and supplemented with Foetal Bovine Serum
1
1
(
10 %), penicillin (200 U mL ), streptomycin (200 µg mL ) and glutamine (2 mM). After reaching
confluence, the cells were harvested by trypsinization and seeded in a 96 well plate at a density of
000 cells per well and left overnight at 37 °C with 5 % CO , 20 % O . Cells were then washed twice
5
2
2
with 100 µl PBS and 160 µl of fresh media added. Cells were spiked with relative controls (PBS and
free Fitc‐CM‐ Dextran) and polymersome solutions containing 0.1 mg/mL Fitc‐CM‐dextran in 0.7
mg/ml polymersome and incubated for a further 5 h at the conditions stated above. The aqueous
content was then removed and each well washed twice with 200 µl of PBS and replaced with a
Page 10 of 27

further 200 µl of PBS before fluorescence was recorded using a BMG Labtech Omega micro plate
reader (excitation wavelength 485 nm and emission collected at 520 nm). Fluorescence values then
used to work out concentration of FITC taken up by cells by using standard calibration curves
established for free FITC Dextran and FITC encapsulated inside each polymersome.
2
.6.2. Confocal Imaging to determine uptake mechanism
Hela cells were cultured in Hams F‐12 nutrient mixture and supplemented with Foetal Bovine Serum
1
1
(
10 %), penicillin (200 U mL ), streptomycin (200 µg mL ) and glutamine (2 mM). After reaching
4
–1
confluence, the cells were harvested by trypsinization and seeded at a density of 2 × 10 cells mL in
a 96‐well plate. The cells were incubated at 37 °C with O /CO /air (20:5:75, v/v/v) overnight and then
2
2
in the presence of either free DOX‐HCl or polymersomal DOX‐HCl (14.4 ug/ml) for 5 h, following
which time the media was removed and each well washed twice with 200 µl PBS (pH = 7.2) with 180
µl media replaced along with 20 µl CellLight® Reagent BacMam 2.0 GFP and the well plate incubated
overnight in the conditions described above. The images were recorded on an inverted Leica SP5
confocal microscope, using an excitation wavelength of 470 nm and collecting the emission between
4
3
3
94 and 560 nm.
. Results and Discussion
.1 Characterisation of monomer 7
Monomer 7 was synthesised according to the procedure shown in Scheme 1. Cholesteryl
chloroformate was first reacted in a 1:14 stoichiometry with ethylene diamine in an overnight
reaction to yield the corresponding amide (3). The structure of this intermediate compound was
1
confirmed by MS ([M+H] = 474.3 m/z) and H NMR (data displayed in Figure 2). The additional peaks
at 2.8 and 3.2 ppm, not present in the precursor compound 1 were attributed to the methylene
protons associated with the new ethyl chain bridging the amide and amine functionalities. In
addition, the proton on position C‐2 of the steroidal cyclopentanoperhydrophenanthrene ring was
Page 11 of 27

shifted upfield from 4.7ppm to 4.5 ppm indicative of the increased shielding provided by the amine
that replaced the acid chloride. Compound 3 was then reacted with compound 6 in dry organic
1
solvent at room temp for 1h. The successful synthesis of 7 was again confirmed using MS and H
NMR spectroscopy. The base peak at 564 Da was consistent with the sodium adduct of the
+
+
1
molecular ion i.e. M + 23 (Na ion). The H NMR spectrum is shown in Figure 2. These data confirm
product formation with the olefinic protons of the methacrylate moiety clearly observed as
diastereotropic peaks at 5.36 and 5.76 ppm with the additional methyl protons from the methacrylic
unit as a singlet at 3.19ppm. The detailed NMR characterisation of compound 7 can be seen in Figure
3
with the corresponding peak allocation in Table 1.
3
.2. Characterisation of Polymers 11 – 16
The polymers were synthesised according to scheme 2 with the specific molar ratios employed given
1
in Table 2. The polymers were characterised using H NMR as shown in Figure 4. Figure 4 shows the
stacked spectra of the three individual monomers (7, 9 and 10) in addition to polymer 13 and clearly
indicates successful polymer formation. All the olefinic resonances present at ~5.4 and ~6.0 ppm in
each of the monomers are absent in the polymer spectrum indicating the formation of a new
carbon‐carbon sigma bond. In addition, the spectrum of the polymer contains peaks corresponding
to each of the monomers. Moreover, the resonances in the up‐field region are much broader in the
polymer spectrum than for the corresponding monomers. Such peak broadening is common for
protons in or near the backbone of polymers and is due to an averaging of the chemical shift
anisotropies. The spectrum of 13 is also dominated by the large characteristic PEG protons at ~3.5
ppm due to the high molar incorporation of this monomer within the polymer chain.
3
3
.3 Characterisation of Polymersomes
.3.1 Dynamic Light Scattering (DLS)
Page 12 of 27

The polymersome formulations all had a hydrodynamic radius of < 600 nm rendering them suitable
for cellular uptake via endocytosis. There was a general increase in size with increasing cholesteryl
2
content, through linearity was poor with an R of 0.78. A significant increase in hydrodynamic radius
was observed for polymersome 16 (Table 3). This particular polymersome contained no carbon‐10
chains, only PEG and cholesteryl, thus implying that the alkyl chain plays a significant role in the
interdigitation of the polymersome bilayer. We have previously shown polymersomes to be size
tuneable using this method of preparation however, for this study all parameters were kept
consistent so as to allow a direct comparison between the hydrodynamic radius of polymersomes.
Contrary to our results, the incorporation of cholesterol into polymer domains has been
documented to cause reduced membrane thickness of synthesised nanoparticles (Huang et al. 2013;
Magarkar et al. 2012; Yin et al. 2014). However, when incorporated into polymersomes it would
appear to have the opposite effect, perhaps due to the small size of the polymersomes to begin
with. Size is hugely important in ensuring polymersomes can reach and be internalised into target
cells, if too small they will be unable to encapsulate significant amounts of active agent and if too
large they may be quickly cleared by renal excretion and have issues crossing the cell membrane
(Tanner et al. 2011; Jaskiewicz et al. 2012). It has been considered that polymersomes with a
diameter of around 100nm are the most suitable for drug carrier systems due to their ability to
evade detection by RES and an appropriate size for retention in tumour vasculature by the EPR
effect (Jia et al. 2014). All polymers produced polymersomes of desired nano‐size with good
polydispersity.
Polydispersity Index (PDI) was measured for each of the polymersome samples. Results ranged
between 0.37 ‐ 0.76, this indicates a relatively good degree of polydispersity. However, some degree
of aggregation of particles is implied and again in particular for polymer 16, this could suggest that
rather than a large increase in polymersome hydrodynamic radius, instead we are seeing an increase
in aggregation between polymersomes or the creation of cholesteryl domains.
Page 13 of 27

3
.3.2. Encapsulation Efficiencies (% EE)
In order to establish the encapsulating ability of polymersomes prepared from polymers 11 ‐16, the
fluorescent probe Fitc‐CM‐Dextran (4 kDa) was used as a hydrophilic model dye which accumulates
in the aqueous core of the system at a loading capacity of 12.5 % wt:wt (wt of fluorophore: total DDS
wt). As can be seen from Table 3, the encapsulation efficiencies of all the polymersomes tested did
not vary significantly, this is as expected due to the hydrophilic component of these DDS remaining
unchanged. The PEG is responsible for the creation of the inner hydrophilic core of this system, and
it is anticipated that any hydrophilic component will reside within this hydrophilic environment. The
results detailed in table 3 ranged between 52 – 60 % encapsulation efficiency. These results are in
keeping with a previous study where we determined the encapsulation efficiency of polymersomes
prepared from polymer 11 to have a % EE of 53 % at 12.5 % wt: wt loading. We have also shown that
this % EE can be increased to > 70 % if the amount of drug loading is significantly reduced (Martin et
al. 2015).
3
.3.3. FITC Dextran Release from Polymersomes
Release of hydrophilic fluorescent Fitc‐CM‐dextran from polymersomes was established under sink
conditions at 37 °C. A maximum release rate was observed within the first hour but this burst release
then gave rise to a more constant rate observed from 2 ‐ 24 hours. The percentage release value at
2
4 hours was 56 % (± 13.5) (Figure 5).
3
.3.4 Scanning Electron Microscope (SEM) Imaging
Blank polymersomes (i.e. no drug encapsulated) prepared from polymer 13 were analysed by SEM.
Figure 6). The images reveal several clusters of sizes in the region of 100 nm to 200 nm with all of
(
the polymersomes having the expected spherical morphology. The sizes of polymersomes observed
were in the expected range and correspond with the results from DLS which show the same
polymersome to have a hydrodynamic radius of 197 nm.
Page 14 of 27

3
3
.4. In‐Vitro Evaluation of Polymersomes
.4.1. Cellular Uptake of Polymersomes with Varying amounts of Cholesterol
Cellular uptake was established in Hela cells by encapsulating Fitc‐CM‐Dextran (MW = 4KDa) into the
hydrophilic core of the polymersomes. Fitc‐CM‐Dextran was chosen as it is known to have poor
cellular uptake due to its high water solubility and negative charge. All polymersomes were prepared
as described with the dye incorporated within the core. Following incubation the media was
removed and the cells washed with PBS before being analysed for intracellular Fitc‐CM‐Dextran
content. The results of this study are shown in Figure 7 and have been compared to free FITC‐CM‐
Dextran. Polymersomes formulated with the 12% w/w polymer (polymer 13) showed the highest
uptake of Fitc‐CM‐Dextran by HeLa cells. This was more than double the uptake observed with any
of the other polymersomes containing cholesteryl and a ten‐fold increase on the cellular uptake of
un‐encapsulated Fitc‐CM‐Dextran. All of the polymersomal systems prepared displayed an enhanced
uptake when compared with the intracellular concentration of free Fitc‐CM‐Dextran within the HeLa
cells (0.297 µg/ml). The lowest cellular uptake was observed for the polymersome containing no
decyl units (prepared from polymer 16), which may be due to the rigidity of the polymersome and
greater hydrodynamic radius than any of the other prepared DDSs or the potential formation of
aggregates preventing endocytosis. Indeed, this only serves to highlight the significance of tailoring
and fine tuning the polymers prior to formulation into the DDS.
3
.4.2. Confocal Imaging to determine uptake mechanism
In order to confirm the mechanism of cellular uptake, the late stage endosomal green fluorescent
protein stain (CellLight® Reagent BacMam 2.0 GFP) was incubated in Hela cells alongside a
polymersome formulated from polymer 13 that contained DOX‐HCl. DOX‐HCl was chosen as its
absorbance and emission spectra enable simultaneous detection of both the encapsulated drug as
well as the GFP endosomal stain. When comparing the bright field images of the cells with no DOX‐
HCl (A) to that of the free DOX‐HCl (D) and polymersomal DOX‐HCl (G), there is a clear difference in
Page 15 of 27

the number of living cells (Figure 8). This is not unsurprising given the toxicity of DOX‐HCl to the HeLa
cell line. When we consider the DOX‐HCl emission (560 – 620nm) from images B, E and H there
appears to be a difference in the morphology of the cells that were exposed to free DOX‐HCl (E)
when compared to the polymersomal DOX‐HCl (H). The cells exposed to free DOX‐HCl have only
minimal evidence of any late stage endosomes, whereas those exposed to the polymersomal DOX‐
HCl display a significant amount of late stage endosomes in all cells. This confirms that the
mechanism of transport between the two forms of DOX‐HCl is very different, with the polymersomal
system transcending the cellular membrane via endocytosis whereas the free DOX‐HCl does not. The
mechanism of transport for the free DOX‐HCl is most likely to be passive diffusion given the
relatively low molecular weight of the drug.
4
. Conclusion
In conclusion, the present study successfully demonstrates how cholesterol can be incorporated
within polymersomal drug delivery systems to significantly increase cellular uptake. By the
preparation and analysis of polymersomes containing between 0 and 39 % wt/wt cholesteryl we can
conclude that a 12% wt /wt incorporation leads to a ten‐fold increase in cellular uptake of
encapsulated drug within Hela cells. In addition to the improved cellular uptake we have shown that
the mechanism of transport of these systems is via endocytosis. The size of the DDSs increases with
increasing cholesteryl content, and the presence of flexible ligands within the random co‐polymer,
such as the decyl chain, is required to enable the size of the polymersome to remain > 300nm. This
data suggests that polymersomes are yet to reach their full capabilities in drug delivery systems and
there is still much work required to reach their full potential.
Acknowledgements
The authors would like to thank the University of Ulster, the Department for Employment and
Learning (DEL) Northern Ireland and the Dowager Countess Eleanor Peel Trust (grant number JWP
1
5/05‐81) for their financial support.
Page 16 of 27

5
. References
Alibolandi, M. Sadeghi, F. Sazmand, S.H. Shahrokhi, S.M. Seifi, M. Hadizadeh, F. Synthesis and self‐
assembly of biodegradable polyethylene glycol‐poly (lactic acid) diblock copolymers as
polymersomes for preparation of sustained release system of doxorubicin, Int. J. Pharm.
Investig. 5 (2015) 134–41.
Bangham, A. Liposomes ‐ the Babaham connection, Chem. Phys. Lipids. 64 (1993) 275–285.
Bartfai, T. Lees, G. V. Drug Discovery: From Bedside to Wall Street, Academic Press, 2010.
Dahan, A. Miller, J.M. Amidon G.L., Prediction of solubility and permeability class membership:
provisional BCS classification of the world’s top oral drugs, AAPS J. 11 (2009) 740–6.
Fomina, N. Sankaranarayanan, J. Almutairi, A. Photochemical mechanisms of light‐triggered release
from nanocarriers, Adv. Drug Deliv. Rev. 64 (2012) 1005–20.
Gandhi, N.S. Tekade, R.K. Chougule, M.B. Nanocarrier mediated delivery of siRNA/miRNA in
combination with chemotherapeutic agents for cancer therapy: current progress and
advances, J. Control. Release. 194 (2014) 238–56.
Huang, Z. Teng, W. Liu, L. Wang, L. Wang, Q. Dong. Y.Efficient cytosolic delivery mediated by
polymersomes facilely prepared from a degradable, amphiphilic, and amphoteric copolyer.
Nanotech. 24 (2013) 265104.
Jaskiewicz, K. Larsen, A. chaeffel, D. S Koynov, K. Lieberwirth, I. Fytas, G. Landfester, K. Kroeger, A.
Incorporation of nanoparticles into polymersomes: size and concentration effects, ACS Nano.
6
(2012) 7254–62.
Jia, L. Cui, D. Bignon, J. Di Cicco, A. Wdzieczak‐Bakala, J. Liu, J. Li MH. Reduction‐responsive
cholesterol‐based block copolymer vesicles for drug delivery, Biomacromolecules. 15 (2014)
2
206–17.
Page 17 of 27

Khan, F. Katara, R. Ramteke, S. Enhancement of bioavailability of cefpodoxime proxetil using
different polymeric microparticles, AAPS PharmSciTech. 11 (2010) 1368–75.
Kolli, S. Wong, S.‐P. Harbottle, R. Johnston, B. Thanou, M. Miller A.D. pH‐triggered nanoparticle
mediated delivery of siRNA to liver cells in vitro and in vivo, Bioconjug. Chem. 24 (2013) 314–
3
2.
Lee, J.C. Bermudez, H. Discher, B.M. Sheehan, M.A. Won, Y.Y. Bates, F.S. Discher, D.E. Preparation,
stability, and in vitro performance of vesicles made with diblock copolymers, Biotechnol.
Bioeng. 73 (2001) 135–45.
Lee, J.S. Feijen, J. Polymersomes for drug delivery: design, formation and characterization., J.
Control. Release. 161 (2012) 473–83.
Lin, J.J. Silas, J.A. Bermudez, H. Milam, V.T. Bates, F.S. Hammer, D.A. The effect of polymer chain
length and surface density on the adhesiveness of functionalized polymersomes, Langmuir.
2
0 (2004) 5493–500.
Magarkar, A. Karakas, E. Stepniewski, M. Róg, T. Bunker, A. Molecular dynamics simulation of
PEGylated bilayer interacting with salt ions: a model of the liposome surface in the
bloodstream, J. Phys. Chem. B. 116 (2012) 4212–9.
Martin, C. Aibani, N. Callan, J.F. Callan, B. Recent advances in amphiphilic polymers for simultaneous
delivery of hydrophobic and hydrophilic drugs, Ther. Deliv. 7 (2016) 15–31.
Martin, C. Dolmazon, E. Moylan, K. Fowley, C. McHale, A.P. Callan, J.F. Callan, B. A charge neutral,
size tuneable polymersome capable of high biological encapsulation efficiency and cell
permeation, Int. J. Pharm. 481 (2015) 1–8.
Miyata, K. Christie, R.J. Kataoka, K. Polymeric micelles for nano‐scale drug delivery, React. Funct.
Polym. 71 (2011) 227–234.
Page 18 of 27

Morrow, T. Felcone, L.H. Defining the difference: What Makes Biologics Unique, Biotechnol. Healthc.
(2004) 24–9.
1
Pasparakis, G. Manouras, T. Vamvakaki, M. Argitis, P. Harnessing photochemical internalization with
dual degradable nanoparticles for combinatorial photo‐chemotherapy, Nat. Commun. 5
(
2014) 3623.
Sengupta, P. Basu, S. Soni, S. Pandey, A. Roy, B. Oh, M.S. Chin, K.T. Paraskar, A.S. Sarangi, S. Connor,
Y. Sabbisetti, V.S. Kopparam, J. Kulkami, A. Muto, K. Amarasiriwardena, C. Jayawardene, I.
Lupoli, N. Dinulescu, D.M. Bonventre, J.V. Mashelkar, R.A. Sengupta, S.Cholesterol‐tethered
platinum II‐based supramolecular nanoparticle increases antitumor efficacy and reduces
nephrotoxicity, Proc. Natl. Acad. Sci. 109 (2012) 11294–11299.
Tanner, P. Baumann, P. Enea, R. Onaca, O. Palivan, C. Meier, W.Polymeric vesicles: from drug carriers
to nanoreactors and artificial organelles, Acc. Chem. Res. 44 (2011) 1039–49.
Wu, S.Y. McMillan, N.A.J. Lipidic systems for in vivo siRNA delivery, AAPS J. 11 (2009) 639–52.
Xu, L. Wempe, M.F. Anchordoquy, T.J. The effect of cholesterol domains on PEGylated liposomal
gene delivery in vitro, Ther. Deliv. 2 (2011) 451–60.
Yildiz, I. Impellizzeri, S. Deniz, E. McCaughan, B. Callan, J.F. Raymo, F.M.Supramolecular strategies to
construct biocompatible and photoswitchable fluorescent assemblies, J. Am. Chem. Soc. 133
(
2011) 871–9. doi:10.1021/ja107341f.
Yin, H. Kang, H.C. Huh, K.M. Bae, Y.H. Effects of cholesterol incorporation on the physicochemical,
colloidal, and biological characteristics of pH‐sensitive AB₂ miktoarm polymer‐based
polymersomes, Colloids Surf. B. Biointerfaces. 116 (2014) 2128–37.
Zhi‐Yao, H. Bing‐Yang, C. Via‐Wei, W. Jiao, L. Edwards, C.K. Xiang‐Rong, S. Gu, H. Yong‐Mei, X. Zhi‐
Yong, Q. Recent development of poly(ethylene glycol)‐cholesterol conjugates as drug delivery
systems, Int. J. Pharam. 469 (2014) 168‐ 178.
Page 19 of 27

Figure 1. Polymersome formation with incorporated cholesteryl
Figure 2. Stacked 1H NMR (in CDCl ) of starting material (1), intermediate (3) and monomer
3
5
.
Page 20 of 27

Figure 3. Monomer 7 with specific protons identified for NMR analysis listed alphabetically
from a – j.
Figure 4. Stacked NMR of polymer 13 and monomers 7, 9 and 10.
Page 21 of 27

Figure 5. In-vitro release profile of Fitc-CM-dextran from polymersome prepared from
0
polymer 13 carried out under sink conditions at 37 C.
Figure 6. SEM images of polymersome prepared from polymer 13
Page 22 of 27

Figure 7. Hela cellular uptake of polyrmersomes encapsulating Fitc-CM dextran with varying
amounts of cholesteryl compared with free Fitc-CM-dextran
Figure 8. Confocal images of Hela cells incubated with either no drug (A-C), DOX-HCl (D -E)
or polymersomal DOX-HCl (G-H). All images have been incubated with CellLight® Reagent
BacMam 2.0 GFP as a late stage endosomal stain. Images A D and G are bright field
images. All remaining images are fluorescence emission with an excitation wavelength of
4
74 and 480 nm, emission collected between 490 and 540 (C,F,I) and 560 – 620 (B, E,H).
Page 23 of 27

Scheme 1. Synthesis of monomer 7
Page 24 of 27

Scheme 2. Synthesis of polymer 14 (for illustration purposes; exact structure may vary).
Page 25 of 27

Table 1. Spectral position and confirmation of specific NMR peaks aligned to Figure 3.
Spectral shift
ppm)
No. of
protons
multiplicity
Assigned H
Proton assigned
from Figure 3
(
NHCH CH NH
6
5
5
5
4
.61
1
2
1
1
1
s
d
s
s
s
2
2
i
C=CH2
.73 and 5.36
h
c
j
‐
C=CHCH ‐C
.29
.01
.50
2
‐
CH CH NHCOO‐
2 2
‐
(‐CH ‐) ‐ CHCO
2 2 ‐
d
NH CH CH NHCOO‐
3
3
.43‐3.29
.36‐3.20
2
2
m
m
2
2
2
e
f
NH CH CH NHCOO‐
2
2
2
3
2
.19
3
s
‐CH C=CH
g
3
2
.32‐0.67
39
broad
Steroidal backbone
CH CH CH CH(CH )
3 2
b
0
.67
6
s
a
2
2
2
Table 2. Different molar ratios of cholesteryl and decyl used to create polymers and
subsequent w/w values
Polymer
number
Molar ratio*
%w/w
decyl
cholesteryl
decyl
0.6
cholesteryl
PEG
79
76
73
69
64
61
1
1
1
1
1
1
1
2
3
4
5
6
0
0
21
18
15
9
0.075
0.15
0.3
0.525
0.45
0.3
6
12
22
31
39
0.45
0.6
0.15
0
4
0
*
molar ratio compared to 1Meq PEG
Page 26 of 27

Table 3. Size, PDI and encapsulation efficiency of varying polymers
Polymer number Size (nm)
PDI
% encapsulation
efficiency
1
1
1
1
1
1
1
2
3
4
5
6
177 ±6.6
103 ± 5.5
197 ± 7.4
295 ± 60
299 ± 53
586 ± 47
0.53 ± 0.08
0.76 ± 0.13
0.37 ± 0.15
0.39 ± 0.10
0.53 ± 0.03
0.76 ± 0.13
52 ± 10
60 ± 12
59 ± 3
ND
ND
ND
ND = Not determined n = 3
Page 27 of 27