Synthesis and characterization of a multiarm poly(acrylic acid) star polymer for application in sustained delivery of cisplatin and a nitric oxide prodrug

Article abstract of DOI:10.1002/pola.26059

Functionalized polymeric nanocarriers have been recognized as drug delivery platforms for delivering therapeutic concentrations of chemotherapies. Of this category, star-shaped multiarm polymers are emerging candidates for targeted delivery of anticancer drugs, due to their compact structure, narrow size distribution, large surface area, and high water solubility. In this study, we synthesized a multiarm poly(acrylic acid) star polymer via macromolecular design via the interchange (MADIX)/reversible addition fragmentation chain transfer (MADIX/RAFT) polymerization and characterized it using nuclear magnetic resonance (NMR) and size exclusion chromatography. The poly(acrylic acid) star polymer demonstrated excellent water solubility and extremely low viscosity, making it highly suited for targeted drug delivery. Subsequently, we selected a hydrophilic drug, cisplatin, and a hydrophobic nitric oxide (NO)-donating prodrug, O²-(2,4-dinitrophenyl) 1-[4-(2-hydroxy)ethyl]-3- methylpiperazin-1-yl]diazen-1-ium-1,2-diolate, as two model compounds to evaluate the feasibility of using poly(acrylic acid) star polymers for the delivery of chemotherapeutics. After synthesizing and characterizing two poly(acrylic acid) star polymer-based nanoconjugates, poly(acrylic acid)-cisplatin (acid-Pt) and poly(acrylic acid-NO (acid-NO) prodrug, the in vitro drug release kinetics of both the acid-Pt and the acid-NO were determined at physiological conditions. In summary, we have designed and evaluated a polymeric nanocarrier for sustained-delivery of chemotherapies, either as a single treatment or a combination therapy regimen.

Full text of DOI:10.1002/pola.26059

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Synthesis and Characterization of a Multiarm Poly(acrylic acid) Star
Polymer for Application in Sustained Delivery of Cisplatin and a Nitric
Oxide Prodrug
Shaofeng Duan, Shuang Cai, Yumei Xie, Taryn Bagby, Shenqiang Ren, M. Laird Forrest
University of Kansas, 2095 Constant Ave, Lawrence, Kansas 66047
Correspondence to: M. L. Forrest (E-mail: mforrest@ku.edu)
Received 4 February 2012; accepted 16 March 2012; published online
DOI: 10.1002/pola.26059
ABSTRACT: Functionalized polymeric nanocarriers have been
recognized as drug delivery platforms for delivering therapeutic
concentrations of chemotherapies. Of this category, star-shaped
multiarm polymers are emerging candidates for targeted deliv-
ery of anticancer drugs, due to their compact structure, narrow
size distribution, large surface area, and high water solubility. In
this study, we synthesized a multiarm poly(acrylic acid) star
polymer via macromolecular design via the interchange
(MADIX)/reversible addition fragmentation chain transfer
(MADIX/RAFT) polymerization and characterized it using nuclear
magnetic resonance (NMR) and size exclusion chromatography.
The poly(acrylic acid) star polymer demonstrated excellent
water solubility and extremely low viscosity, making it highly
suited for targeted drug delivery. Subsequently, we selected a
hydrophilic drug, cisplatin, and a hydrophobic nitric oxide (NO)-
donating prodrug, O2-(2,4-dinitrophenyl) 1-[4-(2-hydroxy)ethyl]-
3-methylpiperazin-1-yl]diazen-1-ium-1,2-diolate, as two model
compounds to evaluate the feasibility of using poly(acrylic acid)
star polymers for the delivery of chemotherapeutics. After syn-
thesizing and characterizing two poly(acrylic acid) star polymer-
based nanoconjugates, poly(acrylic acid)–cisplatin (acid–Pt) and
poly(acrylic acid–NO (acid–NO) prodrug, the in vitro drug release
kinetics of both the acid–Pt and the acid–NO were determined at
physiological conditions. In summary, we have designed and
evaluated a polymeric nanocarrier for sustained-delivery of che-
motherapies, either as a single treatment or a combination ther-
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apy regimen. 2012 Wiley Periodicals, Inc. J Polym Sci Part A:
Polym Chem 000: 000–000, 2012
KEYWORDS: conjugated polymer; drug delivery systems; revers-
ible addition fragmentation chain transfer; star polymer; water-
soluble polymer
INTRODUCTION Star polymer architectures are a new gener-
ation of branched polymeric materials that consist of a single
core and multiple connecting arms or chains. Star polymers
usually have highly condensed and globular structures, tail-
orable size, and large surface areas for conjugation of drugs
and imaging agents, which makes them suited as potential
delivery platforms. Various star polymer carriers have been
synthesized and used for the delivery of small-molecule che-
motherapeutics, including doxorubicin1 (conjugation) and
paclitaxel2 (encapsulation), proteins, such as insulin,3 as well
as genetic materials, such as DNAs4 and siRNAs.5 Herein, we
developed a multiarm poly(acrylic acid) star polymer (Fig. 1)
via MADIX/reversible addition fragmentation chain transfer
(RAFT) polymerization for long sustained release of multiple
chemotherapeutics simultaneously as a chemo ‘‘cocktail’’.
renal toxicity, which limits its use. The toxicity of cisplatin is
dependent on the maximum concentration of free drug in
the plasma. Slow and confined release of cisplatin into
tumors can largely alleviate these toxicities. In our previous
studies, we synthesized a hyaluronan–cisplatin conjugate for
controlled release of subcutaneously delivered cisplatin using
the biodegradable and biocompatible polymer hyaluronan.6
The conjugates achieved superior pharmacokinetics, reduced
systemic toxicity, and enhanced anticancer efficacy in rodents
compared to unbound intravenous cisplatin.7–10 However, a
major drawback of this hyaluronan-based delivery platform
and other linear polymers is the relatively high intrinsic vis-
cosity of the vehicle, which makes it challenging to adminis-
ter a high concentration of the chemotherapy within a small
injection volume. Therefore, we sought to develop another
drug carrier with desired solubility and viscosity characteris-
tics that could sustain cisplatin over several days.
Cisplatin is a water soluble, platinum-based chemotherapeu-
tic that has been widely used for many years in the clinic as
a first-line chemotherapy for head and neck squamous cell
carcinoma, ovarian cancer, and non-small cell lung cancer.
The major side effect of cisplatin infusion chemotherapy is
Polymer carriers can overcome problems with drug solubil-
ity and stability. We have developed a nitric oxide (NO)-
donating prodrug, similar to other drugs in this class, it
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NMR (400 MHz) and 13C NMR (100 MHz) spectra were col-
lected on a Bruker DRX 400 spectrometer using compounds
dissolved in CDCl3, MeOD or D2O. Chemical shifts were refer-
enced to d 7.28 and 77.0 ppm for 1H NMR and 13C NMR spec-
tra, respectively. High-resolution mass spectrometry (HRMS)
data were generated after flow injection analysis and man-
ually matching peaks on an Applied Biosystems Mariner TOF
spectrometer with a turbo-ionspray source. The cell culture
media were purchased from MediaTech Inc (Manassas, VA).
Synthesis
Synthesis of MADIX/RAFT Agent
The MADIX/RAFT agent was synthesized based on the proce-
dure reported previously (Fig. 2).19 The core starting mate-
rial, pentaerythritol, was esterified with 2-bromopropionyl
bromide and O-ethylxanthic acid (potassium salt), succes-
sively, to form a four-arm sulfur compound that was used as
the MADIX/RAFT initiator agent.
FIGURE 1 Chemical structure of multiarm poly(acrylic acid)
star polymer.
4-arm Core Initiator 1. Pentaerithol (1.36 g, 10 mmol) was
dissolved in dryꢀ chloroform (25 mL) and pyridine (2.5 mL)
and cooled to 0 C. The 2-bromo propionyl bromide (10.01 g,
45 mmol) was added dropwise, and the reaction proceeded at
releases NO in vivo upon activation by glutathione-s-transferase
in cells. But, it is both very poorly water soluble and unstable in
serum. NO has been shown to effectively inhibit the proliferation
of many cancer cells, including breast cancer,11 colon cancer,12
non-small cell lung cancer,13 and melanoma.14 It is a gaseous
small molecule with a half life of only seconds.15 To deliver ther-
apeutic concentrations of NO to tumorigenic tissues, a controlled
release strategy is critical. A number of sustained release carriers
have been designed for the delivery of NO, including NO-donor
incorporated ethylene/vinyl acetate polymers,16 S-nitrosothiol
conjugated interpolymers,17 as well as block copolymer of N-
acryloylmorpholine, and N-acryloyl-2, 5-dimethylpiperazine.18
To date, no combinational NO/cisplatin delivery platforms have
been developed for delivering these two chemotherapeutics
simultaneously. Therefore, we designed a nanocarrier-based sys-
tem to generate active NO molecules and DNA-crosslinking plati-
num in a sustained release manner that maintains a relatively
steady level of NO and platinum within the therapeutic window
for a longer period of time during treatment.
ꢀ
ambient temperature (ca. 23 C) for 48 h. The mixture then
was neutralized with aqueous HCl (10 wt%). Then, the organic
phases were washed with water, sodium bicarbonate solution
(5 %), and then brine, followed by drying with sodium sulfate.
The solvent was evaporated under reduced pressure, which
yielded the desired compound. The molecular structure was
verified by 1H NMR and compared with the reported data.
A solution of the intermediate compound (2.028 g, 3.0
mmol) in chloroform (45 mL) was treated with a 10-fold
excess of O-ethylxanthic acid, potassium salt (5.01 g, 30
mmol). The mixture was stirred at ambient temperature for
3 days, and the resulting suspension was filtered and
washed with chloroform. Evaporation of the solvent followed
by purification through a flash column on silica gel using 3 :
7 ethyl acetate:hexanes as eluents yielded the desired com-
1
pound, and the molecular structure was verified by H NMR
and compared with the reported data.
EXPERIMENTAL
Synthesis of 4-Arm Tert-Butyl Acrylate Star Polymer
Materials
The multiarm tert-butyl acrylate star polymer was synthe-
sized based on a modification of Ting et al.’s procedure.20
The tert-butyl acrylate (2.50 g, 19.5 mmol) was treated with
basic alumina and dry a, a, a- trifluorotoluene (10 mL) in a
Unless noted otherwise, all reagents and solvents were pur-
chased from Sigma Aldrich (St Louis, MO) or Fisher Scientific
(Pittsburgh, PA) and used without further purification. 1H
FIGURE 2 Synthesis of MADIX/RAFT agent.
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FIGURE 3 Synthesis of multiarm tert-butyl acrylate star polymers.
50-mL round bottom flask. The RAFT agent 1 (27 mg, 3.25
ꢁ 10ꢂ2 mmol) was added to the solution, followed by the
addition of AIBN (0.53 mg, 3.25 ꢁ 10ꢂ3 mmol), and the
reaction flask was placed on ice and purged with argon for
30 min. The flask then was transferred to a thermostatic oil
et al.6 Poly(acrylic acid) star polymer (50 mg) was dissolved
in dd H2O (25 mL) in a 50-mL round bottom flask. The pH
of the solution was adjusted to 10 using 1-M NaOH solution
and cisplatin (25 mg) was added. The solution was stirred
under argon protection at ambient temperature for 36 h.
The solution then was dialyzed (10-kDa MWCO tubing,
Pierce, Rockford, IL) against distilled water at 4 ꢀC for 2
days with water changes every 6 h to remove the unbound
cisplatin. After dialysis the resulting polymer was concen-
trated by rotary evaporation to obtain the desired multiarm
poly(acrylic acid) star polymer–cisplatin conjugates (Fig. 5).
ꢀ
bath at 70 C for ca. 12 h. The mixture was cooled in an ice
bath and poured into cold diethyl ether to obtain the precipi-
tate followed by concentration under reduced pressure (Fig.
3). The molecular structure and molecular weight of the
resulting polymer were determined by 1H NMR and size
exclusion chromatography (SEC), respectively.
Synthesis of Multiarm Acrylic Acid Star Polymers
Synthesis of a NO-donating Prodrug, O2-(2,4-Dinitro-
phenyl) 1-{[4-(2-(2-hydroxyethoxy))ethyl] piperazin-1-yl}
diazen-1-ium-1,2-diolate
The tert-butyl acrylate star polymer (2.50 g, 19.5 mmol) was
dissolved in dichloromethane (200 mL), and trifluoroacetic
acid (TFA, 75 mL, 975 mmol) was then added. The mixture
was stirred vigorously until the generation of white precipi-
tate ceased. The precipitate was filtered and washed with
dichloromethane and diethyl ether, and then dried under
reduced pressure overnight. The molecular structures and
weights of the resulting polymers (Fig. 4) were determined
The NO prodrug was synthesized according to the procedure
of Chakrapani et al.21 (Fig. 6). The 1-[2-(2-hydroxyethoxy)e-
thyl]piperazine (5.0 g, 27.3 mmol) was dissolved in methanol
(2 mL) and treated with 25% (w/v) methanolic sodium
methoxide (6.2 mL), and ether (20 mL). The resulting solu-
tion was charged with NO at 60 psi and stirred at ambient
temperature for 24 h, leading to the formation of a white
precipitate. The white solid was filtered, washed with ether,
and dried under reduced pressure to afford the sodium 1-[2-
(2-hydroxyethoxy)ethyl] homopiperazin-1-yl] diazen-1-ium-1,
2-diolate. Subsequently, the white solid was dissolved in 5%
1
by H NMR and SEC, respectively.
Synthesis of Poly(acrylic acid) Star Polymer–Cisplatin
Conjugates
The poly(acrylic acid) star polymer–cisplatin conjugates
(acid–Pt) were synthesized according to the procedure of Cai
FIGURE 4 Synthesis of multiarm acrylic acid star polymer from tert-acrylate star polymer.
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FIGURE 5 Synthesis of poly(acrylic acid) star polymer–cisplatin conjugates.
(w/v) ice cold sodium bicarbonate solution (60 mL). The solu-
tion was treated with 1-fluoro-2,4-dinitrobenzene (4.67 g,
25.1 mmol) predissolved in a mixture of t-BuOH (30 mL) and
THF (5 mL). A yellow precipitate formed immediately and
was purified by silica flash chromatography (CHCl3: EtOAc ¼
9 : 1) to afford the desired compound. The molecular struc-
ture was verified by 1H NMR and mass spectrometry.
system equipped with an RID-10A refractive index detector
and a TSK gel multipore Hx-M 7.8 ꢁ 30 cm column using
10-mM LiCl in DMF (0.8 mL/min) as the mobile phase. The
calibration curve was generated using polystyrene standards
ranging from 1,180 to 339,500 g/mol. After deprotection,
the resulting multiarm poly(acrylic acid) star polymer was
highly soluble in methanol, water, DMF, and DMSO. The mo-
lecular weights of the multiarm poly(acrylic acid) star poly-
mers were determined by SEC using polyethylene glycol
(PEG) standards ranging from 3,070 to 66,100 g/mol (Scien-
tific Polymer Products) (Table 1).
Synthesis of Poly(acrylic acid) Star Polymer–NO Prodrug
Conjugates
The poly(acrylic acid) star polymer (50 mg) was dissolved in
25 mL of ice cold DMF: dd H2O (25 : 1, v/v) mixture in a
50-mL round bottom flask (Fig. 7), and EDCIꢃHCl (1.5 equiv.)
and HOBtꢃH2O (1.5 equiv.) were added to the solution.
After 5 min, O2-(2,4-Dinitrophenyl) 1-{[4-(2-(2-hydroxyethox-
y))ethyl] piperazin-1-yl}diazen-1-ium-1,2-diolate (1.0 equiv.)
was added to the solution. The reaction proceeded under ar-
gon at 0 ꢀC for 30 min, followed by ambient temperature
overnight in the dark. The resulting poly(acrylic acid)-nitric
(acid–NO) conjugate was dialyzed using 10-kDa MWCO tub-
ing against a mixture of dd H2O: ethanol (1 : 1, v/v) at ambi-
ent temperature for 12 hours, followed by ethanol for
another 12 h. The desired acid–NO conjugates were obtained
after the evaporation of the solvent under reduced pressure.
Determination of Drug Conjugation
The substitution degree of the NO prodrug on the acid–NO
conjugate was determined by 1H NMR based on the ratio of
the aromatic protons of the NO prodrug to the methylene
protons of the polymer core. The degree of cisplatin substi-
tution was determined by atomic absorption spectroscopy
(AAS) (Varian SpectrAA GTA-110) using platinum standards
ranging from 100 to 450 ppb (Fisher Scientific).6ꢀ The fur-
nace program was as follows: ramp from 25 to 80 C, hold 2
s, ramp to 120 ꢀC, hold 10 s, ramp to 1000 ꢀC, hold 5 s,
ramp to 2700 ꢀC, hold 2 s, cool to 25 ꢀC over 20 s. The
graphite partition tube was cleaned every 40 samples by
baking at 2800 ꢀC for 7 s. Argon was used as the injection
and carrier gas.
1
The molecular structure was verified by H NMR.
Characterization
Size Exclusion Chromatography
Viscosity
The molecular weights and polydispersity indices (PDIs) of
the multiarm tert-butyl acrylate star polymers were deter-
mined using EZStart 7.4 software and a Shimadzu 2010CHT
The viscosity parameters were measured using a Stabinger
viscometer (SVM 3000, Anton Paar) at room temperature. A
series of multiarm poly (acrylic acid) star polymer samples
FIGURE 6 Synthesis of O2-(2,4-Dinitrophenyl) 1-{[4-(2-(2-hydroxyethoxy))ethyl] piperazin-1-yl}diazen-1-ium-1,2-diolate.
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FIGURE 7 Synthesis of poly(acrylic acid) star polymer–NO prodrug conjugates.
RESULTS AND DISCUSSION
(MWs: 64,000, 75,000, 80,300 and 110,000 g/mol) were pre-
pared by dissolving the polymers in dd H2O at three concen-
trations including: 1.0, 3.0, and 10.0 mg/mL.
Synthesis of Acid–NO and Acid–Pt Conjugates
The poly (tert-butyl acrylate) star polymers were synthe-
sized via MADIX/RAFT polymerization mediated by xan-
thates in a, a, a- trifluorotoluene, using AIBN as an initiator.
The MADIX/RAFT polymerization approach is of special in-
terest to polymer and drug delivery scientists compared to
other synthetic strategies involving free radical reactions,
including NMP, ATRP, and RAFT, as a wide range of potential
monomers could be readily used to generate well-controlled
polymeric architectures. The polymerization resulted in 90%
conversion of the monomers after a reaction duration of
approximately 14 h. Reaction byproducts started to form if
the reaction was allowed to proceed for longer than 20 h.
Subsequent treatment of the poly (tert-butyl acrylate) star
polymers of different molecular weights with TFA in
dichloromethane yielded the corresponding acid star poly-
mers. The acid star polymer and its corresponding polymer–
chemotherapeutic conjugates, including acid star polymer–
NO prodrug conjugate (20 wt% drug loading) and acid star
polymer–cisplatin conjugates (8 wt% drug loading) were
successfully synthesized. The acid star polymers were solu-
ble in water, methanol, DMF and DMSO. Besides the multi-
arm poly(acrylic acid) star polymers we developed, various
other polymeric systems have also been explored for locore-
gional delivery of anticancer drugs, including PEG-graft-a,b-
poly [(N-amino acidyl)-aspartamide] polymers,22 PLGA-4-
In Vitro Release of Drug from Acid Star Polymers
The in vitro release kinetics of cisplatin was determined
according to our previously published procedure.6 The acid–
Pt conjugate (10 mg) was dissolved in 10 mL of phosphate
buffered saline (PBS), transferred to dialysis tubing (10 kDa
MWCO), and placed in a PBS bath (pH 7.4, 37 ꢀC, 4 L) with
or without 10% bovine serum albumin and 0.2% sodium az-
ide. Two hundred microliter aliquots were collected from the
ꢀ
tubing at predetermined intervals and stored at ꢂ80 C until
analysis by AAS. The AAS produced a linear concentration
curve from 10 to 450 ng/mL (R2 ¼ 0.9998), with a limit of
detection of 5 ng/mL and a limit of quantification of 10 ng/
mL (5% standard deviation).
The release half-life of NO from NO–prodrug and acid–NO
conjugates was determined in cell culture. MCF-7 cells were
seeded in 96-well plates 24 h before drug treatment (100
lL/well). On the following day, cells were treated with 20
lM of either the NO prodrug or the multiarm polymer based
NO prodrug conjugate, acid–NO. Fifty microliters of cell cul-
ture media were collected from each plate at 10 min, 2, 7,
22, 48, and 96 h, post treatment to determine the nitrite
content (N ¼ 5). The Griess reaction was performed accord-
ing to the manufacturer’s protocol. The absorbance was
measured at 535 nm using a UV microplate reader. The ni-
trite concentration and the corresponding nitrite oxide levels
were determined using the nitrite standard curve previously
generated.
arm-PEG branched polymeric nanoparticles,23 and
core–shell star polymer carrier with a poly(styrene) core.24
a
Characterization – Size Exclusion Chromatography
The molecular weights of the poly (tert-butyl acrylate) star
polymers and their corresponding acid star polymers were
TABLE 1 Molecular weights and PDIs of star polymers.
Cytotoxicity
Cell growth inhibition was determined in 96-well plates.
Plates were seeded with 3000 cells/well in 100 lL of media
(12 replicates/sample). Drug or conjugate solutions were
applied after 24 h. Resazurin blue in 10 lL of PBS was
applied to each well (final concentration 5 lM) after another
72 hours. After 4 h, the well fluorescence was measured
(ex/em 560/590) (SpectraMax Gemini, Molecular Devices),
and the IC50 concentration was determined as the midpoint
between drug-free medium (positive) and cell-free (negative)
controls.
Poly(tert-butyl acrylate)
Star Polymersa
Poly(acrylic Acid)
Star Polymersb
Mw,SEC
Mn,SEC
PDI
Mw,SEC
Mn,SEC
PDI
148,991
115,697
83,788
a
130,122
97,107
71,209
1.14
1.19
1.16
86,073
67,112
54,007
72,321
55,927
39,992
1.19
1.20
1.31
Polystyrene was used as a MW standard.
PEG was used as a MW standard.
b
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FIGURE 8 SEC traces of poly(tert-butyl acrylate) star polymers (Left panel, MWs: 148,991, 115,697, and 93,891 Da) and poly(acrylic
Acid) star polymers (Right panel, MW: 86,073, 67,112, and 54,007 Da).
determined by SEC using polystyrene and PEG as standards,
respectively (Fig. 8). The PDIs of poly (tert-butyl acrylate) star
polymer and acid star polymer with increasing molecular
weights were calculated and reported in Table 1. The PDIs
were similar to values reported by other groups using
MADIX/RAFT to form structured polymers, including four-arm
star polymers reported by Stenzel et al.19 (PDIs: 1.20–1.44).
The PDI was slightly higher than ideal in part due to using
linear PEGs and polystyrenes as molecular weight standards,
since standards with similar architecture were not available.
Also, the highly charged nature of the polymers may have led
to some interactions with the column stationary phase.
Characterization – Viscosity
The viscosity of acid star polymers slightly increased with
increases in either the molecular weight (54.0–110.7 kDa) or
the concentration (1–10 mg/mL) of the polymer (Fig. 10).
The viscosity of the acid–NO conjugates was also evaluated
and compared to the acid star polymer itself at three differ-
ent concentrations. The acid star polymer selected for the
drug conjugation had a molecular weight of 72.3 kD. Based
on our ongoing studies, star polymers with a molecular
weight close to 75 kD exhibit advantageous patterns of lym-
phatic drainage and retention, compared to star polymers of
higher or lower molecular weights, thus, the 72.3-kD star
polymer drug carrier may be a potential candidate for local-
ized drug delivery applications. It is worth noting that none
of the acid star polymers tested exhibited high viscosity com-
pared to other polymeric injectables for localized drug deliv-
ery, including hyaluronic acid, which demonstrated a three-
fold increase in viscosity relative to acid star polymers with
a similar molecular weight at 10 mg/mL (data not shown).
The FDA recommends that injectables have a viscosity of
less than 50 cP, which may be readily injected using a 25- or
27-ga needle. The viscosities of our materials were less than
2 cP, and they can be easily injected using a 31-ga needle.
Thus, these polymers are highly suited for a locally adminis-
tered chemotherapeutics due to their low viscosity at high
concentrations, which allows the use of small-bore needles
and low injection volumes.
Characterization – NMR
The molecular structures of the star polymers and NO pro-
1
drug were verified using H NMR, 13C NMR and HR-MS (Fig.
9). All data were consistent with the published results (pu-
rity >99%).
O2-(2,4-Dinitrophenyl) 1-{[4-(2-(2-hydroxyethoxy))ethyl]
piperazin-1-yl}diazen-1-ium-1,2-diolate
1H NMR (CDCl3, 400 MHz): d ¼ 1.69 (brs, 1H), 2.70 (t, J ¼
5.3 Hz, 2H), 2.80 (t, J ¼ 5.1 Hz, 4H), 3.63–3.66 (m, 2H),
3.69–3.75 (m, 8H), 7.68 (d, J ¼ 9.3 Hz, 1H). 8.47 (dd, J ¼
9.2, 2.7 Hz, 1H), 8.90 (d, J ¼ 2.7 Hz, 1H); 13C NMR (CDCl3,
100 MHz): d ¼ 50.3, 51.5, 57.0, 61.9, 68.0, 72.3, 117.6,
122.2, 129.1, 137.2, 142.3, 153.9. HRMS (ESI) Calculated for
C
14H21N6O8 (MþH)þ: 401.1421; Found: 401.1413.
Poly (tert-butyl acrylate) Star Polymer
In Vitro Release of Platinum from Acid–Pt
1H NMR (CDCl3, 400 MHz): d ¼ 4.65 (q, 8H), 4.25 (q, 4H),
4.03 (q, 8H), 2.25 (brs), 1.86 (brs), 1.65–1.27 (brs, overlap).
The release kinetics of cisplatin from the acid star polymer
backbone was determined in PBS (pH 7.4) at 37 ꢀC with or
without 10% serum. The half-life was determined by fitting
the release data to either a zero order linear regression
model (release of Pt in PBS without serum) or a first order
decay model (release of Pt in serum-containing PBS) using
GraphPad 5 (R2 > 0.97 for all fits). The acid–Pt conjugates
demonstrated an extended shelf-life in PBS with a platinum
release half-life of approximately 120 days [Fig. 11(A)],
which is significantly more stable than other sustained deliv-
ery platforms of cisplatin, including cisplatin-incorporating
polymeric micelles (release half-life: ca. 4 days),25 and dex-
Poly(acrylic acid) Star Polymer
1H NMR (CDCl3, 400 MHz): d ¼ 4.66 (q, 8H), 4.30 (q, 4H), 4.14 (q,
8H), 2.25 (brs), 1.86 (brs), 1.65–1.27 (brs, overlap), 1.30 (t, 12H).
Poly(acrylic acid) Star Polymer–NO Prodrug Conjugate
1H NMR (CDCl3, 400 MHz): d ¼ 8.90 (d, J ¼ 8.4 Hz, 1H), 8.57
(dd, J ¼ 8.4 Hz, 1H), 7.68 (d, J ¼ 13.2 Hz, 1H), 4.66 (q, 8H),
4.30 (q, 4H), 4.14 (q, 8H), 3.73 (m, 8H), 3.65 (m, 2H), 2.79 (m
4H), 2.70 (m, 2H), 2.73 (brs), 1.81–1.48 (brs, overlap).
Poly(acrylic acid) Star Polymer–Cisplatin Conjugate
1H NMR (CDCl3, 400MHz): d ¼ 4.68 (q, 8H), 4.38 (q, 4H),
2.55–2.37 (bs), 2.06–1.89 (bs), 1.84–1.66 (bs), 1.44 (t, 3H),
1.20 (d, 3H).
tran-based cisplatin conjugates (release half-life: ca.
2
days).26 The presence of serum expedited the drug release
from the acid polymers, which is likely due to the
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FIGURE 9 1H NMR spectra of poly (tert-butyl acrylate) star polymer (top panel, solvent: CDCl3), and poly(acrylic acid) star polymer
(bottom panel, solvent: MeOD).
competitive binding between the platinum and proteins pres-
ent in the serum. The conjugates were able to sustain the
release of cisplatin over 9 days (95% complete) with a
release half-life of approximately 36.7 h in serum-containing
PBS [Fig. 11(B)], suggesting satisfactory stability in plasma
in vivo. If this delivery platform could be translated into the
clinic, it may be used as an adjuvant or maintenance chemo-
therapy post-surgery, releasing a steady concentration of
platinum for an extended period of time, which may eradi-
cate the residual disease and potential nanometastases or
FIGURE 10 Viscosity measurements of (A) a poly(acrylic acid) star polymer at concentrations of 1, 3, and 10 mg/mL, and (B) a star
polymer NO prodrug conjugate (acid–NO, 72.3 kDa).
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FIGURE 11 Release of platinum from acid–Pt conjugates in (A) PBS, and (B) PBS with 10% serum at 37 ꢀC (N ¼ 3). The release half-
lives were determined to be 120 days and 36.7 h, using a linear regression or a first order decay model in GrapPad 5, respectively.
micrometastases in the surrounding tissues and draining
lymph nodes.
may be a potential candidate for the delivery of synergistic
cisplatin and NO combination chemotherapy.
Cytotoxicity of Acid–NO and Acid–Pt Conjugates
In Vitro Release of NO from Acid–NO
The in vitro antiproliferative activities of cisplatin, NO-donat-
ing prodrug, and their star polymer-based conjugates, acid–
Pt and acid–NO, were evaluated in cell culture using a
human breast cancer cell line, MCF-7 (Table 2). Acid–NO
conjugates demonstrated a similar IC50 to the free NO–pro-
drug. Release of NO from the polymeric matrix inhibited the
growth of cancer cells. The acid–Pt appeared to be less cyto-
toxic than cisplatin in the cells. This is likely due to the slow
release of the active drug, which was not unexpected for a
sustained-delivery platform. One of the obstacles that has
not yet been overcome in infusion chemotherapy is the poor
tumor penetration and accumulation of small-molecule anti-
cancer agents. Usually, the majority of the chemotherapeutics
have been cleared from systemic circulation, via the kidneys,
before a significant concentration of the anticancer agent is
achieved in the tumorigenic tissues. However, the acid–Pt
and acid–NO nanoconjugates may greatly enhance the intra-
tumoral drug concentration compared to the normal tissues
due to the enhanced permeability and retention (EPR) effect,
The release kinetics of NO from both the prodrug (NO–pro-
drug) and the carrier–NO prodrug conjugate (acid–NO) were
determined in cell culture media. The half-lives were deter-
mined be 7.4 and 5.3 h for NO–prodrug and acid–NO, respec-
tively, by fitting the release data to a first order decay model
using GraphPad 5 (R2 > 0.97 for all fits, Table 2). The acid–
NO exhibited a shorter release half-life compared to the NO–
prodrug, which is largely due to the initial burst release of
NO (Fig. 12). The poly(acrylic acid) star polymer backbone
created a more acidic microenvironment surrounding the
conjugates once they were solubilized in cell culture media,
which triggered the liberation of NO immediately. Without
the burst release, the acid–NO demonstrated a similar t1/2 as
the NO–prodrug. Another NO-donating prodrug in preclinical
studies, JS-K,11,27,28 has a NO release half-life of approxi-
mately 3.2 h (data not shown), which is shorter than the
release t1/2 of either the NO–prodrug or the acid–NO. All of
the aforementioned NO–prodrugs tested exhibited signifi-
cantly longer half-lives of NO release compared to the gase-
ous NO, which has a t1/2 of 0.05–0.18 ms in blood.29 Accord-
ing to Fetz et al.,30 pretreatment of head and neck cancer
cells using NO-donors, including S-nitroso-N-acetyl-penicill-
amine, reverted the cells’ cisplatin resistance via the modula-
tion of survivin. Thus, a drug delivery platform that gener-
ates fast-releasing NO, along with slow-releasing platinum,
TABLE 2 IC50s and release half-lives of acid-Pt and acid-NO
conjugates.
IC50 in MCF-7
Release
Drugs/Conjugates
Cells (lM)
Half-Life (h)
Cisplatin
Acid–Pt
NO–prodrug
Acid–NO
a
20 6 4
>100
–
36.7a
7.4b
5.3b
48 6 28
64 6 10
FIGURE 12 Release of NO from NO–prodrug and acid–Pt con-
jugates in cell culture media at 37 ꢀC (N ¼ 3). The half-lives
were determined be 7.4 and 5.3 h for NO–prodrug and acid–
NO, respectively, by fitting the release data to a first order
decay model using GraphPad 5 (R2 > 0.97 for all fits).
Release half-life was determined in PBS with 10% serum at 37 ꢀC.
Release half-lives were determined in cell culture media with MCF-7
b
cells.
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3 Chen, X.; Wu, W.; Guo, Z.; Xin, J.; Li, J. Biomaterials 2011,
32, 1759–1766.
in which nanoformulations tend to penetrate tumors more
effectively via leaky, fenestrated tumor blood vessels. In
addition, the acid–Pt and acid–NO formulations could be
given subcutaneously as a local injection adjacent to the tu-
mor to eradicate potential metastasis in the tumor-draining
lymph nodes; whereas, it is clinically infeasible to inject free
cisplatin or NO subcutaneously due to the severe tissue dam-
age that the anticancer agents would create. After local injec-
tion of the acid–Pt, or the acid–NO, the nanocarriers diffuse
into the surrounding cutaneous tissues and enter the lym-
phatic vessels filled with lymph fluid; subsequently, they fol-
low the lymph and reach the sentinel lymph node, the first
tumor draining lymph node, and deliver the chemotherapeu-
tics to the metastases. Furthermore, both of the star poly-
mer-based conjugates could be given simultaneously as a
localized combination therapy. NO has been shown to over-
come chemoresistance in a cisplatin-insensitive human head
and neck squamous cell carcinoma cell line.30 The concur-
rent administration of both the cisplatin- and NO-releasing
conjugates may minimize the incidence of acquired chemore-
4 Zhou, Y. M.; Ishikawa, A.; Okahashi, R.; Uchida, K.; Nemoto,
Y.; Nakayama, M.; Nakayama, Y. J. Control Release 2007, 123,
239–246.
5 Cho, H. Y.; Srinivasan, A.; Hong, J.; Hsu, E.; Liu, S.; Shrivats,
A.; Kwak, D.; Bohaty, A. K.; Paik, H. J.; Hollinger, J. O.; Matyjas-
zewski, K. Biomacromolecules 2011, 12, 3478–3486.
6 Cai, S.; Xie, Y.; Bagby, T. R.; Cohen, M. S.; Forrest, M. L. J.
Surg. Res. 2008, 147, 247–252.
7 Cai, S.; Xie, Y.; Davies, N. M.; Cohen, M. S.; Forrest, M. L. J.
Pharm. Sci. 2010, 99, 2664–2671.
8 Cohen, M. S.; Cai, S.; Xie, Y.; Forrest, M. L. Am. J. Surg.
2009, 198, 781–786.
9 Cai, S.; Xie, Y.; Davies, N. M.; Cohen, M. S.; Forrest, M. L.
Ther. Deliv. 2010, 1, 237–245.
10 Xie, Y.; Aillon, K. L.; Cai, S.; Christian, J. M.; Davies, N. M.;
Berkland, C. J.; Forrest, M. L. Int. J. Pharm. 2010, 392,
156–163.
11 McMurtry, V.; Saavedra, J. E.; Nieves-Alicea, R.; Simeone,
A. M.; Keefer, L. K.; Tari, A. M. Int. J. Oncol. 2011, 38,
963–971.
12 Riganti, C.; Miraglia, E.; Viarisio, D.; Costamagna, C.; Pes-
carmona, G.; Ghigo, D.; Bosia, A. Cancer Res. 2005, 65,
516–525.
sistance during therapy and maximize the efficacy of the
combination treatment. Similar to cisplatin, NO has also been
found to sensitize cancer cells to ionizing radiation,31 which
may be used as an adjuvant combination regimen post radia-
tion therapy. In addition, NO was recently found to increase
the antitumor activity of other chemotherapeutics, for exam-
ple, doxorubicin.32 This discovery can be further explored
using a combinational polymer-based, sustained-release dox-
orubicin (hyaluronan-doxorubicin)33 and NO (acid–NO) deliv-
ery platform that our laboratory developed for treating can-
cers that are responsive to doxorubicin therapy, including
certain types of breast, lung and ovarian cancers.
13 Maciag, A. E.; Chakrapani, H.; Saavedra, J. E.; Morris, N.
L.; Holland, R. J.; Kosak, K. M.; Shami, P. J.; Anderson, L.
M.; Keefer, L. K. J. Pharmacol. Exp. Ther. 2011, 336,
313–320.
14 Mijatovic, S.; Maksimovic-Ivanic, D.; Mojic, M.; Timotijevic,
G.; Miljkovic, D.; Mangano, K.; Donia, M.; Di Cataldo, A.; Al-
Abed, Y.; Cheng, K. F.; Stosic-Grujicic, S.; Nicoletti, F. J. Cell.
Physiol. 2011, 226, 1803–1812.
15 Hakim, T. S.; Sugimori, K.; Camporesi, E. M.; Anderson, G.
Physiol. Meas. 1996, 17, 267–277.
16 Momin, E. N.; Schwab, K. E.; Chaichana, K. L.; Miller-Lotan,
R.; Levy, A. P.; Tamargo, R. J. Neurosurgery 2009, 65, 937–945.
17 Li, Y.; PI., L. Mol. Pharm. 2010, 7, 254–266.
18 Jo, YS; van der Vlies, A. J.; Gantz, J; Thacher, T. N.; Antoni-
jevic, S.; Cavadini, S.; Demurtas, D,; Stergiopulos, N.; Hubbell,
J. A. J. Am. Chem. Soc. 2009, 131, 14413–14418.
CONCLUSION
In summary, we have successfully synthesized a multiarm
poly(acrylic acid) star polymer architecture suited for the mul-
timodal delivery of both hydrophilic and hydrophobic chemo-
therapeutics, as either a single-drug chemotherapy or a combi-
national regimen. This strategy has laid the foundation for
future investigations of the delivery of chemo-cocktails using
multiple anticancer agents that possess a synergism in vivo.
19 Stenzel, M. H.; Davis, T. P.; Barner-Kowollik, C. Chem. Com-
mun. (Camb) 2004, 1546–1547.
20 Ting, S. R.; Gregory, A. M.; Stenzel, M. H. Biomacromole-
cules 2009, 10, 342–352.
21 Chakrapani, H.; Kalathur, R. C.; Maciag, A. E.; Citro, M. L.;
Ji, X.; Keefer, L. K.; Saavedra, J. E. Bioorg. Med. Chem. 2008,
16, 9764–9771.
ACKNOWLEDGMENTS
22 Xue, Y.; Tang, X.; Huang, J.; Zhang, X.; Yu, J.; Zhang, Y.;
Gui, S. Colloids Surf. B Biointerfaces 2011, 85, 280–288.
This work was supported by awards from the National Insti-
tutes of Health K-INBRE (P20 RR016475), the American Cancer
Society (RSG-08-133-01-CDD), and the Department of Defense
Prostate CDMRP. SC acknowledges an Eli Lilly predoctoral fel-
lowship. SD and SC contributed equally to this work.
23 Ding, H.; Yong, K. T.; Roy, I.; Hu, R.; Wu, F.; Zhao, L.; Law,
W. C.; Zhao, W.; Ji, W.; Liu, L.; Bergey, E. J.; Prasad, P. N.
Nanotechnology 2011, 22, 165101.
24 Kowalczuk, A.; Stoyanova, E.; Mitova, V.; Shestakova, P.;
Momekov, G.; Momekova, D.; Koseva, N. Int. J. Pharm. 2011,
404, 220–230.
25 Uchino, H.; Matsumura, Y.; Negishi, T.; Koizumi, F.; Hayashi,
T.; Honda, T.; Nishiyama, N.; Kataoka, K.; Naito, S.; Kakizoe, T.
Br. J. Cancer 2005, 93, 678–687.
REFERENCES AND NOTES
1 Etrych, T.; Strohalm, J.; Chytil, P.; Rihova, B.; Ulbrich, K.
J Drug Target 2011, 19, 874–889.
26 Ichinose, K.; Tomiyama, N.; Nakashima, M.; Ohya, Y.; Ichi-
kawa, M.; Ouchi, T.; Kanematsu, T. Anticancer Drugs 2000, 11,
33–38.
2 Li, X.; Qian, Y.; Liu, T.; Hu, X.; Zhang, G.; You, Y.; Liu, S. Bio-
materials 2011, 32, 6595–6605.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 000, 000–000
9

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27 Shami, P. J.; Saavedra, J. E.; Bonifant, C. L.; Chu, J.; Udupi,
V.; Malaviya, S.; Carr, B. I.; Kar, S.; Wang, M.; Jia, L.; Ji, X.;
Keefer, L. K. J. Med. Chem. 2006, 49, 4356–4366.
W.; Stauber, R. H.; Knauer, S. K. Int. J. Cancer 2009, 124,
2033–2041.
31 Wang, X.; Zalcenstein, A.; Oren, M. Cell Death Differ. 2003,
10, 468–476.
28 Kiziltepe, T.; Hideshima, T.; Ishitsuka, K.; Ocio, E. M.; Raje, N.;
Catley, L.; Li, C. Q.; Trudel, L. J.; Yasui, H.; Vallet, S.; Kutok, J. L.;
Chauhan, D.; Mitsiades, C. S.; Saavedra, J. E.; Wogan, G. N.; Kee-
fer, L. K.; Shami, P. J.; Anderson, K. C. Blood 2007, 110, 709–718.
32 de Luca, A.; Moroni, N.; Serafino, A.; Primavera, A.; Pastore,
A.; Pedersen, J. Z.; Petruzzelli, R.; Farrace, M. G.; Pierimarchi,
P.; Moroni, G.; Federici, G.; Sinibaldi Vallebona, P.; Lo Bello, M.
Biochem. J. 2011, 440, 175–183.
29 Rassaf, T.; Preik, M.; Kleinbongard, P.; Lauer, T.; Heiss, C.; Stra-
uer, B. E.; Feelisch, M.; Kelm, M. J. Clin. Invest. 2002, 109, 1241–1248.
33 Cai, S.; Thati, S.; Bagby, T. R.; Diab, H. M.; Davies, N. M.;
Cohen, M. S.; Forrest, M. L. J. Control Release 2010, 146,
212–218.
30 Fetz, V.; Bier, C.; Habtemichael, N.; Schuon, R.; Schweitzer,
A.; Kunkel, M.; Engels, K.; Kovacs, A. F.; Schneider, S.; Mann,
10
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