Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer

Article abstract of DOI:10.1016/j.jconrel.2013.04.026

The co-delivery of drug combination at a controlled ratio via the same vehicle to the cancer cells is offering the advantages such as spatial-temporal synchronization of drug exposure, synergistic therapeutic effects and increased therapeutic potency. In an attempt to develop such multidrug vehicle this work focuses on functional biodegradable and biocompatible polypeptide-based polymeric micelles. Triblock copolymers containing the blocks of ethylene glycol, glutamic acid and phenylalanine (PEG-PGlu-PPhe) were successfully synthesized via NCA-based ring-opening copolymerization and their composition was confirmed by ¹H NMR. Self-assembly behavior of PEG-PGlu ₉₀-PPhe₂₅ was utilized for the synthesis of hybrid micelles with PPhe hydrophobic core, cross-linked ionic PGlu intermediate shell layer, and PEG corona. Cross-linked (cl) micelles were about 90 nm in diameter (ξ-potential = -20 mV), uniform (narrow size distribution), and exhibited nanogels-like behavior. Degradation of cl-micelles was observed in the presence of proteolytic enzymes (cathepsin B). The resulting cl-micelles can incorporate the combination of drugs with very different physical properties such as cisplatin (15 w/w% loading) and paclitaxel (9 w/w% loading). Binary drug combination in cl-micelles exhibited synergistic cytotoxicity against human ovarian A2780 cancer cells and exerted a superior antitumor activity by comparison to individual drug-loaded micelles or free cisplatin in cancer xenograft model in vivo. Tunable composition and stability of these hybrid biodegradable micelles provide platform for drug combination delivery in a broad range of cancers.

Full text of DOI:10.1016/j.jconrel.2013.04.026

COREL-06711; No of Pages 10
Journal of Controlled Release xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Biodegradable hybrid polymer micelles for combination drug therapy
in ovarian cancer
Swapnil S. Desale , Samuel M. Cohen , Yi Zhao , Alexander V. Kabanov a,1, Tatiana K. Bronich a,⁎
a
b
a
a
Department of Pharmaceutical Sciences and Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska Medical Center,
Omaha 68198-5830, USA
b
Department of Pathology and Microbiology, University of Nebraska Medical Center/Eppley Cancer Center, Omaha 68198-3135, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The co-delivery of drug combination at a controlled ratio via the same vehicle to the cancer cells is offering
the advantages such as spatial–temporal synchronization of drug exposure, synergistic therapeutic effects
and increased therapeutic potency. In an attempt to develop such multidrug vehicle this work focuses on
functional biodegradable and biocompatible polypeptide-based polymeric micelles. Triblock copolymers
containing the blocks of ethylene glycol, glutamic acid and phenylalanine (PEG–PGlu–PPhe) were successfully
Received 9 March 2013
Accepted 26 April 2013
Available online xxxx
Keywords:
1
synthesized via NCA-based ring-opening copolymerization and their composition was confirmed by H NMR.
Combination drug delivery
Cross-linked polymer micelles
Ovarian cancer
Cisplatin
Paclitaxel
Self-assembly behavior of PEG–PGlu90–PPhe25 was utilized for the synthesis of hybrid micelles with PPhe
hydrophobic core, cross-linked ionic PGlu intermediate shell layer, and PEG corona. Cross-linked (cl) micelles
were about 90 nm in diameter (ξ-potential = −20 mV), uniform (narrow size distribution), and exhibited
nanogels-like behavior. Degradation of cl-micelles was observed in the presence of proteolytic enzymes
Controlled release
(cathepsin B). The resulting cl-micelles can incorporate the combination of drugs with very different physical
properties such as cisplatin (15 w/w% loading) and paclitaxel (9 w/w% loading). Binary drug combination in
cl-micelles exhibited synergistic cytotoxicity against human ovarian A2780 cancer cells and exerted a superior
antitumor activity by comparison to individual drug-loaded micelles or free cisplatin in cancer xenograft
model in vivo. Tunable composition and stability of these hybrid biodegradable micelles provide platform
for drug combination delivery in a broad range of cancers.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
development of acquired drug resistance. No new small molecule plat-
inum drug has gained international marketing approval since 1999
indicating a shift in focus in the last decade away from drug design
and toward drug delivery [5]. Among drug delivery systems, those
based on self-assembled block copolymer micelles have great potential
for delivery of anticancer drugs [6]. The advantages of polymer micelles
include their small size, long circulation times, and ability to circumvent
renal excretion and extravasate at sites of enhanced vascular perme-
ability. However, disintegration of the polymer micelles is a major
drawback for their application in drug delivery. In order to overcome
this limitation, we have proposed hydrophilic polyelectrolyte micelles
with core composed of a network of cross-linked polyanion chains
and PEG shell that can encapsulate CDDP with high capacity (up to 35
w/w%) [7,8]. We have demonstrated significant improvement of drug
safety and drug delivery using such CDDP-loaded cross-linked micelles.
However, single drug therapy agents directed to individual targets
frequently show limited efficacies. Hence, multi-component therapies
and multi-targeted drug combinations are the key to most cancer treat-
ments [9]. Combination chemotherapy using sequential administration
of paclitaxel (PTX) with a platinum-based regimen is currently the stan-
dard first-line therapy for ovarian cancer resulting in increased success
rates in eradicating tumors and/or longer remission periods [10]. These
Ovarian cancer is the most lethal gynecologic malignancy in women,
which causes nearly 15,000 deaths in the United States every year. Its
high death rate is a result of the fact that most patients (>75%) are
diagnosed at an advanced stage of disease at which point the 5-year
survival rate is less than 30% [1,2]. The vast majority of ovarian cancers
are epithelial in origin and characterized by the rapid growth and
spread of solid intraperitoneal tumors and formation of large volumes
of ascitic fluid. The standard treatment for patients with advanced
ovarian cancer is maximal surgical cytoreduction followed by systemic
platinum-based chemotherapy [2]. Cytotoxic action of platinum com-
pounds such as cisplatin (cis-dichlorodiamminoplatinum (II), CDDP) is
mediated through the formation of platinum–DNA adducts, which in
turn inhibits DNA replication and/or transcription and results in apo-
ptosis and necrosis [3,4]. Despite its success, the use of CDDP is greatly
limited by severe dose limiting side effects, rapid elimination, and
⁎
Corresponding author. Tel.: +1 402 559 9351; fax: +1 402 559 9365.
E-mail address: tbronich@unmc.edu (T.K. Bronich).
Current address: Department of Pharmaceutical Sciences and Center for Nanotech-
1
nology in Drug delivery, University of North Carolina, Chapel Hill 27599-7362, USA.
0168-3659/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jconrel.2013.04.026
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
lease (2013), http://dx.doi.org/10.1016/j.jconrel.2013.04.026

2
S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
clinical benefits of the combination therapy can be possibly maximized
by controlled delivery of the desired drug ratio to the in vivo target.
Combining drugs in one delivery carrier is a well-suited strategy for
controlling the pharmacokinetics and co-delivery of the desired drug
ratio in vivo. The drug loading and structure of such carriers can be
tuned to control the drug release rates, maximize the therapeutic
potency and minimize drug-associated toxicities. Nanoscale delivery
carriers such as liposomes, nanoparticles, and polymeric micelles have
been explored for co-delivery of anticancer drugs [11–13]. However,
co-incorporation of drug molecules with different physicochemical
properties, such as hydrophilic CDDP and hydrophobic PTX, has been
challenging. As an attempt to address this challenge, we report a design
of multi-compartment cross-linked micelles based on hybrid triblock
copolymers, poly(ethylene glycol)-block-poly(L-glutamic acid)-block-
poly(L-phenylalanine) (PEG–b–PGlu–b–PPhe) for simultaneous loading
and delivery of binary CDDP and PTX combinations. Such micelles have
(DMF) in nitrogen atmosphere at 40 °C. BGlu-NCA (2 mmol, the feed
molar ratio of mPEG-NH to BGlu-NCA was 1:100) dissolved in 5 mL
2
of anhydrous DMF was added dropwise and the solution was stirred
for 24 h. The aliquot of the reaction mixture was precipitated using
excess of diethyl ether, dried under vacuum, and the composition of
PEG–PBGlu diblock copolymer was determined by 1H NMR from the
peak intensity ratios of the methylene protons of PEG and the phenyl
3
protons of the γ-benzyl groups (400 MHz in CDCl :δ = 3.5 (s, 4H,
\OCH CH \), 5.0 (m, 2H, benzyl), 7.3 (m, 5H, aryl)). The calculated
2
2
amount of the monomer for the third block, Phe-NCA, dissolved in
anhydrous DMF was then injected dropwise and mixture was stirred
under a N atmosphere at 40 °C for additional 24 h. The product
2
(PEG–b–PBGlu–b–PPhe) was precipitated by diethyl ether, purified by
repeated precipitation in diethyl ether and dried in a vacuum. The
benzyl groups of PEG–b–PBGA–b–PPhe were removed in the presence
of NaOH to obtain PEG–b–PGlu–b–PPhe. Polymer sample was dissolved
in 10 mL of THF followed by the addition of 5 mL of 1 N NaOH. After
stirring for 10 h at 40 °C THF was removed at reduced pressure, the re-
sidual solution was neutralized by 1 M HCl and dialyzed using a dialysis
membrane (MWCO 3500 Da) against distilled water for 24 h. After di-
alysis the product was dried, washed 2–3 times with ethanol followed
1
) a hydrophobic core formed by PPhe chains, which serves as a reser-
voir for PTX solubilization, 2) an anionic layer, which incorporates
CDDP through coordination with the carboxylic groups of PGlu, and
3
) an outer PEG shell stabilized, which stabilizes micelles in aqueous
dispersion. The crosslinks incorporated into PGlu block layer ensure
that the PEG–b–PGlu–b–PPhe micelles remain stable until they encoun-
ter proteases, which degrade the micelles by cleaving the polypeptide
chains of the block copolymers. This work demonstrates that such bio-
degradable hybrid micelles carrying CDDP and PTX drug combination
exert a superior antitumor efficacy in treatment of ovarian cancer in
xenograft mouse tumor model.
2
by lyophilization. By varying the feed molar ratio of mPEG-NH to
Phe-NCA (1:10 and 1:30), two copolymers with targeted compositions
PEG–b–PBGlu(100)–b–PPhe(10) and PEG–b–PBGlu(100)–b–PPhe(30)
were synthesized. The concentration of carboxylate groups in the copol-
ymer samples was estimated by potentiometric titration. Self-assembly
behavior of PEG–b–PGlu–b–PPhe copolymers was examined using
pyrene as a hydrophobic fluorescence probe [14].
2
. Material and methods
Materials
PEG–b–PGlu–b–PPhe block copolymers with different ratios of
Preparation of cross-linked polymeric micelles
Micelles were prepared by directly dissolving PEG–b–PGlu–b–PPhe
copolymer in aqueous media at a concentration of about 1 mg/mL.
EDC in water (0.2 eq with respect to the amount of carboxylate groups)
was added dropwise to this solution and allowed to stir for additional
10 min at r.t. An aqueous solution of cross-linker, 1,2-ethylenediamine
(0.1 eq) was then added to the dispersion of micelles to achieve 20% of
cross-linking degree. This degree represents the maximum theoretical
amount of cross-linking that could take place, rather than the precise
extent of amidation. The reaction mixture was allowed to stir overnight
at r.t. Byproducts of the cross-linking reaction were removed by exhaus-
tive dialysis of the reaction mixtures against distilled water.
glutamic acid and phenylalanine units (90:7 and 90:25) were synthe-
sized as described below. α-Amino-ω-methoxy poly(ethylene glycol)
−
1
(
2 w w n
mPEG-NH , M = 5000 g mol , M /M = 1.05) was purchased
from Creative PEGWorks Inc., (NC, USA). CDDP was purchased from
Acros Organics. L-Glutamic acid γ-benzyl ester (BGlu), L-phenylalanine
(
Phe), 1,2-ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarb-
odiimide hydrochloride (EDC), paclitaxel, and other chemicals were
purchased from Sigma-Aldrich (St Louis, MO) and were used without
further purification. Fetal bovine serum (FBS), RPMI 1640 medium, pen-
icillin, streptomycin, Trypsin–ethylenediaminetetraacetic acid (EDTA)
(
0.5% trypsin, 5.3 mM EDTA tetra-sodium) and other chemicals were
Physicochemical methods of characterization
purchased from Invitrogen (Carlsbad, CA, USA).
The 1H NMR spectra for the monomers and copolymers were
N-carboxyanhydrides (NCA) of BGlu and Phe
3 2
acquired in CDCl or D O at 25 °C using a Bruker 400 MHz spectrome-
ter. Effective hydrodynamic diameters (Deff) and ζ-potential of micelles
were determined by dynamic light scattering (DLS) using a Zetasizer
Nano ZS (Malvern Instruments Ltd., Malvern, UK). All measurements
were performed in automatic mode at 25 °C. Software provided by
the manufacturer was used to calculate the size, polydispersity indices
(PDI), and ζ-potential of micelles. All measurements were performed
at least in triplicate to calculate the mean values ± SD. Morphology of
the micelles was characterized by transmission electron microscopy
(TEM) and atomic force microscopy (AFM) as previously described [24].
BGlu (0.020 mol) or Phe (0.026 mol) and anhydrous tetrahydrofuran
THF) were added into a dried glass reactor to form a suspension.
Triphosgen (0.023 mol) was separately dissolved in fresh anhydrous
THF and injected drop wise into reaction mixture. Nitrogen was bubbled
through the mixture during synthesis. The mixture was heated at about
(
4
0 °C with constant stirring till mixture became transparent. The solu-
tion was precipitated in n-hexane and then stored at −20 °C overnight
in order to allow complete precipitation of γ-benzyl L-glutamate-N-
carboxyanhydride (BGlu-NCA) or L-phenylalanine -N-carboxyanhydride
(Phe-NCA). The white solids obtained were collected and purified
Drug loading
further by repeated precipitation with n-hexane. The resulting NCA
monomers were dried under vacuum for 24 h and characterized by the
proton nuclear magnetic resonance ( H NMR).
A solution of PTX in ethanol (2 mg/mL) was added dropwise into
the aqueous dispersion of cl-micelles (1 mg/mL) and the mixture was
stirred at r.t. overnight in an open-air system to allow slow evaporation
of ethanol [15]. The residual ethanol was then removed at reduced pres-
sure. The precipitate of unbound PTX was removed by centrifugation.
The aqueous dispersion of PTX-loaded cl-micelles was mixed with an
aqueous solution of CDDP (1 mg/mL) at pH 9.0 at 0.5 molar ratio of
1
PEG–b–PGlu–b–PPhe block copolymer
2
Monoaminomethoxypoly(ethylene glycol) (mPEG-NH ) (0.02 mmol)
was dissolved under stirring in 10 mL of anhydrous dimethylformamide
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
3
2
CDDP to carboxylate groups followed by incubation at 37 °C for 48 h.
Unbound CDDP was removed by Ultracon filter units (MWCO
monitored every second day. Tumor volume (V = 0.5 × L × W ) was
estimated by measuring two orthogonal diameters (longer dimension:
L, and smaller dimension: W) of the tumor using electronic calipers.
1
0,000 Da, Millipore). Pt content in cl-micelles (Pt194/Pt195) was
assayed on a Nexion ion coupled plasma-mass spectrometer (NexION
00Q, PerkinElmer) calibrated with Pt (2–100 ng/mL) and Holmium
3
Animals were sacrificed when tumor volume exceeded 3000 mm ,
greatest tumor dimension exceeded 20 mm, tumor became necrotic,
or animal exhibited a body weight loss of more than 20%. Euthanasia
3
as the internal standard. Samples were diluted in 0.1 N HCl. PTX levels
were determined by high-performance liquid chromatography (HPLC)
analysis under isocratic conditions using an Agilent 1200 HPLC system,
a diode array detector set at 227 nm. As stationary phase a Nucleosil
C18 column was used (250 mm × 4.6 mm), and a mobile phase of
acetonitrile/water mixture (55/45, v/v) was applied at a flow rate of
was performed by CO
by day 45.
2
asphyxiation. All other animals were sacrificed
Apoptosis and proliferation
1
mL/min.
Tumors from mice that received different treatments were excised
at day 14 (2–3 mice per group). The tumors were dissected and fixed
in 10% neutral buffered formalin. Then, the tissues were processed
routinely into paraffin, sectioned at a thickness of 4 μm. Proliferating
and apoptotic cells were detected using an antibody against Ki-67 and
caspase-3, respectively. Visualization was done by incubation with
DAB + (brown, for Ki-67) and Permanent red (for caspase-3)
Release studies
Drug release from the cl-micelles was examined in PBS (pH 7.4),
acetate buffered saline (ABS, pH 5.5, 0.14 M NaCl), and ABS in the
presence of cathepsin B (10 units/mL) by dialysis method using a
membrane with 3500 Da cutoff. The concentrations of PTX and Pt(II)
released were determined by HPLC and inductively coupled plasma
mass spectrometry (ICP-MS), respectively, and expressed as a percent-
age of the total PTX or Pt(II) available vs. time.
(
DAKO) for 2 min. After being rinsed with distilled water, the sections
were counterstained with hematoxylin. For quantification of Ki-67
and caspase-3 expression, the area of positive cells was determined
(
Image J) in 5 random high power fields (20× magnification) and
divided by the total area of cells for each field of slice.
Cell culture and cytotoxicity assay
Sample preparation and drug content measurement in tissue
A2780 human ovarian carcinoma cells were provided by Dr. P. Rogers
(
Institute of Cancer Research, University of Bristol, UK). Cells were
Known weights of thawed tissue, collected from the animals
sacrificed at different days during treatment, were decomposed by
wet-ashing in screw-capped vials with six volumes of concentrated
nitric acid, overnight heating, and stirring at 65 °C. An iridium internal
standard was added prior to digestion. Total platinum concentrations
were determined by ICP-MS using iridium correction. Calibration
range for the assay was platinum 2–100 ng/mL with extrapolation to
platinum 1000 ng/mL. Necessary dilutions were made when the plati-
num concentration exceeded the calibration range. Assay sensitivity
was 0.8 ng of Pt/mL, with inter- and intraday assay variability not
exceeding 5%. For PTX measurements, tissue sample (200 mg) was
spiked with 50 μL diazepam (internal standard, I.S.) to achieve a final
concentration about 25 μg/mL, then 2 mL of tert-butyl methyl ether
maintained in RPMI 1640 medium with 2 mM glutamine supplemented
with 10% (v/v) FBS in the presence of penicillin and streptomycin
(100 U/mL and 0.1 mg/mL, respectively) at 37 °C in a humidified atmo-
sphere containing 5% CO
Technologies) after 80% confluence. Cells seeded in 96-well plates (5000
cells/well) 24 h prior the experiment were exposed to various doses
2
. Cells were harvested with trypsin–EDTA (Life
(0–10 μg/mL on CDDP or PTX basis) of CDDP alone, polymeric micelles
alone, CDDP-loaded cl-micelles, PTX-loaded cl-micelles and (CDDP +
PTX)-loaded cl-micelles for 72 h at 37 °C, followed by washing with
PBS, and maintaining in RPMI 1640 medium with 10% FBS for additional
2
4 h. Cytotoxicity of drug-loaded cl-micelles was assessed by a standard
MTT assay [16] and the IC50 values were calculated using GraphPad
Prism software. Combination index (CI) analysis based on Chou and
Talalay method [17] was performed using CompuSyn software for
CDDP and PTX combinations, determining synergistic, additive, or antag-
onistic cytotoxic effects against A2780 breast cancer cells. Values of
CI b 1 demonstrate synergism while CI = 1 and CI > 1 values represent
additive and antagonistic effects of drug combination, respectively.
(
TBME) was added and tissue was homogenized. Homogenized sam-
ples were centrifuged at 3000 g for 10 min, and 1 mL of supernatant
TBME layer was collected and dried under air. Residue was
reconstituted using 1 mL of mobile phase and 20 μL of it was injected
into the HPLC system. Samples of PTX were prepared by directly
dissolving PTX and I.S. in mobile phase and measured in HPLC system
to calculate recovery rate.
Animal studies
Upon arrival, animals were placed in a facility accredited by the
Association for Assessment and Accreditation of Laboratory Animal
Care. Food and reverse osmosis water were available ad libitum
throughout the study. Treatments were administered by tail vein
injection. Drug amount was calculated based on the average animal
body weight. The University of Nebraska Medical Center Institutional
Animal Care and Use Committee approved all animal protocols. A xeno-
Blood chemistry and histopathology
Blood from the sacrificed animals was collected in EDTA tubes and
analyzed for blood cell count and liver enzymes using Vetscan VS
(Abaxis). Fixed tissues were processed, sectioned, inserted into tissue
cassettes, dehydrated in 70% ethanol overnight, and paraffin embedded
(UNMC Tissue Sciences Facility, Omaha, NE). Serial 5 μm sections were
stained with either hematoxylin and eosin (H&E) or by immunohisto-
chemistry (IHC). For histopathological diagnosis, H&E-stained slides
were examined by light microscopy and photomicrographs were
taken using a Nikon camera mounted on a Nikon Eclipse 600 microscope
(both Nikon Instruments, Melville, NY) with Adobe Elements 3.0 soft-
ware (Adobe Systems, San Jose, CA). For IHC detection of cl-micelles in
tissues, rabbit monoclonal antibody to PEG (anti-PEG methoxy group;
Epitomics, Burlingame, CA) was used. Goat anti-rabbit secondary anti-
body conjugated with fluorescence label AF 488 was used for the detec-
tion followed by counterstaining with Mayer's hematoxylin. Stained
slides were visualized using confocal imaging (Carl Zeiss LSM 510).
graft human ovarian carcinoma model was used as previously described
6
[
8–18]. Briefly, A2780 cells (5 × 10 cells/site) were subcutaneously
transplanted into the flanks, one above each hind limb, of four-week-
old female athymic (Ncr-nu/nu) mice (National Cancer Institute).
3
When the tumors reached a size of about 200–400 mm (12–15 days
after transplantation) animals were randomized (5 treatment groups,
n = 8) and treated with free CDDP or CDDP/cl-micelles or PTX/
cl-micelles or (CDDP + PTX)/cl-micelles at an equivalent dose of
4
5
mg/kg CDDP, or 1 mg/kg PTX or (4 mg/kg CDDP + 1 mg/kg PTX) or
% dextrose solution. Treatments were administered via tail vein injec-
tions at 4-day intervals. Animal body weight and tumor volume were
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
Statistical analysis
Table 1
Characteristics of the PEG–PGlu–PPhe triblock copolymer.
Statistical comparisons except animal studies were carried out
using Student t-test. For the antitumor study and toxicity studies,
group means for tumor volume and body weights were evaluated
using repeated measures analysis of variance with the Bonferroni
post-test. Survival was estimated using Kaplan–Meier analysis and
compared using log-rank test. P values less than 0.05 were considered
significant. Analysis of variance with Bonferroni test and Kaplan–
Meier analysis with log-rank test were performed using GraphPad
Prism 5 (GraphPad Software, Inc.).
Polymer
Feed molar ratio (mmol)
Repeating unit
ratio
Mw (g/mol)
a
PEG BGlu-NCA Phe-NCA PEG PGlu Phe
PEG–PGlu92–PPhe
PEG–PGlu90–PPhe25 0.02
7
0.02
2
2
0.2
0.6
114 92
114 90
7
19,700
25 22,400
a
Calculated from 1H NMR spectra.
They are further denoted as PEG–PGlu90–PPhe
7
and PEG–PGlu90–
PPhe25, respectively.
3
. Results and discussion
Synthesis and characterization of cl-micelles
Synthesis and characterization of PEG–b–PGlu–b–PPhe
We anticipated that the incorporation of hydrophobic PPhe block
into triblock copolymer confers amphiphilic properties and facilitates
the formation of micellar aggregates in an aqueous medium. Indeed,
the formation of small (intensity-average diameter of approximately
90 nm) particles with relatively narrow particle size distribution
(PDI = 0.16) and net negative charge (ξ-potential = −22 mV) was
detected in aqueous solutions of PEG–PGlu90–PPhe25 copolymer. In
contrast, the formation of large aggregates with a broad size distribu-
The synthesis of the amphiphilic hybrid polypeptide-based triblock
copolymers PEG–b–PGlu–b–PPhe is illustrated in Fig. 1.
The copolymers were synthesized via sequential ring-opening
polymerization of BGlu-NCA and Phe-NCA monomers using amino-
−
1
w
terminated PEG (M = 5000 g mol ) as macroinitiator in three
stages. At first, PEG–b–PBGlu diblock copolymer was prepared. The
length of PBGlu block was set constant by using feed molar ratio of
tion was observed only in relatively concentrated PEG–PGlu90–PPhe
7
mPEG-NH
2
to BGlu-NCA at 1:100. The second step was polymerization
solutions. Differences in the association behavior of PEG–PGlu–PPhe
block copolymers were further confirmed by fluorescence measure-
ments using pyrene as a probe (Fig. S2). Using this approach, we
determined that the critical micelle concentration (cmc) for PEG–
PGlu90–PPhe25 was low (62.5 mg/L). In contrast the onset of aggrega-
of Phe-NCA initiated by PEG–b–PBGlu copolymer. The length of PPhe
block was varied by adjusting the feed molar ratio of NCA monomers,
BGlu-NCA:Phe-NCA (10:1 and 10:3). At the final step, the deprotection
of the glutamate residues was carried out by alkali hydrolysis to obtain
PEG–b–PGlu–b–PPhe. The chemical composition of two resulting
7
tion of PEG–PGlu90–PPhe chains was detected at concentrations
1
triblock copolymers was determined by H NMR analysis (Supporting
higher than 1000 mg/L. These data indicate that increase in the
length of the hydrophobic PPhe block from 7 to 25 units is accompa-
nied by an order of magnitude decrease in the cmc value. A similar
trend in the dependency of cmc on the length of core-forming block
was observed earlier for other amphiphilic block copolymers [19]. In
dilute solution, hydrophobic and π–π stacking interactions of the
phenylalanine residues play a major role in driving self-assembly
of Phe-based peptides like amyloid β peptide [20] or synthetic poly-
peptides [21]. A red shift of the emission peak from the normal
information, Fig. S1) and further confirmed by Fourier transform
infrared spectroscopy (FTIR) (data not shown). The polymerization
degrees of PGlu and PPhe blocks are summarized in Table 1.
Notably, PEG–b–PGlu–b–PPhe maintained the same degree of
polymerization after complete removal of the benzyl protecting groups.
These copolymers had a constant PEG block (114 repeating units), prac-
tically identical length of anionic PGlu block (about 90 repeating units),
and differed in the length of PPhe blocks (7 and 25 repeating units).
Fig. 1. Scheme for the synthesis of PEG–b–PGlu–b–PPhe polymer via ring-opening NCA-based polymerization.
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
5
phenylalanine fluorescence maximum (around 280 nm) has been
Table 2
Summary of characterization data for cl-micelles by TEM, AFM, and DLS.
previously observed upon assembly in amyloid system [20]. In the
present case, the fluorescence spectra obtained from 0.01% solutions
of PEG–PGlu–PPhe copolymers displayed a peak at 320 nm that is
attributable to π–π stacking interactions of phenyl units; though the in-
a
b
c
Sample
TEM
av, nm
45 ± 3.3
AFM
av, nm
47 ± 6.8
DLS
eff, nm
90 ± 1.2
D
D
Hav, nm
D
Cl-micelles
8.7 ± 3.7
tensity of the fluorescence was lower for PEG–PGlu90–PPhe
7
copolymer
(
Fig. S3). Notably, the fluorescence spectrum for PEG–PGlu90–PPhe
7
Data are expressed as mean ± SD.
a
Number-average diameter (Dav), n = 27. A drop of the sample solution was
allowed to settle on a Formvar precoated copper grid and the sample was allowed to
air-dry.
was recorded below the cmc. It is possible that some loose pre-micelle
aggregates of copolymer chains with short PPhe blocks might exist
below experimentally determined cmc and the observed fluorescence
characteristics may reflect the local association of aromatic units in
such pre-aggregates. Interestingly, it was previously reported that pep-
tide–PEG amphiphiles containing the hydrophobic sequence of four Phe
b
Number-average height (Hav) and diameter (Dav), n = 55. cl-Micelles were
deposited from aqueous solutions at pH 6.5 onto APS mica surface and allowed to
dry in vacuum.
c
Intensity-average hydrodynamic diameter(Deff) for DLS was determined at pH 7.4,
n = 5.
−
1
residues conjugated to PEG of molar mass 5000 g mol
(PEG–PPhe
4
using our nomenclature) exhibit self-assembly behavior despite its
relatively short peptide sequence with cmc value of 950 mg/L in
water. It seems that the insertion of ionic PGlu block into PEG–b–
PGlu–b–PPhe triblock copolymer provides for additional negative con-
tribution to micellization and increasing cmc. This may be explained,
on the one hand, by stabilization of the single polymer chains in solu-
tion, and, on the other hand, by destabilization of multimolecular
micelles due to the strong electrostatic repulsion of PGlu chains in the
micelle corona. Overall, the optimal hydrophilic–lipophilic balance
Such behavior was indicative of ionization and swelling of the
intermediate layer formed by the cross-linked PGlu chains in the
shell of the micelles. The kinetic stability of cl-micelles was further
analyzed in the presence of urea (Fig. 2B). Urea, a small hydrophilic
molecule, is widely used as protein denaturant due to its ability to
form stronger attractive dispersion interactions with the protein side
chains and backbone than does water. Therefore, it was expected that
urea is able to destabilize PEG–PGlu90–PPhe25 micelles by weakening
the hydrophobic interactions in PPhe core region as well as by
disrupting hydrogen-bonding interactions between polypeptide chains.
Indeed, for non-cross-linked micelles the addition of 8 M urea led to
significant increase in the size and PDI of the aggregates. In the mean-
time, only little changes in the average size of cl-micelles were detected
in the presence of urea (Fig. 2B). The observed limited swelling of PEG–
PGlu90–PPhe25 cl-micelles can be thus directly related to a restricted
mobility of the PGlu chains in the shell and provides additional evidence
that the physical stability of micelles can be greatly enhanced via
(
HLB) in terms of numbers of Phe groups with respect to PEG and
PGlu chains is essential for the formation of multilayer micelles with
well-defined and distinct domains: hydrophobic PPhe core, the inter-
mediate ionic PGlu layer, and hydrophilic nonionic PEG outer shell.
The PEG–PGlu90–PPhe25 micelles were further utilized as “core–shell”
templates for synthesis of cl-micelles. The cross-linking was achieved via
condensation reactions between the carboxylic groups of PGlu chains in
intermediate layer and the amine groups of ethylenediamine in the
presence of a water-soluble carbodiimide, EDC. The targeted extent of
cross-linking (20%) was controlled by the molar ratio of cross-linker
to carboxylic acid groups of the Glu residues. The cl-micelles displayed
an effective diameter of about 90 nm (ξ-potential = −20 mV) and
were uniform (monomodal, narrow size distribution) as determined
by DLS. It is important to note that cl-micelles had comparable sizes
with the precursor PEG–PGlu90–PPhe25 micelles (90 ± 1.2 nm) indi-
cating that the micelles retained their integrity and the formation of
cross-links was limited to intramicellar reactions. It is likely that the
outer PEG shell of the micelles prevented the undesirable intermicellar
reactions. The dimensions and morphology of cl-micelles were further
characterized by TEM and tapping-mode AFM in air. Based on AFM
and TEM images PEG–PGlu90–PPhe25 cl-micelles had a spherical mor-
phology. As expected the number-average particle height (Hav) and
diameter (Dav) values of dehydrated cl-micelles were reduced com-
pared to the hydrodynamic diameters Deff determined by DLS
(Table 2). Upon adsorption on mica surface cl-micelles exhibited a
high diameter versus height aspect ratios (Dav/Hav) suggesting substan-
tial flattering of the particles. This observation was in agreement
with the expected flexible character of the PEG–PGlu shell of the
cl-micelles. The electrostatic interactions of the negatively charged
cl-micelles with the positively charged amino-modified mica surface
may induce additional flattering of cl-micelles.
Interestingly, the hybrid PEG–PGlu90–PPhe25 cl-micelles were
characterized by a significantly lower aspect ratio (ca. 6) compared to
PEG-poly(methacrylic acid) (PEG-PMA) cl-micelles containing entirely
hydrophilic cross-linked PMA cores [22]. For example, PEG-PMA133
and PEG-PMA75 cl-micelles were characterized by dimension aspect
ratios of 13.3 and 15.0, respectively. The observed topological differ-
ences can be explained by the presence of relatively rigid hydrophobic
PPhe cores in the PEG–PGlu90–PPhe25 cl-micelles that rendered them
more stiff and deceased their deformation at the substrate surface.
The pH-induced dimensional changes (nanogel-like behavior) of the
cl-micelles were evident from an increase in the micelle size and net
negative charge (Fig. 2A).
Fig. 2. Swelling behavior (A) and stability (B) of cl-micelles. (A) The effective diameter,
D
eff (●) and ζ-potential (■) of cl-micelles as a function of pH. (B) Changes in Deff of
cl-micelles (●) and non-cl-micelles (■) upon treatment with different concentrations
of 8 M urea.
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
covalent cross-linking. Importantly, the cross-linking did not prevent
the degradation of cl-micelles by proteolytic enzymes. The enzymatic
biodegradability of PEG–PGlu90–PPhe25 cl-micelles was determined by
incubating the micelles with cathepsin B followed by the analysis
of the reaction mixture using size exclusion chromatography (SEC)
Table 3
Physicochemical characteristics of cl-micelles before and after drug loading.
a
b
Sample
Deff (nm)
PDI
ζ-Potential (mV) LC (w/w%)
Cl-micelles
PTX/cl-micelles
CDDP/cl-micelles
90 ± 1.2 0.14 −20.0 ± 1.2
105 ± 2.8 0.23 −19.1 ± 1.5
71 ± 3.3 0.19
76 ± 4.0 0.24
–
8.4
(
Fig. S4). A gradual decrease in the UV absorption of the micelle peak
−3.7 ± 1.0
−5.7 ± 0.47
22.4
(CDDP + PTX)/cl-micelles
24.8c
was observed in SEC chromatograms over the incubation time, which
almost disappeared after 24 h. Furthermore, decrease in light scattering
intensity along with the drastic increase of PDI was detected by DLS of
the cl-micelles dispersions already after 12 h of incubation with cathep-
sin B suggesting structural disintegration of such micelles (Table S1).
However, further studies will be necessary to characterize the degrada-
tion products and determine whether drug incorporation can alter the
degradation pattern of the cl-micelles. Overall, these results imply that
the reinforcing cross-links within the shell of PEG–PGlu90–PPhe25
micelles can permanently suppress their dissociation. Resulting cl-
micelles can withstand environmental challenges and sink conditions
encountered after systemic administration and, consequently, provide
means for temporal control over drug disposition. On the other hand,
enzymatic degradation of the polypeptide-based building blocks of
cl-micelles reduces the risk of polymer accumulation inside cells and
can also facilitate specific intracellular drug release.
Data are expressed as mean ± SD (n = 3).
a
Effective diameter (Deff), polydispersity index (PDI) and ζ-potential were determined
in water (pH 6.5).
b
Pt and PTX contents were determined by ICP-MS and HPLC, respectively. Loading
capacity (LC) is expressed as the mass of incorporated drug per mass of drug loaded
cl-micelles (w/w).
c
Total LC for the combination of CDDP (15.4%) and PTX (9.4%).
as compared to single CDDP/cl-micelle formulation (15% vs. 23%). This
apparent difference in drug loading is not yet understood and needs
to be investigated further. We hypothesize that the presence of hydro-
phobic solute in the core of the micelle affects the conformation of the
polypeptide chains at the PPhe core–PGlu shell interface. Specifically,
in more hydrophobic environment PGlu chains can transition from ran-
dom coil to α-helical conformation leading to structural condensation
in this region which may restrict space for the loading CDDP molecules.
Validation of this hypothesis is of considerable interest and is ongoing in
our laboratory.
Drug loading and release from cl-micelles
PEG–PGlu90–PPhe25 cl-micelles have a well-defined structure with a
central hydrophobic core formed by PPhe chains, swollen intermediate
layer of a cross-linked PGlu network and external PEG shell surrounding
the core and intermediate layer. Each of these three nanodomains con-
tributes to the utility of cl-micelles as drug nanocarriers. A PPhe core can
serve as a cargo for water-insoluble drugs. An ionic intermediate layer
can incorporate hydrophilic drug molecules through electrostatic or
covalent bonding while PEG shell in addition to stabilizing micelles in
dispersion also allows minimizing interactions of the micelles with
blood components. Therefore, such cl-micelles would provide unique
opportunity to simultaneously incorporate and deliver multiple chemo-
therapeutic drugs with very different physical properties and modes of
action inside a single polymeric carrier in order to achieve a synergistic
effect and enhance the efficacy of treatment. In the present study
cl-micelles with binary drug combination of hydrophilic CDDP and
hydrophobic PTX was prepared. At first, PTX was solubilized into the
PPhe cores of the cl-micelles using micelle extraction method. According
to this method, PTX dissolved in ethanol was added to the aqueous
dispersion of cl-micelles upon agitation followed by evaporation of
organic solvent and removal of unbound PTX. Under these conditions
PTX loading capacity (the net amount of drug loaded into a carrier)
was about 9 w/w% weight as was quantified by HPLC. The size and
ζ-potential of the PTX-loaded cl-micelles were comparable to those of
empty cl-micelles indicating that the PTX incorporation into PPhe
cores did not affect the macroscopic characteristics of the pre-formed
cl-micelles. As the next step, CDDP was incubated with the aqueous
dispersions of PTX-loaded cl-micelles for 48 h at pH 9.0 [7]. Such condi-
tions were chosen to maximize the swelling of PGlu cross-linked layer
The release of the drugs from cl-micelles was studied by equilibrium
dialysis at 37 °C at either pH 7.4 (PBS) or pH 5.5 (ABS), which reflect
conditions encountered in plasma and in intracellular compartments
(lysosomes), respectively. The kinetic profiles of drug release from
(CDDP + PTX)/cl-micelles are presented in Fig. 3. As seen from these
data sustained but temporally distinct release of Pt(II) species and PTX
was observed. Notably, PTX release was much faster than that of
Pt(II), which is expected since PTX is physically entrapped into the
PPhe core. In contrast, CDDP binds with PGlu chains through electro-
static and coordination interactions and its release usually proceeds
via ligand exchange reactions with chloride or other biologically abun-
dant anions, thus delaying its liberation from the cl-micelles. Pt(II)
release from the cl-micelles was also a pH-dependent process. Indeed,
Pt(II) species were liberated from the micelles faster at pH 5.5 than at
pH 7.4 (Fig. 3B), probably, due to protonation of carboxylic groups of
PGlu, which weakens the drug and micelle electrostatic coupling. For
example, during 24 h (CDDP + PTX)/cl-micelles released 16.7 ± 5.5%
of loaded Pt(II) at pH 5.5 and only 8.8 ± 3.6% at pH 7.4, respectively.
Notably, pH changes practically did not affect the release of PTX.
Also, noteworthy that Pt(II) release from (CDDP + PTX)/cl-micelles
at the acidic pH was further accelerated in the presence of cathepsin
B in the media (Fig. 3C). Cathepsin B is a lysosomal thiol-dependent
protease [23] and is also extracellularly present in pathological tissues
such as tumors and sites of inflammation [24,25]. These results indicate
that degradation of cl-micelles can further facilitate the drug release
once located within targeted tissue and cells.
In vitro cytotoxicity of the drug-loaded cl-micelles
(Fig. 2A) and increase the accessibility of the bulk of the carboxylate
groups to CDDP. As expected, the net negative charge and particle size
of cl-micelles decreased upon CDDP loading consistent with neutraliza-
tion and condensation of PGlu segments by CDDP (Table 3).
The cytotoxicity of drug combination loaded into cl-micelles was
determined in human ovarian carcinoma A2780 cells using MTT assay.
Calculated IC50 values are summarized in Table 4.
1
5 w/w% loading was achieved with respect to CDDP while total
Importantly, cl-micelles alone were nontoxic to the cells up to the
concentration of 5 mg/mL. CDDP/cl-micelles displayed lower cytotoxic
activity than free CDDP. Since unloaded cl-micelles did not affect the
cell survival in the entire range of concentrations the cytotoxic activity
of CDDP/cl-micelles was mediated by the Pt(II) species slowly released
from the micelles. However, we found that (CDDP + PTX)/cl-micelles
were significantly more effective in killing A2780 ovarian cancer cells
than CDDP/cl-micelles. The dose of CDDP in cl-micelles was indeed
reduced nearly by 30-fold when used in combination with PTX (at
loading values for binary drug combination was about 25% and
corresponded to 10:1 molar ratio (CDDP:PTX) of drugs in the
micelles. (CDDP + PTX)/cl-micelles maintained their spherical mor-
phology as was confirmed by TEM (Fig. S5). Notably, (CDDP + PTX)-
loaded cl-micelles were stable in aqueous dispersions, exhibiting no
aggregation or precipitation with loading capacity preserved for as
long as month (Table S2). Interestingly, the loading capacity of
cl-micelles with respect to CDDP was decreased after loading of PTX
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
7
Table 4
Comparison of IC50 values for various drug-loaded cl-micelles and free CDDP
against A2780 cells as determined by the MTT assay.
IC50 (nM)
Free CDDP
1.5
CDDP/cl-micelles
PTX/cl-micelles
3.9
0.17
0.14
6.3
a
(CDDP + PTX)/cl-micelles
a
CDDP/cl-micelles + PTX/cl-micelles
a
Calculated with respect to CDDP.
and CDDP/cl-micelles were injected 4 times at 4-day intervals at an
equivalent dose of 4 mg-CDDP/kg determined as the maximum toler-
ated dose upon this treatment schedule [18]. Animals injected with
(
CDDP + PTX)/cl-micelles received 4 mg/kg CDDP and 1 mg/kg PTX
equivalents per dose. The changes in the relative tumor volume,
body weight and animal lifespan are shown in Fig. 4.
Tumor burden was significantly decreased by both free CDDP
(
P b 0.05) and CDDP/cl-micelle (P b 0.05) treatments compared to
control (Fig. 4A). However, tumors in the animals treated with CDDP/
cl-micelles remained relatively smaller than in animals treated with
free CDDP between days 0 and 18. As a result, increased survival of
the animals was observed in mice treated with CDDP/cl-micelle com-
pared to CDDP alone (Fig. 4C). Notably, treatment with PTX-loaded
cl-micelles did not have any substantial effect on the tumor growth
and only a minor increase in survival was observed over controls. In
contrast, nearly complete inhibition of tumor growth was observed
for the mice treated with (CDDP + PTX)/cl-micelles from days 0 to
1
4, which translated into significantly increased overall survival of the
animals treated with (CDDP + PTX)/cl-micelle compared to either
CDDP/cl-micelles (P b 0.05) or CDDP alone (P b 0.05) (Fig. 4A and C).
On the second day post-treatment animals injected with either
(
CDDP + PTX)/cl-micelles or CDDP/cl-micelles displayed significantly
higher levels of Pt in tumors compared to free CDDP treatment group
P b 0.05) which may be attributed to the enhanced permeability and
(
retention (EPR) effect (Fig. 5A) [26].
To elucidate the mechanism underlying the enhanced antitumor
activity of (CDDP + PTX)/cl-micelles, the tumors were excised post-
treatment (on 2nd day after last injection) and processed for Ki-67-
Caspase-3 assay to examine the effect of each treatment on the induc-
tion of apoptosis (Fig. 6).
The number of caspase-3 positive cells were significantly higher in
tumors from mice that received (CDDP + PTX)/cl-micelles compared
to tumors in CDDP/cl-micelles (P b 0.05) or PTX/cl-micelles (P b 0.05)
or free CDDP (P b 0.05) (Fig. 6 and Fig. S6). This assay further corrobo-
rates the superior antitumor efficacy of binary drug combination cl-
micelle formulation over single drug-loaded micelles or CDDP alone.
Since the levels of Pt accumulation in tumors were comparable for the
treatments with both single or binary drug formulations, we rationalize
that an increase in therapeutic efficacy of (CDDP + PTX)/cl-micelles
can be related to highly synergistic interactions between CDDP and
PTX simultaneously delivered to the tumors. Indeed, the average weight
ratio of the CDDP and PTX in the tumors of the animals treated with
(CDDP + PTX)/cl-micelles was determined to be 4:1, the same as the
drug loading ratio in cl-micelle, indicating the spatial–temporal syn-
chronization of drug exposure. CDDP and PTX are highly suited for com-
bination chemotherapy because they have distinct mechanisms of
action. CDDP binds to DNA base pairs creating adducts, cross-links,
and strand breaks that inhibit DNA replication. Unrepairable DNA
damage often results in activation of the apoptotic pathway. Paclitaxel
acts by binding to intracellular β-tubulin, which leads to microtubule
Fig. 3. In vitro drug release profiles for Pt (●) and PTX (■) from cl-micelles in (A) PBS,
pH 7.4; (B) ABS, pH 5.5; and (C) ABS in the presence of cathepsin B (10 units/mL) at
37 °C. Data are expressed as mean ± SD (n = 3).
1
0:1 CDDP/PTX molar ratio) to achieve the same cytotoxic effect pro-
duced by the CDDP loaded cl-micelles. The CI value at IC50 for this
CDDP + PTX)/cl-micelles combination was 0.078, indicating strong
(
synergistic cytotoxicity against A2780 ovarian cancer cells. Notably, the
combination of two separate micellar formulations (CDDP/cl-micelle +
PTX/cl-micelle) applied at the same drug ratio elicited a cytotoxicity
comparable to that of the CDDP/cl-micelle formulation (Table 4) and
displayed antagonistic CI value of 1.2 at IC50. These data suggest that
synchronized delivery of this drug combination in the same cl-micelle
formulation may play an essential role in the cytotoxic activity.
Antitumor activity
2
stabilization, G –M arrest and apoptosis [27]. CDDP is thought to be
relatively non-cell cycle specific in terms of its cell-killing effects and
tends to synergize or have additive cytotoxicity with agents that alter
mitosis (PTX) [28,29]. Apart from the mechanism basis, synergistic
effect greatly depends on drug dosages, combination ratios, cell lines
Motivated by the enhanced in vitro efficacy of (CDDP + PTX)/
cl-micelle formulation, we evaluated its antitumor efficacy in vivo in
mice bearing A2780 human ovarian cancer xenografts. Free CDDP
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
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S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
Fig. 5. Tissue distribution of platinum in different treatment groups as determined
ICP-MS. (A) Mice were sacrificed at day 14 of the treatment with (CDDP + PTX)/
cl-micelles or CDDP/cl-micelles or free CDDP. (B) Significant reduction in the Pt
levels in various organs of mice treated with (CDDP + PTX)/cl-micelles was confirmed
by comparing Pt levels at day 14 and day 45. Values indicated are means ± SEM
(n = 3), *P b 0.05.
It should be emphasized that PTX and CDDP may also interact
synergistically with respect to toxicity to the normal tissues as well.
However, this was not the case in our studies. Fig. 4B shows that
either single or binary drug loaded cl-micelles did not induce body
weight loss while the same dose of the free CDDP produced a consider-
able body weight loss (P b 0.05), which indicated systemic toxicity of
free CDDP. In the kidney, the primary target organ of CDDP toxicity
[
33,34], single or binary drug loaded cl-micelles showed significantly
Fig. 4. In vivo antitumor efficacy of (CDDP + PTX)/cl-micelles in A2780 human ovarian
cancer xenograft-bearing female nude mice. Relative changes in (A) tumor volume and
lower Pt levels than the free CDDP (Fig. 5A). Reduced Pt accumulation
(B) body weight were measured following intravenous administration of (CDDP +
PTX)/cl-micelles(●) or CDDP/cl-micelles (■) or PTX/cl-micelles (▲) or free CDDP (▼)
or 5% dextrose (♦). Drug formulations were injected in100 μL at a dose of 4 mg
CDDP or 1 mg PTX equivalents/kg body weight 4 times at 4-day intervals as indicated
by the arrows. Values indicated are means ± SEM (n = 8). (C) Kaplan–Meier analysis
of overall survival in (CDDP + PTX)/cl-micelles group (1) or CDDP/cl-micelles group
(2) or PTX/cl-micelles group (4) or free CDDP group (3) or control group (5). Tumor
volume and body weight are normalized with respect to tumor volume or body weight
at day 0. *P b 0.05.
and intervention schedules. Indeed, synergy between CDDP and PTX
was reported to be highly schedule dependent [30,31]. Profound syner-
gistic effects were observed when PTX was administered prior to CDDP
[
28–30] while the sequence of CDDP before PTX had less antitumor
activity in vitro [31], and induced more profound neutropenia in vivo
32]. One can speculate that the ability of (CDDP + PTX)/cl-micelles
[
to supply drugs in a temporally controlled fashion by releasing PTX sig-
nificantly faster than CDDP may contribute into observed improvement
of antitumor efficacy of drug combination.
Fig. 6. Ki-67-caspase-3 apoptosis assay. Quantification of caspase-3 positive cells in tumor
tissue from mice from various groups. Data are presented as mean ± SD (n = 5 random
microscopic fields for each tumor slice).
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
lease (2013), http://dx.doi.org/10.1016/j.jconrel.2013.04.026

S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
9
in kidney after (CDDP + PTX)/cl-micelles or CDDP/cl-micelle treatment
4. Conclusions
ameliorated CDDP-induced nephrotoxicity as confirmed by tissue histo-
pathology analysis.
In the present investigation, micelles based on biodegradable and
biocompatible amphiphilic PEG–b–PGlu–b–PPhe triblock copolymers
were evaluated as potential carriers for co-delivery of chemotherapeutic
drugs with various physicochemical properties and modes of action.
These nanostructures were designed to have multicompartment mor-
phology for drug loading and were cross-linked at an intermediate
layer within the polymer micelles to ensure their prolonged stability
upon systemic administration. Hydrophilic CDDP and hydrophobic
PTX were loaded in such micelles with high efficiency, exhibited differ-
ential release profiles and synergistic cytotoxic effect against ovarian
cancer cells. The binary drug combination simultaneously delivered
using cl-micelles exhibited superior antitumor efficacy over single
drug cl-micelle analogues in vivo in synergistic manner. Altogether,
this study demonstrates a fundamental possibility for simultaneous
delivery of chemotherapeutics and molecular targeting agents via single
well-defined and structurally tunable polymeric nanocarrier.
Light microscopic examination of H&E stained kidney sections
from sacrificed animals indicated focal tubular basophilia with regen-
eration in kidneys from all animals treated with free CDDP, while no
histopathological changes were observed in kidney from animals in
the (CDDP + PTX)/cl-micelle-treated or CDDP/cl-micelles-treated
groups compared with the control (Fig. 7). Reduced nephrotoxicity
of (CDDP + PTX)/cl-micelles or CDDP/cl-micelles is likely due to its
macromolecular size coupled with slow release of the drug, which
leads to a lower renal tubular drug exposure. This is a significant
result considering renal toxicity is the most severe, dose-limiting toxic-
ity with CDDP treatment [35]. (CDDP + PTX)/cl-micelles showed very
high Pt accumulation in spleen and liver (Fig. 5A), but this amount
reduced significantly (P b 0.01) with time (Fig. 5B). Despite much
higher exposure of liver and spleen to CDDP in the (CDDP + PTX)/
cl-micelle or CDDP/cl-micelles treatment, there was no significant
change in hepatic toxicity markers of blood chemistry (Table S3), indi-
cating no obvious toxicity to the liver. There was no evidence of liver
or splenic toxicity by histopathology (data not shown). In spleen, the
micelles (either free or localized inside macrophages) were detected
predominantly in the red pulp with little to no staining in the lymphoid
region (Fig. S7). Free CDDP treatment has previously been reported to
have a remarkable effect on immunological function due to its ability
to reduce lymphocytes in the thymus and the spleen with changes in
splenic white pulp being the most significant [36]. The absence of
Acknowledgments
This work was supported by the National Institute of Health grant
CA116590 (T.K.B.). We acknowledge the assistance of the Nanomaterials
Core Facility of the Center for Biomedical Research Excellence (CoBRE),
Nebraska Center for Nanomedicine supported by the Institutional
Development Award (IDeA) from the National Institute of General
Medical Sciences of the National Institutes of Health under grant
number P20GM103480. We also thank NMR, Electron Microscopy and
Nanoimaging Cores (University of Nebraska Medical Center). The
authors are grateful to Dr. Larisa Poluektova for assistance with IHC
studies and Dr. Hardeep Oberoi for help with animal studies. We
acknowledge assistance of Jinjin Zhang and Zhengyuan Zhou for the
help in preparation of illustration for this paper.
(
CDDP + PTX)/cl-micelles in lymphoid regions of white pulp may
thus limit free-drug exposure to these follicles. Collectively, these results
demonstrate that biodegradable PEG-polypeptide hybrid cl-micelles
carrying CDDP and PTX drug combination exerted superior antitumor
efficacy, both in terms of tumor inhibition and survival, which could
be attributed to the preferential simultaneous accumulation, and
increased potency.
Fig. 7. Light microscopy images (original magnification 100×) of hematoxylin and eosin-stained kidney sections of kidney. Tissue samples were collected at day 14.
Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
lease (2013), http://dx.doi.org/10.1016/j.jconrel.2013.04.026

1
0
S.S. Desale et al. / Journal of Controlled Release xxx (2013) xxx–xxx
micelles: comparative pharmacokinetics, antitumor activity, and toxicity in mice,
Appendix A. Supplementary data
Int J Nanomedicine 7 (2012) 2557.
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20] M.J. Krysmann, V. Castelletto, A. Kelarakis, I.W. Hamley, R.A. Hule, D.J. Pochan,
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Please cite this article as: S.S. Desale, et al., Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer, J. Control. Re-
lease (2013), http://dx.doi.org/10.1016/j.jconrel.2013.04.026