Multi-Ligand Multi-Drug pHsensitive Polymersomes to

Treat Ovarian Cancer

BE C183/283
Naveen Bains
Nico Etcheverry
Calvin Lee
Richard Seo
March 14, 2014

Abstract
Ovarian cancer is the unregulated cell growth of ovarian cells leading to the formation of tumors which can
eventually metastasize and spread to other areas of the body. Each year, approximately 22,000 patients are
diagnosed with ovarian cancer and about 14,000 women are killed from the disease making it the fifth highest
cause of cancer-related death in women. The cancer is clinically categorized into four different stages
according to how far the cancer has spread from the ovaries, with the survival rate diminishing at each
increasing stage. Although surgery is a possible option to remove localized ovarian tumors at early stages, the
most common treatment for advanced cancers is the intravenous administration of chemotherapeutics.
Chemotherapeutic drugs act by directly killing the cancer cells through a variety of different mechanisms, such
as intercalating with DNA to inhibit DNA replication (doxorubicin) or inhibiting mitotic cell division by preventing
the disassembly of microtubules (paclitaxel). Unfortunately, these drugs by themselves are inherently nonspecific and also kill a large number of healthy cells inducing a wide range of unpleasant side effects for the
patient such as nausea, hair loss, neuropathy, fatigue, and increased risks of infection. Increasing the
specificity of chemotherapeutic agents to reduce these side effects is an important challenge in treating cancer.
We therefore propose the design of a novel polymersome nanoparticle coated with a combination of folate,
luteinizing hormone-releasing hormone (LHRH), and APRPG peptide targeting ligands to specifically target
receptors overexpressed on ovarian cancer cells and deliver the encapsulated chemotherapeutic drugs
doxorubicin and paclitaxel. This proposal will develop a novel triple ligand bearing polymersome for the specific
targeting of ovarian cancer cells. This novel drug delivery device will improve upon existing drug delivery
methods because it uses multiple ligands for targeting and decreasing systemic delivery of the drugs to healthy
tissues. The potential synergistic combinatorial effect of using all three ligands should further decrease the
possible targeting of other tissues that express the receptors. The multi-drug facet of the nanocarrier will allow
for attacking different growth mechanisms of the tumors, further decreasing tumor growth and metastasis. The
proposed experiments in this research proposal should provide better insight with regards to optimal dosing,
ligand surface density, and polymer concentration for increased antitumor efficiency, biodistribution, specified
cytotoxicity, and degradability of the polymersome.

Multi-Ligand Multi-Drug pH-sensitive Polymersomes to Treat Ovarian Cancer

Specific Aims
Ovarian cancer is the unregulated cell growth of ovarian cells leading to the formation of tumors which can
eventually metastasize and spread to other areas of the body. Each year, approximately 22,000 patients are
diagnosed with ovarian cancer and about 14,000 women are killed from the disease making it the fifth highest
cause of cancer-related death in women.(1) The cancer is clinically categorized into four different stages
according to how far the cancer has spread from the ovaries, with the survival rate diminishing at each
increasing stage. Although surgery is a possible option to remove localized ovarian tumors at early stages, the
most common treatment for advanced cancers is the intravenous administration of chemotherapeutics.
Chemotherapeutic drugs act by directly killing the cancer cells through a variety of different mechanisms, such
as intercalating with DNA to inhibit DNA replication, in the case of doxorubicin, or inhibiting mitotic cell division
by preventing the disassembly of microtubules, in the case of paclitaxel. Unfortunately, these drugs by
themselves are inherently non-specific and also kill a large number of healthy cells inducing a wide range of
unpleasant side effects for the patient such as nausea, hair loss, neuropathy, fatigue, and increased risks of
infection. Increasing the specificity of chemotherapeutic agents to reduce these side effects is an important
challenge in treating cancer. We therefore propose the design of a novel polymersome nanoparticle coated
with a combination of folate, luteinizing hormone-releasing hormone (LHRH), and APRPG peptide targeting
ligands to specifically target receptors overexpressed on ovarian cancer cells and deliver the encapsulated
chemotherapeutic drugs doxorubicin and paclitaxel. We can achieve this proposal with the following specific
aims.
Specific Aim 1. Synthesize and characterize pH-sensitive polymersomes coated with folate, LHRH, and
APRPG targeting ligands and containing the drugs doxorubicin and paclitaxel.
1.1.
1.2.
1.3.
1.4.

Synthesize polyethylene glycol (PEG) - polylactic acid (PLA) block copolymers with either a folate
ligand, a LHRH ligand, an APRPG peptide ligand, or no ligand attached to the hydrophilic PEG chain.
Characterize the formation of block copolymers using NMR, FTIR, and mass spectrometry.
Synthesize polymersomes using the synthesized PEG-PLA block copolymers and load the
polymersomes with paclitaxel and doxorubicin.
Characterize the formation of polymersomes using DLS, zeta potential, optical microscopy, TEM, and
X-ray photoelectron spectra (XPS), and quantify the drug loading efficiencies using HPLC.

Specific Aim 2. Evaluate the effects of using combinations of ligands for targeting and determine the optimal
surface ligand density to specifically target ovarian tumor cells.
2.1.
2.2.

Determine the effects of single ligands versus combinations of ligands on tumor specificity and drug
biodistribution using in vivo models of mice with ovarian tumors.
Optimize the polymersome surface ligand densities to specifically target ovarian tumor cells without
major targeting to other organs and tissues.

Specific Aim 3. Evaluate and quantify the delivery of doxorubicin and paclitaxel to ovarian tumor cells and the
biocompatibility of the polymersomes.
3.1.
3.2.
3.3.

Quantify the uptake of the polymersomes and the release of doxorubicin and paclitaxel to tumor cells
using in vitro studies with ovarian cancer cell lines.
Optimize the polymersome parameters to release doxorubicin and paclitaxel only in the ovarian tumor
intracellular environment.
Quantify the cytotoxicity of the polymersomes, the block copolymers, and the degradation products.

If these aims are successful, this novel drug delivery device will improve upon existing drug delivery methods
because it uses multiple ligands for targeting and decreasing systemic delivery of the drugs to healthy tissues.
The potential synergistic combinatorial effect of using all three ligands should further decrease the possible
targeting of other tissues that express the receptors. The multi-drug facet of the nanocarrier will allow for
attacking different growth mechanisms of the tumors, further decreasing tumor growth and metastasis. The
proposed experiments in this research proposal should provide better insight with regards to optimal dosing,
ligand surface density, and polymer concentration for increased antitumor efficiency, biodistribution, specified
cytotoxicity, and degradability of the polymersome.

Research Strategy
Significance
Ovarian Cancer
Ovarian cancer is the deadliest cancer of the female reproductive system with current estimates from the
American Cancer Society stating that about 14,270 women will die from the disease in 2014. Furthermore, the
cancer is prevalent enough to the point where approximately 1.4% of all women will be diagnosed with ovarian
cancer at some point in their life.(1) Defined as unregulated cell growth originating in the ovaries, ovarian
cancer leads to the development of tumors that cause a variety of unpleasant symptoms including fatigue,
bloating, abdominal pain, difficulty eating, weight loss, constipation and back pain. Ovarian cancer is clinically
categorized by the extent that the cancer has spread from its local origin in the ovaries. When the cancer is
diagnosed early while it is still confined to the ovaries, patients have a 91.9% survival rate. Unfortunately, due
to the nonspecificity of the associated symptoms and the lack of an effective non-invasive detection method,
61% of ovarian cancer diagnoses are not made until the cancer has already metastasized and spread to other
areas of the body. At this point, the five-year survival rate drops significantly to only 27.3%.(2)
The three major treatment options for ovarian cancer include surgery, radiotherapy, and chemotherapy.
Gynecologic oncologists can attempt to treat the disease by performing an oophorectomy (removal of the
fallopian tube and ovary) or a hysterectomy (removal of the entire uterus) in order to physically remove the
cancerous tissues. However, such an invasive procedure will leave the patient unable to bear children and may
be futile if the cancer has already spread. Radiotherapy involves using radiation to damage DNA and kill
cancer cells, but this treatment is non-specific and will also kill healthy cells. In addition, there is a maximum
safe lifetime dosage of radiation which cannot be exceeded for any one area of the body. The remaining option
is chemotherapy, a treatment that utilizes drugs to kill cancer cells. Unfortunately chemotherapeutic drugs are
also non-specific and will end up killing healthy cells. This leads to multiple harmful side effects that can
significantly diminish the patients quality of life such as nausea, hair loss, neuropathy, fatigue, and an
increased risk of infection. Due to the late detection of most ovarian cancers and the side effects associated
with the non-specificity of chemotherapeutic drugs, there exists a significant need to develop a drug delivery
system to effectively and specifically kill ovarian cancer cells.
Utilizing Ligands in Receptor-Mediated Endocytosis
A common method to control the release of drugs involves encapsulating the drug in a nanoparticle. If the
nanoparticle contains a ligand for a particular cell-surface receptor, it will bind to the receptor and get taken into
the cell via receptor-mediated endocytosis. At this point, the nanoparticle is in an endosome within the cell
where the pH is lower than extracellular conditions, potentially triggering the release of the drug from the
nanoparticle depending on the engineering of the nanoparticle. Because of this, utilizing ligands that bind with
high affinity to receptors overexpressed on ovarian cancer cells can allow for increased spatial control of drug
release. We will attempt to take advantage of this to design a drug carrier that can more specifically target
cancer cells while minimizing the collateral damage done to healthy cells.
Often cancer cells overexpress certain receptors involved in bringing more nutrients as a result of their larger
metabolic requirements as they continue to grow. In the case of ovarian cancer, it has been found that ovarian
cancer cells overexpress folate receptors, luteinizing hormone-releasing hormone receptors, and the V3
integrin. Folate plays an important role in cell division due to its role as a coenzyme in the synthesis of
nucleotides which is required for DNA replication.(3) The V3 integrin is involved in tumor angiogenesis, a
process that leads to the development of new blood vessels to bring in more nutrients to the tumor, and it can
be targeted by the peptide APRPG.(4) Cheung et al. has made progress in elucidating the role of LHRH
receptors in the cell adhesion required as a first step in metastasis of ovarian cancer cells.(5) Additionally,
luteinizing hormone-releasing hormone receptors have been found to be involved in autocrine signaling to
induce cell proliferation. Approximately 80% of ovarian cancer cells have been found to overexpress LHRH
receptors.(6) Due to the overexpression of these three membrane receptors on ovarian cancer cells, we
hypothesize that a drug carrier coated with ligands for all three of these receptors will have a greater chance of
binding to an ovarian cancer cell, allowing the chemotherapeutic drugs to be specifically delivered to ovarian
carcinoma upon endocytosis.
We acknowledge that this increased specificity for ovarian cancer cells will not necessarily prevent the drug
carrier from similarly binding to and killing healthy cells. While these receptors are overexpressed in ovarian
tumors, they are still expressed to some extent in healthy cells. We hope to overcome this by running
experiments to determine the optimal density of ligands. One possibility is using a relatively low number of
ligands so that any one ligand has a low probability of binding to its particular receptor on a healthy cell, but all
1

three in synergy have a higher chance of binding to the ovarian cancer cell where all three receptors are
overexpressed. If experiments are successful, the ligand concentrations on our drug carrier can be fine-tuned
to effectively kill ovarian carcinoma while hopefully minimizing the harmful side effects associated with killing
healthy cells.
Chemotherapeutic Resistance
Through mechanisms that have not yet been fully elucidated, cancer cells can eventually become resistant to
certain chemotherapeutic drugs. For example, we will consider the resistance of ovarian cancer cells to the
drugs doxorubicin and paclitaxel. Paclitaxel acts by stabilizing microtubules and preventing them from
disassembling which is required for mitotic cell division. It has been shown that paclitaxel-resistant ovarian
cancer cells undergo mutations leading to the overexpression of class III -tubulin. This modified -tubulin is
inherently less stable and can overcome the stabilizing effects of paclitaxel, thereby diminishing its cytotoxic
effects.(7) Doxorubicin is another chemotherapeutic drug that acts by intercalating with DNA, inhibiting DNA
replication and inducing cell apoptosis. In the case of doxorubicin-resistant ovarian cancer cells, it has been
shown that they overexpress a membrane transporter called P-glycoprotein which pumps doxorubicin out of
the cell thereby preventing the drug from entering the nucleus and inducing its cytotoxic effects.(8) It would
follow that a cocktail treatment of multiple chemotherapeutic drugs may be effective, since if the cell line
develops resistance to the apoptotic pathway of one of the drugs, the other drug may still have a chance to kill
the cell through a different mechanism.
Polymersomes as Drug Carriers
For the specific drug carrier, we will utilize a block copolymer of polyethylene glycol (PEG) and polylactic acid
(PLA) to form a polymersome. The PEG group acts to increase the carriers circulation time by reducing its
clearance and degradation by the bodys immune system. The pH-sensitive PLA contains an ester group which
will be hydrolyzed at the lower pH of the endosomes, releasing the chemotherapeutic drugs contained within
upon disassembly of the polymersome. Additionally, such a polymersome will self-assemble in such a way that
it contains a hydrophilic core with a hydrophobic membrane. This allows for the loading of multiple drugs. In
our case, the relatively hydrophilic doxorubicin will be loaded into the core of the polymersome with the
hydrophobic paclitaxel loaded into the membrane. This delivery of multiple drugs can possibly help in killing
ovarian cancer cells despite their eventual development of resistance to specific apoptotic mechanisms as
mentioned earlier. Additionally, in the case of doxorubicin, utilizing a drug-carrier that is endocytosed gives the
drug a greater chance of reaching the nucleus and inducing its cytotoxic effect whereas the free drug that
relies only on passive diffusion is more suspectible to removal from the cell by the P-glycoproteins of
doxorubicin-resistant cells.(9)
A similar amphiphilic nanoparticle capable of delivering both hydrophobic and hydrophilic drugs is a liposome,
a vesicle made of lipids. The polymersome is advantageous over the liposome in our case since polymers
possess a wider range of tunable properties, such as functional groups and molecular weights, which can be
controlled during synthesis. Additionally, incorporating PEG into a liposome would require conjugation to the
lipids hydrophilic head whereas polymersome can incorporate PEG into every monomer comprising the
vesicle.(10) As a result, in our case polymersome synthesis will be more efficient.
We therefore propose the formation of a PEG-PLA polymersome that encapsulates the chemotherapeutic
drugs doxorubicin and paclitaxel and is coated with folate, LHRH, and APRPG. We hypothesize that such a
drug carrier can target ovarian cancer cells with increased specificity due to the multiple targeting ligands while
also killing the cancer cells more effectively due to the utilization of two drugs that induce cell apoptosis
through different mechanisms.
Innovation
In this proposal, we utilize multiple innovative techniques in treating ovarian cancer, including:
1.) The use of three different targeting ligands to specifically target multiple receptors overexpressed in
ovarian cancer cells.
2.) Modifying individual ligand functionalizing ratios and the drug carrier surface ligand density to reduce
targeting of non-cancer cells with receptors for these individual ligands, such as folate and liver cells.
3.) The co-delivery of multiple chemotherapeutic drugs through the use of an amphiphilic polymersome
decorated with multiple targeting ligands.

Approach
Preliminary Data / Previous Results
Individual ligand (LHRH, APRPG, folate)
specificities
Previous work has shown the specificities
of LHRH, APRPG, and folate receptors
with regards to various organs and tissues
(b)
in the body. Previous studies have shown (a)
that all of these ligands are specific for
ovarian tumors. However, each ligand
also has varying affinity levels towards
other tissues. Based on an in vivo study
conducted on mice with xenografts of
A2780 human ovarian carcinoma, LHRH
can also target healthy ovaries due to
endogenous LHRH receptor expression in
ovaries.(11) Folate receptor expression
was investigated for several mammalian
(c)
(d)
species and for both healthy tissues and Figure 1: (a) LHRH biodistribution results. (b) APRPG biodistribution results. (c)
carcinomas
in
humans
using
a Folate receptor biodistribution results for healthy mouse and humans. (d)
quantitative radioligand binding assay. Folate receptor biodistribution results for human healthy tissues and
This study shows that folate receptor carcinoma.
levels are relatively high in the kidneys for multiple species, but that they are high in the lungs only for
humans.(12) APRPG peptide was investigated using in vivo imaging using fluorescence probes attached to
nanoparticles injected into SKOV3 cancer-bearing mice. These results show that APRPG has relatively high
affinity towards the liver and minor affinity towards the spleen.(4) These results can be seen in Figure 1.
Characterization of PEG-PLA nanoparticles loaded with paclitaxel and doxorubicin
Previous work has been done for regular PEG-PLA polymersomes without targeting ligands. The previous
results show that the final polymersomes had diameters around 100 nm and membrane thicknesses around 10
nm. The ideal loading capacity of paclitaxel was 1 mole of drug to 5 moles of copolymers, which was a
concentration of approximately 140 M. The ideal loading capacity of doxorubicin was 1 mole of drug to 1 mole
of copolymers, which was a concentration of approximately 80 M.(9)
Development of Single-Ligand Bearing Polymersomes and Micelles
A lot of research has been conducted involving the surface functionalization of polymersomes or micelles to
target specific receptors on tumor cells. Previous work has shown that a cysteine modified APRPG conjugated
to PEG-PLA polymersomes can successfully target receptors expressed during angiogenesis and efficiently
reduce ovarian tumor size.(4) With regards to targeting cancer cells, a plethora of studies have been
conducted involving the use of folate as a targeting receptor, and its attachment to cross-linked biodegradable
micelles resulted in increased cytotoxicity to KB carcinoma cell lines.(13) The attachment of this ligand has
been well documented and has help reduced tissue toxicity of healthy organs. There are very few studies
applying multiple ligand polymersome structures at the moment, and innovating in this area of drug delivery
may lead to enhanced targeting and even lower systemic toxicity.
Experimental Plan
Specific Aim 1
The PEG-PLA diblock copolymers must first be synthesized with their respective ligand molecules. First, the
base PEG-PLA diblock copolymers are synthesized using ring-opening polymerization. These copolymers are
then conjugated to LHRH, folate, or APRPG ligands using various conjugation chemistry techniques. The
reagents necessary for the reactions can be purchased from chemical manufacturers, like Sigma Aldrich or
Fisher Scientific, unless otherwise stated.
Research groups in the past have successfully synthesized X-PEG-PLA diblock copolymers, where X is either
a maleimide, methoxy, or carboxyl group. Synthesis of these various copolymers can be achieved using ringopening polymerization, with X-PEG-OH as the macroinitiator and tin salts, such as stannous octoate, as the
catalyst.(1416) The L-lactide and X-PEG-OH are first dissolved at 60C in toluene under a nitrogen
atmosphere to minimize moisture. The amounts of the reagents can be adjusted depending on the desired
amount of product and relative length of the PLA chain. The stannous octoate, of which a catalytic amount is
3

also dissolved in toluene, is then added to the mixture and

refluxed at 110C for 4 hours under nitrogen. The solvent is
then evaporated using a rotary evaporator under vacuum,
and the remaining material is heated to 140C for 1 hour
under nitrogen. This reaction mixture is then cooled and
dissolved in dichloromethane. The final polymer product is
purified by adding chilled diethyl ether until there is a 50:50
mixture of dichloromethane-diethyl ether, filtering, and
drying under vacuum. This reaction is repeated for each
different functional group on the PEG to form methoxy-PEGPLA (mPEG-PLA), maleimide-PEG-PLA (mal-PEG-PLA),
and carboxy-PEG-PLA (COOH-PEG-PLA). This reaction
can be seen in Figure 2a.
The synthesized COOH-PEG-PLA copolymers can then be
further modified with different functional groups for further
conjugation with the ligands. NHS-PEG-PLA copolymers
can be obtained by reacting COOH-PEG-PLA with DCC and
NHS with a DMAP catalyst at neutral pH and room
temperature. NH2-PEG-PLA copolymers can be obtained by
reacting the NHS-PEG-PLA copolymers with a diamine
compound, such as ethylene diamine, at neutral pH and
room temperature. These reactions can be seen in Figure 2b.

(a)

(b)
Figure 2: Schematics showing the steps to synthesize
functionalized PEG-PLA block copolymers. (a) Ringopening polymerization reaction. (b) Functionalizing
PEG-PLA with NHS ester and amine groups.

To synthesize APRPG-PEG-PLA, the reaction of a thiol functional group to a maleimide group will be taken
advantage of. A modified APRPG peptide containing a cysteine residue (the alanine residue is substituted with
a cysteine and a glycine) to take advantage of the maleimide is obtained from Shanghai Apeptide Co., a
company located in Shanghai, China, that specializes in producing peptide constructs. The mal-PEG-PLA and
modified APRPG are dissolved in PBS and allowed to react at room temperature for 8-12 hours with stirring.
The product is then purified using ultracentrifugation, dialyzed against water, and then lyophilized.(4, 14)
To synthesize LHRH-PEG-PLA, the reaction of an amine functional group with a reactive NHS ester group will
be taken advantage of. A synthetic analog of LHRH is used, where the sequence of native LHRH is modified
by replacing the glycine residue at position 6 with a lysine residue to yield the superactive, degradationresistant Lys-6-des-Gly-10-Pro-9-ethylamide LHRH analog.(17) The amine side chain on the lysine residue of
the LHRH analog can then react with NHS-PEG-PLA to form LHRH-PEG-PLA with a stable amide bond linking
the LHRH to the PEG. The product is then purified using ultracentrifugation, dialyzed against water, and then
lyophilized.
To synthesize folate-PEG-PLA, the reaction of a reactive NHS ester group with an amine functional group will
be taken advantage of. The - and -carboxylic acids on folate are first activated using DCC and NHS and
then reacted with NH2-PEG-PLA at neutral pH and room temperature. This will produce a mixture that can be
separated using ion-exchange chromatography and identified using carboxypeptidase G 2, which will react with
only the folate bound via the -carboxyl group.(18) Only folate-PEG-PLA with the folate conjugated via the carboxyl group is kept. The product is then purified using ultracentrifugation, dialyzed against water, and then
lyophilized.
To ensure that the block copolymers are properly synthesized, each of the copolymer molecules must be
characterized using C13- and H1-nuclear magnetic resonance (NMR), fourier transform infrared (FTIR), and
mass spectroscopy. C-NMR, H-NMR, and FTIR spectroscopy can identify the functional groups and bonds
present in the copolymers. Mass spectroscopy can identify the structure, the number average and weight
average molecular mass, and the polydispersities of the copolymers.
The various PEG-PLA copolymers can then be mixed to form polymersomes, depending on what ligands are
needed on the polymersomes. For example, if only LHRH ligand is desired, then LHRH-PEG-PLA and mPEGPLA are used as the copolymers for forming polymersomes; if folate and APRPG ligands are desired, then
folate-PEG-PLA, APRPG-PEG-PLA, and mPEG-PLA are used. The relative numbers of ligand-PEG-PLA to
mPEG-PLA will determine the maximum surface ligand density on the polymersomes. The ratio of mPEG-PLA
to ligand-PEG-PLA used in previous experiments have been 9:1(4), but these research projects only included
the use of a single targeting ligand. Due to the complexity of the polymersomes ligand distribution described in
4

this proposal, varying formulations of 10%, 20%, and 30% ligand-PEG-PLA to 90%, 80%, 70% mPEG-PLA,
respectively, will be tested for vesicle formation and cell targeting prowess, respectively. The ratios of APRPG,
folate, and LHRH ligands will also be varied as an experimental condition, with their combined amounts equal
to the above-mentioned 10%, 20%, 30% percentages.
Formation of polymersomes will utilize the solvent evaporation technique, which involves placing the block
copolymers in both organic and aqueous solvents. While organic solvents are known to affect the tertiary
structures of many proteins, they do not affect protein primary structure. So, the use of smaller peptide
sequences in this proposal should definitely weaken the concern for structural decomposition of the targeting
ligands. The block copolymer (0.02 M) is placed in 2 mL of tetrahydrofuran (THF), an organic solvent. 3 mL of
PBS are quickly added to the mixture, followed by vortexing for 1 min at 200 rpm. The THF is then evaporated
using a rotary evaporator leaving the newly formed polymersome suspensions with an estimated concentration
of ~0.7 M. This is a rapid method for producing polymersomes, and the parameters that influence size,
formation, and stability of the polymersomes can be easily controlled.(9, 19)
Polymersome formation and surface ligand distribution will be confirmed via dynamic light scattering (DLS),
zeta potential, optical microscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectra
(XPS). DLS measures the sizes and polydispersities of the polymersomes. Zeta potential measures the
surface charges on the polymersomes. Optical microscopy and TEM can visualize the morphology and
structure of the polymersomes. XPS can identify the the surface functional groups present on the
polymersomes, which can identify which ligands are present on the outer surface.
After polymersomes are formed, the drugs can then be loaded sequentially, with paclitaxel loaded before
doxorubicin. Paclitaxel can be loaded into the hydrophobic membrane via microinjection. Excess paclitaxel is
removed by dialysis. Doxorubicin can be load into the interior of the polymersome using a pH-gradient
technique developed for liposomes. The polymersomes are hydrated in acidic citrate buffer, causing the
interior of the polymersomes to be acidic. This solution is then dialyzed against a neutral buffer containing DOX,
which creates the pH gradient. Due to the pH gradient, the neutral DOX can permeate the membrane and get
protonated due to the acidic interior. Once inside the core, most of the DOX precipitates, which allows the
continuation of DOX permeation from the outside solution.(20) Excess doxorubicin is removed via dialysis.
Drug loading efficiencies can be measured using high performance liquid chromatography (HPLC). Efficiency
of paclitaxel loading is measured before doxorubicin is loaded.
Finally, to obtain a monodisperse population of polymersomes, the final products after polymersome formation
and drug loading are subjected to sonication, freeze-thaw cycles, and serial size extrusion using filter sizes of
0.4, 0.2, and 0.1 m.
Based on previous results done for regular PEG-PLA polymersomes, the final polymersomes had diameters
around 100 nm and membrane thicknesses around 10 nm. The ideal loading capacity of paclitaxel was 1 mole
of drug to 5 moles of copolymers, which was a concentration of approximately 140 M. The ideal loading
capacity of doxorubicin was 1 mole of drug to 1 mole of copolymers, which was a concentration of
approximately 80 M.(9) We expect our polymersomes to have similar dimensions and drug loading
efficiencies. A potential limitation of this approach could be inefficiencies in the conjugation reactions, which
would increase cost and lower final yields. Another potential limitation could be the lack of control of whether
the ligands appear on the outside or inside leaflet of the polymersome membranes, but this can be identified
via the characterization techniques, especially XPS, and dealt with by repeating the protocols more carefully.
Specific Aim 2
Before examining the effects that the triple ligand bearing polymersome have on ovarian cancer cells in vivo,
steps will be taken in order to determine the optimal surface ligand density for the ligand bearing polymersome.
While previous experiments have used an mPEG-PLA to ligand-PEG-PLA ratio of 9:1, little research had been
done in determining surface ligand density for polymersomes with multiple ligands.(4) We plan to construct
polymersomes that include 8:2 and 7:3 polymer to polymer-ligand ratios. So the polymersome surface ligand
density should be ideally 10%, 20%, and 30%. One must consider that it is very likely that many of the ligands
will be facing the interior so obtaining an actual 10% surface ligand density would require a greater ratio than
1:9. Protocols described in Specific Aim 1 should account for the analysis and characterization of the
polymersomes with respect to the exact density of ligands on the surface. Utilizing polymersomes that are too
ligand-dense can inhibit binding, and using polymersomes that do not have a high enough surface ligand
density can also limit targeting, so finding the optimal density value is important. Altering the ratios of folate,
LHRH, and APRPG relative to one another is also a high priority in order to optimize even more so and
5

produce a targeting polymersome that is more

effective at targeting ovarian cancer cells.
Ovarian cancer cells may overexpress some
receptors for certain ligands more than others,
so accounting for this in vitro can lead to a more
optimized targeting polymersome. The different
ligand ratios within the confines of the 10%, 20%,
and 30% ligand bearing polymersomes that will
be tested are depicted in Figure 3a.

Folate LHRH APRPG

1
1
1
2
1
1
1
2
1
1
1
2
2
1
2
1
2
2
2
2
1
5
1
1
1
5
1
1
1
5
5
1
5
1
5
5
(a)
5
5
1

Experimental
Negative Control 0
Negative Control 1a
Negative Control 1b
Negative Control 1c
Negative Control 2a
Negative Control 2b
(b) Negative Control 2c

Folate LHRH APRPG

( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )

Ligand present
Ligand absent

In order to assess which one of the 39 possible

groups is most effective for intracellular tumor
3: Tables showing the experimental conditions for the multiple
cell uptake, an in vitro screening process will be Figure
ligand studies. (a) The relative amounts of ligands used. (b) Which
applied. More specifically, the fluorescent probe ligands will be present on the polymersome surface.
coumarin-6 will be encapsulated within the many
different possible polymersome constructs for use in detecting uptake of the nanoparticles by the ovarian
cancer cell line SKOV3. SKOV3 cells will be seeded at a density of 1.5x10 6 cells in 24 well plates, and they will
be allowed to grow to 70% confluency prior to treatment with the coumarin-loaded polymersomes.(21) The
cells will be treated with said coumarin containing polymersomes for three hours at 37C. Coumarin-6 will be
purchased from Sigma Aldrich, and this dye will fluoresce at the appropriate absorbance wavelengths. The
fluorescence data from the different samples will be captured using fluorescence microscopy, and image
capturing software will allow for quantification of the fluorescence. The brighter the fluorescence, the more
polymersomes are absorbed, leading us to a conclusion on what nanoparticle combination is ideal for use in
the upcoming in vivo experiments.
In order to examine the tumor specificity of the ligand bearing polymersomes with regards to ovarian tumor cell
targeting, the biodistribution of the polymersomes will be examined using an in vivo mouse model. The mice
selected for use in these experiments will be athymic female BALB/c mice, and they will be inoculated with
SKOV3 cells (2x106 cells/0.2 mL) in the legs.(4) SKOV3 cells are a subtype of ovarian cancer cells. These
mice will serve as the in vivo ovarian cancer tumor model, and antitumor efficiency experiments as well as
biodistribution assays will be performed two weeks after inoculation.
Prior to continuing on with the experiments, steps must be taken to determine the appropriate control groups
and reasons for their inclusion. The experimental group will consist of the polymersome with the three ligands,
folate, APRPG, and LHRH. The polymersome control groups for the biodistribution experiment are listed by the
ligands they contain, as seen in Figure 3b. We hypothesize that the experimental group will have a better
biodistribution and antitumor efficiency relative to the other controls due to the variation in the ligands and the
synergistic effects that occur as a result.
In order to image the distribution of the polymersomes in vivo, Xenolight DiR fluorescent dye will be utilized in
order to provide visualization of all of the different polymersome variations in vivo in their respective mouse
models. DiR is a lipophilic, near-infrared fluorescent cyanine dye that allows for staining of the cytoplasmic
membrane. Its chemical formulation is C63H101IN2, and its absorption/emission spectra is 748/780 nm.(22)
Previous research has demonstrated the use of DiR-PEG-PLA for in vivo imaging (4), and this particular
copolymer can replace some of the mPEG-PLA used in polymersome formation for imaging purposes. This lab
group successfully constructed a polymersome with an APRPG-PEG-PLA to PEG-PLA ratio of 1 to 9 with
regards to weight, and our initial biodistribution and antitumor efficiency assays will use the optimal ratio
determined in the in vitro optimization of surface ligand density experiments described above. Also, the ligand
ratios relative to one another that were the most efficient in vitro will also be used for the experimental group as
well as 1:1 for the negative controls 2a and 2b. For further clarification, the biodistribution assay uses
polymersomes made up of 90% DiR-PEG-PLA and the respective ligand-PEG-PLA.
With regards to the biodistribution assays of the different ligand bearing polymersomes, 0.5 mg of DiR per kg
of mouse will be delivered systemically using a tail vein injection and the mice will be sacrificed at 6 hours and
48 hours (n=3) to obtain samples of the heart, liver, spleen, lung, kidney, and tumor tissues.(4) The DiR will
fluoresce upon excitation using infrared light, and this data will show where the particles accumulate within the
mouse. We expect there to be a greater accumulation of the polymersomes with all three ligands in the ovarian
cancer cells relative to the other control groups.
6

After examining the biodistribution of the particles, further experiments will help assess the antitumor efficiency
of the polymersomes. Depending on the results of the biodistribution assays, certain groups will be selected for
continued analysis, and assuming that the experimental group was the most effective, it will be compared to
the following control groups: saline, polymersome with no targeting ligands, free doxorubicin, and free
paclitaxel. The tumors in the mice will be allowed to grow to an estimated size of 0.5 cm2, and a single tail-vein
injection will be done prior to performing the assay to figure out what the maximum tolerable dose is. The max
tolerable dose occurs when the mouse goes over the weight loss decrease threshold of more than 5%. The
maximum dosage for doxorubicin and paclitaxel in drug loaded PEG-PLA polymersomes have been previously
determined to be 2.5 mg/kg and 2.5 mg/kg, respectively, when co-delivered.(9) The maximum tolerable doses
of free doxorubicin and paclitaxel are 1.5 mg/kg and 1.0 mg/kg. The different treatment solutions (n=20) will be
given once every 2 days over a 16 day treatment period, and the cancer bearing mice will be sacrificed to
obtain the heart, liver, spleen, lung, kidney, and tumor tissues for H&E staining and TUNEL staining. The tumor
tissues will also be evaluated with a caliper to determine the tumor volume using the formula: tumor volume
(cm3) = (tumor length x tumor width2)/2.(4) The volumes of the tumor tissues will be plotted over time, and the
ligand-bearing polymersome should show the greatest decrease in size
As well as expecting a decrease in tumor size over time, a reduction in systemic tissue damage as well as
increased apoptosis of the tumor tissue is expected for the triple ligand bearing polymersome, and the
following H&E and TUNEL assays will help confirm these results. A commercially available Tdt-mediated dUTP
nick end labeling (TUNEL) kit will be used according to
instructions to determine the apoptosis occurring in the tumor, and
the commercially available H&E assay kit will be used to assess
the viability of other organs throughout the body. Also, the data for
these experiments will be reported with a P < 0.05. One way
ANOVAs with LSD will be used for multiple group analysis, and
unpaired t-tests will be applied for two group comparisons.(4)
The biggest concern involves the possibility of many of the ligands
ending up on the interior of the polymersome and not the exterior.
This could skew the results especially if the 10% group is
expressing more ligands on the surface, and the 20% group is
expressing more ligands in the interior. While we assume that the
sheer number of nanoparticles most likely mitigates the possibility
of any statistically significant differences between test groups, our
lab group will take careful consideration with respect to
polymersome assembly and characterization.
Specific Aim 3
The release profiles of doxorubicin and paclitaxel have already
been achieved in literature through fluorescence for doxorubicin
and HPLC for paclitaxel, which can
be seen in Figure 4.(9)
PEG-PLA diblock copolymers have
been demonstrated in literature to
exhibit varying clearance rates
depending on the compositional
characteristics of the copolymer
such as the relative PEG
content.(23) PEG increases stability
and circulation times, and this is
illustrated in Figure 5c. A larger
denoting number, such as PEG30,
indicates higher PEG content and
yields slower clearance with PGE1
representing amount in plasma.
This
number
reflects
the
percentage of PEG in which the
nanoparticle was assembled. The

Figure 4: Experimentally determined release

profiles of doxorubicin and paclitaxel from PEGPLA polymersomes as well as non-degradable
polymers. Doxorubicin release during dialysis
was determined by fluorescence. Paclitaxel
release was determined by HPLC.(9)

(a)

(b)
(c)
Figure 5: (a) Table showing the different properties of different PEG molecules. (b)
Clearance rates of various PEG-PLA copolymers obtained with HPLC. Nanoparticle
size was characterized with the oil-in-water solvent diffusion method. (c) Total body
clearance (CL) for various PEG-PLA copolymers.

Uncleaved Release time Release time

esters [%] pH ~ 5.5 [hr] pH ~ 7.4 [hr]
90
2.6
5.6
80
5.5
11.9
70
8.7
19.0
60
12.5
27.3
50
17.0
37.0
40
22.5
48.9
30
29.5
64.3
20
39.5
85.9
(b)
10
56.5
122.9
( )

(a)
(c)

[
[

]
]

Ideal PEG-PLA
configuration
PEG18
PEG30
PEG30
PEG30
PEG30
PEG30
PEG30
PEG30
PEG30
(

Clearance
[ml/h*kg]
47.9
21.5
21.5
21.5
21.5
21.5
21.5
21.5
21.5

Figure 6: This (a) graph and (b) table presents the ideal PEG-PLA confirmations for which clearance will occur at rates where plasma
concentration will be significantly higher at the time where disassembly occurs at pH5.5 than at the time where disassembly occurs at
pH7.4. However, for fraction of uncleaved esters remaining less than 0.5, plasma concentration is very low for either pHs, suggesting
that PEG30 may be inadequate for maintaining drug delivery efficacy. (c) Equations used for the model.

relative PEG content can be then adjusted to exhibit a particular clearance rate
so that it is cleared after endocytosis in order to maintain drug carrier efficacy.
This PEG content should also ideally exhibit clearance rate so that the majority
of it is cleared before polymer degradation in non-tumor regions (pH ~ 7.4) to
minimize cytotoxicity. PEG-PLA clearance data can be seen in Figure 5.
We want to synchronize PEG-PLA clearance with drug carrier degradation rate,
which is in turn modeled by ester cleavage rate. The pH-sensitive ester linkage
cleaves according to first-order kinetics. We will model certain thresholds of ester
degradation percentages as the point at which all the drugs loaded into the
polymersome are released (i.e. at 50% degradation, the polymersome
disassemble and all drugs are released). Ideally, we will then be able to select a
copolymer PEG content percentage that exhibits clearance rate that coincides
with a LOW clearance at a time that threshold ester degradation percentage for
pH ~ 5.5 is reached and HIGH clearance at a time at that threshold ester
degradation percentage for pH ~ 7.4 is reached. This way, drug will essentially
be cleared slowly enough so that they will have time to effectively degrade and
release their drugs inside the target ovarian cancer cells but quickly enough that
they will be cleared before much of the polymersomes release their contents in
non-tumor regions. The results of our model and clearance time, as well as the
equations used for the model, can be seen in Figure 6.
The PEG-PLA copolymer has been established in literature as
biocompatible and low in extracellular cytotoxicity.(24) However,
doxorubicin and paclitaxel are cytotoxic, and literature exploring the
cytotoxicity of PEG-PLA nanofibers loaded with paclitaxel and
doxorubicin has demonstrated that the two drugs in conjunction
demonstrate increased cytotoxicity relative to the individual drugs
themselves.(24) Also, the cytotoxic effects of the incorporation of
multiple ligands (folate, APRPG, and LHRH) are unknown. We plan to
assess the cytotoxicity of the drug loaded polymersome in both ovarian
cancer cells as well as normal cells. We will perform an MTT assay as
well as flow cytometry analysis against A2780 human ovarian cancer
cells and healthy ovarian tissue to determine cytotoxicity. We will also
perform cytotoxicity analysis on liver, spleen, and other cells that exhibit
receptors for any of the ligands presented on the drug carrier
polymersome.
Figure 7 differs from our proposed model in that it was treated was
treated against breast cancer cells as opposed to A2780 human ovarian
cancer cells, and it does not incorporate multiple ligands. We hope to

Figure 7: MTT assay

determined cytotoxicity of
doxorubicin and paclitaxel
delivered by both nondegradable and PEG-PLA
polymersomes.(9)

Figure 8: Cytotoxicity of various drugloaded ber mats to the rat Glioma C6

cells vs. time: (a) PEGPLA blank ber
mat, (b) 1.0 wt% PTX/PEGPLA ber mat,
(c) mixture of 0.5 wt% DOX/PEGPLA
ber mat and 0.5 wt% PTX/PEGPLA ber
mat, (d) (0.5 wt% DOX + 0.5 wt%
PTX)/PEGPLA composite ber mat, and
(e) 1.0 wt% DOX/PEGPLA ber mat. The
total drug contents in the culture media
were kept at 80 g/ml for all tests.(24)

achieve similar results for similar drug loading percentages, as it will suggest that the multiple ligands have
minimal cytotoxic effects.
Figure 8 indicates that PEG-PLA nanoparticles are biocompatible. We hope to achieve similar results for
similar drug loading percentages, as it will suggest that: 1) the PEG-PLA polymersome is biocompatible, 2) the
multiple ligands have minimal cytotoxic effects, and 3) the cytotoxicity of dual drug incorporation is consistent
regardless of the means they were loaded onto the drug.
Conclusions
The completion of these specific aims will result in the development of a novel triple ligand bearing
polymersome for the specific targeting of ovarian cancer cells. This novel drug delivery device will improve
upon existing drug delivery methods because it uses multiple ligands for targeting and decreasing systemic
delivery of the drugs to healthy tissues. The potential synergistic combinatorial effect of using all three ligands
should further decrease the possible targeting of other tissues that express the receptors. The multi-drug facet
of the nanocarrier will allow for attacking different growth mechanisms of the tumors, further decreasing tumor
growth and metastasis. The proposed experiments in this research proposal should provide better insight with
regards to optimal dosing, ligand surface density, and polymer concentration for increased antitumor efficiency,
biodistribution, specified cytotoxicity, and degradability of the polymersome.

References
1.
What are the key statistics about ovarian cancer? (available at
http://www.cancer.org/cancer/ovariancancer/detailedguide/ovarian-cancer-key-statistics).
2.
SEER Stat Fact Sheets: Ovary Cancer (available at http://seer.cancer.gov/statfacts/html/ovary.html).
3.
A. J. Clifford et al., The dynamics of folic acid metabolism in an adult given a small tracer dose of 14Cfolic acid., Adv. Exp. Med. Biol. 445, 239251 (1998).
4.
Y. Wang et al., Specific cell targeting with APRPG conjugated PEG-PLGA nanoparticles for treating
ovarian cancer., Biomaterials 35, 98392 (2014).
5.
L. W. T. Cheung, S. Yung, T.-M. Chan, P. C. K. Leung, A. S. T. Wong, Targeting gonadotropin-releasing
hormone receptor inhibits the early step of ovarian cancer metastasis by modulating tumor-mesothelial
adhesion., Mol. Ther. 21, 7890 (2013).
6.
C. Grndker, G. Emons, Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer., Reprod.
Biol. Endocrinol. 1, 65 (2003).
7.
S. Mozzetti et al., Class III -tubulin overexpression is a prominent mechanism of paclitaxel resistance in
ovarian cancer patients, Clin. Cancer Res. 11, 298305 (2005).
8.
H. C. Arora et al., Nanocarriers enhance Doxorubicin uptake in drug-resistant ovarian cancer cells.,
Cancer Res. 72, 76978 (2012).
9.
F. Ahmed et al., Shrinkage of a rapidly growing tumor by drug-loaded polymersomes: pH-triggered
release through copolymer degradation., Mol. Pharm. 3, 34050 (2006).
10. D. E. Discher, A. Eisenberg, Polymer vesicles., Science (80-. ). 297, 96773 (2002).
11. S. S. Dharap et al., Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.,
Proc. Natl. Acad. Sci. U. S. A. 102, 129627 (2005).
12. N. Parker et al., Folate receptor expression in carcinomas and normal tissues determined by a
quantitative radioligand binding assay., Anal. Biochem. 338, 28493 (2005).
13. J. Xiong et al., Folate-conjugated crosslinked biodegradable micelles for receptor-mediated delivery of
paclitaxel, J. Mater. Chem. 21, 5786 (2011).
14. U. S. Toti, B. R. Guru, A. E. Grill, J. Panyam, Interfacial Activity Assisted Surface Functionalization: A
Novel Approach To Incorporate Maleimide Functional Groups and cRGD Peptide on Polymeric
Nanoparticles for Targeted Drug Delivery, Mol. Pharm. 7, 11081117 (2010).
15. S. Aryal, C. Hu, L. Zhang, PolymerCisplatin Conjugate Nanoparticles for Acid-Responsive Drug
Delivery, ACS Nano 4, 251258 (2009).
16. X. Zhang et al., Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate.,
Biomaterials 26, 21218 (2005).
17. P. M. Conn, E. Hazum, Luteinizing hormone release and gonadotropin-releasing hormone (GnRH)
receptor internalization: independent actions of GnRH., Endocrinology 109, 20405 (1981).
18. S. Wang, R. J. Lee, C. J. Mathias, M. a Green, P. S. Low, Synthesis, purification, and tumor cell uptake
of 67Ga-deferoxamine--folate, a potential radiopharmaceutical for tumor imaging., Bioconjug. Chem. 7,
5662 (1996).
19. F. Ahmed, D. E. Discher, Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered
controlled release vesicles., J. Control. Release 96, 3753 (2004).
20. T. D. Madden et al., The accumulation of drugs within large unilamellar vesicles exhibiting a proton
gradient: a survey., Chem. Phys. Lipids 53, 3746 (1990).
21. J. Yin et al., Tetramethylpyrazine inhibits migration of SKOV3 human ovarian carcinoma cells and
decreases the expression of interleukin-8 via the ERK1/2, p38 and AP-1 signaling pathways., Oncol.
Rep. 26, 6719 (2011).
22. XenoLight DiR (2012) (available at http://www.perkinelmer.com/invivoreagents).
23. S. S. Venkatraman, P. Jie, F. Min, B. Y. C. Freddy, G. Leong-Huat, Micelle-like nanoparticles of PLAPEG-PLA triblock copolymer as chemotherapeutic carrier., Int. J. Pharm. 298, 21932 (2005).
24. X. Xu, X. Chen, Z. Wang, X. Jing, Ultrafine PEG-PLA fibers loaded with both paclitaxel and doxorubicin
hydrochloride and their in vitro cytotoxicity., Eur. J. Pharm. Biopharm. 72, 1825 (2009).

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