Stabilization of Resveratrol in Blood Circulation by Conjugation to mPEG and mPEG-PLA Polymers: Investigation of Conjugate Linker and Polymer Composition on Stability, Metabolism, Antioxidant Activity and Pharmacokinetic Profile

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Resveratrol is naturally occurring phytochemical with diverse biological activities such as chemoprevention, anti-inflammatory, anti-cancer, anti-oxidant. But undergoes rapid metabolism in the body (half life 0.13h). Hence Polymer conjugation utilizing different chemical linkers and polymer compositions was investigated for enhanced pharmacokinetic profile of resveratrol. Ester conjugates such as α-methoxy-ω-carboxylic acid poly(ethylene glycol) succinylamide resveratrol (MeO-PEGN-Succ-RSV) (2 and 20 kDa); MeO-PEG succinyl ester resveratrol (MeO-PEGO-Succ-RSV) (2 kDa); α-methoxy poly(ethylene glycol)-co-polylactide succinyl ester resveratrol (MeO-PEG-PLAO-Succ-RSV) (2 and 6.6kDa) were prepared by carbodiimide coupling reactions. Resveratrol-PEG ethers (2 and 5 kDa) were synthesized by alkali-mediated etherification. All polymer conjugates were fully characterized in vitro and the pharmacokinetic profile of selected conjugates was characterized in rats. Buffer and plasma stability of conjugates was dependent on polymer hydrophobicity, aggregation behavior and PEG corona, with MeO-PEG-PLAO-Succ-RSV (2 kDa) showing a 3h half-life in rat plasma in vitro. Polymer conjugates irrespective of linker chemistry protected resveratrol against metabolism in vitro. MeO-PEG-PLAO-Succ-RSV (2 kDa), Resveratrol-PEG ether (2 and 5 kDa) displayed improved pharmacokinetic profiles with significantly higher plasma area under curve (AUC), slower clearance and smaller volume of distribution, compared to resveratrol.

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Citation: Siddalingappa B, Benson HAE, Brown DH,
Batty KT, Chen Y (2015) Stabilization of Resveratrol
in Blood Circulation by Conjugation to mPEG and
mPEG-PLA Polymers: Investigation of Conjugate
Linker and Polymer Composition on Stability,
Metabolism, Antioxidant Activity and Pharmacokinetic
Profile. PLoS ONE 10(3): e0118824. doi:10.1371/
journal.pone.0118824

Academic Editor: Dominique Delmas, UMR
INSERM U866, FRANCE

Received: September 9, 2014

Accepted: January 22, 2015

Published: March 23, 2015

Copyright: © 2015 Siddalingappa et al. This is an
open access article distributed under the terms of the
[Creative Commons Attribution License, which permits](http://creativecommons.org/licenses/by/4.0/)
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.

Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.

Funding: Curtin University contributed research
space and equipment, and scholarship funds. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.

Competing Interests: The authors have declared
that no competing interests exist.

RESEARCH ARTICLE
# Stabilization of Resveratrol in Blood Circulation by Conjugation to mPEG and mPEG-PLA Polymers: Investigation of Conjugate Linker and Polymer Composition on Stability, Metabolism, Antioxidant Activity and Pharmacokinetic Profile

Basavaraj Siddalingappa 1¤ *, Heather A. E. Benson 1, David H. Brown 2, Kevin T. Batty 1,
Yan Chen 1

1 School of Pharmacy, CHIRI Biosciences, Curtin University, Perth, Western Australia, 2 Department of
Chemistry, Curtin University, Perth, Western Australia

¤ Current address: Graduate College of Biomedical Sciences, Western University of Health Sciences,
Pomona, CA, United States of America

- bsiddalingappa@westernu.edu

## Abstract

Resveratrol is naturally occurring phytochemical with diverse biological activities such as

chemoprevention, anti-inflammatory, anti-cancer, anti-oxidant. But undergoes rapid metab
olism in the body (half life 0.13h). Hence Polymer conjugation utilizing different chemical

linkers and polymer compositions was investigated for enhanced pharmacokinetic profile of
resveratrol. Ester conjugates such as α-methoxy-ω-carboxylic acid poly(ethylene glycol)

succinylamide resveratrol (MeO-PEGN-Succ-RSV) (2 and 20 kDa); MeO-PEG succinyl
ester resveratrol (MeO-PEGO-Succ-RSV) (2 kDa); α-methoxy poly(ethylene glycol)-co
polylactide succinyl ester resveratrol (MeO-PEG-PLAO-Succ-RSV) (2 and 6.6kDa) were

prepared by carbodiimide coupling reactions. Resveratrol-PEG ethers (2 and 5 kDa) were

synthesized by alkali-mediated etherification. All polymer conjugates were fully character
ized in vitro and the pharmacokinetic profile of selected conjugates was characterized in

rats. Buffer and plasma stability of conjugates was dependent on polymer hydrophobicity,

aggregation behavior and PEG corona, with MeO-PEG-PLAO-Succ-RSV (2 kDa) showing

a 3h half-life in rat plasma in vitro. Polymer conjugates irrespective of linker chemistry pro
tected resveratrol against metabolism in vitro. MeO-PEG-PLAO-Succ-RSV (2 kDa), Res
veratrol-PEG ether (2 and 5 kDa) displayed improved pharmacokinetic profiles with

significantly higher plasma area under curve (AUC), slower clearance and smaller volume

of distribution, compared to resveratrol.

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 1 / 22

Stabilization of Resveratrol by Polymer Conjugation

Introduction

Resveratrol is a naturally occurring polyphenolic phytochemical with beneficial biological activities such as anti-inflammatory, anticancer, antioxidant, and cardio protection [1–3]. Diverse biological activities of resveratrol are due to its intervention in multiple inflammatory
pathways [2]. More recently, resveratrol has been confirmed to be an allosteric activator of
SIRT1 [4], an enzyme that belongs to the sirtuin family of nicotinamide adenine dinucleotide
(NAD+)-dependent deacetylases. SIRTI1 is known to be involved in many fundamental cellular processes including gene silencing, DNA repair, and metabolic regulation, which are all related to aging. Price and his colleagues [4] showed that activation of SIRT1 by resveratrol
increased mitochondrial biogenesis and improved mitochondrial function. Hence resveratrol is
also considered to be a potential therapeutic candidate for treating age-related disorders and
increasing lifespan of individuals. It is not surprising that resveratrol is widely used as a nutritional supplement for various health benefits. However the health benefits of resveratrol are
equivocal because of its poor pharmacokinetic profile [5–7].
The pharmacokinetic profile of resveratrol after oral administration in humans was described
as high absorption but very low bioavailability due to extensive first-pass metabolism and short
elimination half-life [8]. This rapid metabolism pattern has been seen in other species including mouse, rat, pig and dog [9–12]. After intravenous administration to rats, the elimination
half-life of resveratrol (plasma) was 0.13 h [6]. Since resveratrol is polyphenolic in nature, hydroxyl groups of resveratrol were found to be targets for metabolism via conjugation and accordingly various conjugated sulfate and glucuronide metabolites of resveratrol were detected
in the plasma; however, resveratrol-3-O-sulfate and resveratrol-3-O-glucuronide were found
to be the major metabolites [13]. Although the understanding of biological activities of these
metabolites remains elusive, the shorter plasma residence of resveratrol has been reported to be
inadequate to elicit biological activities in vivo [14].
Resveratrol was found to be effective in certain in vivo animal models where the native un-metabolized resveratrol is accessible at the site of action. Resveratrol has shown a significant chemopreventive effect in azoxymethane induced carcinogenesis in a rat colon cancer model when
given orally in drinking water [14]. Similarly resveratrol was also found to be an effective chemopreventive agent in a mouse skin cancer model with 7, 12-dimethylbenzanthracene
(DMBA) as an initiator, and 12-O-tetradecanoylphorbol-13-acetate [TPA] as tumour promoter. The number of tumours per mouse was significantly reduced compared to controls [15].
Such effects were attributed to the presence of the native resveratrol at the site of action, as the
resveratrol did not pass through the systemic circulation to reach the site of action. However
there was a reported discrepancy between in vitro and in vivo anti-leukemic effects of resveratrol. Despite showing a strong anti-leukaemic effect in the in vitro studies, resveratrol failed to
show a promising anti-leukaemic effect in vivo and did not protect mice injected with leukaemia cells [16]. The inability of resveratrol to arrest leukaemia cell differentiation in the mice
was attributed to its rapid metabolism, and was in stark contrast to in vitro observations. In a
study conducted by Wenzel et al. to evaluate the chemopreventive and antioxidant activities of
resveratrol, the researchers concluded that chemopreventive parameters such as levels of certain phase-I and phase-II metabolizing enzymes, and total antioxidant activity were unaltered
in rats after 8 weeks of resveratrol treatment at various doses given with food (particularly at
300 mg high dose) [7]. The lack of native resveratrol in the body was considered to be responsible for the inactivity of reseveratrol.
Polymer conjugation is a strategy used to improve the circulation time of peptides/proteins
and small drug molecules. Studies have shown that conjugation with polymers significantly reduced the elimination rate of peptides/proteins by proteolytic degradation, as well as

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 2 / 22

Stabilization of Resveratrol by Polymer Conjugation

immunogenic and antigenic responses, thereby prolonging circulation times [17–18]. Polymer
conjugation approaches have successfully improved safety, efficacy and pharmacokinetic profiles of small molecule drugs such as doxorubicin, norfloxacine, campothecine and paclitaxel

[17–23]. However most of these drugs are metabolically stable and the feasibility of employing
polymer conjugation to protect small molecules such as resveratrol against metabolism needs
to be fully explored. Structure-activity relationship studies of resveratrol revealed that hydroxyl
groups in the structure are essential for its biological activity [24–25]. Hence resveratrol delivery approaches based on polymeric conjugation should not permanently block hydroxyl
groups and, even if permanent conjugation is considered, only selected hydroxyl groups should
be modified with appropriate polymers and with minimum impact on the biological activity
of resveratrol.
The major limitation in developing polymeric conjugates of small molecules is the absence of
functional groups amenable for conjugation and the reported poor stability of conjugates in
plasma due to their hydrolysis by plasma esterases and carboxypeptidases. This is also a major
reason that polymeric conjugates were largely exploited as prodrugs for improving solubility of
small molecules such as paclitaxel [23].
The objective of our investigation was to evaluate various polymer conjugation technologies
for stabilizing resveratrol against metabolism and consequent impact on resveratrol’s pharmacokinetics profile and in turn its biological effects in vivo. Resveratrol polymer conjugates were
prepared using ester and ether linker chemistry with different molecular weight of polymers.
In this study, efforts were made to understand the effect of the physicochemical properties of
polymers and chemistry of linkers on the stability of conjugates in buffer and plasma. Finally
the selected polymer conjugates were also evaluated for in vitro antioxidant activity, metabolism and pharmacokinetic profile in Wistar rats.

Materials and Methods

Materials

trans-Resveratrol (99%: DND Pharma-Tech Co., Inc, Shanghai, China) was used throughout the
study as this isomer was reported be more active than the cis-isomer. α-Methoxy-ω-carboxylic
acid poly(ethylene glycol) succinylamide acid (MeO-PEGN-SuccOH) of molecular weight
(MW) 2 kDa and 20 kDa, methoxypoly(ethylene glycol)bromide (MeO-PEG-CH2CH2Br), 2
kDa and 5 kDa were obtained from IRIS Biotech GmbH (Germany). Methoxypoly(ethylene glycol) (MeO-PEG-OH, MW 2 kDa), methoxypoly(ethylene glycol)-co-polylactide (MeO-PEGPLA-OH, Mw 2 kDa) and succinic anhydride were purchased from Sigma-Aldrich Ltd. (Sydney,
Australia). MeO-PEG-PLA-OH 6.6KDa was purchased from Akina Inc (Indiana, USA). N,
N'-Diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC.HCl) were obtained from GL Biochem (Shanghai, China). Rat liver
microsomes (20mg/mL protein) were obtained from Invitrogen (Mulgrave, Australia). UDPglucuronosyltransferases (UGT A) and UGT B solutions were purchased from BD Bioscience
(San Jose, CA, USA). Dimethyl yellow was obtained from Sigma. 0.1 M acetate buffer pH 4.5
and 0.1 M phosphate buffer pH 7.4 were prepared in-house. All remaining chemicals such as potassium carbonate, dichloromethane (DCM), pyridine, toluene and diethyl ether were of analytical grade and used without any further purification. Male Wistar rats (180–220g) and rat plasma
were obtained from the Animal Resource Centre, Murdoch, Western Australia. The animal
studies were approved by the Animal Ethics Committee of Curtin University and were performed according to the Australian Code of Practice for the care and use of animals for
scientific purposes.

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Stabilization of Resveratrol by Polymer Conjugation

Synthesis of functionalized polymer and Resveratrol-Polymer conjugates.
Synthesis of MeO-PEGO-SuccOH. MeO-PEG-OH (2 kDa, 5g, ca 2.5 mmol) was dissolved
in toluene (50 mL) and succinic anhydride (1.2g, 12 mmol) was added to the solution. The resulting solution was heated at 90°C for 4 h. The solution was concentrated to 10 mL in vacuo
then cooled to room temperature. Diethyl ether (150 mL) was added to precipitate the MeOPEGO-SuccOH. The precipitate was collected by filtration and air-dried.

Synthesis of MeO-PEG-PLAO-SuccOH. MeO-PEG-PLA-OH, (Mw 6.6kDa, 1 g, ca 0.15
mmol) was dissolved in toluene (10 mL) and an excess of succinic anhydride (0.2 g, 2 mmol)
was added to mixture. The reaction mixture was refluxed at 90°C for 4 hours in a round bottom
flask fitted with a condenser. At the end of the reaction, the mixture was cooled to room temperature and diethyl ether (100 mL) was added to precipitate the MeO-PEG-PLAO-SuccOH.
The precipitate was isolated by filtration and characterized by NMR. A similar process was
used to prepare MeO-PEG-PLAO-SuccOH Mw 2 kDa.

Synthesis of Resveratrol-PEG conjugates. DIC (0.1 g, 0.79 mmol) was added to a solution
of MeO-PEGN-SuccOH (Mw 2 kDa, 1 g, ca. 0.5 mmol) and excess resveratrol (0.25 g, 1.09
mmol) in pyridine (2 mL) and the resulting solution was stirred in darkness, at room temperature for 24 h. The solution was filtered (0.44 μm filter) and the filtrate was diluted with diethyl
ether (150 mL). The resulting precipitate was then washed with a solution of ethyl acetate/
diethyl ether (20 mL/80 mL), followed by a solution of ethyl acetate/diethyl ether (50 mL/50
mL), and then dried in vacuo to afford an off-white material (300 mg, 30% yield). A similar
procedure was followed for preparation of resveratrol conjugates with MeO-PEGN-SuccOH 20
kDa and MeO-PEGO-SuccOH 2 kDa.

Synthesis of Resveratrol-PLA-PEG conjugates. MeO-PEG-PLAO-SuccOH, (Mw 6.6kDa,
1 g, ca 0.15 mmol) was dissolved in pyridine (5.0 mL). The excess of resveratrol (0.15g, 0.65
mmol) and EDC.HCl (0.15 g, 0.78 mmol) were added to the mixture. The reaction mixture was
stirred at room temperature for 48 hours under dark conditions. At the end of the reaction, the
mixture was added to diethyl ether (150 mL) and the product was isolated by filtration. The
product was dissolved in DCM (50 mL) and was extracted with 0.1 M HCl (50 mL) twice to remove any unreacted EDC.HCl and its urea by-product. Finally DCM layer was evaporated
in vacuo to obtain an off-white powder (500 mg, 50% yield). The MeO-PEG-PLAO-SuccResveratrol, Mw 2 kDa was also prepared with the same procedure.

Synthesis of Resveratrol-PEG ethers. MeO-PEG-CH2-CH2-Br, (Mw 2 kDa, 1 g, 0.5 mmol)
and resveratrol (0.15 g, 0.657 mmol) were dissolved in of DMF (5 mL). K2CO3 (0.25 g, 1.81
mmol) was added to solution and mixture was stirred at room temperature for 24 hours in
dark. At the end of the reaction, the mixture was added to DCM (50 mL), solution was filtered
to remove potassium bromide produced in the reaction and also unreacted K2CO3. The filtrate
was extracted with 0.1 M HCl solution (50 mL) to remove traces of KBr and K2CO3. The extraction process was repeated two more times and DCM layer was evaporated in vacuo to yield
an off white to slightly brown product (500 mg, 50%). The resveratrol-PEG ether with MeOPEG-Br Mw 5 kDa was also prepared with the same procedure.

Characterization of resveratrol-polymer conjugates.
Characterization of polymer conjugates and their precursors by 1 H NMR. Dried powder
samples of reaction products obtained from Section 2.2 to 2.6 were analysed by 1 H NMR to elucidate the chemical structures of compounds/mixtures. All 1 H NMR analyses were performed
on a Bruker AVN400 spectrometer (400.1 MHz for 1 H) with spectra referenced to
solvent signals.

Characterization of Polymer conjugates by LC-MS. The LC-MS analysis of polymer conjugates was performed to confirm formation of conjugates, extent of drug conjugation to polymer and extent of polymer functionalization. All the LC-MS experiments were performed on a

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Stabilization of Resveratrol by Polymer Conjugation

Shimadzu LC-MS 2020 system (Kyoto, Japan) as per our previously published method [26].
The system consists of a binary pump 20AD, auto injector SIL 20 AC HT, column oven CTO
20A, PDA detector SPD M 20A, mass analyser MS2020 and communication unit CBM 20A.
Data were acquired, processed and analysed using chromatographic software LABSOLUTIONS. Chromatographic elution was performed using a linear gradient with water and acetonitrile as the mobile phase system. The percentage of acetonitrile in the mobile phase was: at
1 min 40%, 4 min 95% and 8 min 40%. A Zorbax SBC-18 column was employed for separation
at column oven temperature of 30°C. The mass detector parameters were set as follows: DL
temperature 300°C, interface temperature 350°C and the heating block temperature was maintained at 220°C, with drying gas nitrogen flow at 10 L/min. The selected ion monitoring (SIM)
was carried out with positive ions 700, 720, 730, 900, 1000 for PEG fragments and 227 for
resveratrol [26].

Polymeric micelles preparation and characterization. A solution of 40 mg of MeO-PEGPLAO-Succ-RSV conjugate in acetonitrile (2 mL) was prepared. The stock solution was diluted
with water (4 mL). The acetonitrile was removed by bubbling inert nitrogen gas at room temperature in the dark. The final volume of micellar/nanoparticle dispersion was adjusted to the
required volume with water. For pharmacokinetic studies, final concentration of conjugate in
micelle dispersion was 10 mg/mL and for critical micellear concentration measurement, the
dispersion was diluted accordingly to obtain a series concentration of conjugate in solution
below and above the CMC. The micelles of MeO-PEGO-Succ-RSV were formed spontaneously
after dispersing in water.

Measurement of micelles size. Particle size and size distribution of the resveratrol polymer
conjugate micelles were measured by a Zetasizer (3000HS, Malvern Instruments, UK), using a
quartz cell with the automatic mode at 25°C and a detection angle of 90°. Mean diameters (Zaverage) were obtained after 10 repeated measurements.

Determination of critical micellar concentration (CMC) values for resveratrol polymer
conjugates. CMCs for various micelles-forming polymer conjugates of resveratrol were determined by dye solubilization technique using dimethyl yellow as UV probe (27). Both absorbances at fixed wavelength and shift in absorbance maxima of dimethyl yellow were monitored
as the function of concentration of polymer conjugate. A concentrated solution of dimethyl
yellow (10 mg/mL) was prepared in acetone. The initial concentrated MeO-PEG-PLAO-SuccRSV micelle solutions were prepared by diluting 125 μL of polymer conjugate stock solution
(20 mg/mL) in acetonitrile to 100 mL with water as per previous section. After removing acetonitrile, this solution was further diluted with water to obtain a series of solutions with various
concentrations of polymer conjugates (1–25 mg/L). The MeO-PEGO-Succ-RSV solutions were
prepared by dissolving 40 mg of conjugate in 10 mL of watewith further serial dilution to produce solutions of concentrations from 0.005 to 0.4% (50 to 4000 mg/L). These polymer conjugate solutions were added separately to vials containing dimethyl yellow and sonicated for 10
minutes. The solutions were stored in the dark for 3 h to equilibrate. These solutions were then
scanned for max using a UV-Visible spectrophotometer between 300 and 510 nm. The change
in wavelength of maximum absorption and absorbance at fixed wavelength 441nm were noted
against polymer conjugate concentration. The CMC values were determined from a plot of the
log of concentration (log C) of polymer conjugates versus max (or absorbance at 441 nm).

Micelle morphology by atomic force microscopy (AFM) and field emission scanning
electron microscopy (FESEM). Atomic Force Microscopy was used to visualise the conjugate
samples. All measurements were taken in Tapping Mode operation with a Digital Instruments
Dimension 3000 SPM system (Veeco, Santa Barbara, USA). Standard silicon Tapping Mode
probes type NCH (NanoWorld, Switzerland) with resonant frequency 330 kHz, spring constant 42 N/m and tip radius < 8 nm. The AFM samples were prepared by drop casting the

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Stabilization of Resveratrol by Polymer Conjugation

micelle solution onto a freshly cleaved mica substrate. The sample was placed in a desiccator attached to a vacuum pump to remove excess solvent.
Morphological evaluation of micelles was performed using FESEM instrument, Zeiss Neon 40
ESB (Oberkocken, Germany). For FESEM, one drop of micelle solution was mounted on a carbon-coated stub. Excess sample was removed carefully and the stud was dried in air. A Polaron
E5100 sputter-coater was used to sputter coat the samples with chromium. Samples were then
examined under the FESEM instrument with an accelerating voltage of 1 kV.

Stability of resveratrol-polymer conjugates in physiological relevant buffers. The stability of resveratrol polymer conjugate was evaluated at different pH buffers 4.5 (acetate buffer
0.1M) and 7.4 (phosphate buffer 0.1M) to mimic lysozomal and blood pH. The resveratrol
polymer conjugates of different Mw and linkers were incubated in the two buffers at a concentration of 10 mg/mL at 37°C, the samples were withdrawn at 0, 1, 2, 4, 6 and 24 hours and were
diluted with water/acetonitrile (50:50) and were analysed by HPLC [28] using an Agilent Technologies chromatographic system 1100 series (Santa Clara, CA, USA) with dual fluorescence
and PDA detectors. Briefly, reversed phase chromatographic separation was employed for
analysis with xxx column. The HPLC gradient system consisted of solvent A, buffer pH 7.0
with triethyl amine-formic acid (0.05 and 0.2% v/v in water) and solvent B, methanol. A 30
min linear gradient was programmed as follows: 0 min 5% B, 4 min 20% B, 7 min 20% B, 16
min 55% B, 18 min 55% B, 18.5 min 95% B, 23 min 95% B, 24 min 5% B. The flow rate was
maintained at 1 mL/min and the injection volume was 50 μL. The HPLC analysis was performed at ambient temperature. Altech Apollo C18 150 X 4.6 mm, 5μ column was employed
for seperation. This HPLC method was validated to precisely separate and quantify the transresveratrol in the analyte mixture. There was at least two min difference in retention times for
the trans and cis isomers of resveratrol. The UV spectrum of the trans isomer is very different
from the cis isomer, thus any change to the trans isomer could have been detected. Although
quantification was performed using the fluorescence detector at an excitation wavelength of
320nm and emission at 400 nm, simultaneous UV chromatograms were also recorded for specificity and peak purity purposes.
A similar procedure was used to study the stability of MeO-PEG-PLAO-Succ-RSV in buffers;
micelles were prepared as per section 2.8 and diluted 1:5 with buffer solutions and incubated at
37°C. All samples were protected against light during the experiments.
Stability of resveratrol-polymer conjugates in rat plasma. The stability of various resveratrol polymer conjugates in rat plasma was tested in order to assess the stability of the ester linkages towards plasma esterases. The MeO-PEGN-Succ-RSV, 2 kDa, 20 kDa and MeO-PEGOSucc-RSV 2 kDa (MW quoted in the paper is the size of polymer only. Mw for RSV is 228 Da)
at the 10 mg/mL were incubated in plasma at 37°C. The micelles of MEO-PEG-PLAO-SuccRSV 2 kDa and 6.6 kDa were prepared and, finally the micelles were mixed with plasma and
incubated at 37°C. Aliquots of plasma (0.1 mL) were withdrawn at regular intervals and mixed
with acetonitrile (0.9 mL). The mixtures were centrifuged at 10,000 rpm for 5 minutes. The supernatant (0.25 mL) was diluted with water (0.25 mL) and analysed by HPLC [28]. All samples
were protected against light during the experiments.

In vitro preliminary metabolism studies of resveratrol and its polymer conjugates in rat
liver microsomes. Resveratrol has been reported to undergo only phase II metabolism, such as
glucuronidation and sulfation (6–8), therefore the metabolism studies with rat liver microsomes were conducted in the presence of uridine diphosphoglucuronic acid (UDPA). The
stock solutions of resveratrol (0.13 mM) and polymer conjugates (0.13 mM resveratrol equivalents) were prepared in 10% v/v DMSO in water. The rat liver microsomes, (UDP-glucuronosyltransferases (UGT) solution A (contains UDPA) and UGT solution B were thawed at 37°C.
The 200 μl of UGT solution B, 80 μl of UGT solution A and 660 μl of water were mixed, and 10

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Stabilization of Resveratrol by Polymer Conjugation

μl of the test substance stock solution (resveratrol or conjugates) was added to the mixture. The
mixture was kept at 37°C. The reaction was initiated by adding 50 μl of microsomes (1mg protein). Samples (250μl) from reaction mixtures were withdrawn at 0, 10, 20, 30, 40 and 60 min
and mixed with equal volumes of cold acetonitrile. The mixtures were centrifuged at 7000 × g
for 10 min and supernatants were collected and analyzed by HPLC. Incubations of test substances in reaction mixtures without microsomes were used as a control. All samples were protected against light during the experiments.

In vitro antioxidant assay of resveratrol and its polymer conjugates. The free radical
scavenging effects of resveratrol and its polymer conjugates were evaluated using 1,1-diphenyl2-picrylhydrazyl (DPPH) inhibition assay. Both DPPH solution (2 x 10 -4 M) and a series of solutions of resveratrol and resveratrol-PEG ester conjugates were prepared in methanol. The
DPPH solution of 1mL was mixed with 1 mL of resveratrol solution. Final concentration of
DPPH was maintained at 1 x 10 -4 M. The procedure was repeated for other resveratrol solutions
of various concentrations and also for resveratrol-PEG ester solutions. The concentration of
resveratrol in testing solutions ranged from 21–877 μMol/L and 210 to 8770 μMol/L for polymer conjugates. After 30 min incubation at room temperature, the absorbance of these solutions was determined at 518 nm using a Hewlett Packard 8452A, UV-VIS spectrophotometer
(Waldbronn, Germany). The DPPH concentration remaining in solution was calculated by
comparing with control, a standard solution of DPPH without any resveratrol or its conjugate.
The IC 50 values were determined from the plot of % DPPH remaining against concentration
of RSV and its equivalent conjugates.

Preliminary pharmacokinetic study of resveratrol and its polymer conjugates in Wistar
rats with IV injection. Pharmacokinetic studies were conducted with male Wistar rats. Rats
were acclimatized and housed at 22°C under 12h light/dark cycle with free access to sterilized
commercial food pellets and water in the animal research facility (Curtin University) for at
least 3 days prior to experiments. All animal experiments were approved by the Animal Ethics
Committee of Curtin University (Approval no: R37–10 Amendment Approval No: A47–10
and A56–10) and were performed according to the Australian Code of Practice for the
care and use of animals for scientific purposes. In order to minimize pain and suffering, the
dosing and bleeding procedures were conducted under anesthesia (isoflurane at 5% for the induction and 2% for the maintenance, in combination with 1.5% oxygen). At the end of the
study, rats were scarified by anesthetic euthanasia using 5% isofluorane with 1.5% oxygen.
Each treatment group was assigned 5–6 rats and received one of the following treatments: resveratrol solution, MeO-PEG-PLA-Succ-RSV 2 kDa and 6.6 KDa, RSV-PEG ethers 2 kDa and
5 kDa via intravenous injection into the tail vein. A resveratrol solution (6 mg/mL), prepared
in 20% hydroxyl-propyl-β-cyclodextrin, was dosed to Wistar rats at 10 mg/kg body weight.
Polymeric micelles of the MeO-PEG-PLA-Succ-RSV 2 kDa and 6.6 kDa at a concentration of
10 mg/mL of conjugate were prepared as per section 2.8 and dosed to rats at a dose of 2 mg/kg
of resveratrol equivalent. The 20 mg/mL solution of RSV-PEG ethers 2 kDa and 5 kDa was prepared by dissolving ether conjugates in water and dosed at 2 mg/kg of resveratrol equivalent.
Blood (300 μL) was collected from the tail vein at 0.08, 0.5, 1, 2 and 4 hours into EDTA coated
tubes. The blood samples were immediately stored in ice and protected from light until centrifuged (12000 × g at 4°C for 10 min). The separated plasma (100 μL) was mixed with an equal
volume of acetonitrile and centrifuged (12000 × g at 4°C for 12 min). The supernatant was collected and the concentrations of resveratrol and resveratrol equivalent (polymer conjugates)
were determined using a validated HPLC method [28]. Metabolites were identified using
LC-MS/MS (Applied Biosystems, 4000 QTRAP, Toronto, Canada) [26].

Statistical and pharmacokinetic analyses. Data analysis and representation (mean ± SD)
were performed with SigmaPlot version 12 (Systat Software, Inc., Chicago, IL).

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Stabilization of Resveratrol by Polymer Conjugation

Pharmacokinetic analysis was performed using Kinetica Version 5.0 (Thermo Fisher Scientific,
Inc., Waltham, MA). Non compartmental analysis of the plasma concentration-time data was
used to estimate the area under the curve (AUC0-1; log-linear trapezoidal method), elimination half-life (t½), apparent clearance (CL; Dose�AUC0-1), mean residence time (MRT) and
volume of distribution (Vss; CL×MRT). Statistical analysis of pharmacokinetic parameters was
performed using unpaired t-test with Graphpad Prism software (version 6.05). Pharmacokinetic parameters obtained for each group were analyzed against the resveratrol group and p values
were reported.

Results and Discussion

Synthesis of succinic acid terminated polymers for resveratrol
conjugation

The esterification of MeO-PEG-OH and MeO-PEG-PLA-OH with succinic anhydride was
chosen for coupling of polymers to hydroxyl groups of resveratrol. The esterification was
achieved by ring opening reaction mediated by heat in an inert solvent toluene. This was a
modified method adopted from Kuang et al [29]. The reaction worked well for both MeOPEG-OH and MeO-PEG-PLA-OH irrespective of their polymer chain length and composition.
The success of esterification was confirmed by the presence of two triplets at 2.3 and 2.4 ppm
in NMR, which are characteristic triplet peaks of ethylene protons of succinic acid. In addition,
the esterification of MeO-PEG-OH was supported by LC-MS analysis [26] as there was absence
of MeO-PEG-OH peak in LC-MS chromatogram but MeO-PEGO-SuccOH peak was detected,
indicating 100% conversion of MeO-PEG-OH to MeO-PEGO-SuccOH.

Synthesis and characterization of resveratrol-Polymer conjugates

Resveratrol-PEG conjugates were synthesized by carbodiimide coupling, using a modified
method reported by Greenwald et al. for the PEGylation of 6-hydroxyquinoline [30]. The reaction was conducted with excess resveratrol and MeO-PEGN-SuccOH (2 kDa) in the presence
of diisopropylcarbodiimide and pyridine, which afforded a mixture of resveratrol-PEG conjugates (Fig. 1A), with a final yield of 30%. The low yield was due to the loss of compound(s) during repeated washing with diethyl ether/dichloromethane to remove un-reacted resveratrol
from the mixture. The NMR analysis of the product (s) suggested that the obtained mixture included two mono-PEGylated resveratol conjugates 4'-MeO-PEGN-Succ-RSV (ca. 50%) and
3-MeO-PEGN-Succ-RSV (ca. 35%) as the major products, and what we believe is a disubstituted derivative 3,4'-di(MeO-PEGN-Succ)-RSV as a minor product. The identification of the
mixture, and the substitution pattern, was based on the 1 H NMR spectra obtained for the
resveratrol-PEG conjugate mixture (see Fig. 2 and Figs. A and B in S1 File). The 1 H NMR spectra revealed that the conjugate mixture contained no free resveratrol, as indicated by the absence of a signal at ca. 6.1 ppm in Fig. 2(a). This signal, observed in Fig. 2(b), is due to H-4 of
free resveratrol (see Fig. B in S1 File for assigned spectra). As a control, a solution of resveratrol
and MeO-PEGN-SuccOH in d6-DMSO was analysed by 1 H NMR spectroscopy. The chemical
shifts of the signals for resveratrol in the mixture were identical to that shown in Fig. 2(b) for
resveratrol in d6-DMSO. The 1 H NMR spectra for the resveratrol-PEG conjugate mixture
also indicated that no free MeO-PEGN-SuccOH remained in the material. The signals corresponding to the succinyl moiety in MeO-PEGN-SuccOH were absent in the spectra for the
resveratrol-PEG conjugate mixture (see Fig. A in S1 File). The assignment of signals corresponding to 3-MeO-PEGN-Succ-RSV and 4'-MeO-PEGN-Succ-RSV in Fig. B in S1 File was
based on a comparison with the spectra for resveratrol and 2D NMR spectra (COSY).

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Stabilization of Resveratrol by Polymer Conjugation

Fig 1. Synthesis of (A) resveratrol-PEG ester conjugates, (B) MeO-PEG-PLAO-Succ-RSV conjugates
and (C) resveratrol-PEG ether conjugates.

doi:10.1371/journal.pone.0118824.g001

Additional minor signals remain unassigned in Fig. B in S1 File. We believe these signals are
due to a third product, 3,4'-di(MeO-PEGN-Succ)-RSV which was formed as a minor component. To confirm this assumption a sample of the resveratrol-PEG conjugate mixture, formed
in the synthesis of MeO-PEGN-Succ-RSV, in d6-DMSO was treated with water and then heated at 90°C for ca. 1 week, to hydrolyse the phenolic ester linkages. The 1 H NMR spectrum for
the sample, shown in Fig. 2(c) (and Fig. A in S1 File), revealed the majority of the material had
hydrolysed to afford free resveratrol. The remaining signals shown in Fig. 2(c) can be attributed
to residual 3-MeO-PEGN-Succ-RSV and 4'-MeO-PEGN-Succ-RSV. Interestingly, the hydrolysis of the resveratrol-PEG conjugate did not produce MeO-PEGN-SuccOH as the other hydrolysis product, though this may have formed as an intermediate. The 1 H NMR spectra for
the solution after the hydrolysis reaction correlate with the formation of a succinimideterminated PEG chain. Fig. A(d) in S1 File displays a singlet at ca. 2.6 ppm which is consistent
with the succinimidyl ring methylene protons, and the absence of the succinyl ester amide protons observed for MeO-PEGN-SuccOH (c.f. Figs. A(a) and A(d) in S1 File). The formation of a
succinimide ring is consistent with literature observations for the hydrolysis of the succinyl
ester amide moiety [31–33]. Integration of the signals for resveratrol and the succinimide

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Stabilization of Resveratrol by Polymer Conjugation

Fig 2. The down field region of 1 H NMR spectra (400 MHz) for: (a) a solution of the MeO-PEGN-Succ-RSV (2 kDa) conjugate mixture in d6-DMSO; (b)
a solution of resveratrol in d6-DMSO; and (c) the solution from (a) after treatment with water and heat (90°C) for ca. 1 week). In (c) the signals for free
resveratrol are highlighted in red and those for the remaining resveratrol-PEG conjugates are highlighted in blue. Full width versions of these spectra are
provided in S1 File, along with a comparison with the MeO-PEG-Succ-OH starting material.

doi:10.1371/journal.pone.0118824.g002

terminated PEG in Fig. A(d) in S1 File indicated that there was more succinimide-terminated
PEG than free resveratrol, which supports our assumption that the minor component of the
resveratrol-PEG conjugate was the disubstituted product 3,4'-di(MeO-PEGN-Succ)-RSV. The
full blown 1 H NMR spectra of MeO-PEGN-Succ-RSV, MeO-PEGN-Succ-OH, MeO-PEGNSucc-RSV after heat treatment are shown in Figs. C, D, E in S1 File respectively.
The 1 H NMR spectra for the other PEG ester conjugates of resveratrol showed that the conjugation pattern was similar to that of MeO-PEGN-Succ-RSV (2 kDa). However, the extent of
PEG conjugation to resveratrol varied between PEGs of different MW and acid functional
groups. The conjugation was almost ca 100% for MeO-PEGN-SuccOH 2 kDa, ca. 70% and
50% respectively for MeO-PEGO-SuccOH 2 kDa and MeO-PEGN-SuccOH 20 kDa, However,
in the synthesis of MeO-PEGO-Succ-RSV 2 kDa and MeO-PEGN-Succ-RSV 20 kDa the isolated products contained a significant amount of an N-acyl urea as a by-product, which is formed
by isomerisation of the O-acylisourea"active ester" that results from the reaction of the carbodiimide and the PEG acid [34–35]. This indicated the reaction of the active ester and resveratrol was slow and incomplete.
For synthesis of MeO-PEG-PLAO-Succ-RSV, the successful conjugation was achieved using
EDC as a coupling agent and pyridine as the base (Fig. 1B). The 1 H NMR analysis of MeOPEG-PLAO-Succ-RSV 2 kDa indicated that the product was a mixture of conjugates 4'-MeOPEG-PLAO-Succ-RSV(ca. 50%) and 3-MeO-PEG-PLAO-Succ-RSV (ca. 35%) as the major
products, and a disubstituted derivative, presumably 3,4'-di (MeO-PEG-PLAO-Succ)-RSV, as
a minor product. The extent of conjugation was almost 80%. The substitution pattern for
MeO-PEG-PLAO-Succ-RSV 6.6 kDa was found similar to MeO-PEG-PLAO-Succ-RSV 2 kDa,
but contained certain amount of free resveratrol and unreacted polymer (<1%). The
free resveratrol, however, can be separated by HPLC from conjugates and did not interfere
with the study.
The synthesis of resveratrol-PEG ethers was achieved using PEG-Br in the presence of K2CO3
in DMF (Fig. 1C). Initially the synthesis of the PEG ether was attempted with K2CO3 and acetone [36] at reflux (60°C) but no product was isolated. The combination of K2CO3 and DMF

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Stabilization of Resveratrol by Polymer Conjugation

Table 1. Physicochemical properties and stability data of Resveratrol-polymer conjugates.

CMC
Moles/L

RSV-polymer
conjugates

MeO-PEG-PLAO-SuccRSV 2 kDa

MeO-PEG-PLAO-SuccRSV 6.6 kDa

PLA
Mw

PEG MW
(Daltons)

% Conjugate Remaining
at 24 hr pH 4.5

% Conjugate Remaining
at 24 hr pH 7.4

Plasma t1/2 CMC
mg/L

1000 750 99.2 104 3 hours 4 0.002

4600 2000 78.4 78.7 5 minutes 8 0.0012

MeO-PEGO-Succ-RSV N/A 2000 89.1 64.2 <5 minutes 500 0.25
2 kDa

MeO-PEGN-Succ-RSV N/A 2000 86.4 7.6 <5 minutes N/A N/A
2 kDa

MeO-PEGN-SuccRSV20 kDa

MeO-PEG-RSV ether 2
kDa

MeO-PEG-RSV ether 5
kDa

N/A 20000 88.1 9.3 <5 minutes N/A N/A

N/A 2000 100 100 No hydrolysis
stable

N/A 5000 100 100 No hydrolysis
stable

N/A N/A

N/A: not applicable as conjugates are soluble.

doi:10.1371/journal.pone.0118824.t001

yielded favorable results and conjugation was found to be dependent on amount of K2CO3 in
the reaction mixture. The reaction was monitored by HPLC, which indicated that conjugation
was highest when the concentration of K2CO3 in the reaction mixture was 1.8 mM. The 1 H
NMR analysis of resveratrol-PEG ether 2 kDa revealed that 4'-PEG-RSV was a major product
(ca. 60%), along with 3-PEG-RSV(ca. 30%) and a minor 3,4'-di(PEG)-RSV (ca. 10–15%). The
extent of conjugation was approximately 80%. The pattern of substitution and conjugation
chemistry was similar for the resveratral-PEG ether 5 kDa [37]. The various resveratrol-polymer conjugates synthesized in the study are listed in Table 1, along with their
physicochemical properties.

Characterization of resveratrol-polymer conjugates

Micelles characterization. The resveratrol conjugates consisting of PLA-PEG were formulated
into micelles then characterized both in vitro and in vivo. Detailed micelles characterization
was performed to correlate various physicochemical properties to in vitro and in vivo stability
of micelles. It was interesting to note that MeO-PEGO-Succ-RSV 2 kDa self-aggregated into
polymeric micelles, as the solution turned turbid during the in vitro buffer stability study. The
self-aggregation of MeO-PEGO-Succ-RSV into micelles presumably occurred because of the
hydrophobicity of resveratrol. The solubilizing power of PEG MW 2 kDa was not sufficient to
counter the hydrophobicity of resveratrol, hence self-assembly into micelles was favored. Veronese et al [38] reported similar findings with doxorubicin-PEG (DOX-PEG) conjugates with a
tetra-peptide linker (GFLG). The authors found that the tendency to aggregate into polymeric
micelles was dependent on the molecular architecture and MW of the polymer. The DOXGFLG-PEG with PEG5000 aggregated more readily than with PEG10000. This was attributed
to greater solubilisation effect of PEG10000. In stark contrast to MeO-PEGO-Succ-RSV 2kD,
the MeO-PEGN-Succ-RSV 2 kDa was soluble and formed a clear solution with no cloudiness
even at a high concentration (10 mg/mL). The CMC for MeO-PEG-PLA-Succ-RSV 2 kDa,
MeO-PEG-PLA-Succ-RSV 6.6 kDa and MeO-PEGO-Succ-RSV 2 kDa were found to be 4 mg/
L, 8 mg/L, and 500 mg/L respectively (Fig. 3 and Table 1). This trend clearly indicates that the
CMC is strongly dependent on the hydrophobic content in the chain and MW of polymer. The

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Stabilization of Resveratrol by Polymer Conjugation

Fig 3. Determination of CMC for MeO-PEG-PLAO-Succ-RSV 2kDa. A: plot of log (Concentration mg/L) vs
Absorbance for dimethyl yellow at 441. B: plot of log (Concentration mg/L) vs Absorption maximum (nm) for
dimethyl yellow. Mw quoted is for polymer only. Mw for RSV is 228 Da.

doi:10.1371/journal.pone.0118824.g003

CMC values reduce as the hydrophobic chain length increased in the polymer. This observation was true up to a certain extent of hydrophobic chain length. There are other molecular mechanics that drive the assembly of molecules into micelles which are discussed in detail in the
sections below.
In this study, the CMC values were determined using a UV probe instead of fluorescent probes
such as pyrene, because of interference from resveratrol in fluorescent measurements. Resveratrol has very broad excitation (290–350 nm) and emission (380–420 nm) ranges. These broad
ranges overlap with those of fluorescent probes such as pyrene. The dimethyl yellow employed
in this study has poor aqueous solubility (13 mg/L), which provides an opportunity to study
the UV absorption of dimethyl yellow under a particular wavelength and monitor the change

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Stabilization of Resveratrol by Polymer Conjugation

Table 2. Polymeric micelle size and polydispersibility index of resveratrol-polymer conjugates.

Concentration of polymer (%) Size nm (SD) Polydispersibility index (SD)

Meo-PEG-PLAO-Succ RSV 2 kDa

0.05 170 (0.6) 0.21 (0.05)

0.1 249 (10.4) 0.59 (0.03)

0.2 255 (3.0) 0.49 (0.08)

Meo-PEG-PLAO-Succ RSV 6.6K

0.05 147(3) 0.25(0.08)

0.1 147(6.1) 0.34(0.07)

0.2 165(12.4) 0.75 (0.15)

MeO-PEGO-Succ-RSV 2 kDa

0.1 116 (1.5) 0.12 (0.02)

0.25 136 (1.2) 0.08 (0.05)

0.5 181 (2.5) 0.19 (0.03)

doi:10.1371/journal.pone.0118824.t002

in wavelength of the maximum absorption as a function of surfactant or amphiphilic polymer
concentration [27]. The CMC values obtained by both dye solubilisation and wavelength shift
have correlated well. This indicates efficiency of the dimethyl yellow as an alternative UV
probe in CMC determination.
The micelles size and polydispersibility index are given Table 2. There was no exact correlation
between micelle size and polymer architecture such as chain length and hydrophobic content.
In general, the polymer conjugates with less hydrophobic content formed micelles with a
smaller diameter. The micelles diameter increased with an increase in polymer concentration.
This could be due to the formation of large aggregates at higher concentrations.
Stability of polymer conjugates in buffer. The stability of the polymer conjugates was studied
in buffers of pH 4.5 and 7.4 at 37°C, physiologically relevant to the pH of lysosomes and blood.
All the polymer conjugates of resveratrol were found to be stable in pH 4.5, with nearly 90% of
the polymer conjugates remaining intact after 24 h (Table 1). The MeO-PEGN-Succ-RSV 2
kDa and 20 kDa, were found to be unstable at pH 7.4 with only about 9% polymer conjugates
remaining intact after 24 h, which indicates that the ester link is unstable in slightly alkaline
conditions. Similar results were reported for PEG-scutellarin conjugates and PEG-Taxol conjugates [23,39]. Contrary to this, MeO-PEGO-Succ-RSV 2 kDa was found to be relatively stable
in pH 7.4 with nearly 65% of polymer conjugate found intact after 24 hours. This could be attributed to self assembly of polymer conjugates to micelles. The micelles would form a barrier
against hydrolysis of esters. This was further confirmed from results of micelle forming MeOPEG-PLAO-Succ-RSV conjugates which were found to be stable in pH 4.5 and 7.4 with >
78% polymer conjugates found intact after 24 h. As anticipated, the resveratrol-PEG ether
MeOH-PEG-RSV 2 kDa did not hydrolyze in any of the buffers because of the stability of the
ether linkage.
Stability of resveratrol-polymer conjugates in rat plasma. The effectiveness of the drug-polymer conjugates with an ester link for prolonged circulation in plasma would depend on the stability of the ester linkage in the plasma, as plasma esterase would facilitate hydrolysis. All PEG
ester conjugates of resveratrol, MeO-PEGN-Succ-RSV 2 kDa and 20 kDa, and MeO-PEGOSucc-RSV 2 kDa, irrespective of the linker and polymer chain length, hydrolysed rapidly to
yield free resveratrol when incubated with rat plasma. It is interesting to note that whatever
amount of resveratrol released after hydrolysis was trans isomer. There was no isomeric conversion. The hydrolysis was so rapid that almost no intact polymer conjugate was detected by

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Stabilization of Resveratrol by Polymer Conjugation

HPLC at the initial time point of 0 h. The rate of hydrolysis was more rapid than that reported
for campothecin-20-PEG and taxol-2’-PEG which have plasma t½ of 2 and 0.4 h respectively

[23]. The reason for this rapid hydrolysis is presumably a result of the more reactive phenolicester linkage involved in the PEG-RSV conjugates. Although there are a few reports suggesting
the application of more electronegative acid linkers, such as glycine for campothecine and peptides in the case of doxorubicin, results in greater plasma stability, for resveratrol, PEGylation
via a glycine linker did not improve plasma stability in previous studies [40]. To improve the
plasma stability of our conjugates, micelle-forming polymeric conjugates were explored. Accordingly the MeO-PEG-PLAO-Succ-RSV of different MW and with a PLA/PEG MW ratio of
1:0.75 and 4.6:2, were prepared and evaluated for plasma stability. Their plasma t1/2, MW of
conjugate, MW of PLA/PEG segments are shown in Table 1. The results indicate that MeOPEG-PLAO-Succ-RSV 2 kDa was more stable than the MeO-PEG-PLAO-Succ-RSV 6.6kDa
conjugate. The other micelle forming conjugate MeO-PEGO-Succ-RSV 2 kDa was no different
to MeO-PEGN-Succ-RSV 2 and 20 kDa in terms of plasma stability despite its better stability
in the buffer. It hydrolyzed rapidly with no intact conjugate being detected at the initial
time point.
The plasma stability of the polymeric drug ester conjugate is a complex phenomenon. The enzymatically catalyzed release of various drugs bound to N-(2-hydroxypropyl)methacrylamide
(HPMA)copolymers via ester linkages was found to be dependent on many factors, including
inter- and intra-molecular forces and the hydrophobicity of the drug and polymer [41–43].
Similarly, studies conducted to understand colloidal stability, protein adsorption and phagocytic clearance of PEG-PLA nanostructures, revealed that aggregation into colloidally stable micelles/nanoparticles was most favored when the PEG corona (PEG chain length and surface
density) is high [44–47]. Higher PLA content in the PEG-PLA chain has been reported to be
associated with decreased colloidal stability and reduces tendency to aggregate into micelles. In
addition, higher protein adsorption and phagocytic clearance was observed due to "naked"
PLA patches on the micelles surface [45]. Considering these observations and reports, it can be
concluded that to improve plasma stability of the conjugate, there has to be an optimum hydrophobicity, micelle forming ability and colloidal stability, with adequate surface protection by
the PEG. In this regard, presumably MeO-PEG-PLAO-Succ-RSV 2 kDa had relatively optimum hydrophobicity, forming compact and stable micelles with a relatively strong PEG corona, thereby providing maximum stability against ester hydrolysis. MeO-PEG-PLAO-SuccRSV 6.6kDa, on other hand, with a longer PLA chain length, presumably lead to the formation
of micelles with significantly higher "naked" PLA surfaces thus providing less protection
against ester hydrolysis. Consistent with these assumptions, AFM/FESEM of air dried samples
of MeO-PEG-PLAO-Succ-RSV 2 kDa micelles revealed formation of solid nanoparticle-like
structures and these solid nanostructures could impart additional resistance to enzymatic hydrolysis. The resveratrol-PEG ethers were found to be intact after 5 h incubation with rat plasma, as anticipated because the ether linkage is resistance to enzymatic cleavage.
In vitro antioxidant assay. The in vitro antioxidant profile as assessed by DPPH inhibition
assay is shown in Fig. 4. The IC 50 value of resveratrol was found to be 65 μM, which is in close
agreement with the value reported by Fauconneau et al. [48]. The IC 50 value for resveratrolPEG ether MeO-PEG-RSV, 2 kDa was 2190 μM (equal to 219 μM of resveratrol) and the values
were similar for MeO-PEGN-Succ-RSV 2 kDa and MeO-PEG-PLAO-Succ-RSV 2 kDa. The
profile shows that resveratrol conjugated to polymers is less active than native resveratrol with
regard to antioxidant activity (Fig. 4). There was an almost 3.5 times increase in the IC 50 values when compared to resveratrol. It is, however, anticipated that the resveratrol polymer ester
conjugates would hydrolyze in the body slowly releasing native resveratrol. The decrease in antioxidant activity indicates that all the hydroxyl groups of resveratrol are involved in reaction

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Stabilization of Resveratrol by Polymer Conjugation

Fig 4. DPPH inhibition profile of A: Resveratrol; B: MeO-PEGN-Succ-RSV, 2kDa; C: MeO-PEG-RSV
ether, 2kDa All the data are presented as mean ± SD (n = 3). Mw quoted is for polymer only. Mw for RSV is
228 Da.

doi:10.1371/journal.pone.0118824.g004

with free radicals. Hence, blocking one or more hydroxyl groups would lead to a decrease in activity. The antioxidant activity measured by this assay method was based on direct neutralization of free radicals and may not reflect actual in vivo activity, as resveratrol exerts in vivo
antioxidant activity by many other mechanisms. For instance, it was reported that resveratrol
could mediate the release or production of nitric oxide, which has stronger affinity for O2-[1].

Resveratrol is believed to induce enzymes dismutase and NAD(P)H:quinineoxidoreductase,
both of which scavenge the reactive oxygen species [1].
To produce a successful clinical candidate, there has to be a balance of all biological properties
such as activity, toxicity and metabolism. The best molecule, in terms of biological activity
would not necessarily make it to clinical studies or to the market due to issues such as metabolism [49]. Similarly, having an unmodified resveratrol with higher activity but prone to metabolism would not be useful for clinical studies. Chemically conjugated resveratrol, such as the
resveratrol-PEG ether MeO-PEG-RSV, with a hydrolytically stable link and prolonged plasma
residence time and better pharmacokinetic profile would offer an alternative choice to resveratrol despite the moderate decrease in the antioxidant activity.
In vitro metabolism studies of resveratrol and its polymer conjugates. The metabolism studies conducted by incubation with rat liver microsomes in the presence of the UGT reaction
mixture showed that resveratrol readily underwent metabolism to produce two metabolites,
presumably glucuronides, and the retention times of those metabolites correspond to those observed in rat plasma samples, degradation pattern is shown Fig. 5 and metabolites in Fig. 6.
The metabolism of resveratrol was rapid and almost all of the resveratrol was metabolized
within 10 minutes. The incubations of MeO-PEG-PLAO-Succ-RSV 2 kDa with microsomes
over 60 minutes did not result in any detectable level of metabolites by HPLC and presumably

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Stabilization of Resveratrol by Polymer Conjugation

Fig 5. The degradation profile of A: Resveratrol and B: MeO-PEG-PLAO-Succ-RSV 2 kDa in microsomal incubations with UDPA. C: MeO-PEGNSucc-RSV 2 kDa in microsomal incubation, D: MeO-PEGN-Succ-RSV 2 kDa in incubation buffer without microsomes. E: MeO-PEG-RSV ether 2 kDa in
microsomal incubation. Mw quoted is for polymer only. Mw for RSV is 228 Da.

doi:10.1371/journal.pone.0118824.g005

there was no metabolism occurring within the test period. The incubation samples of MeOPEGN-Succ-RSV 2 kDa showed formation of moderate amount of glucuronide metabolites.
However the control incubations of MeO-PEGN-Succ-RSV 2 kDa without microsomes
showed a significant degree of conjugate hydrolysis to free resveratrol which could be due to
pH. The resveratarol generated in such a way would have been subsequently metabolized
in the incubations containing microsomes under similar pH conditions. In contrast, the
resveratrol-PEG ether MeO-PEG-RSV 2 kDa was stable in microsomal incubations (Fig. 5E).
These observations clearly demonstrate that to provide protection against the metabolism of
resveratrol, the polymer conjugates need to be intact and hydrolyze slowly in the body.
Single dose intravenous pharmacokinetic studies. The plasma concentration profiles of resveratrol, MeO-PEG-PLAO-Succ-RSV 2 kDa, RSV-PEG ethers MeO-PEG-RSV 2 kDa and MeOPEG-RSV5 kDa are presented in Fig. 7. After intravenous dosing of resveratrol at 10 mg/kg,
the plasma concentration profile showed rapid elimination of resveratrol from the plasma.
There were four major metabolites detected by HPLC and characterized by LC-MS/MS. The
four metabolites were identified and assigned to 3- and 4’- monoglucuronides, 3- and 4’- mono
sulphates [28].
Pharmacokinetic parameters for the resveratrol formulations are shown in Table 3, The elimination half-life (t½ = 1.4 h) and clearance (CL = 20 L/h/kg) were comparable to previous

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Stabilization of Resveratrol by Polymer Conjugation

Fig 6. Chromatogram of resveratrol in microsomal incubations at 10 minutes.

doi:10.1371/journal.pone.0118824.g006

Fig 7. Plasma concentration-time profiles for resveratrol formulations in rats. Doses are resveratrol
equivalents and data are mean ± SD. Symbols:MeO-PEG-RSV ether 2 kDa, 2 mg/kg (●; n = 5); MeO-PEGRSV ether 5 kDa, 2 mg/kg (◯; n = 6); MeO-PEG-PLAO-Succ-RSV 2kDa, 2 mg/kg (▲; n = 6); Resveratrol, 10
mg/kg (5; n = 4).

doi:10.1371/journal.pone.0118824.g007

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Stabilization of Resveratrol by Polymer Conjugation

Table 3. Pharmacokinetic parameters for resveratrol and its conjugate formulations in rats.

AUC0-1 t½ MRT ‡ CL ‡ Vss

(μmol.h/L) P value, (h) P value, (h) P value, (L/h/kg) P value, 95% CI

95% CI 95% CI 95% CI (Significance)

(L/kg) P value, 95% CI
(Significance)

MeO-PEG-RSV
ether 5 kDa; 2 mg/
kg † (n = 6)

MeO-PEG-RSV
ether 2 kDa; 2 mg/
kg † (n = 5)

MeO-PEG-PLAOSucc-RSV 2kDa; 2
mg/kg † (n = 6)

Resveratrol; 10
mg/kg † (n = 4)

50 ± 15 0.0003* 5.3 ± 2.1 0.0086 5.9 ± 3.5 0.0361* 0.19 ± 0.06 0.0003* 1.01 ± 0.41 0.009

74 ± 16 0.0001** 5.5 ± 1.4 0.015 7.1 ± 2.0 0.0012 0.12 ± 0.02 0.001* 0.87 ± 0.28 0.0170*

21 ± 17 0.0645 1.7 ± 0.5 0.512 1.9 ± 0.4 0.214 0.58 ± 0.26 0.0004* 1.12 ± 0.60 0.0094

2.4 ± 0.7 Not
applicable

1.4 ± 0.9 Not
applicable

1.3 ± 1.0 Not
applicable

20.1 ± 8.4 Not applicable 26.8 ± 19.1 Not applicable

- 2 mg/kg = 8.8 μmol/kg; 10 mg/kg = 44 μmol/kg. Doses are resveratrol equivalents† and data are mean ± SD.

- MRT: mean residence time;

CL: clearance; Vss: volume of distribution at steady-state.

P values are from comparison of a particular group against resvertrol,

- * **** indicate p <0.05, increasing number of stars implies increasing significance.

doi:10.1371/journal.pone.0118824.t003

reports (t½ = 1.3 h and CL = 11–19 L/h/kg [5, 9, 50]. After two hours, the resveratrol concentration was below the limit of detection, indicating its rapid metabolism. By comparison, MeOPEG-PLAO-Succ-RSV 2 kDa was given to rats at one-fifth of the resveratrol dose (2 mg/kg
equivalent), but resulted in a 9-fold higher AUC, a 34-fold lower apparent CL and 24-fold
lower Vss, suggesting a reduced tissue distribution of the drug that should lead to more persistent retention of the drug in the circulation. However, the lack of detectable conjugate after 2
hr can be attributed to clearance by the mononuclear phagocyte systems (MPS) [47].
The plasma profile of MeO-PEG-PLAO-Succ-RSV 6.6 kDa was considerably different to that
of MeO-PEG-PLAO-Succ-RSV 2 kDa (data not shown). There was no quantifiable polymer
conjugate detected in the plasma, even at initial time point of 5 min. The rapid decline or nonavailability of polymer conjugate could be due to the combined effect of hydrolysis and MPS
clearance. Unlike MeO-PEG-PLAO-Succ-RSV 2 kDa, MeO-PEG-PLAO-Succ-RSV 6.6 kDa
displayed a higher level of metabolites, possibly due to more "naked" PLA on the surface of micelles, which in turn made the conjugate more vulnerable to enzymes and MPS attack. This
lead to hydrolysis of ester and released resveratrol. The latter was subsequently metabolized resulting in a high level of metabolites. These observations correlate with results obtained from
the in vitro plasma stability study.
The resveratrol-PEG ethers MeO-PEG-RSV2 kDa and MeO-PEG-RSV 5 kDa had a higher
AUC and lower apparent CL compared to MeO-PEG-PLAO-Succ-RSV 2kDa, but the Vss was
comparable (Table 3). When compared to the resveratrol group, these two conjugates are statistically significantly (P<0.05) increased in mean residence time, AUC, volume of distribution, clearance and elimination half-life. The prolonged residence time of resveratrol-PEG
ether conjugates in the blood circulation could be attributed to the combined effect of PEG
conjugation and ether linkage, which increased the hydrophilicity of resveratrol and avoided
the phagocyte detection and metabolism. However, eventually the conjugates underwent filtration via the glomerulus. The similarity in pharmacokinetic parameters for resveratrol-PEG
ether conjugates (MeO-PEG-RSV 2 kDa and MeO-PEG-RSV 5 kDa) suggests that an increase

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 18 / 22

Stabilization of Resveratrol by Polymer Conjugation

in MW from 2 kDa to 5 kDa has a marginal effect on kidney filtration [17]. In a previous report, pharmacokinetic studies with radio labelled PEGs of various MW clearly showed that a
significant effect of MW on clearance of PEG can be observed only above a MW of 20 kDa

[51]. Nevertheless, it is plausible that the special conformational arrangements of 3,4’-di(PEG)
may give a more hydrodynamic volume and low kidney filtration 13 . Indeed, Fee et al. have
studied the size comparison between proteins PEGylated with linear and branched chain PEGs

[52] and concluded that branched PEG would have a higher viscosity and larger radius than
proteins PEGylated with linear PEGs of the same molecular weight. Based on the same reasoning, di-substituted resveratrol with a PEG of 2 kDa could have a branched structure and a more
hydrodynamic volume than mono-substituted resveratrol with PEG of 5 kDa. However these
findings need further confirmation using suitable analytical techniques. Overall, the pharmacokinetic profile of resveratrol-PEG ethers is encouraging and further improvement in the pharmacokinetic properties may be achieved by increasing the MW of PEG.

Conclusion

Ester and ether-based PEG and PEG-PLA polymer conjugates (soluble and polymeric micelles)
to improve the pharmacokinetic profile of resveratrol were synthesized and evaluated. Studies
of the buffer stability, plasma stability and in vitro metabolism using rat liver microsomes, and
pharmacokinetics in rat showed that MeO-PEG-PLA-Succ-RSV 2 kDa, RSV-PEG ethers 2
kDa and 5 kDa consistently out-performed other polymers such as PEG linked to resveratrol
via ester bond and resveratrol itself. It was shown that both the type of linkage between the
polymer and drug, and the relatively compact PEG corona on the polymeric micelles are crucial for rendering a stable conjugate system which protects from degradation. For the polymers
containing an ester bond, it is the ability to form micelles that enhances in vitro and in vivo stability. In conclusion, our study demonstrates that polymeric conjugates can be an effective approach to improve the pharmacokinetic profile of resveratrol. Furthermore, the polymer
architecture and linkage can have a strong influence on the stability of conjugates in vitro
and in vivo.
NMR spectra and structures of polymer conjugates are given in S1 File. This material is avail[able free of charge via the Internet at http://www.plosone.org](http://www.plosone.org)

Supporting Information

[S1 File. Fig. A,](http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0118824.s001) 1 H NMR spectra (400 MHz) for: (a) MeO-PEGN-SuccOH; (b) the resveratrolPEG conjugate mixture from the synthesis of MeO-PEGN-Succ-RSV; (c) resveratrol; and (d)
the solution from (b) after treatment with water and heat (90°C) for ca. 1 week). All solutions
are in d6-DMSO. Key: † = d5-DMSO, ‡ = H2O. To identify the low intensity down-field signals
the spectra in (a), (b) and (d) have been expanded vertically. Fig. B, The down field region of the
1H NMR spectra (400 MHz) for: (a) a solution of the resveratrol-PEG conjugate mixture in
d6-DMSO showing the assigned signals for the two major products 4'-MeO-PEGN-Succ-RSV
and 3-MeO-PEGN-Succ—RSV; and (b) a solution of resveratrol in d6-DMSO. Fig. C, 1 H NMR
spectra for the MeO-PEGN-Succ-RSV conjugate mixture in d6-DMSO, recorded at 400 MHz.
Key: † = d5-DMSO, ‡ = H2O. Fig. D, 1 H NMR spectra for MeO-PEGN-Succ-OH in d6-DMSO,
recorded at 400 MHz. Key: † = d5-DMSO, ‡ = H2O. Fig. E, 1 H NMR spectra of the MeOPEGN-Succ-RSV conjugate mixture after treatment with water and heat (90°C) for ca. 1 week,
in d6-DMSO, recorded at 400 MHz. Key: † = d5-DMSO, ‡ = H2O.
(DOCX)

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 19 / 22

Stabilization of Resveratrol by Polymer Conjugation

Acknowledgments

Authors wish to thank Dr Beng Chua, Manager of the Animal Facility at Curtin University, for
his assistance in animal experiments and also Dr Christopher Cruickshank for his help in LCMS analysis.

Author Contributions

Conceived and designed the experiments: BS, HB, DB, YC. Performed the experiments: BS,
DB. Analyzed the data: BS, HB, DB, KB, YC. Contributed reagents/materials/analysis tools: BS,
HB, DB, YC. Wrote the paper: BS, HB, DB, KB, YC.

References

1. Das DK, Maulik N (2006) Resveratrol in cardioprotection: a therapeutic promise of alternative medicine.
Mol Interventions 6: 36–47.

2. Le Corre L, Chalabi N, Delort L, Bignon Y-J, Bernard-Gallon DJ (2005) Resveratrol and breast cancer
chemoprevention: Molecular mechanisms. Mol Nutr Food Res [49: 462–471. PMID: 15786518](http://www.ncbi.nlm.nih.gov/pubmed/15786518)

3. Das S, Das DK (2007) Anti-Inflammatory Responses of Resveratrol. Inflamm Allergy Drug Targets 6:
[168–173. PMID: 17897053](http://www.ncbi.nlm.nih.gov/pubmed/17897053)

4. Hubbard BP, Gomes AP, Dai H, Li J, Case AW, Considine T, et al. (2013) Evidence for a Common
Mechanism of SIRT1 Regulation by Allosteric Activators. Science [339: 1216–1219. doi: 10.1126/](http://dx.doi.org/10.1126/science.1231097)
[science.1231097 PMID: 23471411](http://dx.doi.org/10.1126/science.1231097)

5. Boocock DJ, Faust GES, Patel KR, Schinas AM, Brown VA, Ducharme MP, et al. (2007) Phase I Dose
Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent. Cancer Epidemiol Biomark Prev [16: 1246–1252 PMID: 17548692](http://www.ncbi.nlm.nih.gov/pubmed/17548692)

6. Marier J-F, Vachon P, Gritsas A, Zhang J, Moreau J-P, Ducharme MP, et al. (2002) Metabolism and
Disposition of Resveratrol in Rats: Extent of Absorption, Glucuronidation, and Enterohepatic Recirculation Evidenced by a Linked-Rat Model. J Pharmacol Exp Ther [302: 369–373. PMID: 12065739](http://www.ncbi.nlm.nih.gov/pubmed/12065739)

7. Wenzel E, Soldo T, Erbersdobler H, Somoza V (2005) Bioactivity and metabolism of trans-resveratrol
orally administered to Wistar rats. Mol Nutr Food Res [49: 482–494. PMID: 15779067](http://www.ncbi.nlm.nih.gov/pubmed/15779067)

8. Walle T, Hsieh F, DeLegge MH, Oatis JE, Walle UK (2004) High absorption but very low bioavailability
of oral resveratrol in humans. Drug Metab Dispos [32: 1377–1382. PMID: 15333514](http://www.ncbi.nlm.nih.gov/pubmed/15333514)

9. Sale S, Verschoyle RD, Boocock D, Jones DJL, Wilsher N, Ruparelia KC, et al. (2004) Pharmacokinetics in mice and growth-inhibitory properties of the putative cancer chemopreventive agent resveratrol
and the synthetic analogue trans 3,4,5,4[prime]-tetramethoxystilbene. Br J Cancer 90: 736–744.
[PMID: 14760392](http://www.ncbi.nlm.nih.gov/pubmed/14760392)

10. Kapetanovic I, Muzzio M, Huang Z, Thompson T, McCormick D (2011) Pharmacokinetics, oral bioavailability, and metabolic profile of resveratrol and its dimethylether analog, pterostilbene, in rats. Cancer
Chemother Pharmacol [68: 593–601. doi: 10.1007/s00280-010-1525-4 PMID: 21116625](http://dx.doi.org/10.1007/s00280-010-1525-4)

11. Azorín-Ortuño Ma, Yañéz-Gascón MaJ, Pallarés FJ, Vallejo F, Larrosa M, García-Conesa MT, et al.
(2010) Pharmacokinetic Study of trans-Resveratrol in Adult Pigs. J Agric Food Chem. 58:
[11165–11171. doi: 10.1021/jf102799m PMID: 20886884](http://dx.doi.org/10.1021/jf102799m)

12. Muzzio M, Huang Z, Hu S-C, Johnson WD, McCormick DL, Kapetanovic IM, et al. (2012) Determination
of resveratrol and its sulfate and glucuronide metabolites in plasma by LC—MS/MS and their pharmacokinetics in dogs. J Pharm Biomed Anal [59: 201–208. doi: 10.1016/j.jpba.2011.10.023 PMID:](http://dx.doi.org/10.1016/j.jpba.2011.10.023)
[22079044](http://www.ncbi.nlm.nih.gov/pubmed/22079044)

13. Yu C, Shin YG, Chow A, Li Y, Kosmeder JW, Lee YS, et al. (2002) Human, Rat, and Mouse Metabolism
of Resveratrol. Pharm Res [19: 1907–1914. PMID: 12523673](http://www.ncbi.nlm.nih.gov/pubmed/12523673)

14. Tessitore L, Davit A, Sarotto I, Caderni G (2000) Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21CIP expression. Carcinogenesis 21: 1619–1622. PMID:
[10910967](http://www.ncbi.nlm.nih.gov/pubmed/10910967)

15. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, et al. (1997) Cancer Chemopreventive Activity of Resveratrol, a Natural Product Derived from Grapes. Science 275: 218–220.
[PMID: 8985016](http://www.ncbi.nlm.nih.gov/pubmed/8985016)

16. Gao X, Xu YX, Divine G, Janakiraman N, Chapman RA, Gautam SC, et al. (2002) Disparate In Vitro
and In Vivo Antileukemic Effects of Resveratrol, a Natural Polyphenolic Compound Found in Grapes.
The Journal of Nutrition [132: 2076–2081. PMID: 12097696](http://www.ncbi.nlm.nih.gov/pubmed/12097696)

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 20 / 22

Stabilization of Resveratrol by Polymer Conjugation

17. Monfardini C, Veronese FM (1998) Stabilization of Substances in Circulation. Bioconjug Chem 9:
[418–450. PMID: 9667945](http://www.ncbi.nlm.nih.gov/pubmed/9667945)

18. Veronese FM, Morpurgo M (1999) Bioconjugation in pharmaceutical chemistry. Il Farmaco 54:
[497–516. PMID: 10510847](http://www.ncbi.nlm.nih.gov/pubmed/10510847)

19. Danhauser-Riedl S, Hausmann E, Schick H-D, Bender R, Dietzfelbinger H, Rastetter J, et al. (1993)
Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (AD-70, DOX-OXD). Invest
New Drugs [11: 187–195. PMID: 7505268](http://www.ncbi.nlm.nih.gov/pubmed/7505268)

20. Cao N, Feng S. (2008) Doxorubicin conjugated to d-[alpha]-tocopheryl polyethylene glycol 1000 succinate (TPGS): Conjugation chemistry, characterization, in vitro and in vivo evaluation. Biomaterials
[29:3856. doi: 10.1016/j.biomaterials.2008.05.016 PMID: 18606445](http://dx.doi.org/10.1016/j.biomaterials.2008.05.016)

21. Zhang X, Li Y, Chen X, Wang X, Xu X, Liang Q, et al. (2005) Synthesis and characterization of the
paclitaxel/MPEG-PLA block copolymer conjugate. Biomaterials [26: 2121–2128. PMID: 15576187](http://www.ncbi.nlm.nih.gov/pubmed/15576187)

22. Xie Z, Guan H, Chen X, Lu C, Chen L, Hu X, et al. (2007) A novel polymer—paclitaxel conjugate based
on amphiphilic triblock copolymer. J Control Release [117: 210–216. PMID: 17188776](http://www.ncbi.nlm.nih.gov/pubmed/17188776)

23. Greenwald RB, Gilbert CW, Pendri A, Conover CD, Xia J, Martinez A. (1996) Drug Delivery Systems:
Water Soluble Taxol 2‘-Poly(ethylene glycol) Ester Prodrugs Design and in Vivo Effectiveness. J Med
Chem [39: 424–431. PMID: 8558510](http://www.ncbi.nlm.nih.gov/pubmed/8558510)

24. Ovesna Z, Horvathova-Kozics K (2005) Structure-activity relationship of trans-resveratrol and its analogues. Neoplasma [52: 450–455. PMID: 16284688](http://www.ncbi.nlm.nih.gov/pubmed/16284688)

25. Lee HJ, Seo JW, Lee BH, Chung K-H, Chi DY (2004) Syntheses and radical scavenging activities of
resveratrol derivatives. Bioorg Med Chem Lett [14: 463–466. PMID: 14698182](http://www.ncbi.nlm.nih.gov/pubmed/14698182)

26. Basavaraj S, Benson HAE, Cruickshank C, Brown DH, Chen Y (2011) Development of a liquid
chromatography/mass spectrometry methodology to separate, detect, characterize and quantify PEGresveratrol prodrugs and the conjugation reaction precursors and intermediates. Rapid Commun Mass
Spectrom [25: 1543–1551. doi: 10.1002/rcm.5000 PMID: 21594928](http://dx.doi.org/10.1002/rcm.5000)

27. Voronov A, Vasylyev S, Kohut A, Peukert W (2008) Surface activity of new invertible amphiphilic polyesters based on poly(ethylene glycol) and aliphatic dicarboxylic acids. J. Colloid Interface Sci 323:
[379–385. doi: 10.1016/j.jcis.2008.04.053 PMID: 18501919](http://dx.doi.org/10.1016/j.jcis.2008.04.053)

28. Basavaraj S, Benson HAE, Brown DH, Chen Y (2013) Application of solvent influenced fluorescencequenching and enhancement to develop a highly sensitive HPLC methodology for analysis of
resveratrol-PEG conjugates. Curr Pharm Anal 9:199–207.

29. Kuang J, Yuan J, Zhou M, Yuan W, Sui X, Li Z. (2008) Grafting of PEG onto lanthanum hydroxide nanowires. Mater. Lett 62: 4078–4080.

30. Greenwald RB, Choe YH, Wu D (2003) Selective phenolic acylation of 10-hydroxycamptothecin using
poly (ethylene glycol) carboxylic acid. Bioorg Med Chem Lett [13: 577–580. PMID: 12565975](http://www.ncbi.nlm.nih.gov/pubmed/12565975)

31. Alla A, Rodrıguez-Galán A, Mú ñoz-Guerra S (2000) Hydrolytic and enzymatic degradation of copoly
(ester amide)s based on l-tartaric and succinic acids. Polymer 41: 6995–7002.

32. Anderson BD, Conradi RA, Knuth KE (1985) Strategies in the design of solution-stable, water-soluble
prodrugs I: A physical—organic approach to pro-moiety selection for 21-esters of corticosteroids.
J Pharm Sci [74: 365–374. PMID: 3998996](http://www.ncbi.nlm.nih.gov/pubmed/3998996)

33. Villuendas I, Molina I, Regaño C, Bueno M, Martínez de Ilarduya A, Galbis JA, et al. (1999) Hydrolytic
Degradation of Poly(ester amide)s Made from Tartaric and Succinic Acids: Influence of the Chemical
Structure and Microstructure on Degradation Rate. Macromolecules 32: 8033–8040.

34. Stadler A, Kappe CO (2001) The effect of microwave irradiation on carbodiimide-mediated esterifications on solid support. Tetrahedron 57:3915–20.

35. Bates HS, Jones JH, Witty MJ (1980) Direct observation of an alkoxycarbonylamino acid
O-acylisourea. J Chem Soc Chem Commun 16:773–774.

36. Jiang YL (2008) Design, synthesis and spectroscopic studies of resveratrol aliphatic acid ligands of
human serum albumin. Bioorg Med Chem [16: 6406–6414. doi: 10.1016/j.bmc.2008.05.002 PMID:](http://dx.doi.org/10.1016/j.bmc.2008.05.002)
[18499462](http://www.ncbi.nlm.nih.gov/pubmed/18499462)

37. Biasutto L, Mattarei A, Marotta E, Bradaschia A, Sassi N, Garbisa S, et al. (2008) Development of
mitochondria-targeted derivatives of resveratrol. Bioorg Med Chem Lett [18: 5594–5597. doi: 10.1016/j.](http://dx.doi.org/10.1016/j.bmcl.2008.08.100)
[bmcl.2008.08.100 PMID: 18823777](http://dx.doi.org/10.1016/j.bmcl.2008.08.100)

38. Veronese FM, Schiavon O, Pasut G, Mendichi R, Andersson L, Tsirk A, et al. (2005) PEG−Doxorubicin
Conjugates: Influence of Polymer Structure on Drug Release, in Vitro Cytotoxicity, Biodistribution, and
Antitumor Activity. Bioconjug Chem [16: 775–784. PMID: 16029018](http://www.ncbi.nlm.nih.gov/pubmed/16029018)

39. Zhou QS, Jiang XH, Yu JR, Li KJ (2006) Synthesis and Characterization of PEG-scutellarin Conjugates,
a Potential PEG Ester Prodrug for the Oral Delivery of Scutellarin. Chin Chem Letters 17: 85–88.

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 21 / 22

Stabilization of Resveratrol by Polymer Conjugation

40. Biasutto L, Marotta E, Mattarei A, Beltramello S, Caliceti P, Salmaso S, et al. (2009) Absorption and metabolism of resveratrol carboxyesters and methanesulfonate by explanted rat intestinal segments. Cell
Physiol Biochem [24:557–66. doi: 10.1159/000257512 PMID: 19910696](http://dx.doi.org/10.1159/000257512)

41. Pan H, Kopečková P, Liu J, Wang D, Miller S, Kopecek J et al. (2007) Stability in Plasmas of Various
Species of HPMA Copolymer—PGE Conjugates. Pharm Res [24: 2270–2280. PMID: 17899324](http://www.ncbi.nlm.nih.gov/pubmed/17899324)

42. Pan H, Kopečková P, Wang D, Yang J, Miller S, Kopeček J (2006) Water-soluble HPMA copolymer—
prostaglandin E1 conjugates containing a cathepsin K sensitive spacer. J Drug Target 14:425–35.
[PMID: 17092842](http://www.ncbi.nlm.nih.gov/pubmed/17092842)

43. Ding H, Kopečková P, Kopeček J (2007) Self-association properties of HPMA copolymers containing
an amphipathic heptapeptide. J Drug Target [15: 465–474. PMID: 17671893](http://www.ncbi.nlm.nih.gov/pubmed/17671893)

44. Riley T, Govender T, Stolnik S, Xiong CD, Garnett MC, Illum L, et al. (1999) Colloidal stability and drug
incorporation aspects of micellar-like PLA—PEG nanoparticles. Colloids Surf B 16: 147–159.

45. Gref R, Lück M, Quellec P, Marchand M, Dellacherie E, Harnisch S, et al. (2000) ‘Stealth’ corona-core
nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain
length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids Surf B [18: 301–313. PMID: 10915952](http://www.ncbi.nlm.nih.gov/pubmed/10915952)

46. Jain JP, Kumar N (2010) Self Assembly of Amphiphilic (PEG)3-PLA Copolymer as Polymersomes:
Preparation, Characterization, and Their Evaluation As Drug Carrier. Biomacromolecules 11:
[1027–1035. doi: 10.1021/bm1000026 PMID: 20178378](http://dx.doi.org/10.1021/bm1000026)

47. Li Y, Qi XR, Maitani Y, Nagai T (2009) PEG-PLA diblock copolymer micelle-like nanoparticles as
all-trans-retinoic acid carrier: in vitro and in vivo characterizations. Nanotechnology 20: 055106. doi:
[10.1088/0957-4484/20/5/055106 PMID: 19417337](http://dx.doi.org/10.1088/0957-4484/20/5/055106)

48. Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon JM, et al. (1997) Comparative
study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci [61: 2103–2110 PMID: 9395251](http://www.ncbi.nlm.nih.gov/pubmed/9395251)

49. Selick HE, Beresford AP, Tarbit MH (2002) The emerging importance of predictive ADME simulation in
drug discovery. Drug Discov Today [7: 109–116. PMID: 11790621](http://www.ncbi.nlm.nih.gov/pubmed/11790621)

50. Das S, Lin HS, Ho PC, Ng KY (2008) The impact of aqueous solubility and dose on the pharmacokinetic
profiles of resveratrol. Pharm Res [25: 2593–2600. doi: 10.1007/s11095-008-9677-1 PMID: 18629618](http://dx.doi.org/10.1007/s11095-008-9677-1)

51. Mehvar R (2000) Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation. J Pharm Pharm Sci [3: 125–136. PMID: 10954682](http://www.ncbi.nlm.nih.gov/pubmed/10954682)

52. Fee CJ (2007) Size comparison between proteins PEGylated with branched and linear poly(ethylene
glycol) molecules. Biotechnol Bioeng [98: 725–731. PMID: 17461424](http://www.ncbi.nlm.nih.gov/pubmed/17461424)

PLOS ONE | DOI:10 1371/journal pone 0118824 March 23 2015 22 / 22

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