Self-assembling, amphiphilic polymer-gemcitabine conjugate shows enhanced antitumor efficacy against human pancreatic adenocarcinoma

Article abstract of DOI:10.1021/bc400032x

The therapeutic efficacy of gemcitabine is severely compromised due to its rapid plasma metabolism. Moreover, its hydrophilicity poses a challenge for its efficient entrapment in nanosized delivery systems and to provide a sustained release profile. In th

Full text of DOI:10.1021/bc400032x

Article
pubs.acs.org/bc
Self-Assembling, Amphiphilic Polymer−Gemcitabine Conjugate
Shows Enhanced Antitumor Efficacy Against Human Pancreatic
Adenocarcinoma
Deepak Chitkara,§,† Anupama Mittal,§ Stephan W. Behrman,‡ Neeraj Kumar,† and Ram I. Mahato*,§
‡
§Department of Pharmaceutical Sciences and Department of Surgery, University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States
†Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, India 160062
ABSTRACT: The therapeutic efficacy of gemcitabine is severely compromised due to its rapid plasma metabolism. Moreover,
its hydrophilicity poses a challenge for its efficient entrapment in nanosized delivery systems and to provide a sustained release
profile. In this study, gemcitabine was covalently conjugated to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene
carbonate) (PEG-PCC) which could self-assemble into micelles of 23.6 nm. These micelles afforded protection to gemcitabine
from plasma metabolism as evident by negligible amount of gemcitabine and its metabolite dFdU detected in the plasma after 24
h. A controlled release of gemcitabine from the micelles was observed with 53.89% drug release in 10 days in the presence of
protease enzyme Cathepsin B. Gemcitabine conjugated micelles were cytotoxic, showed internalization, and induced cell
apoptosis in MIA PaCa-2 and L3.6pl pancreatic cancer cell lines. These micelles efficiently inhibited tumor growth when injected
intravenously into MIA PaCa-2 cell derived xenograft tumor bearing NSG mice at a dose of 40 mg/kg in terms of reduced tumor
volume and tumor weight (0.38 g vs 0.58 g). TUNEL assay revealed that gemcitabine conjugated micelles induced a much higher
extent of apoptosis in the tumor tissues compared to free gemcitabine. In conclusion, gemcitabine conjugated micelles were able
to enhance the drug payload, protect it from rapid plasma metabolism, and provide a sustained release and showed enhanced
antitumor activity, and thus have the potential to provide a better therapeutic alternative for treating pancreatic cancer.
hydrophilic nature which inhibits a high payload and prolonged
drug release. Moreover, due to its hydrophilicity, its extravasation
into the pancreatic cancer tissues which have a very dense fibrotic
stroma is restricted. This compels a high dosing frequency at high
drug doses which in turn precipitates significant adverse effects.
To overcome these limitations, several derivatives/conjugates of
gemcitabine and new delivery systems have been designed for its
effective delivery into the tumor compartment.
INTRODUCTION
■
Pancreatic ductal adenocarcinoma is the fourth leading cause of
cancer-related deaths, with only 3% of patients surviving for 5
years.1 Surgical resection of the tumor is the only reliable
treatment modality; however, prognosis still remains poor due to
its potential for metastatic spread, high local recurrence, and
chemoresistance, largely due to the presence of cancer stem
cells.2−4 Consequently, the need for chemotherapeutic strategies
is of paramount importance.
Gemcitabine (2′-deoxy-2′,2′-difluorocytidine) is a pyrimidine
antimetabolite that displays anticancer activity by inducing S-
phase arrest and inhibiting DNA synthesis.5,6 Gemcitabine
(Gemzar) is used as the first-line therapy for treating pancreatic
ductal carcinoma; however, its clinical benefit is limited due to its
short plasma half-life resulting from rapid metabolism by cytidine
deaminase, into its inactive metabolite 2′,2′-difluorodeoxyur-
idine (dFdU).7 Its therapeutic efficacy is further diminished by its
Liposomes,8−11 nanoparticles,12−15 lipidic and nonlipidic
derivatives, and polymeric drug conjugates5,16,17 have been
investigated to prevent rapid plasma degradation of gemcitabine
and enhance delivery to the tumor tissue. Its liposomal
formulation showed an appreciable improvement in its cytotoxic
Received: January 16, 2013
Revised: June 7, 2013
© XXXX American Chemical Society
A
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activity against multiple myeloma11 and human pancreatic cancer
cell lines8 along with an increase in half-life and area under the
curve (AUC) in animal studies.18 However, gemcitabine being a
low molecular weight, uncharged molecule at physiological pH
readily diffused out from the liposomal bilayers resulting in low
entrapment efficiencies and rapid release rate.8 Biodegradable
poly(ε-caprolactone) nanoparticles containing magnetite and
gemcitabine also showed promising results in xenograft
models.12,15 Nevertheless, these reported systems either have
low drug encapsulation efficiencies due to high water solubility of
gemcitabine or posed a problem of rapid drug release owing to
faster drug diffusion from the system into the external aqueous
environment.
form stimulus-sensitive micelles for lysosomal delivery. These
micelles have been used for loading 4-(N)-stearoyl gemcitabine,
a lipophilic prodrug.33 Thus, this system is suitable only for
hydrophobic drugs.
In our previous publications,34−36 we used a copolymer of
mPEG and MBC/PCC for delivery of hydrophobic drugs which
get solubilized in the core of the micelles. However, the current
work focuses on delivery of hydrophilic drug, gemcitabine, which
itself is a challenging molecule for encapsulation in a nanocarrier
system since it shows low payloads in nanocarriers and is prone
to plasma metabolism. In this study, we have conjugated
gemcitabine to the carboxyl pendant groups of poly(ethylene
glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)
(PEG-PCC) thus enabling its efficient loading and improving
the plasma stability. This polymer consists of two components, a
biocompatible PEG block and a biodegradable polycarbonate
PCC block. For the carbonate block, the starting monomer used
is 5-methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one carbonate
which is a modification of an intermediate used in the synthesis
of numerous antiviral compounds. This polymer was chosen
since polycarbonates possess biodegradability, low toxicity, and
tunable mechanical properties. Furthermore, they degrade into
carbon dioxide and alcohol, which unlike the degradation
products of more frequently used polymers like poly(L-lactide)
are less acidic, have less effect on microenvironment pH, and as
such will not result in local inflammation.36−38 Further, this
amphiphilic copolymer self-assembles into micelles which were
characterized for plasma stability, drug release, in vitro
cytotoxicity, intracellular fate, and ability to induce apoptosis in
MIA PaCa-2 pancreatic cancer cells and subsequently tested in
vivo in MIA PaCa-2 cell derived xenograft tumor model after
intravenous administration.
Conjugating gemcitabine directly onto the lipidic or polymeric
carrier has provided greater therapeutic advantage. Prodrugs of
gemcitabine were synthesized by conjugating the lipidic chains at
3′ and/or 5′ hydroxyl and/or 4-amino group of gemcita-
bine.19−25 These lipophilic prodrugs were shown to prevent
degradation of gemcitabine by water as well as by metabolic
enzymes. However, their low aqueous solubility necessitated
their formulation into nanoparticles or lipid based carriers.26−28
To meet this end, squalenoyl derivatives of gemcitabine were
explored that self-assembled to form nanoparticles in aqueous
media and showed superior anticancer activity as compared with
free gemcitabine in 60 human tumor cell panel and
subcutaneously grafted L1210 tumor models.23 Although these
nano-assemblies exhibited better pharmacokinetic profiles, their
efficacy was limited by the rapid reticulo-endothelial system
(RES) uptake because of hydrophobic surface and colloidal
nature.22 To reduce opsonization and blood clearance of these
nano-assemblies, the PEGylation approach was used wherein
poly(ethylene glycol)-cholesterol (PEG-chol) or squalene-PEG
was added to form composite nano-assemblies and this was
accompanied by a significant decrease in gemcitabine loading;
however, addition of PEG showed enhanced efficacy on
gemcitabine-resistant leukemia L1210K cells than either of the
non-PEGylated counterparts or free gemcitabine.29 In the
present study, the presence of PEG corona ensures stealth
properties of gemcitabine conjugated polymeric micelles while
the presence of carboxyl groups enable the conjugation of higher
content of gemcitabine to the polymer resulting in higher
payload of gemcitabine. The current approach is so designed that
it could be further exploited for co-delivery of multiple
chemotherapeutic drugs,30 since the polymer bears multiple
carboxylic groups. This will help us achieve higher efficacy or
overcome chemoresistance which might not be feasible in the
previous system.
Polymeric conjugates of gemcitabine have been prepared using
PEG,5,6 poly(N-hydroxyethyl acrylamide) (PHEA),17 polygluta-
mic acid,16,31 and poly(N-(2-hydroxypropyl)methacrylamide)
(HPMA).32 PEG conjugates of gemcitabine have shown to
improve the stability and its pharmacokinetic profile and enhance
cytotoxicity.5,6 However, PEG provides only one reactive
functional group for conjugation with gemcitabine thus resulting
in a reduced payload. To increase the payload, a bicarboxylic
amino acid, aminoadipic acid, was used as a linker wherein a drug
loading of 6.39% w/w was obtained.5 Polyglutamic acid when
conjugated to gemcitabine showed improved stability16 and in
vivo antitumor efficacy in breast tumor bearing mice.31 Similar
advantages of conjugation were also demonstrated by Yang et
al.32 and Cavallaro et al.17 for HPMA and PHEA conjugates,
respectively. In another study, hydrazone bonds were incorpo-
rated in the hydrophilic PEG and hydrophobic stearic acid to
EXPERIMENTAL PROCEDURES
■
Materials. Gemcitabine hydrochloride was purchased from
LC Laboratories (Woburn, MA) and tetrahydrouridine was
purchased from Biovision. 2′-Deoxycytidine, Cathepsin B (5 U/
mL), 2,2-bis(hydroxymethyl) propionic acid, methoxy poly-
ethylene glycol (mPEG; Mn = 5000, polydispersity index (PDI)
= 1.03), stannous 2-ethylhexanoate (Sn(Oct)2), 1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide HCl (EDC), 1-hydroxyben-
zotriazole (HOBT), and benzyl bromide were purchased from
Sigma-Aldrich (St. Louis, MO) and used as received.
LysoTracker Red DND-99 was procured from Molecular Probes,
Invitrogen. DeadEnd Fluorimetric TUNEL (Terminal deoxy-
nucleotidyl Transferase Biotin-dUTP Nick End Labeling)
system was purchased from Promega (Madison, WI). All other
chemicals were of analytical grade.
Methods. Synthesis and Characterization of Polymer Drug
Conjugate. Synthesis of 2-Methyl-2-Benzyloxycarbonyl-Pro-
pylene Carbonate (MBC). To improve the delivery and stability
of gemcitabine, amphiphilic polymer with carboxylic pendant
groups was synthesized as reported earlier by our group.34,39
Briefly, monomer, MBC, was synthesized in a two-step reaction,
wherein a mixture of 2,2-bis(hydroxymethyl)propionic acid
(0.168 mol) and potassium hydroxide (0.169 mol) was reacted
with benzyl bromide (0.202 mol) in dimethylformamide (DMF;
125 mL) at 100 °C for 16 h. The solvent was removed from the
reaction mixture under reduced pressure and the residue
obtained was dissolved in ethyl acetate, washed with water, and
dried over MgSO4. Ethyl acetate was then evaporated to yield a
crude product, which was recrystallized from toluene to give pure
benzyl-2,2-bis(methylol)propionate. In the second step, benzyl
B
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2,2-bis(methylol)-propionate (0.05 mol) was dissolved in
dichloromethane-pyridine mixture (6:1 v/v; 175 mL) and chilled
to −78 °C. Triphosgene (25 mmol in dichloromethane) was
then added dropwise to it over 1 h and the reaction was allowed
to continue for additional 2 h at the room temperature, followed
by quenching with saturated aqueous NH4Cl solution (75 mL).
The organic layer was subsequently washed thrice with
hydrochloric acid (1 M, 100 mL) followed by saturated aqueous
NaHCO3 (300 mL) and dried with Na2SO4. The organic layer
was then evaporated under reduced pressure to give MBC as a
crude product, which was further recrystallized from ethyl
acetate.
Synthesis of PEG-MBC. Polymer, PEG-MBC, was synthesized
by ring-opening polymerization of MBC and mPEG in the
presence of stannous-2-ethyl hexanoate as a catalyst at 130 °C for
16 h under reduced pressure. The crude polymer so obtained was
dissolved in chloroform and purified by precipitation in hexane−
diethyl ether mixture followed by drying under vacuum for 48 h.
Hydrogenation of PEG-MBC. Protective benzyl group of
PEG-MBC was removed by hydrogenation to obtain free
carboxyl group for subsequent conjugation. For this, PEG-
MBC (1 g) was dissolved in a mixture of tetrahydrofuran and
methanol (1:1; 12 mL) containing palladium on carbon (Pd/C)
(200 mg) and charged with hydrogen at a pressure of 55 psi.
Reaction was allowed to continue for 18 h following which Pd/C
was removed by filtration and solvent was evaporated under
reduced pressure to get PEG-PCC.
Conjugation of Gemcitabine and Dodecanol onto PEG-
PCC. Gemcitabine and dodecanol were conjugated to the
carbonyl groups of PEG-PCC polymer using carbodiimide
coupling. For this, PEG-PCC (300 mg) was dissolved in 5 mL of
DMF followed by addition of 223 mg of 1-hydroxybenzotriazole
(HOBT in 1 mL DMF), 317 mg of 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide HCl (EDC in 5 mL DMF) and 340 μL of
diisopropylethylamine (DIPEA). Gemcitabine hydrochloride
and dodecanol were dissolved in DMF containing 20 μL of
DIPEA and added to the reaction mixture. The reaction was
allowed to proceed for 48 h under nitrogen atmosphere at room
temperature. After completion of the reaction, crude product was
purified by precipitation in large excess of cold diethyl ether. The
precipitate was then dissolved in chloroform and precipitated
twice in cold isopropyl alcohol. The precipitate obtained was
then dissolved in acetone and dialyzed against water and
lyophilized to obtain mPEG-b-PCC-g-gemcitabine-g-dodecanol
(mPEG-PCC-g-GC-g-DC).
Characterization of Gemcitabine−Polymer Conjugate
Micelles. Particle Size and Zeta Potential. Particle size and
zeta potential were measured by using Malvern Zetasizer (Nano
ZS series). Micelles (10 mg/mL) were taken in purified water
and analyzed at a scattering angle of 173°. A total of 12
measurements were taken per sample with a time span of 10 s.
Zeta potential of micelles was determined in terms of
electrophoretic mobility by taking an average of 5 measurements
per sample. Data of particle size and zeta potential are reported as
the mean SD of three independent samples.
Particle Morphology (Transmission Electron Microscopy,
TEM). Polymer drug conjugate micelles were visualized using a
JEM-100S (Japan) TEM. Micellar suspension was loaded onto
copper grid and air-dried. It was stained with uranyl acetate (1%
w/v) and visualized under TEM at magnifications ranging from
50K to 100K with an accelerating voltage set at 60 kV.
Critical Micelle Concentration (CMC). CMC of gemcitabine
conjugated polymer was determined using pyrene as a
hydrophobic fluorescent probe as described earlier.33 The
fluorescent intensity was recorded at an excitation wavelength
of 338 and 333 nm and emission wavelength of 390 nm using
SpectraMax M2/M2e spectrofluorimeter (Molecular Devices,
Sunnyvale, CA). The CMC value was obtained from the plots of
intensity ratio (I338/I333) versus logarithm of polymer concen-
tration as intersection point of two tangents drawn to the curve at
high and low concentrations.
Quantification of Gemcitabine Payload in Polymer Drug
Conjugate. To extract out the conjugated drug, formulation (10
mg) was prepared and subjected to alkaline hydrolysis in NaOH
(1 N) at 40 °C for 1 h as reported earlier6 for PEG−gemcitabine
conjugate formulation. Samples were then appropriately diluted
and the content of gemcitabine was determined by HPLC-UV
analytical method. Chromatography was performed on Inertsil
ODS 3 column (4.5 × 250 mm) using methanol and sodium
acetate buffer (20 mM, pH-5.5) as a mobile phase at the ratio of
07:93. Injection volume was kept at 20 μL and analysis was
performed at 268 nm. Payload was calculated using eq 1. The
stability of free gemcitabine was also tested at the alkaline
hydrolysis conditions, i.e., 1 N NaOH at 40 °C for 1 h to
determine any degradation of drug.
Amount of drug loaded
Total weight of formulation
⎛
⎞
⎟
w
w
⎜
Payload %
=
× 100
⎝
⎠
(1)
In Vitro Release of Gemcitabine. In vitro release of
gemcitabine from polymer drug conjugate micelles was studied
in the presence and absence of protease Cathepsin B (5 U/mL).
Micelles formulated in PBS (1 mL; equivalent to 0.5 mg of
gemcitabine) containing 20 μL of enzyme were taken in a dialysis
bag (molecular weight cutoff 2000 Da), clamped at both ends,
and placed in aqueous buffer, PBS (pH 5.5 and 7.4; 5 mL).
Samples (1 mL) were withdrawn at regular time intervals and
replaced with an equivalent amount of the fresh media. The
content of gemcitabine present in the samples was analyzed using
HPLC-UV method as described in the previous section. Samples
containing physically entrapped gemcitabine in mPEG-b-PCC-g-
DC micelles and free gemcitabine were also kept as controls.
Stability Study in Human Plasma. Human plasma (0.5 mL)
was taken in microcentrifuge tubes and polymer drug conjugate
micelles (in PBS; equivalent to 0.2 mg of gemcitabine) or
gemcitabine were added to each tube followed by incubation at
37 0.1 °C. At regular time intervals, plasma samples were taken
to which 10 μL of tetrahydrouridine (10 μg/mL) was added to
Characterization of Polymer and Polymer Drug Conjugate.
1H NMR spectra were recorded on a Bruker (400 MHz, T = 25
°C) using DMSO-d6 as a solvent in a chemical shift range of 0−
12. FTIR of gemcitabine, PEG-PCC, and polymer drug
conjugate was recorded on a Perkin-Elmer spectrometer in a
frequency range of 650−4000 cm−1.
Formulation Development. Gemcitabine conjugated poly-
meric micelles were prepared by nanoprecipitation as described
previously with slight modification.34 Briefly, 20 mg of mPEG-b-
PCC-g-GC-g-DC copolymer was dissolved in acetone (0.5 mL)
and added to 2 mL of phosphate buffered saline (PBS; pH 7.4).
Acetone was then evaporated under reduced pressure followed
by centrifugation at 5000 g for 5 min and filtration through 0.22 μ
filter to obtain the micelles. To assess the physical entrapment of
gemcitabine in micelles, mPEG-b-PCC-g-DC was prepared and
gemcitabine was loaded into it using nanoprecipitation method
at a theoretical loading of 12.8% w/w.
C
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Figure 1. Gemcitabine conjugated amphiphilic polymer. (A) Synthesis scheme. (B) FTIR spectral analysis.
inhibit cytidine deaminase enzyme and 20 μL of 2′-deoxy
cytidine) was added as an internal standard. Gemcitabine and its
metabolite (dFdU) were extracted from plasma by precipitation
using acetonitrile (500 μL) followed by centrifugation at 5000
rpm for 5 min. The supernatant was taken and analyzed using
LC-MS method. Chromatographic separation was performed on
XterraMS C18 column (2.5 μm, 4.6 mm × 50 mm) with
acetonitrile:water (containing 0.1% formic acid) (20:80) as the
mobile phase run at a flow rate of 0.35 mL/min. Injection volume
was kept at 20 μL. Calibration curves for gemcitabine and dFdU
were plotted in the concentration range of 50−5000 ng/mL.
In Vitro Cell Culture Studies. MIA PaCa-2 cells were obtained
as a kind gift from Dr. Fazlul H. Sarkar (Wayne State University,
Detroit, MI) and L3.6pl cells were purchased from ATCC
(Manassas, VA). Both cell lines were maintained in Dulbecco’s
Modified Eagle Medium (DMEM) containing 10% fetal bovine
serum and 1% antibiotic in an incubator at 37 °C/5% CO2.
Cytotoxicity Assay. For cytotoxicity evaluation, cells (5000/
well) were seeded in 96 well culture plates and allowed to attach
at 37 °C/5% CO2. After 24 h, culture media was removed and
fresh media (200 μL) containing gemcitabine or polymer drug
conjugate micelles were added. Cells were incubated at 37 °C/
5% CO2 for 72 h followed by assessment of cell viability by MTT
assay. The absorbance was measured at 560 nm and corrected for
the cell debris by subtracting absorbance at 655 nm. Cell viability
was calculated using eq 2. To assess the toxicity of the blank
copolymer, cytotoxicity of the copolymer without gemcitabine,
i.e., mPEG-b-PCC-g-DC, was also assessed using the same
protocol in the concentration range of 10 μg/mL to 1000 μg/
mL.
D
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Figure 2. Characterization of gemcitabine conjugated polymeric micelles. (A) Particle size analysis. (B) Transmission electron microscopy (TEM)
image. (C) Critical micellar concentration (CMC). (D) gemcitabine payload in the formulation analyzed by HPLC. (D1) gemcitabine standard, (D2)
and (D3) gemcitabine conjugated polymeric micelles before and after alkaline hydrolysis, respectively. D2 shows that the entire amount of drug is
conjugated to the polymer and no free drug is seen.
Absorbance of test sample
Absorbance of control
min. The slides were blotted dry and rTdT incubation buffer (50
μL) was added to the cells followed by incubation of the slides at
37 °C for 60 min inside the humidified chamber to allow the
tailing reaction to occur. The reaction was terminated by
immersing the slides in 2× saline-sodium citrate buffer (SSC) in a
Coplin jar for 15 min at room temperature. Unincorporated
fluorescein-12-dUTP was removed by repeated washings with
PBS. The samples were counterstained with propidium iodide (1
μg/mL) for 15 min at room temperature in the dark followed by
washing with PBS. The samples were immediately analyzed
under Olympus fluorescence microscope using the standard
fluorescein filter set to view the green fluorescence of fluorescein
at 520 20 nm and red fluorescence of propidium iodide at >620
nm.
In Vivo Evaluation in Xenograft Models. All animal
experiments were performed in accordance with the NIH animal
use guidelines and protocol approved by the Institutional Animal
Care and Use Committee (IACUC) at the University of
Tennessee Health Science Center (UTHSC, Memphis, TN).
Heterotopic xenograft flank tumors were established in 6−8
week old male NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ (NSG) mice
by subcutaneous injection of 2 × 106 MIA PaCa-2 pancreatic
cancer cells suspended in 1:1 serum-free media and Matrigel (BD
Biosciences, CA). When the tumor volume had reached 200
mm3 animals were randomly divided into three groups with each
group containing six animals and samples were administered via
tail vein once a week for three weeks. Group 1 was kept as the
control and received normal saline, group 2 received free
gemcitabine as aqueous solution (40 mg/kg), and group 3
received gemcitabine conjugated polymeric micelles equivalent
to free gemcitabine (40 mg/kg). Tumor sizes were measured
daily using digital vernier calipers in two perpendicular axis and
reported as tumor volume (V = 1/2[a × b2], a = longest axis, b =
shortest axis). Body weight of the animals was recorded every
day. At the end of the study (i.e., day 20), tumor tissues were
excised and weighed.
Cell viability(%) =
× 100
(2)
Subcellular Fate of Gemcitabine Polymer Conjugate
Micelles. The intracellular localization of gemcitabine conjugated
micelles into the lysosomes and endosomes was determined
using LysoTracker Red DND-99 (an acidic organelle dye, Ex/
Em 577/590 nm). MIA PaCa-2 cells (4 × 104 cells/well) were
plated in a 24-well cell culture plate in 1 mL of media and
incubated overnight for attachment. Next day, the cells were
treated with free gemcitabine and gemcitabine conjugated
micelles at a concentration of 100 nM for 24 and 72 h. The
control cells were left untreated. After the predetermined
incubation period, the cells were washed with PBS (pH 7.4)
and incubated with the media containing LysoTracker Red
DND-99 (4 μL of 1 mM DMSO solution in 40 mL of media) for
30 min in the dark. Thereafter, the cells were washed thrice with
PBS and fixed for 20 min using 10% buffered formaldehyde. The
images were observed in Zeiss fluorescent microscope (Axio)
using standard Cyt3 filter set at 20× magnification to detect the
formation of lysosomes.
Apoptosis Measurement. The extent of apoptosis induced by
free gemcitabine and gemcitabine conjugated micelles in
pancreatic cancer cells were studied by TUNEL assay using
manufacturer’s protocol. Briefly, MIA PaCa-2 cells (4 × 104
cells/well) were plated in chamber slides and incubated
overnight for attachment. The cells were then treated with the
media containing free gemcitabine and gemcitabine conjugated
micelles at a concentration of 100 nM for 72 h while the control
cells were left untreated. Thereafter, the cells were fixed using
10% buffered formaldehyde followed by washing twice with PBS.
The cells were permeabilized by immersing the slides in 0.2%
Triton X-100 solution for 5 min followed by incubation with
equilibration buffer (100 μL) consisting of 200 mM potassium
cacodylate (pH 6.6 at 25 °C), 25 mM Tris-HCl (pH 6.6 at 25
°C), 0.2 mM DTT, 0.25 mg/mL BSA, and 2.5 mM cobalt
chloride, and allowed to equilibrate at room temperature for 10
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Figure 3. Release of gemcitabine from aqueous solution vs methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-
gemcitabine-graft-dodecanol) polymer drug conjugate micelles after containing equivalent gemcitabine content (A) in PBS at pH 5.5 and 7.4 in the
presence and absence of protease Cathepsin B, and (B) in human plasma, (B1) Gemcitabine, and (B2) dFdU.
Histological Analyses. At the end of the treatment (i.e., day
20), three representative tumor tissues were harvested per
experimental group and fixed with 10% buffered formalaldehyde.
The fixed tissues were embedded in paraffin and thin sections of
4 μm were obtained. To detect the extent of tumor cell apoptosis,
the slides were stained with TUNEL and counterstained with
propidium iodide dye (Invitrogen) and visualized under Zeiss
fluorescence microscope (Axio).
−CH2−CH2−O−), 1−1.5 (m, protons of PCC units −CH3 and
dodecanol units −CH2). The FTIR spectra of PEG-PCC shows
the characteristic peaks at 3400 cm−1 for stretching vibrations of
−OH group, 2874 cm−1 for −CH stretching vibrations for
symmetric and antisymmetric modes of methylene groups, 1746
cm−1 for carbonyl group, and 1091 cm−1 for C−O−C stretching
vibration of repeated −O−CH2−CH2− units of PEG (Figure
1B). Gemcitabine shows characteristics peaks for amide bands at
1675 cm−1 and 3250 cm−1 for stretching vibration of −NH2.
After conjugation with PEG-PCC, characteristics peaks were
obtained at 3400 cm−1 (stretching vibrations of −OH group),
1749 cm−1 (for carbonyl group), 1669 cm−1 (amide band), and
1091 cm−1 (for C−O−C stretching vibrations).
Formulation Development. Upon nanoprecipitation,
gemcitabine conjugated polymer self-assembled into micelles
with the mean particle size of 23.6 4 nm and PDI of 0.097 as
observed by dynamic light scattering. This indicates the
formation of a nanosized formulation with a highly homoge-
neous particle size distribution. TEM images of the formulation
revealed the formation of spherical micelles (Figure 2A and B).
CMC is an important determinant to evaluate the micelle
forming ability of the gemcitabine conjugated copolymer. CMC
determined by fluorescence spectroscopy using pyrene as the
probe was 1.56 × 10−3 mg/mL (Figure 2C). HPLC-UV analysis
after alkaline hydrolysis of gemcitabine conjugated micelles
showed a peak at 13.25 min corresponding to gemcitabine
(Figure 2D3). Further, free gemcitabine was stable under alkaline
hydrolysis conditions. A gemcitabine payload of 12.8% w/w was
found in the micelles.
Statistical Analysis. Statistical evaluation was performed by
using Student’s t test. The significance level was set at p < 0.05.
RESULTS
■
Synthesis and Characterization of Polymer Drug
Conjugate. Polymer drug conjugate was synthesized by direct
coupling of gemcitabine onto the free carboxylic acid groups
present on the hydrophobic block of the amphiphilic copolymer,
PEG-PCC as illustrated in Figure 1A. Gemcitabine and
dodecanol were coupled to PEG-PCC using EDC and HOBT
coupling chemistry and purified conjugate was characterized by
1H NMR and FTIR. In 1H NMR spectra of PEG-PCC, protons
corresponding to −CH2−CH2−O− of PEG were observed at δ
3.4−3.6, and −CH2− units of PCC δ 4.2−4.4 and −COOH were
observed at δ 12−14 as reported earlier by our group.39 Average
molecular weight of PEG-PCC was 8680 Da with 23 PCC units
as determined using integral value corresponding to protons of
−CH2 of PEG and −CH2 of PCC units. After conjugation of
gemcitabine to PEG-PCC, protons corresponding to gemcita-
bine were observed in the NMR spectra: 1H NMR of deuterated
dimethyl sulfoxide (DMSO-d6) of conjugate, δ 9.6 (s, 1H,
−NC(R)H), 8.4 (s, 1H, −NHCO−), 6.0−6.2 (m, 1H, −CH),
In Vitro Gemcitabine Release Studies. In vitro release
studies carried out in PBS (pH 5.5) showed a controlled release
of gemcitabine from the micelles, which remained sustained even
in the presence of proteolytic enzyme Cathepsin B. The presence
)
4.2−4.6 (4nH, proton of PCC −CH2 , 4.0−4.2 (2yH, protons of
dodecanol −CH2−O), 3.4−3.8 (s, 4mH, Protons of PEG unit
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Figure 4. Efficacy of gemcitabine conjugated micelles in pancreatic cancer cell lines. (A) Cytotoxicity profiles in MIA PaCa-2 and L3.6pl after 72 h of
treatment. (B) Intracellular fate of gemcitabine conjugated micelles shown by staining with LysoTracker Red DND-99 after 24 and 72 h of treatment.
Magnification: 20×. (C) Apoptosis measurement in MIA PaCa-2 using TUNEL staining. The image shows cells after treatment with (C1) Control,
(C2) free gemcitabine as aqueous solution, and (C3) gemcitabine conjugated micelles. Images show the overlay of bright field, PI (red for cell nucleus)
and TUNEL staining (green for apoptosis). Magnification: 10×.
of enzyme in the release media increased the rate of drug release.
The difference in release was less visible initially as 9.08% was
released with enzyme while 6.80% was released without enzyme
in the first 2 h. However, this difference became more
pronounced with time, since 13.67% and 39% release was
observed on days 1 and 10, respectively in the absence of the
enzyme, while under the influence of enzyme, 18.05% and
53.89% gemcitabine was released on days 1 and 10, respectively
(Figure 3A). The rate of gemcitabine release from the conjugated
micelles was higher at pH 7.4 compared to pH 5.5. To assess the
ability of micelles to physically entrap and control the release of
gemcitabine, the dialysis bag method was used similar to that of
gemcitabine conjugated micelles. Gemcitabine solution was kept
as a control for assessing the hindrance caused by dialysis
membrane to the diffusion of free drug to the external media. It
was observed that entire gemcitabine was diffused out within 4 h
and there was no difference in the diffusion of free gemcitabine
and physically entrapped gemcitabine indicating the inability of
micelles to physically entrap and control the release of
gemcitabine (Figure 3A). This was expected since gemcitabine
is a hydrophilic molecule and has a high tendency to remain in
the outer aqueous phase and not in the core of the micelles,
which is hydrophobic.
gemcitabine and dFdU concentrations which gave peaks at
retention times of 4.3 and 7.2 min, respectively, while retention
time of internal standard was obtained at 3.5 min. Free
gemcitabine on incubation with plasma showed a drastic
reduction in its concentration from 200 μg/mL to 3 μg/mL
while the concentration of its metabolite dFdU was increased
from 0 to 95 μg/mL within 24 h. However, in the case of
gemcitabine conjugated polymeric micelles, only 3 μg/mL of
intact gemcitabine was detected in plasma and 4.5 μg/mL of
dFdU in 24 h (Figure 3B). Further, plasma samples at 24 h were
hydrolyzed by alkaline degradation and analyzed by LC-MS
method and conjugated gemcitabine was recovered indicating
that the conjugated gemcitabine did not metabolize or change in
the conjugate.
In Vitro Cytotoxicity Assay. Cytotoxic activity of
gemcitabine after conjugating to the amphiphilic copolymer
was evaluated by incubating MIA PaCa-2 and L3.6pl pancreatic
cells with free gemcitabine or gemcitabine conjugated micelles
for 72 h and determining the cell viability using MTT assay. The
formulations were found to be cytotoxic against both MIA PaCa-
2 and L3.6pl cells with IC50 values of 149.3 and 741.2 nM,
respectively, while free gemcitabine showed IC50 values of 7.6
and 105.9 nM, respectively. We have also determined the toxicity
of the blank polymer, i.e., mPEG-b-PCC-g-DC (without
gemcitabine) on MIAPaCa-2 and L3.6pl cells and these polymers
were found to be nontoxic (Figure 4A).
Plasma Stability. Stability of micelles in plasma was studied
by determining the amount of intact gemcitabine released from
the formulation and its metabolite dFdU formed after incubation
in human plasma. LC-MS method was used to determine
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Figure 5. In vivo efficacy of gemcitabine polymer conjugate in MIA PaCa-2 derived xenograft tumors in NSG mice. Tumors were developed by
subcutaneous injection of 2 × 106 cells in NSG mice. When tumor size reached 200 mm3 animals were treated with gemcitabine or gemcitabine
conjugated micellar at a dose of 40 mg/kg once weekly for three weeks via intravenous injection through tail vein (indicated by arrow). (A) Change in
tumor volume. (B) Change in body weight. (C) Images of tumors excised. Data represented as the mean SE (N = 6).
Figure 6. Detection of apoptosis induced in the tumor tissues by gemcitabine conjugated micelles in comparison to free drug and control as indicated by
the green fluorescence of TUNEL. Red spots mark the cells after staining with propidium iodide. Tissues were excised from the animals at the end of the
study, i.e., day 20.
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Intracellular Fate. Subcellular fate of gemcitabine con-
jugated polymeric micelles compared to free gemcitabine was
studied using LysoTracker Red DND-99 which primarily
concentrates in the acidic organelles of the cells. Figure 4B
shows the comparative formation of lysosomes and endosomes
after treatment of cells with free and conjugated gemcitabine for
24 and 72 h. The control (untreated cells) exhibited few red spots
indicating the presence of basal level of lysosomes in the cells;
however, in the case of gemcitabine treated cells significantly
enhanced punctate fluorescence colocalized with LysoTracker
Red was observed indicating the formation of lysosomes.
Significantly higher fluorescence intensity was seen in
gemcitabine conjugated polymeric micelle treated cells com-
pared to the cells treated with aqueous solution of gemcitabine
after 24 h, and the difference became even more predominant
after 72 h of treatment.
Apoptosis Measurement. Apoptosis is considered to be the
key mechanism underlying chemotherapy induced cell death.
Figure 4C shows TUNEL assay images of the MIA PaCa-2 cells
after incubation with free gemcitabine or gemcitabine conjugated
micelles for 24 h. Cells were counterstained with propidium
iodide which showed red fluorescence. No apoptosis was
observed in the control cells, while both free gemcitabine and
gemcitabine conjugated micelles were able to induce apoptosis in
the cells as indicated by the green fluorescence of TUNEL.
In Vivo Efficacy in Xenograft Model. Gemcitabine
conjugated micelles were tested in MIA PaCa-2 cell derived
xenograft model in NSG mice. Animals treated with gemcitabine
conjugated micelles showed significantly reduced tumor growth
rate as compared to gemcitabine solution or the control group
(Figure 5A). Further, it was observed that, after injecting
gemcitabine conjugated polymeric micelles, the decrease in
tumor volume was maintained for at least 4 days before again
showing an increase in contrast to gemcitabine solution wherein
tumor volume exhibited a continuously increasing pattern until
the last injection, and no significant difference was observed
between animals treated with free gemcitabine and the control
group (untreated). Figure 5B shows the change in body weight of
the animals, which was less than 5% for both free gemcitabine
and gemcitabine conjugated micelles indicating tolerance of
doses given to animals. At the end of the study tumors were
excised, weighed, and photographed (Figure 5C) which clearly
showed significant difference in the tumor weights of groups
treated with gemcitabine conjugated micelles compared to free
gemcitabine or the control group (0.38 g versus 0.58 g). The
extent of apoptosis in the tumor tissues as indicated by TUNEL
assay (Figure 6) was also in agreement with these results, since
not much difference was observed between free gemcitabine
treated and the control group, while the group treated with
gemcitabine conjugated polymeric micelles induced a signifi-
cantly higher apoptosis which might be responsible for a better
antitumor activity of the conjugate compared to the free drug.
efforts are being made to devise an effective delivery system for
gemcitabine considering its high chemotherapeutic potential for
treating pancreatic cancer, which is, however, not yet completely
realized clinically owing to its poor pharmacokinetics, rapid
plasma metabolism, and extremely high hydrophilicity. Bio-
conjugation to a polymeric carrier provides an attractive strategy
to deliver gemcitabine to the tumor tissue after systemic
administration protecting it from plasma metabolism and
passively delivering the drug to the tumor compartment. In
line with this approach, several lipidic conjugates,26,28 squalenoyl
derivatives,20,21,23 and PEGylated conjugates of gemcitabine29
have been prepared and explored for enhanced anticancer
properties. However, these systems were limited by poor
solubility, uptake by the RES, and/or lower gemcitabine payload.
In the present study, we synthesized a self-assembling,
amphiphilic copolymer PEG-PCC with several carboxyl pendant
groups on the hydrophobic block and conjugated gemcitabine to
these carboxyl groups via EDC/HOBT coupling. The presence
of PEG ensures the stealth properties of gemcitabine conjugated
micelles, while the presence of carboxyl groups enable the
conjugation of higher content of gemcitabine to the polymer
resulting in higher payload compared to previously reported
PEG conjugates.5,6 Additionally, we also attached a lipid,
dodecanol, to impart requisite hydrophobicity and thus assist
in self-assembly and micellization to a very small size (∼23 nm)
compared to other nanocarrier systems of gemcitabine, thus
further ensuring its reduced RES uptake. PEG-PCC copolymer
showed poor solubility in organic solvents such as chloroform,
acetone, and dichloromethane, but was soluble in THF and
DMSO. After conjugation of gemcitabine and dodecanol to the
PCC block, its solubility showed marked increase in acetone,
CHCl3, and CH2Cl2. 1H NMR and FTIR confirmed the
conjugation of gemcitabine to the copolymer. Gemcitabine
content determined by HPLC-UV assay showed a drug loading
of 12.8% and also indicated absence of any unconjugated drug
(Figure 2D2). This could be attributed to the presence of several
carboxylic groups in the hydrophobic PCC segment of
copolymer PEG-PCC for conjugation with gemcitabine thus
ensuring an appreciable loading of the drug which is otherwise
difficult to obtain in the case of hydrophilic drugs. Previously
reported PEGylated derivatives of gemcitabine have shown a
comparatively poor loading of the drug ranging from 0.98% to
6.79% w/w.5,6 In this study, gemcitabine conjugated polymer
self-assembled into nanosized micelles of 23.6 nm (Figure 2A)
indicating their ability to escape from the RES system and
extravasate into the tumor compartment. Although our
formulation needs improvement in terms of payload compared
to gemcitabine−squalene nanoassemblies which showed a pay
load of 41% w/w,21,23,24 these gemcitabine conjugated micelles
offer several additional advantages over the squalenoylated
nanoassemblies. These micelles showed a much smaller micellar
size as compared to gemcitabine−squalene nanoassemblies (23
nm vs 140 nm). Further, PEG outer shell can provide stealth
effect to gemcitabine conjugated micelles. In contrast,
gemcitabine−squalene nanoassemblies were rapidly removed
from the bloodstream by the reticulo-endothelial system
(RES),22 thus requiring PEG coating to reduce opsonization
and blood clearance.29 PEGylation was achieved in squalenoy-
lated gemcitabine nanoassemblies by addition of PEG−squalene
or PEG−cholesterol in the weight ratio of 1:0.7 to form
composite nanoassemblies. Though addition of PEG−squalene
in the formulation resulted in decrease in effective gemcitabine
loading in the composite nanoassemblies, however, the
DISCUSSION
■
A viable strategy for treating pancreatic ductal adenocarcinoma is
the need of the hour due to its growing prevalence and high
mortality rate. Surgery, radiation, and chemotherapy constitute
the major treatment modalities; however; each suffers its own
specific limitations mainly arising from local recurrence and
metastatic spread of the disease. Chemoresistance in pancreatic
cancer results from the desmoplasmic tumor microenvironment
which inhibits the infiltration of anticancer drugs, particularly
hydrophilic molecules like gemcitabine. Extensive research
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significance of PEG cannot be overruled since the PEGylated
gemcitabine nanoassemblies showed improved efficacy over the
non-PEGylated nanoassemblies.29 The presented formulation
exhibits a balance between a moderate drug loading (12.8% w/
w) and PEG content (∼55%).
release is not entirely dependent on the enzymatic activity but is
enhanced in its presence. However, the linkage of drug to
polymer may break in vitro without enzyme being present in the
media by hydrolysis leading to the release of gemcitabine.
Further, it was evident from this data that the drug could
dissociate itself from the polymer drug conjugate in an acidic and
enzyme-rich environment to elicit its therapeutic action, along
with the ability of the synthesized conjugate to sustain and
control the release of gemcitabine, which is otherwise a challenge
in the case of hydrophilic drugs. Cathepsin B is a lysosomal
protease which can enzymatically cleave the amide bonds
between the drug and the polymer and thereby release the drug
from the system. Previous reports on polymeric conjugates and
prodrugs of gemcitabine have also shown the role of cathepsins in
degradation of the amide bond and release of the drug
moiety.20,45,46 We have not tested the mechanism on how
cathepsin B will interact with the micelles and result in
subsequent drug release. For cathepsin B to act on the linkage
of drug and polymer, the micelles should get dissociated. Since
micelles exist in a dynamic equilibrium with the molecularly
dissolved copolymer molecules (unimers) in the solution, it is
expected that the enzyme may act on dissociated copolymer
leading to release the drug. Furthermore, the polymer backbone
contains the degradable carbonate bonds in the hydrophobic
segment of the copolymer, which can undergo hydrolysis in
water, liberating the gemcitabine conjugated oligomers or
monomers which could be further acted upon by enzyme
resulting in the release of free gemcitabine. Other polymeric
conjugates and lipidic derivatives of gemcitabine, which aimed to
prolong the release of this hydrophilic molecule, exhibited as
much as 40−50% of the drug release within the initial 48 h,5,6 but
in the present study, the release was much sustained since more
than 40% of the drug still remained in the micelles even after 10
days. A possible explanation for slow release could be strong
affinity between the conjugated drug and the hydrophobic core
of the micelles as also stated by Rijcken et al.47 The presence of
dodecanol in the synthesized polymer could also have imparted
additional stability to the system, since lipids have a tendency to
stabilize the micellar structures. It is also well-recognized that
fatty acid chains stabilize and hold together the phospholipid
bilayers of the cell membrane by a variety of forces such as van
der Waals interactions.33 Thus, our conjugate holds a better
potential to reduce the dosing frequency and overall dose of
gemcitabine and hence moderate the systemic adverse effects and
enhance patient compliance over the previously reported
gemcitabine conjugates.
Since gemcitabine conjugated micelles are intended for
intravenous route of administration, it is important that the
formulation remains stable and does not release the drug in the
plasma which would otherwise undergo plasma metabolism and
would not reach therapeutically effective levels in the tumor
tissue. Once in the systemic circulation, the effect of blood
dilution on the stability of micellar formulation could be
alleviated by designing polymers which show a low CMC
value, which in the present case was 1.56 μg/mL. This CMC
value is much lower than reported with frequently used nonionic
surfactants, Tween 80 and Tween 20 (both ∼300 μg/mL),40
Pluronic block copolymers (∼25−299 μg/mL),41 or with other
synthetic PEG based amphiphilic polymers designed to deliver
gemcitabine as micelles (∼4 μg/mL and ∼70 μg/mL),33,42,43 and
thus the formulation is expected to be stable against dilution.
Chemically, gemcitabine is a stable molecule;44 however, on
intravenous administration it undergoes rapid metabolism by
cytidine deaminase enzyme to its inactive metabolite, dFdU.
Thus, one objective of the work was to improve the in vivo
stability toward metabolism in plasma and hence to enhance its
delivery in vivo. Upon incubation with human plasma,
gemcitabine conjugated micelles still retained more than 98%
of the drug for 24 h (Figure 3B) indicating that attaching the
polymer at N4 of gemcitabine imparted protection to the drug
from plasma deamination. This shows a significant improvement
over the previous attempts to curtail drug metabolism whereby
PEGylated and linoleic acid based conjugates of gemcitabine,
when incubated in plasma, still exhibited 30−40% of drug release
within 24 h.5,25 This could be attributed to the presence of
conjugated gemcitabine inside the core of the micelles thereby
decreasing its accessibility to the enzyme activity and/or
hydrolysis and ensuring a sustained and targeted release of the
drug. Polymer drug conjugates are taken up by the cells via
endocytosis, and once inside the cells, these must be internalized
by the lysosomes and endosomes so as to ensure the degradation
of the conjugate to facilitate the release of active moiety and
exhibit its resultant therapeutic benefit.6 Incubation of MIA
PaCa-2 pancreatic cells with the conjugate for 24 h showed
higher fluorescence intensity, signifying enhanced formation of
lysosomes and endosomes inside the cells and better internal-
ization of gemcitabine via drug polymer conjugate vs free drug
(Figure 4B). Increase in the fluorescence intensity after 72 h also
indicates the elevated retention of gemcitabine via conjugated
micelles with time. PEGylated gemicitabine conjugates reported
by Sahoo et al. also showed similar behavior and were retained
inside the lysosomes for a longer duration of time.6 Once inside,
the conjugate shall be degraded inside the acidic and enzyme-rich
environment of these organelles and thus enable release of
gemcitabine. To confirm the liberation of the free drug from the
conjugate, drug dissociation from the polymer conjugate was
studied in the presence and absence of cathepsin B (a cysteine
protease) at acidic pH 5.5 (to mimic the lysosomal compart-
ment). Previous studies have shown that gemcitabine remains
stable under these conditions.16 We observed that 54% of the
total conjugated drug was released from the conjugate in 10 days
while 39% release was observed in absence of Cathepsin B.
Further, the release at pH 7.4 was faster than at pH 5.5 with
50.8% release in 10 days. The results indicate that the drug
The amount and rate of drug release from the conjugate has
direct influence on the therapeutic efficacy of the drug. MTT
assay indicated that the bioconjugation with the amphiphilic
copolymer did not compromise its cytotoxic activity (Figure 4A).
Free gemcitabine showed lower IC50 values, which is in
agreement with the previous reports for PEG−gemcitabine
conjugates.5 This difference mainly arises due to the slower
uptake of the conjugate which enters the cells via the endocytosis
pathway and, second, gemcitabine has to be dissociated from the
conjugate inside the cells before showing its activity. Since the
polymer is biodegradable and byproducts can form during the
hydrolysis of the polymer backbone, we have tested the toxicity
of the blank polymer (mPEG-b-PCC-g-DC) to confirm any
nonspecific toxicity. Results indicated that the polymer and its
byproducts were nontoxic to the cells; thus, the cell killing
observed in the cytotoxicity assays was due to the gemcitabine.
Another confirmation of the preserved therapeutic efficacy of the
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drug was observed by carrying out the TUNEL assay which
specifically marks the apoptotic cells within a cell population.
Similar to free gemcitabine, these conjugates were also able to
induce significant apoptosis in the pancreatic cancer cells (Figure
4C). Recently, Maksimenko et al. reported much higher IC50
values in 4 different cancer cell lines and comparable/slightly less
apoptotic fractions in MIA PaCa-2 cells upon treatment with
isoprenoyl conjugates of gemicitabine compared to the free
drug.48 The evidence of a clear therapeutic benefit which a
nanoformulation could yield over free drug is difficult to observe
in in vitro cell culture studies due to the lack of the biological
dynamism and absence of barriers that a drug has to bypass
before reaching its target tissue in vivo. On the contrary, in the
case of cell culture studies, the drug immediately gets into direct
contact with the cells which diminishes the advantages offered by
nanocarriers in terms of protecting the drug from the hostile GI
environment, a different uptake mechanism via Peyer’s patches,
and thus ensuring a higher absorption and exploiting the EPR
effect. To understand if gemcitabine conjugated polymeric
micelles offered any therapeutic advantages over the free drug,
we carried out the in vivo evaluation in xenograft model of
pancreatic cancer in NSG mice after intravenous injection
(equivalent to 40 mg/kg of gemcitabine). Gemcitabine is usually
administered in mice via intraperitoneal route and intravenous
route at a dose ranging widely from 7 mg/kg to 500 mg/
kg.23,31,48−51 In the present study, intravenous route was used for
free gemcitabine since we wanted to compare the results with the
conjugated micelles that were also administered by intravenous
route. We have selected a dose of 40 mg/kg based on a previous
report for polymer−gemcitabine conjugate,31 even though this
dose is not the maximal tolerable dose (MTD). Compared to
free gemcitabine, gemcitabine conjugated micelles efficiently
decreased the tumor growth and final tumor weight when
administered at the same equivalent dose (Figure 5). This could
be attributed to the decreased plasma metabolism of the
conjugated gemcitabine owing to blockage of the site of
deamination and hindrance provided by the polymeric carrier
to the cytidine deaminase enzyme. These results are in
agreement with the previous study wherein gemcitabine
conjugates have shown superior antitumor activity over free
drug.5,31,48 Further, the nanosize of the micelles could enable
enhanced permeation and retention into the tumor compart-
ment. The outer PEG shell of the micelles could prevent its
uptake by the RES system and increase its mean residence time in
the central compartment. Gemcitabine conjugated micelles
suppress the tumor growth more dramatically right after
injection compared to free drug. This could be due to faster
release of gemcitabine from the micelles in the highly degradable
lysosomal environment of the tumor cells compared to only
53.89% gemcitabine release from the micelles in vitro in 10 days.
This warrants a thorough investigation of the pharmacokinetic−
pharmacodynamic profile of the formulation since it could not be
predicted solely from in vitro drug release as this may change in in
vivo conditions (in tumor microenvironment or in tumor cells).
The efficacy of gemcitabine conjugated micelles is modest as
compared to the previous work reported by Harrison et al.52 and
could be attributed to the dosing schedule and more particularly
to the different strain of mice (athymic nude mice versus NSG
mice) which can affect the pharmacokinetic properties/
metabolism of the system in vivo. Further, there is no difference
in the control group and free gemcitabine which could be due to
the inefficacy of the free drug to reach the tumor compartment at
the therapeutic level to show any effect. This is also expected and
has been reported previously wherein free gemcitabine treated
group did not show any tumor reduction.52 The antitumor
efficacy of gemcitabine mainly results from its ability to induce
apoptosis. High antitumor efficacy of squalenoyl−gemcitabine
conjugate against aggressive metastatic leukemia model was
mainly attributed to induction of apoptosis by the active
triphosphate form of gemcitabine after getting incorporated
into the DNA.5 Herceptin-conjugated gemcitabine chitosan
nanoparticles have been shown to exhibit superior antiprolifer-
ative activity against pancreatic cancer along with an enhanced S-
phase arrest, leading to apoptosis in comparison with free
gemcitabine.53 In the present study also, the underlying
apoptosis mechanism for enhanced antitumor activity of the
conjugate was investigated by the TUNEL assay conducted on
the tumor tissue samples (Figure 6). While free gemcitabine
induced negligible apoptosis in the tumor cells, the gemcitabine
polymer conjugate treated group showed a significantly high
number of apoptotic cells. Further, the conjugate did not cause
adverse systemic side effects as indicated by the weight change
profile of the animals, where the weight of the experimental
animals was almost similar to that in the control group.
CONCLUSION
■
Gemcitabine was conjugated to an amphiphilic copolymer PEG-
PCC, bearing multiple carboxylic groups, thus ensuring a high
payload of the drug. Gemcitabine conjugated polymer self-
assembled into nanosized micelles which released the drug in a
controlled fashion and provided enhanced stability in human
plasma since the drug was conjugated to the hydrophobic core of
the micelles thereby making it inaccessible to metabolism and
improving its stability. The micelles showed considerable
reduction in cell viability of pancreatic cancer cell lines by
enhanced internalization and apoptotic cell death. In vivo
evaluation in pancreatic tumor model demonstrated enhanced
antitumor activity of the gemcitabine conjugated micelles
compared to free drug without inducing any significant toxicity.
This might be attributed to the apoptotic cell death induced by
micelles in addition to the presence of outer PEG shell which
would have provided long circulation and enhanced permeation
and retention in the tumor compartment. The system needs to
be further evaluated for in vivo pharmacokinetics and
biodistribution to confirm this fact. In a nutshell, the system
offers potential for additional advantages such as loading of
another hydrophobic drug in the core for combination therapy
and modification of the hydrophobic block by attaching different
ligands viz. cationic chains for miRNA delivery. The strategy
outlined in this work not only provides a promising approach to
be developed as an alternative therapy for an effective pancreatic
cancer treatment, but could also be explored for delivery of other
difficult-to-deliver hydrophilic drug molecules particularly which
have shown high therapeutic potential but modest clinical benefit
due to delivery challenges associated with them.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 402-559-5422; Fax: 402-559-9543; E-mail: ram.mahato@
unmc.edu; Skype: ram.mahato.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Kosten foundation is duly acknowledged for providing financial
support.
ABBREVIATIONS
■
CMC, critical micelle concentration; dFdU, 2′,2′-difluorodeox-
yuridine; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide HCl; HOBT, 1-hydroxybenzotriazole; MBC, 2-methyl-2-
benzyloxycarbonyl-propylene carbonate; mPEG, methoxy poly-
ethylene glycol; PBS, phosphate buffered saline; PDI, poly-
dispersity index; PEG-PCC, poly(ethylene glycol)-block-poly(2-
methyl-2-carboxyl-propylene carbonate); TEM, transmission
electron microscopy; TUNEL, terminal deoxynucleotidyl trans-
ferase biotin-dUTP nick end labeling
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