Functional poly(ε-caprolactone)s via copolymerization of ε-caprolactone and pyridyl disulfide-containing cyclic carbonate: Controlled synthesis and facile access to reduction-sensitive biodegradable graft copolymer micelles

Article abstract of DOI:10.1021/ma302499a

Pyridyl disulfide-functionalized cyclic carbonate (PDSC) monomer was obtained in four straightforward steps from 3-methyl-3-oxetanemethanol and exploited for facile preparation of functional poly(ε-caprolactone) (PCL) containing pendant pyridyl disulfide (PDS) groups via ring-opening copolymerization with ε-caprolactone. The results showed that PDS-functionalized PCL polymers were prepared with controlled molecular weights and functionalities. The exchange reaction between PDS-functionalized PCL and thiolated poly(ethylene glycol) (PEG-SH) at a PEG-SH/PDS molar ratio of 2/1 afforded PCL-g-SS-PEG graft copolymers in high yields. The dynamic light scattering (DLS) analyses showed that PCL-g-SS-PEG copolymer self-assembled into micelles with a diameter of 110-120 nm and a low polydispersity (PDI) in phosphate buffer (pH 7.4, 10 mM). PCL-g-SS-PEG micelles while sufficiently stable under physiological conditions were prone to rapid shell shedding and aggregation under a reductive condition. Doxorubicin (DOX) was loaded into PCL-g-SS-PEG micelles with a decent drug loading content of 10.1 wt %. Notably, in vitro release studies revealed that ca. 82.1% DOX was released in 12 h under a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell nucleus whereas only ca. 17.5% DOX was released in 24 h under nonreductive conditions. Confocal microscopy observation indicated that DOX was delivered into the nuclei of HeLa cells following 8 h incubation with DOX-loaded PCL-g-SS-PEG micelles. MTT assays in HeLa cells demonstrated that DOX-loaded PCL-g-SS-PEG micelles retained high antitumor activity with low IC₅₀ (half-maximal inhibitory concentration) of 0.82-0.95 μg DOX equiv/mL while blank PCL-g-SS-PEG micelles were nontoxic up to a tested concentration of 1.0 mg/mL. This study presents a versatile and controlled synthesis of PDS-functionalized biodegradable polymers and reduction-sensitive biodegradable graft copolymer micelles that are of particular interest for active intracellular drug release.

Full text of DOI:10.1021/ma302499a

Article
pubs.acs.org/Macromolecules
Functional Poly(ε-caprolactone)s via Copolymerization of
ε‑Caprolactone and Pyridyl Disulfide-Containing Cyclic Carbonate:
Controlled Synthesis and Facile Access to Reduction-Sensitive
Biodegradable Graft Copolymer Micelles
Wei Chen,†,‡ Yan Zou,† Junna Jia,† Fenghua Meng,† Ru Cheng,† Chao Deng,† Jan Feijen,†,‡
and Zhiyuan Zhong*,†
†Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department
of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University,
Suzhou, 215123, P. R. China
‡Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, MIRA Institute for Biomedical Technology
and Technical Medicine, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
ABSTRACT: Pyridyl disulfide-functionalized cyclic carbonate
(PDSC) monomer was obtained in four straightforward steps
from 3-methyl-3-oxetanemethanol and exploited for facile
preparation of functional poly(ε-caprolactone) (PCL) con-
taining pendant pyridyl disulfide (PDS) groups via ring-
opening copolymerization with ε-caprolactone. The results
showed that PDS-functionalized PCL polymers were prepared
with controlled molecular weights and functionalities. The
exchange reaction between PDS-functionalized PCL and
thiolated poly(ethylene glycol) (PEG-SH) at a PEG-SH/
PDS molar ratio of 2/1 afforded PCL-g-SS-PEG graft copolymers in high yields. The dynamic light scattering (DLS) analyses
showed that PCL-g-SS-PEG copolymer self-assembled into micelles with a diameter of 110−120 nm and a low polydispersity
(PDI) in phosphate buffer (pH 7.4, 10 mM). PCL-g-SS-PEG micelles while sufficiently stable under physiological conditions
were prone to rapid shell shedding and aggregation under a reductive condition. Doxorubicin (DOX) was loaded into PCL-g-SS-
PEG micelles with a decent drug loading content of 10.1 wt %. Notably, in vitro release studies revealed that ca. 82.1% DOX was
released in 12 h under a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell
nucleus whereas only ca. 17.5% DOX was released in 24 h under nonreductive conditions. Confocal microscopy observation
indicated that DOX was delivered into the nuclei of HeLa cells following 8 h incubation with DOX-loaded PCL-g-SS-PEG
micelles. MTT assays in HeLa cells demonstrated that DOX-loaded PCL-g-SS-PEG micelles retained high antitumor activity with
low IC50 (half-maximal inhibitory concentration) of 0.82−0.95 μg DOX equiv/mL while blank PCL-g-SS-PEG micelles were
nontoxic up to a tested concentration of 1.0 mg/mL. This study presents a versatile and controlled synthesis of PDS-
functionalized biodegradable polymers and reduction-sensitive biodegradable graft copolymer micelles that are of particular
interest for active intracellular drug release.
maleimide28 groups have been reported. In particular, design
INTRODUCTION
■
of functional cyclic carbonate monomers has received recent
Aliphatic polyesters and polycarbonates, such as poly(ε-
caprolactone) (PCL), polylactide (PLA), poly(lactide-co-
interest.29,30 We have designed 2,4,6-trimethoxybenzylacetal,
(methyl)acryloyl, or vinyl sulfone-functionalized cyclic carbo-
glycolide) (PLGA), and poly(trimethylene carbonate)
nate monomers that provided versatile access to pH-sensitive
(PTMC), are among the most important synthetic biodegrad-
degradable micelles and polymersomes,31,32 photo-cross-linked
able materials.1−3 They have been widely applied for absorbable
biodegradable micelles,33−35 biodegradable injectable hydro-
gels,36 and functional biodegradable polymers and coatings.37
In recent years, taking advantage of high redox potential in
the cytoplasms and nuclei of cancer cells, reduction-sensitive
degradable micelles and nanoparticles have been actively
orthopedic devices,4 microparticles for controlled protein
release,5,6 cell and tissue scaffolds,7,8 drug-eluting stents,9 and
nanoparticles for targeted and controlled drug release.10,11
However, these traditional biodegradable polymers are lack of
reactive groups, which renders them difficult to develop
advanced drug delivery systems and bioactive tissue scaffolds.
In the past decade, various functional aliphatic polyesters and
polycarbonates with pendant hydroxyl,12,13 carboxyl,14,15
amine,16,17 allyl,18−20 alkyne/azide,21−24 acryloyl,25−27 and
Received: December 5, 2012
Revised: January 17, 2013
Published: January 30, 2013
© 2013 American Chemical Society
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Scheme 1. Synthesis of Functional PCL Containing Pendant Pyridyl Disulfide Groups and Facile Access to Reduction-Sensitive
Biodegradable Graft Copolymer Micelles for Intracellular Drug Release
developed for efficient intracellular anticancer drug release.38,39
The intracellular doxorubicin (DOX) level and antitumor
EXPERIMENTAL SECTION
■
Materials. 3-Methyl-3-oxetanemethanol (97%, Alfa), hydrobromic
activity of DOX-loaded reduction-sensitive shell-sheddable
micelles were significantly enhanced as compared to reduc-
tion-insensitive controls. It should be noted that pyridyl
disulfide (PDS) is the most versatile thiol−disulfide exchange
functional group used for the construction of disulfide linkages
and cross-links in bioconjugate chemistry.40 For example,
Hoffman et al. designed poly(methacrylic acid-co-butyl acrylate-
co-pyridyl disulfide acrylate) random copolymer for facile
conjugation and active intracellular release of thiol-containing
therapeutic oligopeptide drugs.41 Thayumanavan et al.
developed reduction-responsive polymeric nanogels based on
a random copolymer containing oligoethylene glycol and PDS
units as side-chain functionalities, in which PDS was used to
form disulfide cross-links as well as to introduce TAT peptide
or FITC on nanogel surfaces.42,43
In this paper, we report synthesis of novel functional PCL via
ring-opening copolymerization of ε-CL and pyridyl disulfide-
functionalized cyclic carbonate (PDSC) monomer (Scheme 1).
The postpolymerization modification with thiolated PEG by
thiol−disulfide exchange reaction yielded reduction-sensitive,
amphiphilic, biodegradable graft copolymers that are self-
assembled into stable micellar nanoparticles in water. It is
interesting to note that graft copolymer micelles offer several
advantages over block copolymer micelles; for instance, they
often exhibit enhanced stability with low critical micelle
concentration (cmc),44−46 micellar core and surface properties
can be broadly adjusted by backbone length, graft density, and
graft length, and presence of many hydrophilic grafts per
macromolecule enables conjugation of tunable density targeting
ligands for optimal tumor targeting. However, there are few
reports on biodegradable graft copolymer micelles based on
hydrophobic biodegradable polymers grafted with hydrophilic
polymers,21,44,47 likely due to challenging synthesis. In this
study, synthesis and ring-opening copolymerization of PDSC
monomer, synthesis and self-assembly of PCL-g-SS-PEG graft
copolymer, loading and reduction-responsive release of DOX,
and intracellular release and antitumor activity of DOX-loaded
graft copolymer micelles were investigated.
acid (40%, SCRC), sodium hydrosulfide hydrate (68%, Acros), 2,2′-
dipyridyl disulfide (98%, Alfa Aesar), triethylamine (Et3N, Alfa Aesar,
99%), stannous octoate (Sn(Oct)2, 95%, Sigma), and doxorubicin
hydrochloride (DOX·HCl, 99%, Beijing Zhong Shuo Pharmaceutical
Technology Development Co. Ltd.) were used as received. ε-
Caprolactone (ε-CL, 99%, Alfa Aesar) was dried over CaH2 and
distilled under reduced pressure prior to use. Thiolated PEG (PEG-
SH, Mn = 5.0 kg/mol) was synthesized according to a previous
report.48 Tetrahydrofuran (THF) and toluene were dried by refluxing
over sodium wire under an argon atmosphere prior to distillation.
Isopropanol was dried by refluxing over CaH2 under an argon
atmosphere. Ethyl chloroformate was freshly distilled before use.
Preparation of Pyridyl Disulfide Carbonate (PDSC). Pyridyl
disulfide carbonate (PDSC) was synthesized in four steps (Scheme 1).
The first two steps were carried out according to our previous report.37
Briefly, HBr (40%, 40 mL) was dropwise added into a solution of 3-
methyl-3-oxetanemethanol (10.20 g, 100.0 mmol) in THF (100 mL)
under stirring at 0 °C. The reaction mixture was warmed to room
temperature and stirred for another 5 h. The reaction mixture was then
diluted with H2O (150 mL) and extracted with diethyl ether (4 × 150
mL). The organic phase was dried over anhydrous MgSO4 and
concentrated to give the desired product (bromo-diol) as a white solid.
1
Yield: 17.57 g (96%). H NMR (400 MHz, CDCl3): δ 3.68 (s, 4H,
−C(CH2OH)2), 3.55 (s, 2H, −CH2Br), 2.13 (s, 2H, -(OH)2), 0.93 (s,
3H, −CH3).
Next, bromo-diol (9.15 g, 50.0 mmol) was added under stirring to a
solution of NaSH (8.40 g, 150.0 mmol) in DMF (150 mL). The
reaction mixture was stirred at 75 °C for 20 h. The solvent was
removed by rotary evaporation, the residue was dissolved in 150 mL of
deionized water and extracted with ethyl acetate (3 × 150 mL), and
the organic phase was dried over anhydrous MgSO4 and concentrated
to yield mercapto-diol as a yellowish oil. Yield: 3.54 g (52%). 1H NMR
(400 MHz, CDCl3): δ 3.64 (d, 4H, −C(CH2OH)2), 2.67 (d, 2H,
−CH2SH), 2.27 (s, 2H, −(OH)2), 1.31 (t, 1H, −SH), 0.85 (s, 3H,
−CH3).
Then, to a stirred solution of 2,2′-dithiodipyridine (8.68 g, 39.0
mmol) and a catalytic amount of glacial acetic acid (0.5 mL) in
methanol (75 mL) was added dropwise a solution of mercapto-diol
(3.54 g, 26.0 mmol) in methanol at room temperature. The reaction
mixture was stirred at room temperature for an additional 16 h. The
solvent was evaporated to yield crude product as a yellow oil. The
product was purified by column chromatography using silica gel as
stationary phase and a mixture of ethyl acetate/petroleum ether (v/v =
1
1/3) as eluent. Yield: 4.14 g (65%). H NMR (400 MHz, CDCl3): δ
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8.47, 7.62, 7.41, and 7.17 (m, pyridyl protons), 3.67 (d, 4H,
−C(CH2OH)2), 3.25 (s, 2H, −CH2SS-Py), 0.84 (s, 3H, −CH3).
Finally, to a stirred solution of pyridyl disulfide-diol (4.14 g, 16.9
mmol) and ethyl chloroformate (3.4 mL, 35.5 mmol) in dried THF
(150 mL) at 0 °C was added dropwise a solution of Et3N (5.4 mL,
39.1 mmol) in THF. The reaction was allowed to proceed for 4 h at 0
°C. The reaction mixture was filtered, the filtrate was concentrated
under reduced pressure, and the residues were crystallized in THF/
diethyl ether to yield a white solid. Yield: 3.66 g (50%). 1H NMR (400
MHz, CDCl3): δ 8.51, 7.65, 7.54, and 7.14 (m, pyridyl protons), 4.38−
4.15 (d, 4H, −C(CH2O)2CO), 3.08 (s, 2H, −CH2SS-Py), 1.23 (s, 3H,
−CH3). Elemental Analysis: Calcd for C9H12O5: C, 48.69; N, 5.16; H,
4.83. Found C, 48.70; N, 5.02; H, 4.75. TOF-MS (m/z): calcd for
C11H13NO3S2 271.36; found 271.37.
mM. The samples were gentlely stirred at 37 °C. The changes in
micelle size and PDI were monitored over time by DLS.
Loading and Redox-Triggered Release of DOX. DOX was
loaded into micelles by dropwise addition of 2 mL of phosphate buffer
(10 mM, pH 7.4) to a mixture of 0.2 mL of copolymer solution in
DMF (5 mg/mL) and 10, 20, or 40 μL of DOX solution in DMSO (5
mg/mL) under stirring at room temperature, followed by stirring for 1
h and dialysis against phosphate buffer (10 mM, pH 7.4) with a
molecular weight cutoff (MWCO) of 3500 Da at room temperature in
the dark for 6 h.
The release profiles of DOX from PCL-g-SS-PEG micelles were
studied using a dialysis tube (MWCO 12 000 Da) at 37 °C in two
different media, i.e., PB (100 mM, pH 7.4) or PB (100 mM, pH 7.4)
with 10 mM DTT. In order to acquire sink conditions, drug release
studies were performed at low drug loading contents (ca. 2.9 wt %)
and with 0.6 mL of micelle suspension dialysis against 20 mL of the
same medium. At desired time intervals, 5.0 mL of release media was
taken out and replenished with an equal volume of fresh media. The
amount of DOX released was determined by using fluorescence
(FLS920) measurement (excitation at 480 nm). The release
experiments were conducted in triplicate. The results presented are
the average data. For determination of the drug loading content, DOX-
loaded micellar suspensions were freeze-dried, then dissolved in
DMSO, and analyzed with fluorescence spectroscopy. A calibration
curve was obtained using DOX/DMSO solutions with different DOX
concentrations. To determine the amount of DOX released,
calibration curves were run with DOX/phosphate buffer solution
(pH 7.4, 100 mM) with different DOX concentrations. The emission
at 600 nm was recorded. Release experiments were conducted in
Ring-Opening Copolymerization. The ring-opening copolymer-
ization of ε-CL and PDSC was carried out in toluene at 100 °C using
isopropanol as an initiator and Sn(Oct)2 as a catalyst. The following is
a typical example. In a glovebox under a nitrogen atmosphere, to a
stirred solution of ε-CL (0.757 g, 6.64 mmol) and PDSC (0.200 g,
0.74 mmol) in toluene (8 mL) was quickly added isopropanol stock
solution (0.18 mL, 0.2 M) and Sn(Oct)2 stock solution (0.20 mL, 100
mM). The reaction vessel was sealed and placed in an oil bath
thermostated at 100 °C with magnetic stirring for 24 h. A sample was
1
taken for determination of the monomer conversion using H NMR.
The resulting P(CL-co-PDSC) copolymer was isolated by precipitation
in cold diethyl ether and dried in vacuo at room temperature.
Synthesis of PCL-g-SS-PEG by Thiol−Disulfide Exchange
Reaction. To a solution of P(CL-co-PDSC) 5.4% (50 mg, 20 μmol of
PDS groups) and PEG-SH (200 mg, 40 μmol) in DCM under a
nitrogen atmosphere was added a catalytic amount of acetic acid. The
reaction was allowed to proceed with stirring for 48 h at room
temperature. The resulting polymer was isolated by precipitation from
cold diethyl ether, washed with excess ethanol, and dried in vacuo at
room temperature.
triplicate. The results are presented as the average
standard
deviation.
Drug loading content (DLC) and drug loading efficiency (DLE)
were calculated according to the following formulas:
1
weight of loaded drug
total weight of polymer and loaded drug
Characterization. H NMR spectra were recorded on the Unity
DLC (wt %) =
× 100%
Inova 400 operating at 400 MHz. CDCl3 was used as solvent and the
chemical shifts were calibrated against residual solvent signals. The
molecular weight and polydispersity of the copolymers were
determined by a Waters 1515 gel permeation chromatograph (GPC)
weight of loaded drug
weight of drug in feed
DLE (%) =
× 100%
́
instrument equipped with two linear PLgel columns (500 Å and
Mixed-C) following a guard column and a differential refractive index
detector. The measurements were performed using CHCl3 as the
eluent at a flow rate of 0.5 mL/min at 30 °C and a series of narrow
polystyrene standards for the calibration of the columns.
Confocal Microscopy Observation of HeLa Cells Incubated
with DOX-Loaded Micelles. HeLa cells were plated on microscope
slides in a 24-well plate (5 × 104 cells/well) using DMEM medium
containing 10% FBS. The cells were incubated with prescribed
amounts of DOX-loaded PCL-g-SS-PEG 2 micelles or free DOX at 37
°C and 5% CO2. After incubation for 4 and 8 h, the culture medium
was removed and the cells on microscope plates were washed three
times with PBS. The cells were fixed with 4% paraformaldehyde, and
the cell nuclei were stained with DAPI. Fluorescence images of cells
were obtained with a Nikon Digital Eclipse C1si confocal laser
scanning microscope (CLSM, Nikon).
MTT Assays. The cytotoxicity of PCL-g-SS-PEG micelles and
DOX-loaded PCL-g-SS-PEG micelles was studied by MTT assays
using HeLa cells. Cells were seeded onto a 96-well plate at a density of
1 × 104 cells per well in 100 μL of Dulbecco’s Modified Eagle medium
(DMEM) containing 10% FBS and incubated for 24 h (37 °C, 5%
CO2). The medium was replaced by 90 μL of fresh DMEM medium
containing 10% FBS, and then 10 μL samples of various
concentrations (0.5−10 mg/mL) of the micelle suspensions in
phosphate buffer (10 mM, pH 7.4) were added. The cells were
incubated for another 48 h, the medium was aspirated and replaced by
100 μL of fresh medium, and 10 μL of MTT solution (5 mg/mL) was
added. The cells were incubated for 4 h, and then the medium was
aspirated and replaced by 150 μL of DMSO to dissolve the resulting
purple crystals. The optical densities at 570 nm were measured using a
BioTek microplate reader. Cells cultured in DMEM medium
containing 10% FBS (without exposure to micelles) were used as
controls.
Micelle Formation and Critical Micelle Concentration.
Micelles were typically prepared under stirring by dropwise addition
of 2 mL of phosphate buffer (10 mM, pH 7.4) to 0.2 mL of
amphiphilic PCL-g-SS-PEG copolymer solution (0.5 wt %) in THF at
room temperature. The resulting solution was stirred overnight under
reduced pressure to allow complete evaporation of THF. The size and
size distribution of the micelles were determined by dynamic light
scattering (DLS). The micellar suspension was filtered through a 450
nm syringe filter before measurements. Measurements were carried
out at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments
equipped with a 633 nm He−Ne laser using backscattering detection.
The critical micelle concentration (cmc) was determined using
pyrene as a fluorescence probe. The concentration of graft copolymer
was varied from 2.0 × 10−5 to 0.2 mg/mL, and the concentration of
pyrene was fixed at 1.0 μM. Fluorescence spectra were recorded using
a FLS920 fluorescence spectrometer and an excitation wavelength of
330 nm. Fluorescence emissions at 372 and 383 nm were monitored.
The cmc was estimated as the cross-point when extrapolating the
intensity ratio I372/I383 at low and high concentration regions.
Redox-Triggered Change of Micelle Sizes. The change in size,
size distribution, and light scattering of the micelles in response to 10
mM DTT was followed by DLS at 37 °C. The micelles were prepared
as above. PCL-g-SS-PEG micellar suspension (0.2 mg/mL) was
divided into two aliquots. To one of the aliquots (1.0 mL) was added
10 μL of DTT (1.0 M), which gave a final DTT concentration of 10
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at δ 4.15−4.38 while signals at δ 3.67 owing to methylene
protons next to hydroxyl groups shifted to δ 4.15−4.38 (Figure
1B). The integral ratio of resonances at δ 4.15−4.38 and δ 8.51,
7.65, 7.54, and 7.14 (pyridine protons) was close to the
theoretical value of 1:1, indicating successful synthesis of PDSC
monomer. The structure of PDSC was further confirmed by
mass and elemental analyses. Hedrick et al. reported versatile
synthesis of functionalized cyclic carbonate monomers through
a pentafluorophenyl ester intermediate, which reacting with S-
2-pyridyl-S′-2-hydroxyethyl disulfide could yield 2-(pyridin-2-
yl-disulfanyl)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate.49
However, ring-opening (co)polymerization of pyridyl disul-
fide-functionalized carbonate monomer to synthesize biode-
gradable polymers with pendant PDS groups and further
reduction-sensitive amphiphilic biodegradable graft copolymers
has not yet been reported.
RESULTS AND DISCUSSION
■
Synthesis of PDSC Monomer. Pyridyl disulfide-function-
alized cyclic carbonate (PDSC) was prepared in four
straightforward steps: (i) 3-methyl-3-oxetanemethanol was
treated with HBr to afford bromo-diol; (ii) bromo-diol was
reacted with sodium hydrosulfide to yield mercapto-diol; (iii)
thiol-exchange reaction between mercapto-diol and excess 2,2′-
dipyridyl disulfide gave rise to pyridyl disulfide-diol; and (iv)
similar to reported procedure for other cyclic carbonate
monomers27,31 cyclization of pyridyl disulfide-diol using ethyl
chloroformate in dilute anhydrous THF solution at 0 °C with
dropwise addition of triethylamine furnished PDSC monomer
in decent yields (Scheme 2). PDSC monomer could be readily
Scheme 2. Synthetic Pathway for Pyridyl Disulfide-
Functionalized Cyclic Carbonate Monomer (PDSC)
a
Synthesis of Functional PCL Containing Pendant PDS
Groups. The aim of this study was to provide a versatile
approach to PDS-functionalized biodegradable polymers and
reduction-sensitive biodegradable graft copolymers by post-
polymerization modification with thiol-containing molecules.
To prove our concept, we synthesized PDS-functionalized PCL
as an example (Scheme 3). The results showed that PDSC was
readily copolymerized with ε-CL in toluene at 100 °C using
isopropanol as an initiator and Sn(Oct)2 as a catalyst to afford
1
PDS-functionalized PCL in good yields (Table 1). H NMR
a
showed that signals characteristic of both CL units (δ 4.04,
2.30, 1.64, and 1.38) and PDSC units (δ 8.46, 7.65, 7.10, 4.05,
3.02, and 1.10) were clearly detected (Figure 2). Importantly,
signals due to pyridine protons maintained at δ 8.46, 7.65, and
7.10, indicating that PDS functional groups were intact during
copolymerization and subsequent work-up procedures. The
copolymer compositions could be determined by comparing
signals at δ 2.30 (methylene protons next to carbonyl in CL
units) and 3.02 (methylene protons next to disulfide bond in
PDSC units). The results showed that P(CL-co-PDSC)
copolymers with PDSC contents (FPDSC) ranging from 2.5 to
10.3 mol % were obtained at PDSC monomer feed ratio
(fPDSC) varying from 5 to 20 mol % (Table 1). FPDSC though
Conditions: (i) 40% hydrobromic acid, 0 °C, 5 h, THF (yield 96%);
(ii) sodium hydrosulfide, 75 °C, overnight, DMF (yield 52%); (iii)
2,2′-dithiodipyridine, catalytic amount of glacial acetic acid, room
temperature, overnight, methanol (yield 65%); (iv) ethyl chlorofor-
mate, Et3N, 0 °C, 4 h, THF (yield 50%).
recrystallized from dry THF/diethyl ether to yield pure white
crystals. H NMR spectrum of pyridyl disulfide-diol showed
signals of pyridine protons at δ 8.47, 7.62, 7.41 and 7.17 as well
as resonances at δ 3.67, 3.25, and 0.84 attributable to methylene
protons next to hydroxyl groups, methylene protons neighbor-
ing the disulfide bond, and methyl protons, respectively (Figure
1
1
1A). H NMR of PDSC revealed that signals assignable to the
methylene protons next to the carbonate were clearly detected
lower than feed ratios increased linearly with increasing f PDSC
,
denoting good control over PDS functionality. The number-
average molecular weights (Mn) estimated from 1H NMR end-
group analysis by comparing the integrals of peaks at 2.30
(methylene protons next to carbonyl in CL units) and 3.02
(methylene protons next to disulfide bond in PDSC units) with
δ 1.22 (methyl protons of isopropyl ester end group) were
close to the theoretical values (Table 1). Gel permeation
chromatography (GPC) measurements using polystyrene as
standards showed that these P(CL-co-PDSC) copolymers had
moderate polydispersities (PDI) of 1.33−1.61 and Mn
increased with increasing monomer-to-initiator ratios (Table
1). Therefore, copolymerization of ε-CL and PDSC provides
PDS-functionalized PCL with controlled molecular weights and
functionalities.
Synthesis of PCL-g-SS-PEG Graft Copolymer by
Thiol−Disulfide Exchange Reaction. It is well-known that
the pyridyl disulfide group has a high reactivity toward thiol-
containing molecules via thiol−disulfide exchange reaction to
form disulfide linkages or cross-links.50 Here, two P(CL-co-
PDSC) copolymers with similar PDS functionality (5.2−5.4%)
but different Mn values (12.6 and 22.3 kg/mol; Table 1, entries
2 and 4) were chosen to react with thiolated PEG (Mn = 5000).
The reaction was performed at a PEG-SH/PDS molar ratio of
1
Figure 1. H NMR spectra (400 MHz, CDCl3) of pyridyl disulfide-
diol (A) and PDSC monomer (B).
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a
Scheme 3. Synthesis of P(CL-co-PDSC) Copolymer and Reduction-Sensitive PCL-g-SS-PEG Graft Copolymer
a
Conditions: (i) ring-opening copolymerization of ε-CL and PDSC using isopropanol as an initiator and Sn(Oct)2 as a catalyst in toluene at 100 °C;
(ii) thiol−disulfide exchange reaction with thiolated PEG using catalytic amount of acetic acid in DCM at room temperature.
a
Table 1. Synthesis of Pyridyl Disulfide-Functionalized PCL
Mn (×103)
b
c
f
F
PDI
e
d
e
entry [M]/[I] (%)
(%)
design 1H NMR GPC
GPC
1
2
3
4
5
6
100
100
200
200
200
400
5
10
5
3.2
5.2
12.2
13.0
24.4
25.9
29.1
44.7
12.0
12.6
20.1
22.3
21.4
18.6
19.2
30.9
29.6
29.8
55.5
1.42
1.40
1.55
1.52
1.33
1.61
2.5
10
20
5
5.4
10.3
2.7
a
The copolymerization was carried out in toluene at 100 °C using
isopropanol as an initiator and Sn(Oct)2 as a catalyst at a total
b
monomer-to-initiator ratio of 100/1, 200/1, and 400/1. Molar
1
Figure 3. H NMR spectrum (400 MHz, CDCl3) of PCL-g-SS-PEG
c
fraction of PDSC monomer in feed. Molar fraction of PDSC units in
the resulting copolymer determined by H NMR. Estimated by H
NMR end-group analysis. Determined by GPC (eluent: chloroform;
copolymer (Table 2, entry 2).
d
1
1
e
flow rate: 0.5 mL/min; standards: polystyrene).
(methyl protons of PDSC units) indicated that ca. 86−89%
pyridyl disulfide groups have reacted with thiolated PEG to
afford amphiphilic PCL-g-SS-PEG graft copolymers (Table 2).
The Mn of these two copolymers was calculated by 1H NMR to
be 35.6 and 66.0 kg/mol (denoted as PCL-g-SS-PEG 1 and 2,
respectively). GPC curves of both graft copolymers remained
unimodal with a PDI of 1.95−2.02 (Table 2). The Mn
1
determined by GPC was lower than that calculated from H
NMR, likely due to their graft structure. The deviation of Mn
could be also partly because polystyrene was used as a standard
for GPC measurements. It is evident that PCL-g-SS-PEG graft
copolymers can be readily prepared from PDS-functionalized
PCL via thiol−disulfide exchange reaction.
1
Figure 2. H NMR spectrum (400 MHz, CDCl3) of P(CL-co-PDSC)
Formation and Reduction-Triggered Disruption of
Micelles. Micelles of PCL-g-SS-PEG copolymers were
prepared by the solvent exchange method. Dynamic light
scattering (DLS) measurements showed that PCL-g-SS-PEG
copolymers formed micelles with average sizes of 110−120 nm
and low polydispersities (PDIs) of 0.09−0.12 (Figure 4A). The
critical micelle concentration (cmc) was determined using
pyrene as a probe in 10 mM PB at pH 7.4 (Figure 4B). The
results showed that PCL-g-SS-PEG 1 and 2 had particularly low
cmc of 0.92 and 0.87 mg/L, respectively (Table 2), supporting
copolymer (Table 1, entry 4).
2/1 in DCM at room temperature for 48 h using a catalytic
amount of acetic acid (Scheme 3). The free PEG was removed
by washing with excess ethanol. H NMR displayed that new
signals corresponding to methylene protons of PEG appeared
at δ 3.65 while peaks due to pyridine groups at δ 8.46, 7.65 and
7.10, were diminishing (Figure 3). The comparison of integrals
of resonances at δ 3.36 (methoxyl protons of PEG) and δ 1.02
1
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Table 2. Synthesis of PCL-g-SS-PEG Graft Copolymer
P(CL-co-PDSC)
Mn (×103)
a
b
c
c
d
PCL-g-SS-PEG copolymer
FPDSC (%)
Mn (×103)
PEG conjugation (%)
1H NMR
GPC
PDI GPC
cmc (mg/L)
1
2
5.2
5.4
12.6
86
35.6
66.0
28.6
43.9
2.02
1.95
0.92
0.87
22.3
89
a
b
1
1
Percentage of PEG conjugation determined by H NMR. Determined by H NMR according to the integral ratio of resonances at δ 3.36
c
(methoxyl protons attributed to PEG) and δ 1.02 (methyl protons attributed to initial PDSC units). Determined by GPC (eluent: chloroform; flow
rate: 0.5 mL/min; standards: polystyrene). Critical micelle concentration (cmc) determined using pyrene as fluorescence probe.
d
Figure 4. Size distribution of PCL-g-SS-PEG graft copolymer micelles
determined by DLS (A) and the fluorescence intensity ratio I372/I383 of
pyrene as a function of PCL-g-SS-PEG 2 concentration (B).
Figure 5. Change of particle sizes (A) and PDI and light scattering
intensity (B) of PCL-g-SS-PEG 2 micelles in time in response to 10
mM DTT.
Table 3. DOX Loading Content and Loading Efficiency of
Polymeric Micelles
that amphiphilic graft copolymers form particularly stable
micelles.
The reduction-sensitive property of PCL-g-SS-PEG 2
micelles was studied using DLS by monitoring change of
micelle sizes in response to 10 mM dithiothreitol (DTT) in PB
buffer (pH 7.4, 10 mM). The results showed that micelles were
quickly destabilized by DTT to form large aggregates of about
600 nm in 4 h (Figure 5A), accompanied by decrease of light
scattering intensity and increase of PDI (Figure 5B). These
results are in line with our previous observations for PEG-SS-
PCL micelles.48 In contrast, no change in micelle sizes and
PDIs was discerned after 24 h in the absence of DTT under
otherwise the same conditions. DTT-triggered cleavage of
disulfide bonds in PCL-g-SS-PEG 2 micelles was also confirmed
by GPC in that PCL-g-SS-PEG 2 micelles following treatment
with DTT gave two distributions corresponding to P(CL-co-
PDSC) copolymer (Table 1, entry 4) and PEG, respectively.
Loading and Triggered Release of DOX. DOX was
loaded into PCL-g-SS-PEG micelles at theoretical drug loading
contents (DLC) of 4.8, 9.1, and 16.7 wt %. Interestingly, PCL-
g-SS-PEG 1 and 2 micelles exhibited similar DOX loading levels
and particles sizes (Table 3), indicating that molecular weights
of amphiphilic graft copolymers have little influences on drug
loading behaviors. The drug loading efficiencies (DLE) ranged
from 54.1 to 71.1%, which decreased with increasing DLC.
DLC (wt %)
a
polymer
theory determined
DLE (%)
size (nm)
56.2 0.5
PDI
0.13
PCL-g-SS-
PEG 1
4.8
3.3 0.07
68.2 1.5
9.1
16.7
4.8
5.7 0.09
9.8 0.12
3.4 0.06
60.3 1.6
54.1 2.0
71.1 1.4
68.7 1.5
116.1 2.6
58.9 0.4
0.17
0.23
0.12
PCL-g-SS-
PEG 2
9.1
5.8 0.09
61.5 1.7
55.9 2.1
64.8 1.3
0.14
0.20
16.7
10.1 0.13
108.2 2.1
a
Drug loading content for DOX was determined by fluorescence
measurements.
Moreover, DOX-loaded PCL-g-SS-PEG micelles had low PDIs
of 0.12−0.23 and particle sizes ranging from 56.2 to 116.1 nm
depending on DOX loading levels. The micelle size became
smaller at a low drug loading level likely due to existence of
effective interactions between micellar core and DOX.
The in vitro release of DOX from PCL-g-SS-PEG 2 micelles
was investigated using a dialysis tube (MWCO 12 000 Da) in
PB buffer (pH 7.4, 100 mM) at 37 °C in either the presence or
absence of 10 mM DTT. Interestingly, the results showed that
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DOX was released rapidly in response to 10 mM DTT, in
which ca. 62.1% and 82.0% DOX was released in 4 and 12 h,
respectively (Figure 6). In contrast, minimal drug release (ca.
Figure 6. Reduction-triggered release of DOX from PCL-g-SS-PEG 2
micelles in phosphate buffer (pH 7.4, 100 mM). The release of free
DOX was used as a control.
17.5%) was observed within 24 h in the absence of 10 mM
DTT under otherwise the same conditions. This redox-
triggered drug release behavior has been reported for various
shell-sheddable micelles.48,51−54 This fast drug release is likely
because micelles following shedding of PEG shells are
destabilized and form drug diffusion channels. These
reduction-sensitive biodegradable graft copolymer micelles
with easy synthesis, enhanced stability, and redox-triggered
rapid drug release behaviors are highly interesting as “smart”
vehicles for active intracellular drug release.
Figure 7. CLSM images of HeLa cells following 4 or 8 h incubation
with DOX-loaded PCL-g-SS-PEG 2 micelles and free DOX (10 μg/
mL). For each panel, the images from left to right showed cell nuclei
stained by DOX fluorescence in cells (red), DAPI (blue), overlays of
both images. The scale bars correspond to 25 μm in all the images. (A)
DOX-loaded PCL-g-SS-PEG 2 micelles, 4 h; (B) DOX-loaded PCL-g-
SS-PEG 2 micelles, 8 h; (C) free DOX, 4 h.
Intracellular Drug Release and Antitumor Activity of
DOX-Loaded PCL-g-SS-PEG Micelles. The cellular uptake
and intracellular drug release behaviors of DOX-loaded PCL-g-
SS-PEG micelles were studied using CLSM. The cell nuclei
were stained with DAPI (blue). Interestingly, the results
showed that DOX-loaded PCL-g-SS-PEG micelles delivered
and released DOX into the perinuclei and nuclei regions
following 4 h incubation (Figure 7A), indicating fast internal-
ization of micelles and rapid release of DOX inside cells. At a
longer incubation time of 8 h, DOX was fully delivered into the
nuclei of HeLa cells (Figure 7B). It is remarkable to note that
DOX-loaded PCL-g-SS-PEG micelles displayed a similar
intracellular DOX release rate to free DOX (Figure 7C).
MTT assays revealed that both PCL-g-SS-PEG 1 and 2
micelles were nontoxic to HeLa cells with cell viabilities more
than 98% up to a tested concentration of 1.0 mg/mL (Figure
8A), confirming that these biodegradable graft copolymer
micelles have excellent biocompatibility. DOX-loaded PCL-g-
SS-PEG micelles, however, displayed pronounced antitumor
activity toward HeLa cells following 48 h incubation (Figure
8B). DOX-loaded PCL-g-SS-PEG 1 and 2 micelles had low
IC50 (half-maximal inhibitory concentration) of 0.95 and 0.82
μg DOX equiv/mL, respectively, which was close to that
observed for free DOX (IC50 = 0.46 μg/mL). It should further
be noted that the antitumor activity of graft copolymer micellar
drugs could further be enhanced by installing a tumor-targeting
ligand like aptamer, peptide, and antibody fragment that
facilitates efficient and specific tumor cell uptake of micelles.
These redox-sensitive biodegradable graft polymer micelles
with excellent biocompatibility, low cmc, and intracellular
redox-responsive drug release are highly promising for targeted
cancer therapy.
Figure 8. (A) Cytotoxicity of PCL-g-SS-PEG micelles to HeLa cells
following 48 h incubation. (B) Viabilities of HeLa cells following 48 h
incubation with DOX-loaded PCL-g-SS-PEG micelles and free DOX
as a function of DOX dosages. All the data are presented as the average
standard deviation (n = 4).
CONCLUSIONS
■
We have demonstrated that pyridyl disulfide-functionalized
poly(ε-caprolactone) can be conveniently prepared with
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containing pendant multiple pyridyl disulfide groups. These
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water. Interestingly, these graft copolymer micelles exhibit
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AUTHOR INFORMATION
(27) Chen, W.; Yang, H.; Wang, R.; Cheng, R.; Meng, F.; Wei, W.;
Zhong, Z. Macromolecules 2010, 43, 201−207.
■
Corresponding Author
(28) Onbulak, S.; Tempelaar, S.; Pounder, R. J.; Gok, O.; Sanyal, R.;
Dove, A. P.; Sanyal, A. Macromolecules 2012, 45, 1715−1722.
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*Tel +86-512-65880098; e-mail zyzhong@suda.edu.cn.
Notes
The authors declare no competing financial interest.
(30) Tempelaar, S.; Mespouille, L.; Coulembier, O.; Dubois, P.;
Dove, A. P. Chem. Soc. Rev. 2013, 42, 1312−1336.
(31) Chen, W.; Meng, F. H.; Li, F.; Ji, S. J.; Zhong, Z. Y.
Biomacromolecules 2009, 10, 1727−1735.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (NSFC 51003070, 51103093, 51173126,
and 51273139), the National Science Fund for Distinguished
Young Scholars (51225302), Science and Technology
Innovation Training Program (STITP 201210285020), and a
Project Funded by the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions.
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