Comparison of active and passive targeting of docetaxel for prostate cancer therapy by HPMA copolymer-RGDfK conjugates

Article abstract of DOI:10.1021/mp100402n

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α_vβ ₃ integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α_vβ₃ integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.

Full text of DOI:10.1021/mp100402n

ARTICLE
pubs.acs.org/molecularpharmaceutics
Comparison of Active and Passive Targeting of Docetaxel for Prostate
Cancer Therapy by HPMA CopolymerꢀRGDfK Conjugates
||
Abhijit Ray,†,‡,§ Nate Larson,†,‡,§ Daniel B. Pike,‡, Michele Gr€uner,†,‡,^ Sachin Naik,†,‡,# Hillevi Bauer,†,‡
||
Alexander Malugin,†,‡ Khaled Greish,†,‡ and Hamidreza Ghandehari*,†,‡,
†Department of Pharmaceutics and Pharmaceutical Chemistry, ‡Center for Nanomedicine, Nano Institute of Utah, and
Department of Bioengineering, University of Utah, Salt Lake City, Utah, 84108, USA
^Johannes-Gutenberg-Universit€at Mainz, Mainz, Germany
#Pharmacy Department, The Maharaja Sayajirao University of Baroda, Gujarat, India
ABSTRACT: N-(2-Hydroxypropyl)methacrylamide (HPMA)
copolymerꢀdocetaxelꢀRGDfK conjugate was synthesized,
characterized, and evaluated in vitro and in vivo in comparison
with untargeted low and high molecular weight HPMA co-
polymerꢀdocetaxel conjugates. The targeted conjugate was
designed to have a hydrodynamic diameter below renal thresh-
old to allow elimination post treatment. All conjugates demon-
strated the ability to inhibit the growth of DU145 and PC3
human prostate cancer cells and the HUVEC at low nanomolar
concentrations. The targeted conjugate showed active binding
to Rvβ3 integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding.
Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated
with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates
exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that Rvβ3 integrin targeted
polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising
candidates for prostate cancer therapy.
KEYWORDS: docetaxel, HPMA copolymer, prostate carcinoma, RGDfK, Rvβ3 integrin, DU-145 prostate cancer cell line, PC3
prostate cancer cell line, targeted drug delivery
’ INTRODUCTION
drugs including cisplatin and carboplatin in cancer.7 Docetaxel is
reported to have both antiangiogenic and antitumor efficacy.8
However, treatment with this drug is associated with gastro-
intestinal toxicity9 and can result in aggravated risk of acute and
subacute pulmonary damage.10 Another major problem asso-
ciated with administration of docetaxel is its poor aqueous
solubility, requiring formulation with the nonionic surfactant
polysorbate 80 (Tween 80). Administration of docetaxel is
associated with the occurrence of unpredictable acute hypersen-
sitivity reactions and cumulative fluid retention.11 These adverse
effects have been attributed, in part, to the presence of poly-
sorbate 80 and have consequently initiated research focused on
the development of a less-toxic, better-tolerated polysorbate 80-
free formulation.
Prostate cancer is the leading cause of cancer among men in
the United States and the second leading cause of death with
217,730 new cases and an estimated 32,050 deaths in 2010.1 In
the past decade, much progress in the treatment of prostate
cancer has been reported. However, routine treatment regimens
have frequently changed due to poor prognosis of advanced
disease. Despite standard treatment, the current survival from the
time of diagnosis in patients with metastatic disease of 3.5 years is
unacceptable.2 These results demand the exploration of new
alternatives which overcome drawbacks of current treatment
modalities.
Docetaxel (Taxotere) is a semisynthetic natural product which
was approved by the FDA in 2004 for metastatic and androgen
dependent prostate cancer. Clinical trial results strongly suggest
the use of docetaxel as a first line of treatment for prostate
cancer.3,4 Docetaxel binds to β-tubulin, thereby stabilizing
microtubules and inducing cell-cycle arrest resulting in
apoptosis.5 It is up to five times more potent than paclitaxel in
vitro with regard to tubulin promotion and inhibition of
depolymerization.6 There is incomplete cross resistance between
paclitaxel and docetaxel, and they act synergistically with several
Considerable progress has been made over the past 2ꢀ3
decades in the development of polymeric carriers for targeted
drug delivery to solid tumors.12,13 Due to their macromolecular
Received: December 1, 2010
Accepted: May 20, 2011
Revised:
April 20, 2011
Published: May 21, 2011
r
2011 American Chemical Society
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nature, polymeric systems accumulate passively in target tissues
such as the reticuloendothelial system (RES) (through nonspe-
cific uptake by macrophages) or tumors by a process called the
enhanced permeability and retention (EPR) effect.14,15 The
mechanism of the EPR effect has been summarized according
to the following cascade of events: (i) tumor angiogenesis results
in hypervasculature, providing increased blood flow to the
tumor; (ii) tumor vasculature becomes highly permeable for
macromolecules and plasma proteins due to factors such as
tumor vascular permeability factor, bradykinin, and tumor necrosis
factor; (iii) a less effective functioning of lymphatic drainage
observed in tumors results in long-term retention of macromo-
lecular drugs. These factors result in larger carriers having
decreased renal clearance, thereby taking longer to be eliminated
from the body.16
HPMA copolymers are well characterized, water-soluble,
biocompatible, nonimmunogenic and nontoxic synthetic poly-
meric drug carriers.12,17,18 The in vivo disposition of macromo-
lecules depends to a significant extent on their physicochemical
properties. It has been shown that liver and kidney based
clearance are the major factors affecting the biodistribution of
macromolecules19 and such clearance is a function of molecular
weight. Studies have shown that HPMA copolymers and other
polymeric macromolecules of molecular weights less than ap-
proximately 45 kDa (hydrodynamic diameter <5ꢀ6 nm) are
rapidly filtered by the kidney.16,20ꢀ22 Most macromolecules that
passively target tumors via the EPR effect have sizes larger than
7 nm, in order to overcome the glomerular renal threshold,
thereby resulting in prolonged plasma half-life. Despite signifi-
cant progress made in the field of macromolecular drug delivery,
one challenge encountered in the development of such therapy is
the fate of large macromolecules post treatment.
Another challenge often overlooked while using the EPR
effect as a rationale for drug delivery of macromolecules is the
elevated interstitial fluid pressure (IVF) which reduces convec-
tive transport in the core of the tumor.23,24 It is reported that the
imbalance of the pro- and antiangiogenic factors leads to forma-
tion of chaotic new blood vessels in tumors.25ꢀ27 This leads to
uneven blood distribution, leaving unevenly perfused or unper-
fused regions inside the tumor.28ꢀ31 These factors lead to
interstitial hypertension in the core of the tumor.32ꢀ34 Interstitial
pressure and impaired blood supply reduce the effective trans-
port of anticancer agents in solid tumors.25 Active targeting can
possibly overcome these limitations for drug penetration.35
The tripeptide sequence Arg-Gly-Asp (RGD) has been iden-
tified as a high affinity Rvβ3 selective ligand by phage display.36
The conformationally restrained cyclic RGDfK binds to Rvβ3 up
to 200-fold more avidly than linear peptides.37 RGD peptides
have been conjugated to humanized antibodies,38 liposomes,39ꢀ41
and poly(ethylene glycol)42 to improve the biodistribution and
increase tumor accumulation. The RGD (Arg-Gly-Asp) peptides
have been used to target tumor angiogenesis.43,44 They have
been used for the targeted delivery of chemotherapeutic agents,
gene therapy45 and oncolytic adenovirus.46 They have also been
reported to be tumor penetrating, and its coadministration
enhances the efficacy of anticancer drugs.47 Compared to non-
targeted systems, previous investigations in our laboratory48ꢀ52
have identified actively targeted HPMA copolymerꢀcyclo-RGD
conjugates that increase tumor accumulation. This accumulation
takes place through specific interaction of RGD motifs present in
the copolymer side chains with Rvβ3 integrins overexpressed on
both angiogenic blood vessels37 and a variety of tumor cells
including prostate cancer.53 Enhanced accumulation was also
demonstrated by active targeting in various human prostate
cancer xenografts.48,51 Further, these targeted conjugates have
also been shown to inhibit HUVEC cell migration thereby
delaying neoangiogenesis.54 To enhance efficacy and reduce
toxicity of docetaxel and to compare and contrast the effects of
active vs passive targeting, our present study aims at producing
HPMA copolymerꢀdocetaxelꢀcyclic RGD conjugates for tar-
geted delivery to prostate tumors.
’ METHODS
Chemicals. All chemicals obtained commercially were of
analytical grade and used without further purification. Docetaxel
was obtained from AK Scientific (Mountain View, CA). RGDfK
(MW 604.5) was obtained from New England Peptide Inc.
(Boston, MA) at >95% purity and used as supplied. Methacryloyl
chloride, glycylglycine, and p-nitrophenol were obtained from
Sigma-Aldrich (St. Louis, MO). Glycyl-phenylalanine and leucyl-
glycine were obtained from Bachem Americas, Inc. (Torrance,
CA). N-(2-Hydroxypropyl)methacrylamide (HPMA),55 N-
methacryloylglycylglycyl p-nitrophenyl ester (MA-GG-ONp),56
N-methacryloylglycylphenylalanylleucylglycine (MA-GFLG-OH),57
N-methacryloylglycylphenylalanylleucylglycine p-nitrophenyl ester
(MA-GFLG-ONp),57 and N-methacryloyltyrosinamide (MA-
Tyr)58 were synthesized and characterized according to previously
described methods.
Synthesis and Characterization of Comonomers. N-Metha-
cryloylglycylphenylalanylleucylglycylꢀDocetaxel (MA-GFLG-
Docetaxel). Docetaxel (0.335 g, 4.1 mmol), 4-(dimethylamino)-
pyridine (DMAP, 0.049 g, 4.0 mmol) and MA-GFLG-OH (0.188
g, 4.0 mmol) were dried under vacuum. The reaction mixture was
dissolved under nitrogen in anhydrous N,N-dimethylformamide
(DMF, 5 mL) and cooled with an ice bath (salt/ice) at <0 ꢀC, and
diisopropylcarbodiimide (DIPC, 76 μL, 4.89 mmol) was added
dropwise. The reaction mixture was subsequently stirred for an
hour before the ice bath was removed; the mixture was allowed to
warm up to room temperature and stirred overnight, and
progress was monitored by thin layer chromatography (TLC,
eluent dichloromethane (DCM):methanol (MeOH) (95:5)) for
the disappearance of the starting material and the formation of
MA-GFLG-docetaxel, and further characterized by mass spec-
troscopy (m/z Mþ þ Na = 1272.62). DMF was removed under
vacuum using a rotavapor. The product was purified by silica gel
chromatography and eluted using ethyl acetate (EtOAc):MeOH
(95:5). The product identity was confirmed by Thermo Finne-
gan LTQ FT high resolution mass spectrometry (m/z Mþ1 for
C66H84N5O19þ, calculated 1250.57605 (100%), 1251.57941
(71.4%), found m/z 1250.57487 (100%), 1251.57827 (71.4%)).
N-MethacryloylglycylglycylꢀRGDfK. MA-GG-ONp (0.20 g,
0.623 mmol) and RGDfK (0.376 g, 0.623 mmol) were mixed in a
round-bottom flask, kept in a vacuum desiccator for 1 h followed
by addition of anhydrous dimethyl sulfoxide (DMSO, 5 mL) and
stirred for 18 h at room temperature. DIPEA (200 μL) was then
added to the reaction mixture and further stirred for 4 h. The
progress of the reaction was monitored by mass spectrometry for
the disappearance of RGDfK (m/z Mꢀ1 603.2) .The reaction
mixture was concentrated under high vacuum, followed by
addition of H2O (25 mL). This mixture was partitioned in
diethyl ether (3 ꢁ 25 mL). The organic layer was removed
and the aqueous layer was lyophilized to obtain a white solid.
Product formation was confirmed by Thermo Finnegan LTQ FT
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Scheme 1. Schematic Synthesis and Resulting Structure of HPMA CopolymerꢀRGDfKꢀDocetaxel (P2) Conjugatesa
a Select copolymers contained the monocyclized RGDfK peptide targeting moiety to target docetaxel to Rvβ3 integrins.
Table 1. Characteristics of HPMA Copolymer Conjugates
comonomer feed composition (mol %)
wt %
MA-GFLG- MA-GG-
est Mw
Mw/
hydrodynamic
polymer no.
structure
HPMA
95.5
RGDfK MA-Tyr 3-MPA (kDa)a (SEC) Mn diameter (nm) (QELS) DOCb RGDfKc
a
DOC
P1
P2
P3
P-(GFLG-DC)
2.5
2.5
2.5
2
2
2
4
4
1
32.6
26.9
87.1
2.9
1.9
4.9
3.0
3.0
7.6
6.8
P-(GFLG-DC-RGDfK) 92.5
3
6.84
7.15
5.061
P-(GFLG-DC) 95.5
a Estimated by size exclusion chromatography (SEC). b Determined by HPLC. c Determined by amino acid analysis.
high resolution mass spectrometry (for C35H50N10O11, calcu-
lated m/z 786.36605 (100%), 787.36941 (37.9%), 788.37276
(7%), found m/z 786.38951 (100%), 787.39328 (37.9%),
788.39627 (7%)).
Barbara, CA) with imbedded QELS and an OptiLab rEX
differential refractometer (Wyatt Technologies). The Superose
6 column was previously calibrated with fractions of known
molecular weight HPMA homopolymers. The hydrodynamic
radii were determined by QELS and calculated from the Stokesꢀ
Einstein relation. All data were collected and analyzed using
Wyatt Technology Corporation Astra 5.3.4.13 light scattering
software (Wyatt Technologies).
Docetaxel and RGDfK Content Determination in Conju-
gates. Drug contents of the synthesized copolymers were
determined by enzymatic release59 of free docetaxel and quanti-
fication by HPLC. Briefly, 5.0 mg of the conjugate was dissolved
in 200 μL of DMSO. Ten microliters of this solution was
incubated in 20 μL of buffer A consisting of 0.1 M citrate
phosphate buffer containing 2 mM EDTA at pH 6.0, 0.6 mM
papain and 100 μL of buffer B consisting of 0.1 M citrate
phosphate buffer containing 2 mM EDTA at pH 6.0 and
10 mM glutathione. The mixture was incubated at 37 ꢀC for
24 h. The condition for complete release of the drug was
optimized by varying the concentration of papain (0.1 mM to
1.0 mM) over time (data not shown). An aliquot (50 μL) of the
reaction mixture was removed, diluted in 450 μL of water:
acetonitrile (65:35), evaluated for docetaxel content by HPLC
and compared to calibration standards prepared using serial
dilutions of docetaxel in the mobile phase. Mobile phase
consisted of deionized water (Milli-Q system, Millipore, Billerica,
MA, USA) and HPLC grade acetonitrile (ACN) using the
Synthesis and Characterization of HPMA Copolymer Con-
jugates. HPMA copolymers were synthesized via free radical
copolymerization of comonomers in 10% v/v anhydrous DMSO
in acetone using N,N0- azobisisobutyronitrile (AIBN) as the
initiator (Scheme 1).55 The feed composition of comonomers
for all copolymers is given in Table 1. The weight composition of
the comonomers to solvent was kept at 12.5:87.5 (w/w). The
comonomer mixtures were sealed in an ampule under nitrogen
and stirred at 50 ꢀC for 24 h. Solvent was removed by rotary
evaporation; copolymer precursor was dissolved in methanol,
precipitated and washed in diethyl ether. Copolymer precipitates
were dissolved in deionized water and purified using dialysis tube
molecular weight cutoff (MWCO, 3500, SpectraPor) to remove
small molecular weight impurities. Samples were characterized
for weight average molecular weight (Mw), number average
molecular weight (Mn) and polydispersity (Mw/Mn) by size
exclusion chromatography (SEC) on a Superose 6 column
(10mmꢁ 30 cm) (GE Healthcare, Piscataway, NJ) using a fast pro-
tein liquid chromatography (FPLC) system (GE Healthcare).
The peaks that eluted off the column were monitored via
ultraviolet absorbance (UV), differential refractive index (RI),
and quasi-elastic light scattering (QELS) using a DAWN HE-
LEOS II light scattering instrument (Wyatt Technologies, Santa
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following gradient: 0 min, 35% ACN; 15 min, 65% ACN; 25 min,
75% ACN; 30 min 95% ACN; 39 min, 100% ACN; 40 min 65%
ACN. HPLC analyses were performed with an Agilent series
1100 HPLC (Agilent Technologies, Wilmington, DE, USA)
equipped with an Alltima C18 5 μm 150 ꢁ 4.6 mm column
and a photo diode array detector scanning at 200ꢀ500 nm. A
flow rate of 1.0 mL/min was maintained, and the sample
injection volume was 20 μL. A post time of 5 min was used to
allow column equilibration between samples. UV absorbance at
230 nm was used for quantification of docetaxel. RGDfK content
was determined by amino acid analysis (University of Utah Core
Research Facilities, Salt Lake City, UT).
In Vitro Stability of the Conjugates. The rate of release of
docetaxel from the polymer drug conjugates was evaluated in
phosphate buffer saline (PBS) at pH 7.4 and in cell culture media.
Two milligrams of each of the three copolymers P1, P2 and P3
(Table 1) was incubated in 1 mL of PBS (0.1 M phosphate buffer
in 0.05 M of NaCl at pH7.4) and 1 mL of cell culture medium
(recommended medium for DU145 from ATCC supplemented
with 10% (v/v) fetal bovine serum (FBS) (HyClone, Logan,
UT)), 100 U/mL penicillin, and 100 mg/mL streptomycin
(Sigma). The samples were incubated at 37 ꢀC, and a 100 μL
aliquot was removed at times 0 min, 1 h, 2 h, 6 h, 12 h, and 24 h.
The aliquots were immediately cooled to 4 ꢀC in ice, and then
free docetaxel was extracted with dichloromethane (DCM, 3 ꢁ
100 μL). The organic extract was concentrated under nitrogen
(N2) and then reconstituted in HPLC grade ACN:deionized
water (Milli-Q system) (1:1). The release of the free drug was
analyzed by HPLC and compared to calibration standards
prepared using serial dilutions of docetaxel in the mobile phase.
The mobile phase consisted of deionized water ((Milli-Q
system) and HPLC grade ACN using the following gradient: 0
min, 50% ACN; 10 min, 50% ACN; 10.01 min, 95% ACN; 13
min 95% ACN; 13.01 min, 50% ACN; 15 min 65% ACN. HPLC
analyses were performed with an Agilent series 1100 HPLC
(Agilent Technologies, Wilimington, DE, USA) equipped with
an Alltima C18 5 μm 150 ꢁ 4.6 mm column and a photo diode
array detector scanning at 200ꢀ500 nm. A flow rate of 1.0 mL/
min was maintained, and the sample injection volume was 20 μL.
A post time of 2 min was used to allow column equilibration
between samples. UV absorbance at 230 nm was used for
quantification of docetaxel.
Cell Lines. Human umbilical vein endothelial cells (HUVEC)
and human prostate cancer DU145 and PC3 cell lines were
obtained from American Type Culture Collection (ATCC)
(Manassas, VA). DU145 and PC3 cells were maintained in the
recommended medium from ATCC supplemented with 10%
(v/v) FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin
(Sigma). HUVECs were cultured in endothelial cell growth
media-2 (EGM-2) (Lonza Inc., Allendale, NJ). Cells were
incubated at 37 ꢀC in a humidified atmosphere of 5% CO2
(v/v) and kept in logarithmic phase of growth throughout all
experiments.
In Vitro Cell Growth Inhibition. Cell number and growth
kinetics were assessed by utilizing a water-soluble tetrazolium
salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], as a
component of Cell Counting Kit-8 from Dojindo Molecular
Technologies, Inc. (Sunnyvale, CA). Cells were seeded in 100 μL
of cultured medium at a density of 1 ꢁ 104 (DU145) or 1.5 ꢁ 104
(PC-3 and HUVEC) cells per cm2 into a 96-well microtiter plate.
They were subsequently allowed to adhere for 24 h before
medium was replaced with fresh medium containing various
concentrations of conjugates, free drug, or controls. Due to the
poor water solubility of free docetaxel, stock solutions of con-
jugates, free drug, and controls were prepared in DMSO and
subsequently diluted, resulting in a final concentration of 0.5%
(v/v) DMSO in complete growth medium. No significant
toxicities were observed for any cell line when exposed to 0.5%
DMSO concentrations for the duration of the experiment. The
cytotoxicity of free drug and HPMA copolymerꢀdocetaxel
conjugates was evaluated in two different experimental setups:
(i) continuous incubation with drugs for 72 h (DU145, PC-3);
(ii) short-term incubation or “pulse-chase” experiments where
cells were incubated with drugs for 2 h only, washed with PBS
and then incubated for an additional 70 h (DU145). In pulse-
chase experiments all compounds were added to cells either
immediately after dilution in cell growth medium or after 16 h of
preincubation in the same medium at 37 ꢀC. Following treatment
with drugs, cells were washed once with PBS and WST-8 was
added. The absorbance of colored product at 450 nm, reference
at 630 nm, was measured using a SpectraMax M2 microplate
reader (Molecular Devices, Sunnyvale, CA). The number of
viable cells exposed to the drugs was expressed as a percentage of
untreated (control) cells, concentrationꢀresponse curves were
graphed, and IC50 values were determined by nonlinear regres-
sion analysis using GraphPad Prism v. 5.03 (GraphPad Software
Inc., La Jolla, CA).
Comparative Cell Receptor Binding Assay. The comparative
affinities of free RGDfK and HPMA copolymer conjugates were
assessed using a competitive binding assay to Rvβ3 receptors
present on HUVEC and DU145 cells. HUVEC and DU145 cells
were harvested, washed with PBS, and resuspended in binding
buffer (20 mmol/L tromethamine, pH 7.4, 150 mmol/L NaCl, 2
mmol/L CaCl2, 1 mmol/L MgCl2, 0.1% bovine serum albumin).
Cell suspensions were added to 1.2 μm pore size 96-well Multi-
screen HV filter plates (Millipore, Billerica, MA) at 100,000 cells
per well. They were then coincubated at 4 ꢀC with 0.5 ng of 125I-
echistatin (Perkin-Elmer, Waltham, MA) and increasing RGDfK
peptide equivalent concentrations of copolymer conjugates or
free RGDfK peptide between 0 and 500 μM. Following 20 min
incubation, medium was removed from cells using a Multiscreen
vacuum manifold (Millipore) and cells were washed three times
with binding buffer. Filters were collected and radioactivity was
determined using a Cobra auto-gamma-counter (Canberra In-
dustries, Inc., Meriden, CT). Each experiment was performed in
triplicate, with n = 4 per replicate. Binding percentage relative to
control wells containing only 125I-echistatin was calculated, and
nonlinear regression analysis and determination of IC50 values
was carried out using GraphPad Prism.
In Vivo Efficacy of HPMA Copolymer Drug Conjugates. Six-
week-old athymic (nu/nu) mice were obtained from Charles
River Laboratories (Davis, CA, USA) and used in accordance
with the Institutional Animal Care and Use Committee
(IACUC) of the University of Utah. Mice were anesthetized
using 4% isoflurane mixed with oxygen followed by subcutaneous
injection of 1 ꢁ 107 DU145 cells per flank (n = 5 mice per
treatment group). When the mean tumor size had reached
approximately 50 mm3, the mice were treated with a single dose
of conjugates, free docetaxel, or control (saline injection) via tail
vein injection. Conjugates were prepared in saline, and free
docetaxel required formulation in polysorbate 80:EtOH:saline
(20:13:67, v/v/v) to ensure solubility. The animals were routi-
nely monitored; tumor growth was measured twice weekly, and
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tumor volume was calculated as length ꢁ width2 ꢁ π/6. Tumor
volumes at each time point were normalized by the initial volume
and are reported as mean normalized tumor volume (%) (
standard error of the mean. Animal weights were also measured
at each time point and normalized to initial animal weight
reported as mean ( standard deviation.
Statistical Analysis. Differences in growth inhibition IC50
values were determined by one-way ANOVA. Where differences
were detected, Scheffe’s post hoc analysis was used to test for
significance between groups. In vivo data was analyzed by
repeated measure ANOVA. Where differences were detected,
Scheffe’s post hoc analysis was used to test for significance
between groups. Graphpad Prism v. 5.03 and SPSS v 17
(Chicago, IL) were used for statistical analysis. The significance
level was set at R = 0.05 for all statistical tests.
’ RESULTS AND DISCUSSION
Physicochemical Characteristics of Polymeric Conjugates.
The characteristics of the HPMA copolymer conjugates are listed
in Table 1. The sizes of the copolymer conjugates, i.e., P1 (HPMA
copolymerꢀGFLGꢀdocetaxel (low molecular weight)), P2
(HPMA copolymerꢀGFLGꢀdocetaxelꢀGGꢀRGDfK) and P3
(HPMA copolymerꢀGFLGꢀdocetaxel (high molecular weight))
were controlled by changing the ratio of the total comonomers to
3-mercaptopropionic acid (3-MPA), which acts as a chain
transfer reagent in the presence of a free radical initiator.60
Docetaxel was attached to the HPMA copolymer backbone via
a lysosomally degradable oligopeptide linker glycylphenylalanyl-
leucylglycine, and targeting peptide RGDfK was attached by the
nondegradable dipeptide glycylglycine. The P1 and P2 conju-
gates (with and without Rvβ3 integrin targeting peptide RGDfK)
had a 4% molar equivalent of 3-MPA, while the high molecular
weight conjugate had a 1% molar equivalent of 3-MPA. SEC and
HPLC profiles of the conjugates indicate the absence of small
molecular weight impurities or free docetaxel. Drug content of
each polymer was measured by releasing docetaxel enzymatically
using papain followed by HPLC analysis and was found to be 6.8
wt %, 6.84 wt % and 7.15 wt % for P1, P2 and P3 respectively. The
molecular weights of the copolymers as estimated by SEC on a
Superose 6 column were 32.6 kDa, 26.9 kDa and 87.1 kDa. The
hydrodynamic diameters of the conjugates as measured by quasi
elastic light scattering (QELS) were 3.0 nm, 3.0 nm and 7.6 nm
for P1, P2 and P3 respectively. RGDfK content of P2 was
estimated by amino acid analysis and was found to be 5.061 wt %
of the polymer.
In Vitro Stability of PolymerꢀDrug Conjugates. The in
vitro stability of conjugates was investigated at physiological pH
as well as in cell culture medium at pH 7.4. The three conjugates
P1, P2 and P3 released 14.8 ( 2.6%, 18.6 ( 4.8% and 10.8 (
2.3% in PBS pH 7.4 and 23.4 ( 3.3, 23.2 ( 2.4 and 24.1 ( 2.7 in
cell culture medium in the first 24 h (Figure 1). The higher
release of the conjugates in the medium is likely due to the
presence of serum esterases which cleave the ester bond between
the conjugate and the drug. All copolymer systems under study
had less than 20% release of docetaxel in PBS at pH 7.4 and less
than 25% release in cell culture medium in 24 h, indicating
adequate stability for in vivo accumulation of a portion of intact
conjugates in solid tumors.
Figure 1. Stability of polymerꢀdrug conjugates in PBS, pH 7.4 (A) and
cell culture medium (B) at 37 ꢀC. Diamond represents P1, the
untargeted low molecular weight conjugates; the square represents P2,
the RGDfK targeted low molecular weight conjugate; and the triangle
represents P3, the high molecular weight conjugate. The values of 3
independent measurements are presented as mean ( SD. For sample
characteristics see Table 1.
inhibition curves revealed that the conjugates were 1.4ꢀ2.7 times
less toxic than free docetaxel when cells were incubated for a
continuous 72 h (Table 2). HUVECs were more sensitive to
docetaxel compared to prostate cancer cell lines as almost half of
the drug concentrations were required to achieve the same
magnitude of cell growth inhibition. Besides distinct difference
between cytotoxic potential of free and conjugated forms of
docetaxel, the difference between IC50 values for polymeric
conjugates was found statistically insignificant (p > 0.05) for all
cell lines tested.
Considering different routes of cellular entry and the necessity
of lysosomal degradation of a linker between the polymer back-
bone and a drug, macromolecular therapeutics usually show
50ꢀ200-fold differences in the toxicity compare to a free drug.17
The difference observed between IC50 values of free and con-
jugated forms of docetaxel was expected but was significantly
smaller than that difference reported in the literature for other
macromolecular therapeutics. It is possible that the hydrolysis of
the ester bond and fast drug release from the conjugates are
responsible for high toxicity of the conjugates. The same issue
could play an important role in the efficacy of the conjugates
in vivo as ester bonds could be cleaved by abundant esterases
within the bloodstream.61 In an attempt to mimic in vivo
physiological conditions, DU145 cells in this study were exposed
to the polymerꢀdrug conjugates for 2 h, a time period relevant to
the plasma half-life of typical HPMA copolymerꢀdrug
conjugates.48 In these sets of experiments DU145 cells were
exposed to the drugs for 2 h only and subsequently were washed
with phosphate buffered saline, and incubation continued in
In Vitro Cell Growth Inhibition. All conjugates inhibited
proliferation of prostate cancer cell lines and HUVECs at
nanomolar concentrations (Figure 2AꢀC). Analysis of growth
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Figure 2. Toxicity of docetaxel conjugates toward cultured cells. DU145 (A), PC-3 (B) prostate cancer cells and HUVECs (C) were exposed to the
compounds for a continuous 72 h. In pulse-chase experiments (D) DU145 cells were incubated with the compounds for 2 h only, washed with PBS and
incubated in fresh growth medium for an additional 70 h. The legend for this plot indicates how long compounds were in contact with cell growth
medium. All compounds were added to cells either immediately after dilution in cell growth medium (2 h) or after 16 h of preincubation in the same
medium at 37 ꢀC (16 þ 2 h). The values of 3ꢀ4 independent experiments are presented as mean ( SD. For sample characteristics see Table 1.
Table 2. Calculated IC50 Values (nM)a after Continuous 72 h
the two forms of doxorubicin (Figure 2D). After preincubation
only 4ꢀ5-fold difference between IC50 values of free and
Incubation of Cells with Drugs
conjugated docetaxel was observed most likely due to the release
DU145
PC-3
HUVEC
of the drugs from the conjugates. As in the experiments with 72 h
continuous incubation, no statistically significant difference
between IC50 values for the polymeric conjugates was found.
Thus, given the two variables tested in this experiment, the
release rate of free docetaxel proved to be the determinant
variable that influenced the cytotoxicty of the tested constructs
in vitro. High toxicity of the drug masked the binding advantage
of the RGDfK moiety in vitro.
DOC
P1
2.1 ( 0.5
4.4 ( 1.1*
3.0 ( 0.4*
3.3 ( 0.8*
1.8 ( 0.4
4.4 ( 0.7*
4.3 ( 0.4*
3.9 ( 0.7*
0.8 ( 0.1
2.7 ( 0.9*
2.3 ( 0.4*
1.9 ( 0.5*
P2
P3
a *p < 0.05 vs DOC.
Competitive Binding Studies. Competitive binding studies
demonstrated binding of the targeted HPMA copolymerꢀ
docetaxelꢀRGDfK conjugate (P2) to HUVEC and DU145 cell
lines with IC50 values of 0.5 ( 0.2 μM and 2.6 ( 0.3 μM
respectively (Figure 3). RGDfK peptide alone showed similar
comparative binding affinities of 0.3 ( 0.1 μM and 1.5 ( 0.3 μM
at equivalent peptide concentrations in HUVEC and DU145 cell
lines. Untargeted conjugate (P1) was also evaluated in both cell
lines and showed no evidence of active binding (Figure 3). These
results demonstrate the ability of the targeted conjugate P2
(Table 1) to bind to Rvβ3 integrins of HUVECs and DU145
cells. In both cases, conjugation of RGDfK to the HPMA
copolymer backbone resulted in a small decrease in its binding
affinity as compared to free RGDfK probably due to steric
hindrance of the macromolecular system (Figure 3).
fresh growth medium without drugs until the number of cells was
estimated. Therefore, this experimental setup allowed testing the
influence of two important variables in these drug delivery
systems: drug release rate and the presence of RGDfK targeting
moiety. These experiments revealed a 15ꢀ20-fold difference
between IC50 values of free and conjugated docetaxel when they
were added to DU145 cells immediately after dilution in growth
medium (Figure 2D). This difference is most likely due to the
difference in the mechanisms of cellular internalization between
two forms of the drug, because the stability studies performed
with polymerꢀdrug conjugates revealed that less than 3%
docetaxel was released from polymerꢀdrug conjugates in the
presence of cell culture medium within 2 h (Figure 1). This
suggested that the conjugates were not completely hydrolyzed
within the first two hours. Prolonged 16 h preincubation of the
conjugates in growth medium before the initiation of pulse-chase
experiments significantly changed the observed difference between
In Vivo Antitumor Efficacy of the Conjugates. One focus of
this study was to evaluate whether the efficacy of macromolecules
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Figure 3. Competitive binding of HPMA copolymer conjugates and
free RGDfK peptide binding of HPMA copolymer conjugates with and
without RGDfK was compared to free peptide on (A) HUVEC and (B)
human prostate cancer DU145 cells. Results are expressed as means of
triplicate ( SD. For sample characteristics see Table 1.
with sizes below renal threshold can be enhanced by active
targeting. To this end, we have chosen the cyclic integrin
targeting peptide RGDfK as it has shown the ability to substan-
tially increase tumor accumulation of HPMA copolymers in
prostate tumors.51 Docetaxel was chosen as it is the preferred
drug of choice for patients with metastatic hormone refractive
prostate cancer.9 Efficacy of the conjugates was evaluated in nu/
nu mice bearing DU145 prostate tumor xenografts. A single dose
of each conjugate, free docetaxel, or control was administered.
Physiological saline was used as a negative control and free
docetaxel as positive control. The dose selection for both free
drug and polymeric conjugates was 20 mg/kg and 40 mg/kg
docetaxel equivalent, which corresponds to 60 mg/m2 and 120
mg/m2 in humans weighing 65 kg, as per calculation factors
shown in the literature.62,63 This dose was based on several phase
II/III clinical trials4,64ꢀ72 using docetaxel as a single agent as well
as in combination chemotherapy, where doses varied between 30
mg/m2 and 75 mg/m2 given over several cycles.
Figure 4. (A) Normalized animal weights as a function of time. Data are
expressed as mean ( SEM (n = 5 per treatment group). (B) Efficacy of
free drug and targeted/nontargeted polymerꢀdrug conjugates P1, P2
and P3 at drug equivalent concentration of 20 mg/kg in nu/nu mice
bearing DU145 tumor xenografts. (C) Efficacy of free drug and
targeted/nontargeted polymerꢀdrug conjugates P1, P2 and P3 at drug
equivalent concentration of 40 mg/kg in nu/nu mice bearing DU145
tumor xenografts. Tumor volumes were normalized by their respective
volume on day 0 (approx 50 mm3). Data are expressed as mean ( SEM
(n = 10 tumors per treatment group). For sample characteristics see
Table 1.
immobile for the first 30 min post injection. The lethargy shown
by animals injected with free docetaxel was most likely a result of
toxicity associated with the drug as well as the polysorbate 80
required to dissolve the drug for iv injection. None of the animals
in the control group or those treated with conjugates showed any
signs of acute toxicity. Mouse weights were followed up through
the duration of the study as an overall measurement of the safety
Animals injected with free docetaxel at both 20 mg/kg and 40
mg/kg showed signs of hyperacute toxicity as they were rendered
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of administered compounds. The normalized weights of animals
showed no statistical difference between all groups for the
duration of study (Figure 4A). Animals injected with free
docetaxel showed statistically significant 2.6- and 2.9-fold reduc-
tion in mean tumor size as compared to saline at concentrations
of 20 mg/kg (P < 0.01) and 40 mg/kg (P < 0.001). Although
conjugate P1 (untargeted, low molecular weight HPMAꢀ
docetaxel) at 20 mg/kg demonstrated a 0.9-fold difference in
mean tumor size as compared to saline, this difference was not
statistically significant (P > 0.1). However, treatment animals
with P1 at 40 mg/kg resulted in a 2.3-fold reduction in mean
tumor size as compared to saline (P < 0.05).
Conjugate P2 (RGDfK targeted, low molecular weight HPMA
copolymerꢀdocetaxel) and conjugate P3 (untargeted, high
molecular weight HPMA copolymerꢀdocetaxel) at injected
doses of 20 mg/kg and 40 mg/kg showed reductions in mean
tumor size as compared to saline (P < 0.001) of 3.3-, 3.2-, 3.5-,
and 3.4-fold respectively. While animals injected with both
conjugates P2 and P3 demonstrated reduction in tumor size
greater than the group injected with free docetaxel, only results
with P2 at both 20 mg/kg and 40 mg/kg were statistically
significant. The efficacy of P1 at 20 mg/kg was statistically
inferior to that of P2 at 20 mg/kg and 40 mg/kg (P < 0.01)
and that of P3 at doses of 20 mg/kg and 40 mg/kg (P < 0.05) with
a mean difference in tumor size of 2.5-, 2.4-, 2.3-, and 2.2-fold. P1
at 40 mg/kg had no statistical difference in mean tumor size with
P2 and P3 at 20 mg/kg and 40 mg/kg. P2 and P3 had no
statistical difference at doses of 20 mg/kg or 40 mg/kg.
activity at 20 mg/kg of P3 when compared to free docetaxel was
not statistically significant for the study period of 42 days.
Further, when comparisons were made between active target-
ing (P2) and passive targeting via the EPR effect (P3) at drug
equivalent concentrations of 20 mg/kg, though the mean differ-
ence in tumor size between the groups was not significant, the
mean tumor size reduction was greater in the case of actively
targeted P2 conjugates. However at 40 mg/kg, which corre-
sponds to 120 mg/m2 in humans and is more than 1.5 times the
highest concentration administered in clinical trials of docetaxel
for treatment of prostate cancer, free drug and all three con-
jugates P1, P2 and P3 induced statistically significant tumor size
reduction as compared to saline. The efficacy of P1 was evident at
40 mg/kg because sufficient docetaxel was released from the
conjugate prior to elimination. Administrations of all three
conjugates, unlike free docetaxel, demonstrated no visible signs
of toxicity, and were freely soluble in saline.
The post hoc analysis of the variance of the mean size of the
tumor over the entire study period of 42 days classified the
conjugates and controls into three categories, i.e. those with high
activity, those with moderate activity and those with no activity.
Each compound falls under one or two categories. According to
this analysis, saline falls in the low activity category, P1 at 20 mg/
kg falls under the low and moderate activity categories, P1 at 40
mg/kg and free docetaxel at both 20 mg/kg and 40 mg/kg doses
fall under moderate and high activity categories, and P2 and P3 at
both 20 mg/kg and 40 mg/kg fall under the high activity
category.
The lack of efficacy for P1 at 20 mg/kg in spite of efficacy
observed in free docetaxel can be explained below: (i) low
molecular weight P1 with a hydrodynamic diameter of 3.0 nm
was small enough to be rapidly eliminated by glomerular filtra-
tion, thereby minimizing the persistence time of the conjugate in
blood circulation which is necessary to achieve tumor accumula-
tion due to the EPR effect, and/or (ii) the release of docetaxel
from the conjugate was not fast enough for it to be effective prior
to its elimination. Therefore, free docetaxel at this concentration
was more effective than the untargeted low molecular weight
conjugate. The efficacy of P1 was evident at 40 mg/kg because
sufficient docetaxel could be released from the conjugate prior to
elimination. The only advantage shown by animals injected with
P1 was the lack of adverse hyperacute reaction in response to
injection that was observed for free docetaxel administration,
possibly due to the aqueous solubility acquired by polymer
conjugation.
At 20 mg/kg, P2 showed the largest difference in tumor size
with saline. Conjugate P2, like P1, had a hydrodynamic diameter
of 3.0 nm, which was well below the glomerular renal threshold.
The activity observed in these conjugates most probably is a
consequence of active targeting as they had Rvβ3 integrin
targeting peptide RGDfK linked to the polymer side chains,
thereby allowing them to anchor onto the neovasculature of
angiogenic blood vessels. The RGD peptides have also been
reported to be tumor penetrating, and its coadministration
enhances the efficacy of anticancer drugs.47 RGDfK in conjugate
P2 may have also contributed to a greater efficacy. Conjugate P3,
on the other hand, had a hydrodynamic diameter of 7.6 nm,
which was above the glomerular renal threshold. This increases
the plasma half-life and improves efficacy by passive targeting via
the EPR effect. Administration of P3 at concentrations of 20 mg/
kg had a reduction in mean tumor size difference as compared to
saline (P < 0.001) and P1 (P < 0.05). Again, the difference in
To summarize, administrations of all three conjugates, unlike
free docetaxel, demonstrated no visible signs of toxicity, and were
freely soluble in saline. Actively targeted conjugate P2 demon-
strated the highest tumor size reduction for the duration of the
study as compared to free docetaxel, small nontargeted conjugate
P1 and passively targeted (via EPR) high molecular weight
conjugate P3. All three conjugates were soluble in aqueous media
and did not show any visible signs of hyperacute toxicity in
animals. Further, while P3 does not have a biodegradable
polymer backbone which would result in accumulation of the
macromolecule post treatment, P2 has the advantage of a small
size (3.0 nm), which can allow the dose fraction that does not
reach the tumor to be eliminated.
’ CONCLUSIONS
HPMA copolymerꢀdocetaxel conjugates with sizes of 3.0 and
7.6 nm, which correspond to dimensions below and above the
glomular renal threshold, as well as Rvβ3 integrin targeting
conjugate HPMA copolymerꢀdocetaxelꢀRGDfK of hydrody-
namic diameter 3.0 nm, were successfully synthesized to evaluate
the effect of active targeting with passive targeting. All the
conjugates inhibited proliferation of human prostate cancer
DU145 and PC3 cells as well as HUVEC at nanomolar con-
centrations. Cytotoxicity experiments by pulse-chase method
where the incubation time with free drug and conjugates was
limited to 2 h resulted in a 15ꢀ20-fold difference in activity of
conjugates as compared to free docetaxel. This suggests that the
conjugates were not completely hydrolyzed within the first two
hours. Animals showed no visible signs of toxicity when injected
with conjugates. HPMA copolymerꢀdocetaxel conjugates of
hydrodynamic diameter 7.6 nm and HPMA copolymerꢀ
docetaxelꢀRGDfK of hydrodynamic diameter 3.0 nm demon-
strated the greatest tumor reduction capability with statistically
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significant tumor regression compared to saline. Overall, the
results demonstrate that Rvβ3 integrin targeted low molecular
weight conjugates with improved water solubility, reduced
toxicity and ease of elimination post treatment in vivo are
promising candidates for prostate cancer therapy.
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’ AUTHOR INFORMATION
Corresponding Author
(15) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor
vascular permeability and the EPR effect in macromolecular therapeu-
tics: a review. J. Controlled Release 2000, 65 (1ꢀ2), 271–84.
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(17) Kopecek, J.; Kopeckova, P. HPMA copolymers: origins, early
developments, present, and future. Adv. Drug Delivery Rev. 2010, 62 (2),
122–49.
*Departments of Pharmaceutics & Pharmaceutical Chemistry
and of Bioengineering, Utah Center for Nanomedicine, Nano
Institute of Utah, University of Utah, 383 Colorow Road, Room
343, Salt Lake City, UT 84108, USA. Tel: (801) 587-1566. Fax:
(801) 585-0575. E-mail: hamid.ghandehari@pharm.utah.edu.
Author Contributions
§These authors contributed equally to this work
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Kopecek, J.; Duncan, R. Biocompatibility of N-(2-hydroxypropyl)
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’ ACKNOWLEDGMENT
This research was supported by the National Institutes of
Health Grant R01 EB007171 and the Utah Science Technology
and Research (USTAR) initiative.
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