Synthesis and pH-responsive assembly of methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) with pendant carboxyl groups

Article abstract of DOI:10.1002/pola.26987

pH-responsive methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) bearing pendant carboxyl groups mPEG-b-P(2-CCL-co-6-CCL) was synthesized based on our newly monomer benzyloxycarbonylmethly functionalized ε-caprolactone. Their structure was confirmed by ¹H NMR, ¹³C NMR, and Fourier transform infrared spectrum spectra. In addition, SEC results indicated that the copolymers had a relatively narrow polydispersity. WXRD and DSC demonstrated that the introduction of carboxymethyl groups had significant effect on the crystallinity of the copolymers. Furthermore, the solution behavior of mPEG-b-P(2-CCL-co-6-CCL) has been studied by various methods. The results indicated that mPEG-b-P(2-CCL-co-6-CCL) had a rich pH-responsive behavior and the micelles could be formed by pH induction, and the mPEG-b-P(2-CCL-co-6-CCL) could existed as unimers, micelles or large aggregates in different pH range accordingly. The mechanism of which was supposed to depend on the counteraction between the hydrophobic interaction from PCL and the ionization of the carboxyl groups along the polymer chain. Moreover, the mPEG-b-P(2-CCL-co-6-CCL) copolymers displayed good biocompatibility according to the preliminary cytotoxicity study. Copyright

Full text of DOI:10.1002/pola.26987

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Synthesis and pH-Responsive Assembly of Methoxy Poly(ethylene
glycol)-b-poly(e-caprolactone) with Pendant Carboxyl Groups
Yan Zhang,1,2 Jinhong Li,1 Zhengzhen Du,1 Meidong Lang1
1Shanghai Key Laboratory of Advanced Polymeric Materials, Key Laboratory for Ultrafine Materials of Ministry of Education,
School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
2State Key Laboratory of Molecular Engineering of Polymers (Fudan University), Shanghai 200433, China
Correspondence to: Y. Zhang (E-mail: zhang_yan@ecust.edu.cn) or M. Lang (E-mail: mdlang@ecust.edu.cn)
Received 23 August 2013; accepted 15 October 2013; published online 11 November 2013
DOI: 10.1002/pola.26987
ABSTRACT: pH-responsive methoxy poly(ethylene glycol)-b-
poly(e-caprolactone) bearing pendant carboxyl groups mPEG-b-
P(2-CCL-co-6-CCL) was synthesized based on our newly mono-
mer benzyloxycarbonylmethly functionalized e-caprolactone.
Their structure was confirmed by 1H NMR, 13C NMR, and Fou-
rier transform infrared spectrum spectra. In addition, SEC
results indicated that the copolymers had a relatively narrow
polydispersity. WXRD and DSC demonstrated that the introduc-
tion of carboxymethyl groups had significant effect on the crys-
tallinity of the copolymers. Furthermore, the solution behavior
of mPEG-b-P(2-CCL-co-6-CCL) has been studied by various
methods. The results indicated that mPEG-b-P(2-CCL-co-6-CCL)
had a rich pH-responsive behavior and the micelles could be
formed by pH induction, and the mPEG-b-P(2-CCL-co-6-CCL)
could existed as unimers, micelles or large aggregates in dif-
ferent pH range accordingly. The mechanism of which was
supposed to depend on the counteraction between the hydro-
phobic interaction from PCL and the ionization of the carboxyl
groups along the polymer chain. Moreover, the mPEG-b-P(2-
CCL-co-6-CCL) copolymers displayed good biocompatibility
C
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according to the preliminary cytotoxicity study.
2013 Wiley
Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52,
188–199
KEYWORDS: hydrophilic polymers; micelles; pendant carboxyl
groups; pH-responsive; poly(e-caprolactone); polyesters; water-
soluble
ity, and lack of functional groups along the PCL backbone
have limited its further application. The modification to PCL
has been widely studied in recent years, such as the intro-
duction of pendant groups by ring-opening polymerization of
functionalized caprolactone monomer or by grafting func-
tional groups onto the performed polyester.19,20 Generally,
the hydrophilic groups such as the amino groups, hydroxyl
groups, and carboxyl groups were incorporated into the PCL
to improve its hydrophilicity. For examples, functionalized
PCL bearing azide substituent was obtained based on a-
chloro-e-caprolactone (aCleCL) by reaction with sodium
azide, then the tertiaryamine groups were introduced to the
PCL.21,22 In our previous study, PCL bearing pendant amino
groups has been synthesized by ring-opening polymerization
of c-(carbamic acid benzyl ester)-e-caprolactone and used as
anion drug carrier.23,24 Poly(c-hydroxy-e-caprolactone) was
prepared by ring-opening polymerization of functionalized c-
triethylsilyoxy-e-caprolactone (c-Et3SiOCL) monomer, fol-
lowed by hydrolyzation of the triethylsilanolate groups.25
However, there have been few reports on the pH sensitive
hydrophilic PCL with anionic groups. For example, carboxyl
INTRODUCTION Environmental responsive polymers, the solu-
bility and conformation of which could be adjusted by tem-
perature,1–3 pH,4,5 ionic strength,6,7 and electric or magnetic
fields,8,9 have developed greatly in recent years because of
their prospective uses in biomedical and other applications,
such as drug delivery,10 tissue engineering,11 electro-optical
devices,12 coatings, and textiles.13 Especially, they have been
most thoroughly studied in self-assembly and stimulus trig-
gered drug delivery system due to their distinct advantages,
such as improved drug solubility and controlled release, and
so forth.4 pH-Sensitive self-assemblies could be regulated by
the protonation or deprotonation of the weakly basic or acidic
groups (such as amino groups or carboxyl groups) along the
polymer chain,14–16 and they have been widely exploited as
drug delivery system due to the numerous pH gradients exist-
ing in both normal and pathological states.16
Poly(e-caprolactone) (PCL) is a popular FDA-approved candi-
date material in drug delivery system due to its biodegrad-
ability, biocompatibility, and higher permeability to
drugs.17,18 However, the high crystallinity, poor hydrophilic-
Additional Supporting Information may be found in the online version of this article.
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groups were introduced to the PCL main chain by the ring-
opening polymerization of a-benzyl carboxylate-e-caprolac-
tone. The article focused on the environmental responsive
properties of the polymers having different ratios of carboxyl
to benzyl carboxylate groups on the hydrophobic block.
However, the carboxyl groups were directly suspended on
the PCL backbone without any spacer.26,27
The molecular weight and molecular weight distribution of
mPEG-b-P(2-BCL-co-6-BCL) were determined by size exclu-
sion chromatography (SEC, Waters 1515 HPLC) using THF as
eluent at a flow rate of 1.0 mL min21. The column system
was calibrated by polystyrene standards. However, for
mPEG-b-P(2-CCL-co-6-CCL) copolymers, the molecular weight
and molecular weight distribution were measured by size
exclusion chromatography (PL-GPC 50 Plus, Varian) used
DMF containing 0.01 mol L21 lithium bromide as eluent
against PMMA standards.
Herein, we reported new functional e-caprolactone monomers
bearing benzyloxycarbonylmethly group on 2-/6-position of
caprolactone. Water-soluble copolymers methoxy poly(ethyl-
ene glycol)-b-P(2-carboxymethyl-e-caprolactone-co-6-carboxy-
methyl-e-caprolactone) (mPEG-b-P(2-CCL-co-6-CCL)) were suc-
cessfully synthesized by ring-opening polymerization using
methoxy poly(ethylene glycol) (mPEG) as macroinitiator. The
benzyl carboxylate groups were totally removed and the car-
boxyl groups were attached to the PCL backbone by methyl-
ene spacer, which was beneficial for the further modification
of the polymer and improvement of the hydrophilicity accord-
ingly. Furthermore, the self-assembly of the copolymer by pH
induction have been studied by various methods and the
mechanism was detailed clarified. It was proposed that the
copolymer existed as unimers, micelles, or large aggregates in
different pH range depended on the counteraction between
the ionization of carboxyl groups and the hydrophobic interac-
tion of the PCL. Meanwhile, mPEG-b-P(2-CCL-co-6-CCL) copol-
ymer exhibited good biocompatibility, and nontoxicity
according to MTT assay. Therefore, the water-soluble mPEG-b-
poly(e-caprolactone) bearing anionic carboxyl groups has a
great potential for biomedical application.
Differential scanning calorimetry (DSC) analysis was carried
out on a TA Instruments modulated DSC2910 apparatus
under a nitrogen flow (10 mL min21). The samples were
first heated from 25 to 100 ꢀC, followed by cooling to ꢀ280
ꢀC to erase the thermal history and then heated to 100 C at
21
ꢀ
a rate of 10 C min
.
Wide-angle X-ray diffraction (WXRD) was performed over
the range 2h from 10 to 40o on a Bruker AXS: D8 Focus
equipped with graphite monochromatized Cu Ka radiation.
The critical micelle concentration (CMC) was determined by
fluorescence probe method.28 A predetermined amount of
pyrene solution at a concentration of 6 3 1026 M in acetone
was added into a 10 mL of volumetric flask, and the acetone
was allowed to evaporate. About 10 mL of micelle solution at
different concentrations (1–1024 mg mL21) were then poured
into the volumetric flasks. It should be noted that all the micelle
solution was allowed to equilibrate at room temperature over
night before measurement. The final pyrene concentration was
6 3 1027 M, and k 5 337 nm was chosen as excitation wave-
length. Both excitation and emission slits widths were set at 3
nm. All experiments were performed on a Fluorolog-3-P fluo-
rescence spectrophotometer (Jobin Yvon, France).
EXPERIMENTAL
Materials
mPEG (Mn 5 2000 and 5000 Da, purity: 99.99%) were bought
from Aldrich and were dried in toluene by an azeotropic dis-
tillation before use, (II)22-ethylhexanoate (Sn(Oct)2, 95%)
and charcoal coated with palladium (10 wt %) were obtained
from Aldrich without further purification. Benzyl bromoace-
tate and 3-chloroperoxybenzoic acid (m-CPBA) were pur-
chased from Aladdin and used as received. Cyclohexanone
and morpholine were distilled under reduced pressure before
use. Dulbecco’s modified eagle medium (DMEM) was pur-
chased from Gibco, and fetal bovine serum (FBS) was bought
from Hyclone. Dialysis bag was purchased from Shanghai
Greenbird. Deionized water used in this study was purified
with a Millipore Mill-Q system. Other reagents were obtained
from Shanghai Lingfeng Chemical Reagent and used as
received.
The pKa of mPEG-b-P(2-CCL-co-6-CCL) samples were given
by potentiometric titration. The diblock copolymer was dis-
solved in aqueous NaOH solution at pH 5 13. Then, the solu-
tion was titrated by the dropwise addition of aqueous HCl
solution (0.1 M) until the pH of the solution kept almost
constant. All the pH values of the solution were monitored
by a PH620 pH meter. Moreover, the turbidity of the samples
was measured at varying pH values using a UV spectropho-
tometer (SP-1900UV) at a 600 nm wavelength.
The zeta potential (f) of mPEG-b-P(2-CCL-co-6-CCL) copoly-
mers were measured on a Zetasizer Nano-ZS (Malvern) at
different pH. The pH of the solution was adjusted by using
NaOH or HCl solution according to the requirement.
Characterizations
1H and 13C NMR spectra were performed on a Bruker AV
apparatus at frequencies of 400 MHz, using CDCl3 for mPEG-
b-P(2-BCL-co-6-BCL) and d6-DMSO for mPEG-b-P(2-CCL-co-6-
CCL) as solvents. Fourier transform infrared spectrum (FTIR)
was recorded on a Nicolet 5700 spectrometer in the range
of 4000 to 5000 cm21. The Mass Spectrometry measurement
was performed on a Micromass GUT apparatus using elec-
tron impact (EI) method.
The size and size distribution of micelles were measured by
dynamic light scattering (DLS), using an ALV/5000E laser light
scattering spectrometers at room temperature and the scatter-
ing angle was 90ꢀ. All samples were purified by filtering
through a 0.45 mm filter to remove dust before measurement.
Samples for transmission electron microscopy (TEM) obser-
vation were prepared by placing 1 mg mL21 micelle solution
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on copper-coated grid. The grid was held horizontally for 20
s to allow the colloidal aggregates to settle down. A drop of
2% solution of phosphotungstic acid in phosphate-buffered
saline (PBS) (pH 5 7.0) was then added to provide negative
stain. After 5 min, excess fluid was removed with filter
paper. The samples were then air-dried and loaded in TEM
instrument (JEOL/JEM-1400E XII) operated at an accelera-
tion voltage of 60 keV.
Synthesis of mPEG-b-P(2-BCL-co-6-BCL) Copolymers
Methoxy poly(ethylene glycol)-b-poly(2-benzyloxycarbonyl-
methyl-e-caprolactone-co-6-benzyloxy-carbonylmethyl-e-cap-
rolactone) (mPEG-b-P(2-BCL-co-6-BCL)) diblock copolymers
were synthesized by ring-opening polymerization of 2-BCL
and 6-BCL using mPEG as macroinitiator and Sn(Oct)2 as cat-
alyst.32–35 Typical procedures employed for preparation of
sample 2K-PCCL24 were as follows. mPEG (0.25 g, 0.125
mmol) and BCL (1.0 g, 3.816 mmol, 2-BCL/6-BCL 5 1/9)
were introduced into an argon-purged flame-dried Schlenk
flask equippeꢀd with a magnetic bar. Then, the mixture was
heated to 60 C until the solution became clear. A total of 10
lL Sn(Oct)2 (0.00125 mg, 0.031 mmol) in dry toluene stock
solution was added into the flask under the protection of
argon by using a microliter syringe, and the exhausting–refill-
ing process was repeated for several times to remove toluene.
The Schlenk flask was then sealed with argon. The polymer-
ization reaction was allowed to proceed for 24 h at 130 ꢀC.
After cooling to room temperature, the crude polymer was
precipitated in cold diethyl ether and dried under vacuum.
Synthesis of e-Caprolactone Bearing Pendant
Benzyloxycarbonylmethyl Group
The e-caprolactone bearing pendant benzyloxycarbonyl-
methyl group (BCL) was synthesized by three steps. The fol-
lowing was a typical process to prepare the new monomer.
Briefly, (1) 98 mL (1.0 mol) cyclohexanone and 99 mL (1.2
mol) morpholine were added in 100 mL of dry toluene in a
500 mL round-bottom flask equipped with oil–water sepa-
rator. The mixture was allowed to react at 140 ꢀC used p-
toluenesulfonic acid (0.2 g) as catalyst until the product
water was about 18 mL (1.0 mol). Enamine was obtained
after reduced pressure distillation. (2) Benzyl bromoacetate
(187 g, 0.82 mol) was dropped into the enamine (137 g,
Preparation of mPEG-b-P(2-CCL-co-6-CCL) Copolymers
The copolymers methoxy poly(ethylene glycol)-b-poly(2-car-
boxymethly-e-caprolactone-co-6-carboxymethyl-e-caprolac-
tone) (mPEG-b-P(2-CCL-co-6-CCL)) were obtained by the
catalytic debenzylation of mPEG-b-P(2-BCL-co-6-BCL) in the
presence of hydrogen gas.36 Briefly, in a 100 mL round-bot-
tom flask, mPEG-b-P(2-BCL-co-6-BCL) (0.8 g) was dissolved
in 50 mL mixed solution of tetrahydrofuran and methanol
(volume ratio, 4:1), charcoal coated with palladium (0.16 g)
was then dispersed in this solution. The flask was evacuated,
then connected to a hydrogen depot and filled with hydrogen
gas. The solution was then stirred vigorously with a mag-
netic stirrer and reacted with hydrogen gas at room temper-
ature. After 72 h, the reaction solution was centrifuged at
10,000 rpm to remove the catalyst. The supernatant was col-
lected and concentrated by using a rotary evaporator. The
obtained polymer was dissolved in acetone, and then the
solution was transferred into a dialysis bag (Mw cutoff:
1000–7000 Da) and dialyzed against distilled water for 2
days by replacing the medium with fresh water at a time
interval of about 3–4 h.
ꢀ
0.82 mol) in methanol solution (100 mL) at 80 C. After 1.5
h, the methanol was evaporated, followed by addition of
120-mL redistilled water to hydrolyze the unreacted enam-
ine. After cooling to the room temperature, the reaction
mixture was extracted with diethyl ether. The combined
extracts were dried over Na2SO4 for 24 h and then evapo-
rated. After column chromatography purification, 2-(benzy-
loxycarbonylmethyl)-cyclohexanone (BCP) was obtained
(yield: 44%). (3) m-CPBA (20.46 g, 0.12 mol) was first dis-
solved in dry dichloromethane (50 mL) in a 250 mL round-
bottom flask, BCP (24.28 g, 0.1 mol) in dry dichloromethane
solution (50 mL) was then slowly dropped into the flask in
an ice-water bath under stirring, the solution was allowed
to react for 72 h at room temperature. The reaction solution
was then washed with Na2S2O3-, NaHCO3-, and NaCl-
saturated solution by order to get rid of the unreacted m-
CPBA. The obtained solution was dried over Na2SO4 and the
solvent was evaporated. The resultant product was purified
by column chromatograph and the monomer contained 90%
6-(benzyloxycarbonylmethyl)-e-caprolactone (6-BCL) and
10% 2-(benzyloxycarbonylmethyl)-e-caprolactone (2-BCL)
which was due to the selectivity of Baeyer–Villiger reac-
tion.29,30 It was evident that the secondary or tertiary sub-
stituent was more rapidly to rearrange than the first one
during the peroxidic bond cleavage procedure, according to
the mechanism of the Baeyer–Villiger oxidation.31 The
monomer was analyzed by 1H NMR, 13C NMR, and mass
spectrometry, and the mixture of isomers was used in sub-
Preparation of Polymer Micelles
Polymer micelles were prepared by direct pH-induced micel-
lization method.37 Typically, 50 mg mPEG-b-P(2-CCL-co-6-
CCL) copolymers were dissolved in 35 mL aqueous solution
at pH 5 12. Then the micelles were formed by adjusting the
pH to 4 by adding HCl solution (0.1 M) slowly into the poly-
mer solution under vigorous stirring. After 2 h, the micelle
solution was dialyzed against water at pH 5 4 in a dialysis
bag to remove Na1 and Cl2. Finally, the micelle solution was
fixed at 1 mg mL21, and the pH value was adjusted with HCl
or NaOH solution.
1
sequent polymerizations. H NMR (in CDCl3, 400 MHz, d). d:
7.35 (s, 5H), 5.19 (s, 2H), 4.73–4.79 (m, 1H), 4.30–4.32 (m,
2H), 3.15–3.22 (m, 1H), 2.93–2.99, 2.41–2.47 (m, 1H). 2.56–
2.89 (m, 4H), 1.59–1.96 (m, 6H); 13C NMR (in CDCl3, 400
MHz, d). d: 176.2 (C@O), 174.9, 176.5, 170, 135.9, 135.6,
128.4 (C6H5-) 68.7, 66.7, 41.1, 39.6, 37.5, 34.7, 34.3, 29.7,
28.8, 28.2, 28.1, 22.8; MS (m/z, %) 262 (15), 107 (82), 91
(100), 55 (23).
In Vitro Cytotoxicity
The in vitro cytotoxicity of polymeric micelles was evaluated
by MTT assay with human dermal fibroblast cells (Supplied
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SCHEME 1 Synthetic routes for monomer BCL (a) and mPEG-b-P(2-CCL-co26-CCL) copolymer (b).
by School of Bioengineering, East China University of Science
and Technology). The cells were seeded in 96-well plates
containing 100 lL DMEM complete media (supplemented
with 10% heat-inactivated FBS) at an initial density of 1 3
described in the previous section. A series of mPEG-b-P(2-
CCL-co-6-CCL) copolymers were obtained by ring-opening
polymerization of 2-BCL/6-BCL, initiated with mPEG as
microinitiator and Sn(Oct)2 as catalyst, followed by removal
of the protected groups. The route was shown in Scheme
1(b). The structure of mPEG-b-P(2-CCL-co-6-CCL) copoly-
mers was confirmed by 1H NMR spectrum and FTIR spec-
troscopy. In Figure 1(a), peaks at d 5 3.40 ppm (a) and d5
3.65 ppm (b,c) were assigned to methyl protons
(CH3AOACH2A) and methylene protons (ACH2ACH2AOA)
of PEG repeat units, respectively. Peaks at d 5 5.09 ppm (o,
o0) and d 5 7.33 ppm (p, p0) were attributed to methylene
4
ꢀ
10 cells. The cells were incubated at 37 C under an atmos-
phere of 95% relative humidity and 5% CO2 for 24 h. The
cell culture media was replaced with fresh medium contain-
ing micelle solution at different concentration. After 24 h, 20
lL of 5 mg mL21 MTT solution in PBS was added in each
well, and the plates were further incubated for another 4 h.
Then, the supernatant was removed by centrifugation for 10
min, and 200 lL of DMSO was added in each well. The opti-
cal absorbance was measured in a Bio-Tek Elx800 microplate
reader (Bio-Rad 550) at 499 nm. Cells without materials
were used as control. The relative cell viability (%) was cal-
culated as follows:
protons
(ACH2AC6H5)
and
phenyl
ring
protons
(ACH2AC6H5) of the benzyl carboxylate protected group,
respectively. The peak at d 5 2.56 ppm (j, j0) was assigned to
methylene protons (ACH2COOA) adjacent to the carbonyl of
pendant groups. The peaks at d 5 5.19 ppm (i) and d 5 3.47
ppm (e0) were attributed to the tertiary carbon protons
(ACH(CH2COOCH2C6H5)OA) bearing pendent benzyl carbox-
ylate groups. The peak at d 5 4.05 ppm (i0) was assigned to
the methylene protons (ACH2ACH2AOA) of PCL backbone.
Besides, peaks at d 5 2.14 ppm (e), d 5 2.25 ppm (e0),
d 5 1.56 ppm (f, h, f0, h0) and d 5 1.28 ppm (g, g0) were typi-
cal proton signals of PCL. Compared with Figure 1(a), the
proton signals at 5.09 ppm (o, o0) (ACH2AC6H5) and 7.33
ppm (p, p0) (ACH2AC6H5) disappeared [Fig. 1(b)], indicating
the successful removal of the benzyl carboxylate protection
groups. Even though the peak at d 5 2.57 ppm (j, j0) and sol-
vents peak (d 5 2.50 ppm) overlapped, this did not affect the
structure and molecular weight calculation of the copoly-
mers. Besides, the peak at d 5 3.30 ppm appeared which
may be assigned to the peak of HDO that due to the slow
À
Á
Cell viability ð%Þ5 OD sample =OD control 3100%
(1)
where ODsample represented the absorbance at 499 nm from
sample wells; ODcontrol represented the absorbance at 499
nm from control wells.
RESULTS AND DISCUSSION
Polymer Synthesis
Scheme 1(a) showed the synthetic route for the preparation
of anionic activation of caprolactone monomer and the reac-
tion was a combination of Michael Addition technique and
Baeyer–Villiger reaction, and the two isomers could be
obtained by column chromatography. The structure of the
monomer was confirmed by 1H NMR, 13C NMR, and MS, as
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FIGURE 1 1H NMR spectrum of mPEG-b-P(2-BCL-co26-BCL) (a) in CDCl3, and mPEG-b-P(2-CCL-co26-CCL) (b) in DMSO-d6.
intermolecular rate of exchange.38 In order to confirm the
structure of the polymers, 13C NMR spectrum was also used.
A representative 13C NMR spectrum of the copolymer was
given in Supporting Information Figure S1.
the intensity at 3.65 ppm to that at 7.33 ppm, which related
to the methylene protons (-CH2-CH2-O-) of the EG repeat
units and phenyl ring protons (ACH2C6H5), respectively.
After deprotection, the molecular weights were calculated
according to the following equation:
Figure 2 showed the typical FTIR spectra of mPEG-b-P(2-BCL-
co-6-BCL) and mPEG-b-P(2-CCL-co-6-CCL). Figure 2(a) demon-
strated that the peaks at 1731 and 2870 cm21 were attrib-
uted to the C@O stretching vibration of ester group of the
P(2-BCL-co-6-BCL) and the C-H stretching vibration of methyl-
ene in PEG, respectively, which indicated that a large number
of ester bonds had been introduced into the mPEG molecules.
The peaks at 690–750 cm21 appeared in the fingerprint
region were related to the C-H stretching of phenyl ring. Com-
pared with Figure 2(a), the FTIR spectrum of mPEG-b-P(2-
CCL-co-6-CCL) copolymer [Fig. 2(b)] showed a large broad
peak from 2500 to 3500 cm21, which demonstrated the pres-
ence of hydrogen bonded carboxyl groups. Moreover, the char-
acteristic peaks related to the aromatic CAH stretching (690–
750 cm21) disappeared, which further confirmed the success-
ful removal of the benzyl carboxylate groups.
A series of six copolymers with different hydrophilic/hydro-
phobic blocks were prepared by changing the ratios of [PEG]
to [BCL]. The characteristic properties of these copolymers
were summarized in Table 1. The molecular weight by 1H
NMR could be calculated based on the integration ratio of
FIGURE 2 FTIR spectrum of mPEG-b-P(2-BCL-co26-BCL)
(a) and mPEG-b-P(2-CCL-co-6-CCL) (b) copolymers.
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TABLE 1 Molecular Characterization of mPEG-b-P(2-CCL-co-6-CCL) Copolymers
c
d
d
Mn
Mn
Mw/Mn
P(2-BCL)
Blocke (%)
Samplea
Mn
BD
AD
BD
AD
BD
AD
b
2K-PCCL5
2K-PCCL10
2K-PCCL24
2K-PCCL99
5K-PCCL5
5K-PCCL18
4,000
3,333
4,595
8,290
28,030
6,965
9,764
2,702
3,737
6,130
19,031
5,862
8,127
3,778
5,492
7,040
26,293
8,330
8,751
4,341
1.10
1.13
1.25
1.53
1.05
1.28
1.06
1.13
1.12
1.38
1.14
1.16
16.6
16.6
8.3
6,000
9,304
10,000
34,000
9,000
15,696
39,535
9,540
7.0
13.0
9.0
13,000
16,327
a
d
The copolymer with five P(2-CCL-co-6-CCL) blocks, initiated by
The number-averaged molecular weight measured by SEC.
e
mPEG45
b
.
The percentage of p(2-BCL) block in the mPEG-b-P(2-BCL-co-6-BCL)
The theoretical molecular weight of mPEG-b-P(2-BCL-co-6-BCL)
copolymer according to 1H NMR.
copolymers.
BD means the copolymer before deprotection.
AD means the copolymer after deprotection.
c
The number-averaged molecular weight measured by 1H NMR.
ꢀ
ꢁ
A5:19
A3:65
A4:05
A3:65
Mn5MnðPEGÞ1 15:63
17:83
3MnðPEGÞ (2)
where A5.19, A4.05, and A3.65 means the integration of the
intensity at 5.19, 4.05, and 3.65 ppm according to the 1H
NMR spectroscopy.
It was seen from Table 1. that the relative molecular weights
were smaller than the theoretical value, indicating that the
monomer BCL could not react completely, which may be
attributed to the steric hindrance because of the existence of
the protected benzyl carboxylate groups.23 For mPEG-b-P
(2-BCL-co-6-BCL) copolymers, the molecular weight meas-
ured by SEC were in good agreement with the 1H NMR
results, while for mPEG-b-P(2-CCL-co-6-CCL) copolymers, the
molecular weight was larger than that of obtained by 1H
NMR, which may be ascribed to the increased hydrodynamic
volume and the formation of the intermolecular association
by hydrogen bond between carboxyl groups.20,39 The unimo-
dal distribution of the copolymers from SEC analysis was
particularly significant since it could further demonstrated
that the final product was indeed a kind of block copolymer
rather than the mixture of the mPEG macroinitiator and P
(2-BCL-co-6-BCL)/P(2-CCL-co-6-CCL) copolymers. It could
also found from Table 1 that the mole ratio of P(2-BCL)
block in the mPEG-b-P(2-BCL-co-6-BCL) copolymer was gen-
erally consistent in the mole ratio of 2-BCL in BCL monomer,
indicating that the reactivity of the two isomers of the BCL
monomer was almost the same. The 1H NMR, FTIR, as well
as SEC results indicated that the successful conjugation of
BCL to mPEG.
Crystallinity Study
The crystallinity of the obtained copolymers was thoroughly
investigated through WXRD and DSC measurements. As
shown in Figure 3(a), the WXRD patterns of mPEG and PCL
homopolymers showed two sharp diffraction peaks at
2h 5 19.1 and 23.4ꢀ and 2h 5 21.4 and 23.8ꢀ, respectively,
indicating that mPEG and PCL homopolymers were high
FIGURE 3 WXRD patterns (a) and DSC curves (b) of the copoly-
mers: mPEG, PCL, 2K-PCCL5, 2K-PCCL10, 2K-PCCL24, and
5K-PCCL18
.
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crystalline materials.40 It was obvious that the introduction of
carboxymethyl groups to the PCL backbone significantly
changed the crystallinity of PCL based on the fact that there
were no obvious diffraction peaks appeared, and the same
result occurred in the P(2-CCL-co-6-CCL) copolymer. (Shown in
Supporting Information Fig. S2). It was deduced that the pend-
ant carboxymethyl groups inhibited the mobility of molecular
chain and brought about constraints to the crystallization of
PCL.40,41 Importantly, it was further found that the crystalliza-
tion properties of mPEG-b-P(2-CCL-co-6-CCL) copolymers were
greatly affected by P(2-CCL-co-6-CCL) block length, which was
well consistent with He’s studies.42,43 As the P(2-CCL-co-6-
CCL) block increased, the characteristic peaks of mPEG gradu-
ally diminished and then completely disappeared, reflecting
the randomness in molecular structure of the copolymer that
indicated the copolymer became amorphous. It was possible
that the amorphous P(2-CCL-co-6-CCL) blocks confined the
crystallization of crystalline mPEG blocks.44
FIGURE 4 Potentiometric titration curves for 2K-PCCL5, 2K-
PCCL24, and 5K-PCCL18 copolymers.
DSC curves of mPEG, PCL homopolymers and mPEG-b-P(2-
CCL-co-6-CCL) copolymers were shown in Figure 3(b). It
could be seen that the morphology of mPEG-b-P(2-CCL-co-6-
CCL) changed from semicrystalline to amorphous with the
increased P(2-CCL-co-6-CCL) content in the copolymer, which
proved that the presence of carboxymethyl group reduced the
crystallinity of PCL. Whereas PCL and mPEG homopolymers
showed a single melting peak with a melting temperature
(Tm) at 57.75 and 52.28 ꢀC, respectively. However, mPEG-b-
P(2-CCL-co-6-CCL) copolymers had decreased Tm, for example,
the Tm of 2K-PCCL5 and 2K-PCCL10 shifted down to 46.18
and 35.77 ꢀC, respectively. Meanwhile, the enthalpy of fusion
of the copolymers decreased from 99.09 to 18.63 J g21 when
the degree of polymerization (DP) of P(2-CCL-co-6-CCL) block
increased from 5 to 10, suggesting that the crystallinity of
mPEG-b-P(2-CCL-co-6-CCL) decreased. In addition, with the
content of the P(2-CCL-co-6-CCL) further increased in the
copolymers, mPEG-b-P(2-CCL-co-6-CCL) became amorphous
based on the fact that there was no melting peak observed,
which was well in accordance with the WXRD data. It was
noteworthy that the glass transition temperature (Tg)
increased as the increase of P(2-CCL-co-6-CCL) block in the
range of the testing temperature. It was probably resulting
from two aspects: for one thing, the intermolecular forces
(hydrogen bonding and van der Waals forces) reduced the
flexibility of the molecular chain and interfered with the crys-
tallization; for another, the random character of P(2-CCL-co-6-
CCL) block may be not conducive to crystallize.45 Compared
with 2K-PCCL24, 5K-PCCL18 copolymer showed a single melt-
ing peak and the enthalpy of fusion was 39.86 J g21; more-
over, sharp diffraction peaks at 2h 5 19.1 and 23.4ꢀ was also
observed in WXRD spectrum [Fig. 3(a)], which indicated the
crystallinity properties of 5K-PCCL18 copolymer. It may be
resulting from strong crystallinity of mPEG114, and the rela-
tively short P(2-CCL-co-6-CCL) segments could not restrict the
mPEG block crystallize completely. Therefore, combined with
WXRD and DSC results, it could be concluded that the intro-
duction of carboxymethyl groups could decrease the crystal-
linity of the mPEG-b-P(2-CCL-co-6-CCL) block copolymers. It
was deduced that the pendant groups disturbed the regularity
of the macromolecules and the crystallinity decreased
accordingly.46
Solution Properties of the Polymeric Micelles
The copolymer mPEG-b-P(2-CCL-co-6-CCL) consisted of
mPEG segment, which was soluble in water, and P(2-CCL-co-
6-CCL) segment, which could be soluble in basic solution
because of deprotonation of the carboxyl groups. In alkaline
aqueous solution, the ionized carboxyl groups could endow
the macromolecular with hydrophilicity and the formation of
the micelles could be induced by the pH variation, P(2-CCL-
co-6-CCL) block would become hydrophobic as pH decreased
due to the protonation of the carboxyl groups of the P(2-
CCL-co-6-CCL) block, and then the core was formed by insol-
uble hydrophobic P(2-CCL-co-6-CCL) blocks surrounded by a
shell formed by mPEG accordingly.
Potentiometric Titration
The potentiometric titration curves for 2K-PCCL5, 2K-PCCL24
,
and 5K-PCCL18 were presented in Figure 4. mPEG-b-P(2-CCL-
co-6-CCL) could be dissolved in alkaline aqueous media due to
the deprotonation of carboxylic acid groups of P(2-CCL-co-6-
CCL) block, and the titration was carried out with 0.1 M HCl.
The pKa was defined to determine the ionization capacity of
the acid, and the higher value of pKa, the weaker of the acid.
The pKa value of the mPEG-b-P(2-CCL-co-6-CCL) was calculated
from the derivative value of the titration curve, which corre-
sponded to the inflection point.16,47 It was found that mPEG-b-
P(2-CCL-co-6-CCL) copolymers buffered the solution in the pH
range of 11.7–2.4, which was assigned to the deprotonation of
carboxyl groups of P(2-CCL-co-6-CCL) block. The pKa was thus
determined to be 7.1, 7.15, and 7.25 for 2K-PCCL5, 2K-PCCL24
,
and 5K-PCCL18, respectively, which indicating that the copoly-
mers were relatively weak polyacid. Besides, it was found from
Figure 4 that the chain length of both mPEG and P(2-CCL-co-6-
CCL) had a little effect on the pKa of mPEG-b-P(2-CCL-co-6-
CCL) copolymers. Therefore, it was deduced that the copolymer
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from aqueous phase to the hydrophobic area and the micelles
formed. The CMC values of different copolymers at pH5 4.5
were calculated and summarized in Table 2. It was found that
the CMC value decreased from 205 to 6.3 mg L21 with the
DP of P(2-CCL-co-6-CCL) block increased from 5 to 99 when
the DP of mPEG was fixed at 45, which suggested that strong
capability of micellization and relatively high micelle stability
were achievable at a certain DP of P(2-CCL-co-6-CCL) block, it
was reasonable that the length of hydrophobic P(2-CCL-co-6-
CCL) block segment played a significant role in the stability of
micelles to reduce the CMC value. However, the CMC values
were relatively high with the increase of the length of mPEG.
It was reasonable that longer mPEG chains could provide
more hydrophilicity to the copolymers and led to the increase
of the CMC values.50
FIGURE 5 Plots of the intensity ratio (I1/I3) from fluorescent
emission spectra of pyrene at 337 nm in aqueous solution at
25 ꢀC as function of pH of 2K-PCCL24 copolymer solution.
Size and Morphology
bearing carboxyl groups would display interesting self-
assembly behavior at different solution pH.
The size of the polymeric micelles in aqueous solution was
analyzed by DLS. Table 2 summarized the hydrodynamic
radius (Rh, from the number weighted distribution) of these
copolymers at pH 5 4. It could be found that the Rh was
affected by the length of hydrophilic and hydrophobic
sequences. When the DP of P(2-CCL-co-6-CCL) was short, the
Rh for 2K-PCCL5 and for 5K-PCCL5 were 2.31 and 2.28 nm,
respectively, which indicated that the copolymer existed in
polymer solution as random coils in this condition.51 How-
ever, when the length of the hydrophobic P(2-CCL-co-6-CCL)
block increased, the particles formed, for example, 2K-
PCCL10 had a Rh of 7.3 nm and that of 5K-PCCL18 was 13.1
nm. It was supposed that the length of P(2-CCL-co-6-CCL)
block increased and reduced the hydrophilicity of copoly-
mers accordingly; thus the well-defined micelles were
formed under this condition. Furthermore, it was also found
from Table 2 that the Rh increased with the increase of the
length of hydrophobic P(2-CCL-co-6-CCL) block when the
hydrophilic PEG segment fixed, which may be attributed to
the enlarged core of the polymer micelles.
The hydrophilicity of the copolymers was confirmed by fluo-
rescence method.48,49 Pyrene was a common probe used to
monitor the micropolarity because the intensity ratio of the
first peak to the third peak (I1/I3) in pyrene fluorescence
spectrum was sensitive to the polarity. The I1/I3 of copolymer
as function of pH for the 2K-PCCL24 copolymer aqueous solu-
tion was shown in Figure 5. It was found that the value of I1/
I3 increased gradually as the solution pH increased, indicating
that the microenvironment of pyrene became polar, which
was originated from improved hydrophilicity of the system. It
should be mentioned however that a completely hydrophilic
character was not observed even at pH5 8, as indicated by
the I1/I3 value was about 1.50, due to the fact that the partly
unionized carboxyl groups as well as the hydrophobic charac-
ter of PCL backbone. It was supposed that the COOH groups
of P(2-CCL-co-6-CCL) block exhibited an equilibrium between
the uncharged and charged forms in an aqueous milieu when
the pH value was near its pKa. The charged form became
soluble while the uncharged was insoluble, which would pro-
vide a relatively nonpolar environment around the probe.
When the copolymer existed in the basic environment, the
increased degree of ionization of COOH groups could afford a
more hydrophilic environment for pyrene, and as the pH ele-
vated above 12, I1/I3 increased to 1.60. The I1/I3 value kept
nearly constant with further increased the solution pH, which
reflected that the mPEG-b-P(2-CCL-co-6-CCL) copolymer
became wholly hydrophilic.
TEM was employed to characterize the morphology of the
mPEG-b-P(2-CCL-co-6-CCL) copolymer micelles. Figure 6(a)
depicted the TEM images of 2K-PCCL99 copolymer in
TABLE 2 Size and Zeta potential of Different Copolymers.
CMCa
(mg L21
Zeta Potentialc
(mV)
b
Samples
)
Rh (nm)
2K-PCCL5
2K-PCCL10
2K-PCCL24
2K-PCCL99
5K-PCCL5
5K-PCCL18
205
93
2.31 6 0.84
7.31 6 0.67
21.5 6 0.79
24.72 6 0.90
2.28 6 0.12
13.1 6 0.28
233.8 6 1.55
237.9 6 1.68
240.45 6 0.26
249.85 6 1.13
232.0 6 0.64
236.27 6 0.47
In order to gain insight to the self-assembly of the mPEG-b-
P(2-CCL-co-6-CCL) block copolymers, the fluorescence probe
technique with pyrene as a hydrophobic fluorescence probe
was performed in aqueous solution. The CMC determination
by fluorescence probe method was based on the fact that, at a
CMC, the intensity ratio of the first peak to the third peak I1/
I3 showed a substantial decrease. At low concentration of the
copolymers, the total fluorescence intensity ratio remained
nearly unchanged, and a sharp decrease of intensity ratio I1/
I3 was observed as the concentration of the copolymers
increased, indicating that the surroundings of pyrene changed
32
6.3
174
78
a
It was calculated by fluorescence spectroscopy using pyrene as fluo-
rescent probe at pH 5 4.5.
b
It was measured based on the number weighted distributions at
pH 5 4.0 by DLS at 25 ꢀC.
c
It was determined on a Zetasizer Nano-ZS(Malvern) at pH 5 10.0.
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P(2-CCL-co-6-CCL) blocks existed, which would result in
increased polymer micelles. Moreover, Figure 7 displayed that
the mPEG-b-P(2-CCL-co-6-CCL) with shorter P(2-CCL-co-6-CCL)
block had better hydrophilicity and existed as micelles only at
the extreme pH value, and the hydrophilicity of mPEG domi-
nated, and a Tyndall effect indicated the micelle formation
(images in Fig. 7). The same behavior was also investigated
for serial copolymers initiated by mPEG114
.
In order to gain insight into this phenomenon, additional
information had been extracted from the dependence of the
relative scattering intensity (IS/I0) on pH. IS and I0 stood for
the intensities of scattering light and incident light, respec-
tively.54 As shown in Figure 8(a,b), it was observed that the
intensity ratio IS/I0 was low and nearly kept constant when
the pH was near or above its pKa, which was in agreement
with a small Rh detected by the DLS measurement. When the
micellization started, a strong upturn in IS/I0 was observed
for each copolymer under consideration. While the IS/I0 of
the copolymer with longer P(2-CCL-co-6-CCL) chain (2K-
PCCL99) displayed a sharp increase when the pH value var-
ied from 6 to 4, the IS/I0 of copolymer with shorter P(2-CCL-
co-6-CCL) chain (2K-PCCL5) had an IS/I0 upturn in the pH
range of 4–2, which indicated that the formation of polymer
micelles,55,56 though the IS/I0 of the latter kept low level
during the whole pH range. Furthermore, IS/I0 of 2K-PCCL99
decreased with the decrease of pH value, while the Rh
increased dramatically at pH 5 2 (shown in Fig. 7), which
may be attributed to the formation of large aggregates,57
complete precipitate was observed after 24 h. This result
was well in accordance with the DLS data.
FIGURE 6 (a) TEM images and (b) the number-averaged parti-
cle size of 2K-PCCL99 copolymer in aqueous solution with a
concentration of 1 mg mL21 at pH 5 4.
aqueous solution at pH 5 4. It could be seen from Figure
6(a) that mPEG-b-P(2-CCL-co-6-CCL) copolymer formed the
uniform micelles under acid condition. The micelles exhib-
ited regularly spherical morphology and good dispersity with
an average radius of 12 nm. The average size of the spherical
micelles was confirmed by DLS, which revealed an average
radius of 24 nm [Fig. 6(b)]. The discrepancy of the micelle
size that existed between TEM and DLS was attributed to
the fact that the DLS measured the hydrodynamic radius of
micelles in water, whereas the TEM observation revealed the
size of the micelles in the solid state.52
Turbidimetry was often used to study the association behav-
ior of the polymer.58 As shown in Figure 8(c), at higher pH
value, the polymers were well dissolved in the aqueous solu-
tion and the transmittance could reach almost 100%. How-
ever, it was obvious that the transmittance began to
pH Sensitivity of the Polymer Micelles
DLS was a versatile method to investigate the micelle forma-
tion in aqueous solution. It was seen from Figure 7 that the
mPEG-b-P(2-CCL-co-6-CCL) copolymers could be well dissolved
in basic aqueous solution and existed as unimers,51,53 and the
micelles formed with the decrease of pH value of the solution,
which may be attributed to the variation of hydrophilicity by
the balance between the deprotonation and protonation of car-
boxyl groups. When the pH value of solution below its pKa,
the majority of P(2-CCL-co-6-CCL) blocks existed in its union-
ized form while only a small quantity of carboxyl groups was
ionized, the uncharged P(2-CCL-co-6-CCL) blocks could self-
assemble into core while the charged from still expanded in
water. In addition, the lower the pH value, the less ionized
FIGURE 7 Number-averaged particle size of polymer micelles
as a function of pH for 2K-PCCL5 and 2K-PCCL99 at a concentra-
tion of 1 mg mL21. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
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FIGURE 8 The relative scattering intensity (IS/I0) as a function of pH for (a) 2K-PCCL5 and (b) 2K-PCCL99 at a concentration of 1 mg
mL21. (c) Turbidity versus pH for sample 2K-PCCL5 and 2K-PCCL99 in deionized water at a concentration of 1 mg mL21
.
decrease sharply at around pH 5 6 for the copolymer with
longer P(2-CCL-co-6-CCL) chain (2K-PCCL99) because of the
reduced ionization of carboxyl groups and the obviously
increased hydrophobicity of the polymer, and the solution
became gradually more cloudy from the appearance with the
decreased pH value of the solution accordingly. When the pH
value of solution was lower than 2, the complete precipita-
tion of the copolymer was observed and the transmittance
decreased to about 10% accordingly. However, a remarkable
difference occurred for the copolymers with shorter
P(2-CCL-co-6-CCL) chain (2K-PCCL5) that the transmittance
almost remained constant, which was in good agreement
with the light scattering results, which suggested that
2K-PCCL5 was well dissolved in water.
was noteworthy that the lower the pH value, the lower the
absolute value of f-potential value, that was, the system
became thermodynamic unstable as pH decreased.47 The
decrease of f-potential absolute value could be explained by
protonation of carboxyl groups. Moreover, for pH value lower
than 4, where the formation of unstable aggregates due to
protonation of the carboxyl groups, the f-potential value rap-
idly decreased to the value close to zero. The same behavior
was found for all copolymers in our study. Furthermore, it
was found from Table 2 that the f-potential absolute value
increased with the length of P(2-CCL-co-6-CCL) increased.
The results from f-potential measurements, which were in
agreement with the results from other techniques, indicated
that solution properties of the mPEG-b-P(2-CCL-co-6-CCL)
diblock copolymers were greatly influenced by the pH value
of the solution.
Zeta potential measurement was further performed to study
the mPEG-b-P(2-CCL-co-6-CCL) copolymers solution proper-
ties. Representative experimental results were shown in Fig-
ure 9. It could be find that the f-potential value remained
negative at all pH value under our investigation due to the
ionization of carboxyl groups of P(2-CCL-co-6-CCL) blocks. It
Overall, the pH-induced micellization mechanism could be
summarized in Scheme 2. The existing state of the copoly-
mers in aqueous solution depended on the counteraction of
the balance of the hydrophilicity derived from the carboxyl
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FIGURE 10 Cell viability of human dermal fibroblast treated
with mPEG-b-PCL and mPEG-b-P(2-CCL-co26-CCL) copolymers.
FIGURE 9 Zeta potential versus pH copolymer at a concentra-
tion of 1 mg mL21, sample was 2K-PCCL24
.
fibroblasts cells (HF) and the mPEG-b-PCL was used as con-
trol. Figure 10 showed that the HF cell viability after incuba-
tion against DMEM containing mPEG-b-PCL or mPEG-b-P(2-
CCL-co-6-CCL) copolymer micelle solution at different con-
centration after 24 h. It could be seen that both mPEG-b-PCL
and mPEG-b-P (2-CCL-co-6-CCL) copolymers exhibited no
cytotoxicity to the HF cells even when the concentration of
polymers solution had increased to 500 mg L21 as the viabil-
ity of HF cells kept almost 100%. This result confirmed that
mPEG-b-P(2-CCL-co-6-CCL) copolymers had great potential in
biomedical fields.
groups and the hydrophobilicity of the PCL. When the pH
value was above its pKa, the hydrophilic character of the
ionized carboxyl groups conquered the hydrophobic interac-
tions between the PCL backbones that made the copolymer
well dissolved in alkaline conditions. However, as the pH
value of aqueous solution decreased, partly protonation of
carboxyl groups occurred and the hydrophobic interaction
between the PCL backbone began to dominated, thus the
micellization occurred. It was evident that well dispersed
individual polymeric micelle were observed in the pH range
of 4–6, which was ascribed to the electrostatic repulsion
between the partly ionized carboxyl groups. When the pH
value decreased below 4, the system became unstable and
large aggregates formed and the hydrophilicity came from
PEG shell.
CONCLUSIONS
A series of biodegradable mPEG-b-poly(ester) block copoly-
mers with functional carboxyl groups were successfully syn-
thesized through ring-opening polymerization, followed by
deprotection of the BCL groups. The crystallinity of mPEG-b-
P(2-CCL-co-6-CCL) diminished because of the introduction of
carboxymethyl groups. Moreover, mPEG-b-P(2-CCL-co-6-CCL)
could be well dissolved in alkaline water, and they had good
pH sensitivity based on their composition. Well-defined
micelles could be formed by pH induction and the cytotoxic-
ity study confirmed that the mPEG-b-P(2-CCL-co-6-CCL)
copolymers had no cytotoxicity until the polymeric micelles
concentration reached 500 mg L21. Therefore, the pH-
sensitive mPEG-b-P(2-CCL-co-6-CCL) copolymer micelle
would be identified as a potential candidate carrier for con-
trolled drug delivery, and related work is in progress.
In Vitro Cytotoxicity
In vitro cytotoxicity of mPEG-b-P(2-CCL-co-6-CCL) copoly-
mers were evaluated by MTT assay using human dermal
ACKNOWLEDGMENTS
Financial support from the National Natural Science Founda-
tion of China (20804015, 21274039), the Fundamental
Research Funds for the Central Universities (WD0913008),
Shanghai Key Laboratory Project (08DZ2230500) and Basic
Research Key Program Project of Commission of Science and
Technology of Shanghai (12JC1403000, 12JC1403100) are
gratefully acknowledge.
SCHEME 2 Schematic illustration for pH-induced self-assembly
of mPEG-b-P(2-CCL-co26-CCL) copolymer in aqueous solution.
[Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
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