Enzyme-induced formation of thermoreversible micellar gels from aqueous solutions of multiresponsive hydrophilic ABA triblock copolymers

Article abstract of DOI:10.1021/ma200991d

We report on the synthesis of thermo- and enzyme-responsive hydrophilic ABA triblock copolymers, poly(ethoxydi(ethylene glycol) acrylate-co-4- ((dihydroxyphosphoryl)oxy)butyl acrylate)-b-poly(ethylene oxide)-b- poly(ethoxydi(ethylene glycol) acrylate-co-4-((dihydroxyphosphoryl)oxy)butyl acrylate) (P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA)), and the enzyme-induced formation of thermoreversible micellar gels from their moderately concentrated aqueous solutions at 37 °C. PDEGEA is a thermosensitive water-soluble polymer with a lower critical solution temperature (LCST) at 9 °C in water. The block copolymers were prepared by atom transfer radical polymerization of DEGEA and 4-((di-tert-butoxyphosphoryl)oxy)butyl acrylate and subsequent removal of tert-butyl groups. To seek optimal conditions for enzymatic gelation of aqueous solutions of triblock copolymers, a study of dephosphorylation of a random copolymer P(DEGEA-co-OPBA) by acid phosphatase in water at 37 °C was carried out. The time for the solution to turn cloudy was found to decrease with the decrease of pH from 5.48 to 4.70 and level off from pH 4.39 to 4.23. The cleavage of phosphate groups made the polymer less hydrophilic and decreased the LCST from above to below 37 °C. Therefore, pH 4.4 was selected to conduct the enzyme-induced gelation of 7.9 wt % aqueous solutions of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA). The gelation processes were monitored by rheological measurements; the sol-gel transition temperature decreased and the gel strength increased with the increase of reaction time. The gels formed were thermoreversible; lowering temperature converted the gels to free-flowing liquids. From ¹H and ³¹P NMR spectroscopy analysis, the degree of dephosphorylation was high. The formation of three-dimensional micellar network gels stemmed from the thermosensitive properties of the resultant dephosphorylated triblock copolymers, which was confirmed by a dynamic light scattering study. At a slightly higher pH (4.67), the enzyme-induced gelation was significantly slower, consistent with the observation of the effect of pH on dephosphorylation of the random copolymer by acid phosphatase.

Full text of DOI:10.1021/ma200991d

ARTICLE
pubs.acs.org/Macromolecules
Enzyme-Induced Formation of Thermoreversible Micellar Gels
from Aqueous Solutions of Multiresponsive Hydrophilic ABA
Triblock Copolymers
Jeremiah W. Woodcock, Xueguang Jiang, Roger A. E. Wright, and Bin Zhao*
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States
S Supporting Information
b
ABSTRACT: We report on the synthesis of thermo- and enzyme-responsive hydro-
philic ABA triblock copolymers, poly(ethoxydi(ethylene glycol) acrylate-co-4-((di-
hydroxyphosphoryl)oxy)butyl acrylate)-b-poly(ethylene oxide)-b-poly(ethoxydi(eth-
ylene glycol) acrylate-co-4-((dihydroxyphosphoryl)oxy)butyl acrylate) (P(DEGEA-
co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA)), and the enzyme-induced formation of
thermoreversible micellar gels from their moderately concentrated aqueous solutions
at 37 °C. PDEGEA is a thermosensitive water-soluble polymer with a lower critical
solution temperature (LCST) at 9 °C in water. The block copolymers were prepared
by atom transfer radical polymerization of DEGEA and 4-((di-tert-butoxyphosphoryl)oxy)butyl acrylate and subsequent removal of
tert-butyl groups. To seek optimal conditions for enzymatic gelation of aqueous solutions of triblock copolymers, a study of
dephosphorylation of a random copolymer P(DEGEA-co-OPBA) by acid phosphatase in water at 37 °C was carried out. The time
for the solution to turn cloudy was found to decrease with the decrease of pH from 5.48 to 4.70 and level off from pH 4.39 to 4.23.
The cleavage of phosphate groups made the polymer less hydrophilic and decreased the LCST from above to below 37 °C.
Therefore, pH 4.4 was selected to conduct the enzyme-induced gelation of 7.9 wt % aqueous solutions of P(DEGEA-co-OPBA)-b-
PEO-b-P(DEGEA-co-OPBA). The gelation processes were monitored by rheological measurements; the solꢀgel transition
temperature decreased and the gel strength increased with the increase of reaction time. The gels formed were thermoreversible;
1
lowering temperature converted the gels to free-flowing liquids. From H and 31P NMR spectroscopy analysis, the degree of
dephosphorylation was high. The formation of three-dimensional micellar network gels stemmed from the thermosensitive
properties of the resultant dephosphorylated triblock copolymers, which was confirmed by a dynamic light scattering study. At a
slightly higher pH (4.67), the enzyme-induced gelation was significantly slower, consistent with the observation of the effect of pH
on dephosphorylation of the random copolymer by acid phosphatase.
’ INTRODUCTION
copolymer, forms bridges among micellar cores of other blocks,13ꢀ16
and physically jammed micellar gels, in which discrete spherical
micelles of block copolymers are jammed and packed into an
ordered structure.10,11,17 While the second type of gels typically
form at a polymer concentration of ∼20 wt %, the critical gelation
concentration (CGC) of ABA triblock copolymers with stimuli-
sensitive outer blocks is significantly lower. For example,
Kirkland et al. reported that a 7.5 wt % aqueous solution of
poly(N-isopropylacrylamide)-b-poly(N,N-dimethyacrylamide)-
b-poly(N-isopropylacrylamide) formed a free-standing gel upon
heating.13 We previously reported that the CGC of a thermo-
sensitive ABA triblock copolymer in water was between 3 and 4
wt %.14
Polymer hydrogels have been intensively investigated for
biomedical applications including contact lenses, sustained or
triggered release of drugs and biomolecules, cell culture, and tissue
engineering.1ꢀ9 While chemically cross-linked polymer gels are
being widely used and continuously evaluated, there is a growing
interest in stimuli-responsive block copolymer aqueous micellar
gels.10ꢀ21 These physically cross-linked or jammed micellar gels
can be more advantageous for some applications because of the
in situ gelation of a liquid precursor induced by environmental
changes, allowing, e.g., for minimally invasive administration by
injection via syringe and needle. A notable example of injectable
gel drug delivery systems, reported by Jeong et al., was based on
aqueous solutions of block copolymers that underwent thermo-
induced solꢀgel transitions.12 The polymer solutions were
loaded with a model drug in the sol state at an elevated temperature.
Upon subcutaneous injection at the body temperature, the solutions
formed gels instantaneously that subsequently acted as matrices
for sustained release of drug molecules.
Of particular interest to us are block copolymer aqueous micellar
gels that can respond to two or more external stimuli. Such gels
would offer greater design flexibility and more advantages compared
with those that respond to only one external stimulus. There have
been a number of reports on multiresponsive aqueous polymer
In general, there are two types of stimuli-responsive block
copolymer aqueous micellar gels: three-dimensional network
gels, in which one block, e.g., the central block of an ABA triblock
Received: April 30, 2011
Revised:
June 12, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Thermo- and Enzyme-Responsive ABA Triblock Copolymer P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-
co-OPBA)
micellar gels.18ꢀ21 For example, Li et al. reported the synthesis of
thermo- and redox-sensitive ABA triblock copolymers and demon-
strated the thermally and biochemically induced solꢀgel transi-
tions of aqueous solutions of these copolymers.18a Temperature-
(T-) and pH-sensitive polymer micellar gels are probably the
most studied and used multiresponsive gels.19,20 The block
copolymers used in these studies were usually prepared by either
growing pH-sensitive blocks from or introducing carboxylic acid
or other pH-responsive groups to the chain ends of an ABA
triblock copolymer that could form thermoreversible gels in
water (e.g., PEO-b-PPO-b-PEO).19 Other types of multiblock
copolymers were also employed.20
Scheme 2. Dephosphorylation of P(DEGEA-co-OPBA)-b-
PEO-b-P(DEGEA-co-OPBA) by Acid Phosphatase and
Schematic Illustration of Enzymatically Induced Formation of
Thermoreversible Aqueous Micellar Network Gel
We previously reported thermo- and light- or pH-sensitive
aqueous micellar gels of ABA triblock copolymers in which a
small amount of stimuli-responsive functional groups was in-
corporated and randomly distributed in the thermosensitive outer
blocks.14,21 The LCST of the thermosensitive blocks can be
modified by applying a second external stimulus. While we have
shown that aqueous solutions of such triblock copolymers can
undergo multiple solꢀgelꢀsol transitions in response to envi-
ronmental changes and the phase transitions can be well con-
trolled, from a biological perspective, it is extremely attractive to
use enzymes, a class of highly efficient and specific biocatalysts, to
induce the gelation of aqueous solutions or develop enzyme-
responsive hydrogels. One can imagine that such enzyme-respon-
sive systems can be advantageous in some situations as they can
respond to biological cues, e.g., the overexpression of specific
enzymes for certain tissues or diseases. Nature actually uses enzy-
matic gelation to regulate the self-assembly of biomolecules in
cytosol to locally switch part of the cell between the sol and gel
states to control cell focal adhesion and migration.22,23 Inspired
by nature and motivated by biological perspectives, there have
been many reports in the literature on enzyme-induced forma-
tion of small molecule hydrogels and covalently cross-linked poly-
mer gels as well as enzyme-responsive cross-linked hydrogels.23ꢀ34
For example, Xu et al. used kinase and phosphatase to control
phosphorylation and dephosphorylation of a small molecule
hydrogelator and to regulate the formation of supramolecular
hydrogels.24 Anseth et al. reported human neutrophil elastase-
responsive poly(ethylene glycol) hydrogels for controlled
release.27
Here we present the synthesis of thermo- and enzyme-re-
sponsive hydrophilic ABA triblock copolymers (Scheme 1) and
the enzyme-induced formation of thermoreversible three-dimen-
sional micellar network gels from their aqueous solutions at the
physiological temperature, 37 °C. We utilize the enzyme-cata-
lyzed dephosphorylation,24,35 a common biochemical reaction, in
our gel design; a small amount of hydrophilic phosphate groups is
incorporated in a random distribution fashion into the thermo-
sensitive outer blocks of ABA triblock copolymers. The enzy-
matic dephosphorylation decreases the LCST of thermosensitive
blocks from above to below the experimental temperature, resulting
in the formation of a thermoreversible three-dimensional net-
work (Scheme 2). The multiresponsive triblock copolymers,
poly(ethoxydi(ethylene glycol) acrylate-co-4-((dihydroxy-
phosphoryl)oxy)butyl acrylate)-b-PEO-b-poly(ethoxydi(ethylene
glycol) acrylate-co-4-((dihydroxyphosphoryl)oxy)butyl acrylate)
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(P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA)), were
synthesized by atom transfer radical polymerization (ATRP) of
a mixture of DEGEA and 4-((di-tert-butoxyphosphoryl)oxy)butyl
acrylate (BPBA) from a difunctional PEO macroinitiator and
subsequent removal of tert-butyl groups (Scheme 1). PDEGEA is
a thermosensitive polymer with a LCST of 9 °C in water,21b,36
which belongs to a new class of thermosensitive polymers with a
short oligo(ethylene glycol) pendant from each repeat unit.37 We
show that 7.9 wt % aqueous solutions of P(DEGEA-co-OPBA)-b-
PEO-b-P(DEGEA-co-OPBA) undergo gelation in the presence
of acid phosphatase, an enzyme that catalyzes dephosphorylaton,
at a constant temperature, 37 °C, and the gels formed are thermo-
reversible. To the best of our knowledge, this is the first report on
the formation of physically cross-linked micellar gels of block
copolymers in water induced by an enzyme at the physiological
temperature. Compared with chemically cross-linked hydrogels,
thermoreversible micellar gels have many advantages; a notable
one is the facile removal.
filtered off and the solvent was then removed by a rotavapor. Diethyl
ether was added and the mixture was washed twice with a saturated
aqueous solution of sodium bicarbonate and then once with water. The
organic layer was dried over anhydrous sodium sulfate overnight. After
the removal of sodium sulfate, the solution was concentrated using a
rotavapor. The crude product was purified by column chromatography
using hexanes/ethyl acetate (v/v, 1:1) as eluent. The pure product was
obtained as a nearly colorless liquid. 1H NMR (CDCl3): δ (ppm), 6.38
(d, 1H, CHHdCH), 6.09 (dd, 1H, CHHdCHꢀ), 5.80 (d, 1H,
CHH=CHꢀ), 4.17 (t, 2H, ꢀCOOCH2ꢀ), 3.67 (m, 2H, ꢀCH2OH),
1.77ꢀ1.60 (m, 4H, ꢀCH2CH2CH2CH2ꢀ), 1.46 (s, 1H, ꢀOH); 13C
NMR (CDCl3): δ (ppm) 166.30, 130.67, 128.34, 64.30, 62.07, 28.95,
24.99. MS (ESI+): 145.09 ([M + H+]).
Synthesis of 4-((Di-tert-butoxyphosphoryl)oxy)butyl
Acrylate (BPBA). 4-Hydroxybutyl acrylate (1.13 g, 7.85 mmol) and
di-tert-butyl diisopropylphosphoramidite (3.08 g, 0.0111 mol) were
dissolved in dichloromethane (50 mL) in a 250 mL three-necked round-
bottom flask. The flask was placed into an ice/water bath and a solution
of tetrazole in acetonitrile (0.45 M, 20 mL, 9.0 mmol) was added dropwise
into the flask. The mixture became hazy white in appearance. The
reaction mixture was allowed to warm to room temperature and stirred
overnight. The flask was again placed in an ice/water bath and a 30%
aqueous solution of hydrogen peroxide (5 mL) was added into the flask
in a dropwise fashion. The mixture became clear after the addition of
H2O2. The reaction was kept at 0 °C for additional 3 h. A 10 wt %
aqueous solution of Na2S2O5 (30 mL) was slowly added into the reaction
mixture to quench unreacted hydrogen peroxide (Caution! noxious
fumes). The organic layer was separated, washed with water, and then dried
over anhydrous sodium sulfate. The product was isolated by column
chromatography using hexanes/ethyl acetate (1:1) as eluent. After
drying in vacuum, the pure product was obtained as a colorless liquid
(1.72 g, yield: 65.2%). 1H NMR (CDCl3): δ (ppm), 6.37 (d, 1H,
CHHdCHꢀ), 6.08 (dd, 1H, CHHdCHꢀ), 5.79 (d, 1H, CHHdCHꢀ),
4.16 (t, 2H, ꢀCOOCH2ꢀ), 3.96 (m, 2H, ꢀCH2OPꢀ), 1.74 (m, 4H,
ꢀCH2CH2CH2CH2ꢀ), 1.47 (s, 18H, ꢀOC(CH3)3);13CNMR(CDCl3):
δ (ppm) 166.13, 130.60, 128.36, 82.07, 81.98, 66.13, 63.92, 30.28, 30.23,
29.81, 29.75, 26.86, 24.94. MS (ESI+): 337.18 ([M + H+]).
Synthesis of P(DEGEA-co-BPBA) by Atom Transfer Radical
Polymerization. CuBr (11.3 mg, 0.0788 mmol), ethoxydi(ethylene
glycol) acrylate (DEGEA, 2.03 g, 10.8 mmol), 4-((di-tert-butoxypho-
sphoryl)oxy)butyl acrylate (BPBA, 0.351 g, 1.04 mmol), ethyl 2-bro-
moisobutyrate (EBiB, 10.6 mg, 0.0543 mmol), anisole (2.43 g), and N,
N,N0,N0,N00-pentamethyldiethylenetriamine (29.5 mg, 0.170 mmol)
were added into a 25 mL two-necked flask. After the mixture was
degassed by three freezeꢀpumpꢀthaw cycles, the flask was placed into a
90 °C oil bath. The reaction was monitored by both SEC and 1H NMR
spectroscopy analysis. After the polymerization proceeded for 175 min,
the flask was removed from the oil bath and the solution was diluted with
THF. The mixture was passed through an Al2O3/silica gel column using
THF as eluent. The solution was concentrated via rotary evaporation
and then precipitated three times in hexanes/diethyl ether (v/v = 80: 20,
100 mL). A nearly colorless polymer was obtained. SEC analysis results
(polystyrene standards): Mn,SEC = 12 000 g/mol; polydispersity index
(PDI) = 1.14. The degree of polymerization (DP) of the polymer was
calculated from the monomer conversion and the monomer-to-initiator
ratio. The peaks located in the range of 3.9ꢀ4.4 ppm, which were from
ꢀCH2OOCꢀ of DEGEA and BPBA and ꢀCH2OPꢀ from BPBA were
used as the internal standard. The conversion was calculated from the
integral values of the peaks located at 5.6ꢀ6.0 ppm (CHHdCHꢀ from
both BPBA and DEGEA monomers) at t = 0 and 175 min. The cal-
culated DP was 82. The molar ratio of DEGEA and BPBA units in the
copolymer was 100: 13.3, determined from the 1H NMR spectrum using
the peaks located at 3.9ꢀ4.4 ppm, which were from ꢀCH2OOCꢀ of
DEGEA and BPBA units and ꢀCH2OPꢀ from BPBA units, and
’ EXPERIMENTAL SECTION
Materials. 1,4-Butanediol (99%), di-tert-butyl diisopropylphosphora-
midite (95%), tetrazole solution (∼0.45 M in acetonitrile), and acid
phosphatase (Type II, from potato, lyophilized powder, 1.55 units/mg
solid) were purchased from Aldrich and used as received. Acryloyl chloride
(96%, Alfa Aesar) was used as received. N,N,N0,N0,N00-Pentamethyl-
diethylenetriamine (PMDETA, Aldrich), ethyl 2-bromoisobutyrate (EBiB,
Aldrich), and anisole (99%, Acros) were distilled over calcium hydride
under reduced pressure. CuBr (98%, Aldrich) was stirred in glacial acetic
acid, filtered, and washed with absolute ethanol and diethyl ether. The
purified CuBr was then dried in high vacuum and stored in a desiccator.
Poly(ethylene oxide) (HOꢀPEOꢀOH, MW = 20 000 g/mol, Aldrich)
was end-functionalized via the reaction with 2-bromoisobutyryl bromide
as described in a previous publication,14 yielding a difunctional PEO
macroinitiator, BrꢀPEOꢀBr. Trifluoroacetic acid (99%, Acros), H2O2
solution (30%, Fisher Scientific, stabilized with sodium stannate), Na2S2O5
(99%, Acros), citric acid (99%, Acros), and citric acid trisodium salt
(99%, Acros) were used as received. All other chemicals were purchased
from either Aldrich or Fisher/Acros and used without further purification.
General Characterization. Size exclusion chromatography (SEC)
was carried out at room temperature using PL-GPC 20 (an integrated
GPC system from Polymer Laboratories, Inc.) with a refractive index
detector, one PLgel 5 μm guard column (50 ꢁ 7.5 mm), and two PLgel
5 μm mixed-C columns (each 300 ꢁ 7.5 mm, linear range of molecular
weight from 200 to 2 000 000 according to Polymer Laboratories, Inc.).
The data were processed using CirrusTM GPC/SEC software (Polymer
Laboratories, Inc.). THF was used as the carrier solvent at a flow rate of
1.0 mL/min. Standard polystyrenes with narrow polydispersity indexes
1
(Polymer Laboratories, Inc.) were used for calibration. The H (300
MHz), 13C (75 MHz), and 31P (121 MHz) NMR spectra were recorded
on a Varian Mercury 300 NMR spectrometer. The mass spectrometry
analysis was performed at the Mass Spectrometry Center in the Department
of Chemistry at the University of Tennessee, Knoxville, TN, using a
JEOL (Peabody, MA) AccuTOF-D time-of-flight mass spectrometer
with a DART (direct analysis in real time) ionization source.
Synthesis of 4-Hydroxybutyl Acrylate (OHBA). 1,4-Butane-
diol (26.9 g, 0.299 mol) and triethylamine (10.9 g, 0.108 mol) were
dissolved in dichloromethane (75 mL) in a 250 mL three-necked round-
bottom flask. The flask was then placed in an ice/water bath and the
mixture was stirred with a magnetic stir bar under nitrogen atmosphere.
A solution of acryloyl chloride (6.92 g, 0.0765 mol) in dichloromethane
(20 mL) was added dropwise into the flask. The mixture was allowed to
warm to room temperature and stirred overnight. The precipitate was
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1.2 ppm, which was the methyl peak of DEGEA units. 1H NMR (CDCl3): δ
(ppm), 4.19 (ꢀCOOCH2ꢀ of DEGEA units), 4.05 (ꢀCOOCH2- of
BPBA units), 3.98 (ꢀCH2OP of BPBA units), 3.85ꢀ3.20 (ꢀCOO-
CH2CH2OCH2CH2OCH2CH3 of DEGEA units), 2.32 (ꢀCH2CHC-
OO- of both DEGEA and BPBA units), 2.05ꢀ1.36 (ꢀCH2CHCOOꢀ
of both DEGEA and BPBA units), 1.70 (ꢀCOOCH2CH2CH2CH2OP
of BPBA units), 1.47 (s, ꢀC(CH3)3 of BPBA units), and 1.20 (t, ꢀOC-
H2CH3 of DEGEA units). 31P NMR (CDCl3): δ (ppm), ꢀ9.71.
Synthesis of P(DEGEA-co-OPBA). P(DEGEA-co-BPBA) (0.200 g)
was dissolved in methylene chloride (5 mL) in a 50 mL round-bottom
flask, followed by addition of trifluoroacetic acid (TFA, 2.0 g). After the
reaction mixture was stirred at room temperature for 48 h, the volatiles
were removed by a rotary evaporator. The residue was dissolved in
methylene chloride (25 mL) and the volatiles were evaporated again by a
rotavapor. This process was repeated an additional two times to remove
as much trifluoroacetic acid as possible. The polymer was purified by
precipitation three times in hexanes/diethyl ether (100: 20, v:v) and
then once in hexanes (100 mL). After drying under high vacuum, the
polymer was obtained as a clear sticky solid with a yield of 91% (182.3 mg).
The removal of tert-butyl groups of P(DEGEA-co-BPBA) was evidenced
by the disappearance of the tert-butyl peak at 1.47 ppm in the 1H NMR
process was repeated an additional two times to remove as much
trifluoroacetic acid as possible. The polymer was then dissolved in
THF (5 mL), precipitated in hexanes/diethyl ether (v/v = 80: 20,
100 mL) three times, and dried in high vacuum. The removal of the
1
tert-butyl groups was confirmed by H NMR spectroscopy analysis;
1
the tert-butyl peak located at 1.47 ppm disappeared. H NMR (CD-
Cl3): δ (ppm), 4.18 (ꢀCOOCH2ꢀ of DEGEA units), 4.06 (ꢀCO-
OCH2ꢀ and ꢀCH2OP of OPBA units), 3.90ꢀ3.30 (ꢀCH2CH2Oꢀ
of PEO block and ꢀCOOCH2CH2OCH2CH2OCH2CH3 of DEGEA
units), 2.32 (ꢀCH2CHCOO- of both DEGEA and OPBA units),
2.05ꢀ1.36 (ꢀCH2CHCOOꢀ of both DEGEA and OPBA units), 1.74
(ꢀCOOCH2CH2CH2CH2OP of OPBA units), and 1.19 (ꢀOCH2-
CH3 of DEGEA units). 31P NMR (CDCl3): δ (ppm), 1.02.
Preparation of 0.5 wt % Aqueous Solution of P(DEGEA-
co-OPBA) and Study of pH Dependence of Cloud Point of
P(DEGEA-co-OPBA) in Water. A 0.5 wt % aqueous solution of
P(DEGEA-co-OPBA) was made by dissolving the random copolymer
(33 mg, dried in high vacuum) in a 50 mM aqueous citrate buffer. The
vial was then placed into the water bath of a Fisher Scientific Isotemp
refrigerated circulator. The temperature was gradually increased from
8 °C. At each temperature, the sample was allowed to equilibrate for
20 min. When the solution turned cloudy, the temperature was recorded
as the cloud point. The pH of the solution was then adjusted by using
either 1.0 M KOH or 1.0 M HCl and the cloud point was determined by
visual examination as described above.
spectrum and the shift of the 31P peak from ꢀ9.71 to 1.02 ppm in the 31
P
1
NMR spectra. H NMR (CDCl3): δ (ppm), 4.18 (ꢀCOOCH2ꢀ of
DEGEA units), 4.06 (ꢀCOOCH2ꢀ and ꢀCH2OP of OPBA units),
3.85ꢀ3.20 (ꢀCOOCH2CH2OCH2CH2OCH2CH3 of DEGEA units),
2.32 (ꢀCH2CHCOOꢀ of both DEGEA and OPBA units), 2.05ꢀ1.30
(ꢀCH2CHCOOꢀ of both DEGEA and OPBA units), 1.74 (ꢀCOO-
CH2CH2CH2CH2OP of OPBA units), and 1.18 (t, ꢀOCH2CH3 of
DEGEA units). 31P NMR (CDCl3): δ (ppm), 1.02.
Preparation of 0.5 wt % Aqueous Solutions of P(DEGEA-
co-OPBA) and Study of pH Effect on Acid Phosphatase-
Catalyzed Dephosphorylation. P(DEGEA-co-OPBA) was added
into a preweighed 100 mL round-bottom flask and dried under high
vacuum in a 60 °C oil bath for 3 h. The mass of the dried polymer was
91.5 mg. A 50 mM aqueous citrate buffer with pH of 5.10 (18.235 g) was
added into the flask and the mixture was sonicated in an ultrasonic ice/
water bath to dissolve the polymer. The resultant homogeneous polymer
solution was distributed roughly equally into six glass vials with an inner
diameter of 12 mm. The solutions were then adjusted to desired pH
values by injecting either 1.0 M KOH or 1.0 M HCl aqueous solution via
a microsyringe. An aqueous solution of acid phosphatase with a con-
centration of 1.99 wt % was made by dissolving 14.2 mg of acid phos-
phatase in deionized water (0.698 g). A calculated amount of the enzyme
solution was added into each vial via a microsyringe; the enzyme-to-
polymer mass ratio for all solutions was 7.5:100. The vials were then
placed into a 37 °C water bath. The time at which a solution turned
cloudy was recorded as the clouding time. For the samples with pH of
4.23 and 5.48, the polymers were isolated at the clouding times and the
Synthesis of P(DEGEA-co-BPBA)-b-PEO-b-P(DEGEA-co-
BPBA). Below is a typical procedure for the synthesis of P(DEG-
EA-co-BPBA)-b-PEO-b-P(DEGEA-co-BPBA). CuBr (14.6mg, 0.102 mmol),
difunctional PEO macroinitiator Br-PEO-Br (0.864 g, 0.043 mol),
DEGEA (3.237 g, 17.22 mmol), BPBA (0.610 g, 1.81 mmol), and
anisole (3.486 g) were weighed into a 25 mL two-necked flask, fol-
lowed by the injection of PMDETA (18.8 mg, 0.109 mmol) via a micro-
syringe. The mixture was degassed by three freezeꢀpumpꢀthaw cycles.
The polymerization was started by placing the flask into a 90 °C oil
bath. After 110 min, the flask was removed from the oil bath and the
mixture was diluted with THF. The copper catalyst was removed by
passing the solution through a short neutral aluminum oxide/silica gel
column with THF as eluent. The polymer solution was then con-
centrated via rotary evaporation and precipitated in hexanes/ethyl
ether (v/v = 80: 20, 100 mL). Size exclusion chromatography analysis
results (polystyrene standards): Mn,SEC = 49800 g/mol and the
1
1H NMR spectra were recorded. Below is the H NMR data of the
1
polymer isolated from the experiment at pH = 4.23. 1H NMR (CDCl3):
δ (ppm), 4.18 (ꢀCOOCH2ꢀ of both DEGEA and OHBA units),
3.85ꢀ3.30 (ꢀCOOCH2CH2OCH2CH2OCH2CH3 of DEGEA units
and ꢀCH2CH2OH of OHBA units), 2.32 (ꢀCH2CHCOOꢀ of both
DEGEA and OHBA units), 2.08ꢀ1.29 (ꢀCH2CHCOOꢀ of both DE-
GEA and OHBA units, and ꢀCOOCH2CH2CH2CH2OH of OHBA
units), and 1.19 (ꢀOCH2CH3 of DEGEA units).
polydispersity index (PDI) = 1.12. H NMR spectroscopy analysis
showed that the numbers of DEGEA and BPBA units in the triblock
1
copolymer were 143 and 28, respectively. H NMR (CDCl3): δ (ppm),
4.18 (ꢀCOOCH2ꢀ of DEGEA units), 4.04 (ꢀCOOCH2ꢀ of BPBA
units), 3.97 (ꢀCH2OP of BPBA units), 3.90ꢀ3.30 (ꢀCH2CH2Oꢀ of
PEO block and ꢀCOOCH2CH2OCH2CH2OCH2CH3 of DEGEA
units), 2.32 (ꢀCH2CHCOOꢀ of both DEGEA and BPBA units),
2.05ꢀ1.36 (ꢀCH2CHCOOꢀ of both DEGEA and BPBA units), 1.70
(ꢀCOOCH2CH2CH2CH2OP of BPBA units), 1.47 (s, ꢀC(CH3)3 of
BPBA units), and 1.19 (t, ꢀOCH2CH3 of DEGEA units). 31P NMR
(CDCl3): δ (ppm), ꢀ9.71.
Preparation and Enzyme-Induced Gelation of 7.9 wt %
Aqueous Solutions of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-
co-OPBA). Below is a typical procedure for an enzyme-induced gelation
experiment. P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA) (ABA-1)
was added into a preweighed 3.7 mL vial. The vial was placed into a
larger flask and dried under high vacuum in a 60 °C oil bath for 3 h. The
mass of the dried polymer was 0.115 g. The polymer was then dissolved
in a 50 mM aqueous citrate buffer with a pH value of 4.90 (1.335 g) and
the pH of the solution was adjusted to 4.40 by using a 1.0 M HCl
solution. After acid phosphatase (5.60 mg) was added, the vial was capped
tightly and placed into a 37 °C water bath (t = 0 min). The mixture was
stirred with a magnetic stir bar. After the solution became viscous and the
Synthesis of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-
OPBA). P(DEGEA-co- BPBA)-b-PEO-b-P(DEGEA-co-BPBA) (0.987 g,
Mn,SEC = 49800 g/mol, PDI = 1.12) was dissolved in dry dichlor-
omethane (10 mL) in a 20 mL scintillation vial, followed by the
addition of trifluoroacetic acid (3.813 g). The reaction mixture was
stirred at room temperature for 48 h. The volatiles were then removed
using a rotavapor. The residue was dissolved in dichloromethane
(20 mL) and the volatiles were evaporated again by a rotavapor. This
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stir bar did not move freely, aliquots were begun to take at various time
intervals for rheological measurements. Each time, the vial was removed
from the 37 °C water bath and placed into an ice/water bath. After a
90 μL aliquot was withdrawn, the vial was sealed and immediately placed
back into the 37 °C water bath. The reaction was stopped after the
rheological measurements showed no change in the solꢀgel transition
temperature. The resultant polymer was isolated for 1H NMR spectros-
copy analysis. 1H NMR (CDCl3): δ (ppm), 4.18 (ꢀCOOCH2ꢀ of both
DEGEA and OHBA units), 3.90ꢀ3.30 (ꢀCH2CH2Oꢀ of PEO block,
ꢀCOOCH2CH2OCH2CH2OCH2CH3 of DEGEA units, and ꢀCH2C-
H2OH of OHBA units), 2.32 (ꢀCH2CHCOOꢀ of both DEGEA and
OHBA units), 2.00ꢀ1.34 (ꢀCH2CHCOOꢀ of both DEGEA and OH-
BA units, and ꢀCOOCH2CH2CH2CH2OH of OHBA units), and 1.19
(ꢀOCH2CH3 of DEGEA units).
tering (DLS). The 0.02 wt % aqueous solution of P(DEGEA-coꢀ
OHBA)-b-PEO-b-P(DEGEA-co-OHBA) was prepared by diluting the
corresponding 7.9 wt % polymer solution after the enzymatic depho-
sphorylation with a 50 mM aqueous citrate buffer. The pH value of the
solution was maintained at 4.40. The DLS measurement was conducted
with a Brookhaven Instruments BI-200SM goniometer equipped with a
PCI BI-9000AT digital correlator, a temperature controller, and a solid-
state laser (model 25-LHP-928ꢀ249, λ = 633 nm) at a scattering angle
of 90°. The polymer solution was filtered into a borosilicate glass tube
with an inner diameter of 7.5 mm using a 0.2 μm hydrophilic PTFE filter.
The glass tube was then sealed with a PE stopper. The sample was placed
into the cell holder of the light scattering instrument and gradually
heated. At each temperature, the solution was equilibrated for 20 min
prior to data recording. The time correlation functions were analyzed
with a Laplace inversion program (CONTIN).
Rheological Measurements. Rheological experiments were con-
ducted using a rheometer from TA Instruments (Model TA AR2000ex).
A coneꢀplate geometry with a cone diameter of 20 mm and an angle
of 2° (truncation 52 μm) was employed; the temperature was controlled
by the bottom Peltier plate. In each measurement, 90 μL of a polymer
solution was loaded onto the plate by a micropipet. The solvent trap was
filled with water and a solvent trap cover was used to minimize water
evaporation. Dynamic storage (G0) and loss moduli (G00) of a polymer
solution were measured by oscillatory shear experiments performed at a
frequency of 1 Hz in a heating ramp at a heating rate of 3 °C/min. The
frequency dependences of G0 and G00 of a polymer solution at selected
temperatures were obtained by frequency sweep tests. A strain ampli-
tude of γ = 0.2% was used in all dynamic tests to ensure that the
deformation was within the linear viscoelastic regime.
Dynamic Light Scattering Study of 0.02 wt % Aqueous
Solution of P(DEGEA-coꢀOHBA)-b-PEO-b-P(DEGEA-coꢀ
OHBA). The thermo-induced micellization at a concentration of
0.02 wt % of P(DEGEA-coꢀOHBA)-b-PEO-b-P(DEGEA-coꢀOHBA),
obtained from the enzymatic dephosphorylation of P(DEGEA-co-OP-
BA)-b-PEO-b-P(DEGEA-co-OPBA), was studied by dynamic light scat-
’ RESULTS AND DISCUSSION
Synthesis of Monomer 4-((Di-tert-butoxyphosphoryl)oxy)-
butyl Acrylate (BPBA). BPBA was synthesized via a two-step
procedure as shown in Scheme 3. 4-Hydroxybutyl acrylate (OHBA)
was prepared first through the reaction of acryloyl chloride with a
large excess of 1,4-butanediol. The phosphorylation of OHBA
was conducted using 1.4 equiv of di-tert-butyl diisopropylpho-
sphoramidite in the presence of 1.1 equiv of tetrazole, followed
by oxidation with a 30% aqueous solution of hydrogen peroxide.
The pure product was isolated by column chromatography using
hexanes/ethyl acetate (1:1, v/v) as eluent and the molecular
1
structure was confirmed by H and 13C NMR spectroscopy as
well as mass spectroscopy analysis.
Preparation of Thermo- and Enzyme-Responsive Hydro-
philic ABA Triblock Copolymers and a Random Copolymer.
The thermo- and enzyme-responsive hydrophilic ABA triblock
copolymers, P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA),
were prepared according to the procedure illustrated in Scheme 1.
The precursor polymers, P(DEGEA-co-BPBA)-b-PEO-b-P(DE-
GEA-co-BPBA), were synthesized by ATRP of a mixture of DEGEA
and BPBA from a difunctional PEO macroinitiator with a molecular
weight of 20000 g/mol at 90 °C using CuBr/PMDETA as catalyst.
Figure 1A shows the SEC traces of PEO macroinitiator and an ABA
triblock copolymer P(DEGEA-co-BPBA)-b-PEO-b-P(DEGEA-co-
BPBA) (ABA-1-P in Table 1). The peak shifted to the high
molecular weight region and remained narrow, though there was
a small shoulder peak on the front side, which was likely from the
Scheme 3. Synthesis of 4-((Di-tert-butoxyphosphoryl)oxy)butyl
Acrylate (BPBA)
Figure 1. (A) Size exclusion chromatography traces of PEO macroinitiator and an ABA triblock copolymer P(DEGEA-co-BPBA)-b-PEO-b-P(DEGEA-
1
co-BPBA) (ABA-1-P) and H NMR spectra of (B) ABA-1-P, (C) ABA-1, and (D) the dephosphorylated triblock copolymer obtained from the
enzymatic gelation experiment of ABA-1. CDCl3 was used as solvent in 1H NMR spectroscopy. The sharp peak at 1.76 ppm in part B is from water.
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Table 1. Characterization Data for Multiresponsive ABA Tri-
block Copolymers P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-
co-OPBA), Random Copolymer P(DEGEA-co-OPBA), and
their Precursor Polymers
numbers of DEGEA and
BPBA (or OPBA) unitsc
polymersa
Mn,SEC (g/mol)b
PDIb
ABA-1-P
ABA-1
ABA-2-P
ABA-2
R-1-P
49 800
NA
1.12
NA
143, 28
143, 28
187, 45
187, 45
72, 10
58 900
NA
1.11
NA
12 000
1.14
NA
R-1
NA
72, 10
a ABA-1 and -2 are thermo- and enzyme-responsive ABA triblock
copolymers P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA), which
were obtained by the removal of tert-butyl groups of ABA-1-P and ABA-
2-P (P(DEGEA-co-BPBA)-b-PEO-b-P(DEGEA-co-BPBA)), respectively.
b The number-average molecular weights (Mn,SEC) and polydispersity
indexes (PDI) were measured by SEC using polystyrene calibration.
c The numbers of DEGEA and BPBA (or OPBA) units in the copoly-
mers were calculated from 1H NMR spectra.
Figure 3. pH dependence of cloud point of P(DEGEA-co-OPBA)
(R-1) in a 50 mM citrate buffer at a concentration of 0.5 wt %. The
cloud points were determined by visual examination.
Two ABA triblock copolymers with slightly different molec-
ular weights and compositions were synthesized and used in this
work. The characterization data for these copolymers and their
precursors are summarized in Table 1. To seek optimal condi-
tions for enzymatic gelation of moderately concentrated aqueous
solutions of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA),
we synthesized a random copolymer P(DEGEA-co-OPBA) (R-1).
Note that all precursor polymers had relatively narrow molecular
weight distributions (PDI < 1.15).38
pH Dependence of Cloud Point of P(DEGEA-co-OPBA). We
chose DEGEA as the main component for the thermosensitive
blocks to construct thermo- and enzyme-responsive ABA tri-
block copolymers because the cloud point of PDEGEA in water
is 9 °C.21b The cleavage of hydrophilic phosphate groups from
thermosensitive blocks would yield hydrophobic 4-hydroxybutyl
groups (Scheme 2), decrease the LCST of thermosensitive
blocks, and induce the gelation of moderately concentrated aqueous
polymer solutions at 37 °C, the temperature intended for the
enzymatic reaction.
Since phosphoric acid is a weak acid and the cloud point in
water of a thermosensitive polymer that contains a small amount
of weak acid groups is known to depend on solution pH,39 we
studied the pH dependence of cloud point of a random copo-
lymer P(DEGEA-co-OPBA) (R-1). The cloud points of aqueous
solutions of R-1 in 50 mM citrate buffers at various pH values
were measured (Figure 3). We found that the cloud point of this
polymer at pH = 3.70 was 50 °C. When the pH was g3.80, no
cloud point was observed in the studied temperature range (up to
80 °C). Apparently, the ionization of phosphate groups at pH g
3.80 made the polymer very hydrophilic and caused the LCST to
be above 80 °C or disappear. Note that the pKa1 of H3PO4 is
2.15. With the decrease of pH from 3.70 to 1.49, the cloud point
gradually decreased from 50 to 14 °C. No change was observed
when the pH was further lowered to 1.29.
Figure 2. 31P NMR spectra of (A) P(DEGEA-co-BPBA)-b-PEO-b-
P(DEGEA-co-BPBA) (ABA-1-P), P(DEGEA-co-OPBA)-b-PEO-b-P-
(DEGEA-co-OPBA) (ABA-1), and (C) the dephosphorylated triblock
copolymer obtained from the enzymatic gelation experiment of ABA-1.
product of the coupling reaction. The number-average molecular
weight (Mn,SEC) and polydispersity index (PDI) of ABA-1-P
were 49.8 kDa and 1.12, respectively (relative to polystyrene
standards). The numbers of DEGEA and BPBA units in the
copolymer were determined from 1H NMR spectrum(Figure1B)
using the peaks located at 4.36ꢀ3.90 ppm (ꢀCH2OCO of DEGEA
units, ꢀCH2OCO and ꢀCH2OPꢀ of BPBA units), the peaks at
3.90ꢀ3.30 ppm (ꢀCH2CH2Oꢀ of PEO block and ꢀCOOCH2-
CH2OCH2CH2OCH2CH3 of DEGEA units), and the peaks at
1.35ꢀ1.00 ppm (ꢀCH3 of DEGEA units). They were 143 and 28,
respectively. The tert-butyl groups in the copolymer were then
cleaved using trifluoroacetic acid (TFA), yielding the targeted
thermo- and enzyme-responsive ABA triblock copolymer P-
(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA). Figure 1C
pH Effect on Dephosphorylation of Random Copolymer
P(DEGEA-co-OPBA) at 37 °C. The activity of acid phosphatase
in the catalysis of dephosphorylation reaction is known to be
sensitive to solution pH.40 To seek optimal conditions for enzymatic
gelation of aqueous solutions of ABA triblock copolymers, we
studied how the dephosphorylation of R-1 by acid phosphatase
was affected by solution pH. Six 0.5 wt % aqueous solutions of
R-1 with pH ranging from 4.23 to 5.48 were made in small glass
vials using 50 mM citrate buffers, followed by the addition of acid
phosphatase (the mass ratio of the enzyme to the copolymer was
1
shows the H NMR spectrum of ABA-1; the removal of t-butyl
groups was evidenced by the disappearance of the t-butyl peak in the
1H NMR spectrum. TFA is known not to affect other ester bonds.37f
Parts A and B of Figure 2 display the 31P NMR spectra of ABA-1-P
and ABA-1, respectively. The 31P peak shifted from ꢀ9.7 ppm to
+1.0 ppm, also indicating the successful removal of tert-butyl groups.
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7.5: 100 for all samples). The vials were placed immediately in a
37 °C water bath (t = 0 min). When the solution turned cloudy,
the time was recorded as clouding time (tclouding). We found
that the clouding time decreased with the decrease of pH, from
385 min at pH 5.48 to 96 min at pH 4.93, to 41.4 min at pH 4.70,
and 7.6 min at pH 4.39 (Figure 4). Further lowering the pH did
not change the clouding time much (tclouding = 5.4 min at pH
4.23). Since the cloud point of the copolymer with a very small
amount of uncleaved phosphate groups could depend on the
Enzyme-Induced Gelation of a 7.9 wt % Aqueous Solution
of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA) (ABA-1)
at pH = 4.40 and 37 °C. In light of the observations shown in
Figure 4, we chose pH = 4.4 to study the enzymatically induced
gelation of a 7.9 wt % aqueous solution of P(DEGEA-co-OPBA)-
b-PEO-b-P(DEGEA-co-OPBA) (ABA-1) at 37 °C. The 7.9 wt %
aqueous solution of ABA-1 was made by using a 50 mM citrate
buffer and the pH was adjusted to 4.40. A calculated amount of
acid phosphatase was added into the polymer solution (the mass
ratio of the enzyme to the thermosensitive blocks was 7.5:100,
same as that in the enzymatic cleavage experiment of R-1). The vial
was capped tightly and placed into a 37 °C water bath (t = 0 min).
Note that before the addition of the enzyme, the solution exhib-
ited no change in the viscosity upon heating up to 80 °C.
After 1 h, the solution became viscous and the magnetic stir bar
did not move freely. The stirrer stopped moving after 2 h. Figure 5
shows the optical pictures of the sample at various times. Evidently,
the solution became very viscous after 4.5 h (Figure 5D) and was
almost a gel at 6.5 h (Figure 5E). After the reaction proceeded for
8 h, the sample turned into a gel as it remained immobile when
tilted or inverted (Figure 5F). The gel was thermoreversible;
placing the vial in an ice/water bath transformed the gel into a
free-flowing liquid. To quantitatively monitor the gelation pro-
cess, aliquots were taken at various time intervals (the vial was
placed in an ice/water bath to decrease the solution viscosity)
and oscillatory shear experiments were conducted at a frequency
of 1 Hz in a heating ramp mode at a heating rate of 3 °C/min. A
strain amplitude of γ = 0.2% was used for all aliquots to ensure
that the measurements were taken in the linear viscoelastic regime.
Figure 6 shows the rheological data for the selected aliquots taken
from the sample at 180 (A), 390 (B), 480 (C), and 1314 min (D).38
Clearly, for all these aliquots, below a certain temperature, the
values of dynamic storage modulus G0 and loss modulus G00 were
small and the data points were quite scattered, indicating that
they were liquids.41 With the increase of temperature, G0 and G00
increased and at a certain point G0 became larger than G00,
suggesting that the aliquots turned into gels. The crossover, G0 =
G00, has been commonly employed as an indicator of the solꢀgel
transition.42 Using this simple and convenient method, the sol-to-gel
transition temperatures (Tsolꢀgel) of the aliquots were deter-
mined and a plot of Tsolꢀgel versus time was made (Figure 7A). At
the beginning of the experiment, the Tsolꢀgel decreased relatively
fast, from 53.1 °C at 82 min to 41.0 °C at 180 min. After that, the
1
solution pH, we performed H NMR spectroscopy analysis to
look into the degree of the cleavage of phosphate groups at the
clouding time. We isolated the polymers from the samples with
pH of 4.23 and 5.48 at the clouding times by evaporating water of
a small portion of each sample using a stream of nitrogen flow,
dried them at 60 °C under high vacuum, and then analyzed them
by 1H NMR spectroscopy. The two 1H NMR spectra appeared to
be similar;38 the peak located at 4.06 ppm, which was from
ꢀCH2OCOꢀ and ꢀCH2OPꢀ of OPBA units in the copolymer,
mostly disappeared (the peak of ꢀCH2OCO- shifted to ∼4.2
ppm and overlapped with the peak there while the peak of
ꢀCH2OP- shifted to ∼3.7 ppm and overlapped with the peaks
there). This observation suggested that the degrees of the
cleavage of phosphate groups at the two pH values were quite
similar, though the clouding times were very different.
Figure 4. Plot of clouding time, the time for a 0.5 wt % solution of
P(DEGEA-co-OPBA) (R-1) in a 50 mM aqueous citrate buffer in the
presence of acid phosphatase at 37 °C to turn cloudy, as a function of
solution pH.
Figure 5. Digital optical pictures of a 7.9 wt % solution of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA) (ABA-1) in a 50 mM aqueous citrate
buffer with pH of 4.40 before the addition of acid phosphatase (A) and after the enzymatic reaction at 37 °C for 0 (B), 180 (C), 270 (D), 390 (E),
480 (F), 593 (G), and 1314 min (H).
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Figure 6. Plot of dynamic storage modulus G0 (9), dynamic loss modulus G00 (red 0), and tan δ (blue b) versus temperature for the aliquots taken
from a 7.9 wt % solution of P(DEGEA-co-OPBA)-b-PEO-b-P(DEGEA-co-OPBA) (ABA-1) in a 50 mM aqueous citrate buffer with pH of 4.40 in the
presence of acid phosphatase at 37 °C after 180 (A), 390 (B), 480 (C), and 1314 min (D). The data were collected from temperature ramp experiments
performed by using a frequency of 1 Hz, a strain amplitude of 0.2%, and a heating rate of 3 °C/min.
Figure 7. Plot of solꢀgel transition temperature (Tsolꢀgel) (A) and the maximum value of G0 (G0max) (B) versus reaction time. The values of Tsolꢀgel and
G0max were obtained from oscillatory shear experiments, which were performed in a heating ramp mode using a frequency of 1 Hz, a strain amplitude of
0.2%, and a heating rate of 3 °C/min.
change became slower; the Tsolꢀgel decreased by only 8 °C from
t = 180 to 1740 min. This is reasonable because with the increase
of viscosity, the diffusion of acid phosphatase became slower and
the access to the remaining phosphate groups on the thermo-
sensitive blocks by the enzyme became more difficult, causing the
cleavage reaction to slow down. Note that the Tsolꢀgel at 390 min
was 37.2 °C, in agreement with the visual observation that
the sample was almost a gel. The experiment was stopped after
1740 min at which only a very small difference in Tsolꢀgel from
that at 1314 min was observed.
Figure 7B shows the plot of the maximum value of G0 (G0max),
obtained from the temperature ramp experiments, versus reac-
tion time. G0max increased with time, from 151 Pa at t = 82 min to
821 Pa at 1740 min. Similar to the trend of Tsolꢀgel, the change in
the gel strength was faster at the beginning and became slower
with the increase of reaction time. We further characterized the
final gel sample by frequency sweep experiments (Figure 8). At
25 °C, the solution was a liquid that could flow when tilted. The
G0 was smaller than G00 in the range from 0.1 to 20 Hz and both
exhibited power law dependences on frequency f in the low
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Figure 8. Frequency dependences of dynamic storage modulus G0 (9) and loss modulus G00 (red 0) at (A) 25, (B) 28, (C) 31, and (D) 40 °C of
the final aqueous solution of ABA-1 with pH of 4.40 after the enzymatic reaction at 37 °C for 1740 min. A strain amplitude of 0.2% was used in all
frequency sweep experiments.
frequency region: G0 ∼ f 2 and G00 ∼ f (Figure 8A). This is the
typical rheological behavior of a viscous liquid.41 With the increase
of temperature, the frequency dependences of G0 and G00 evolved
(Figure 8B), and at 31 °C, which is close to the Tsolꢀgel (33 °C),
G0 and G00 were of similar magnitudes in the low frequency region
and G00 scaled with f 0.5 (Figure 8C). This is the signature of the
transition between liquid-like and solid-like behavior.41 At 40 °C,
the sample was a free-standing gel. G0 was significantly greater
than G00 and exhibited a much weaker dependence on frequency
(Figure 8D), which is a characteristic of solid-like behavior.41,42
The plateau modulus GN of the gel can be obtained from the
frequency sweep and it is known that the GN of a transient gel is a
measure of the number density of elastically active polymer
chains:
heating ramp (821 Pa). If the central block of every polymer
chain is elastically active in the gel, a calculation shows that the
GN is 3869 Pa at 40 °C. This means that 28.5% of polymer chains
formed effective network strands that could sustain an external
stress in the gel at this temperature. The relatively low percentage
of polymer chains acting as bridges among micellar cores
indicated the presence of the defects in the network, e.g., the
loops (two end blocks were located in the same micellar core)
and dangling chains (one end block stayed in water instead of in
the micellar cores, see Scheme 2).
1H NMR spectroscopy analysis of the polymer isolated after
the enzymatic gelation experiment showed that the peak located
at 4.06 ppm, which was from ꢀCH2OCOꢀ and ꢀCH2OPꢀ of
OPBA units in the copolymer, almost disappeared (Figure 1D).
This is the same as the dephosphorylation of P(DEGEA-co-
OPBA) (R-1) by acid phosphatase. The 31P NMR spectrum of
the dephosphorylated triblock copolymer is presented in
Figure 2C. Compared with the 31P NMR spectrum of ABA-1,
the peak was nearly invisible. Thus, both 1H and 31P NMR spec-
troscopy analysis suggested that the degree of the cleavage of
phosphate groups was high after the enzymatic reaction at 37 °C
for 1740 min.
To confirm that the acid phosphatase-induced gelation of the
7.9 wt % aqueous solution of ABA-1 at 37 °C originated from the
LCST behavior of the thermosensitive blocks formed after the
cleavage of phosphate groups (i.e., P(DEGEA-coꢀOHBA) blocks),
we conducted a dynamic light scattering study of the depho-
sphorylated triblock copolymer in a 50 mM aqueous citrate buffer
with a concentration of 0.02 wt % at the same pH value (4.40).
GN ¼ υkBT
where υ is the number density of elastically active polymer chains
(number of elastically active bridging chains per unit volume), kB
is Boltzmann constant, and T is the absolute temperature.42,43
The GN is usually evaluated as the G0 value at the frequency
where G00 exhibits the minimum value, because the increase of G00
at higher frequencies indicates a fast relaxation process separate
from the terminal flow process. This method for the determina-
tion of GN is well established for the entangled homopolymer
melts and has also been recently used in the study of thermo-
reversible transient gels.43 The frequency at which G00 exhibited a
minimum value at 40 °C was 12.59 Hz; therefore, the value of GN
was 1103 Pa, close to the modulus in the plateau zone in the
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Figure 9. Scattering intensity at scattering angle of 90° (A) and apparent hydrodynamic size Dh (B), obtained from CONTIN analysis, as a function of
temperature in a dynamic light scattering study of a 0.02 wt % solution of P(DEGEA-coꢀOHBA)-b-PEO-b-P(DEGEA-coꢀOHBA) in a 50 mM aqueous
citrate buffer with pH of 4.40. The triblock copolymer was obtained after the enzymatic dephosphorylation of ABA-1 in a 7.9 wt % solution proceeded at
37 °C for 1740 min.
Figure 10. Plot of solꢀgel transition temperature (Tsolꢀgel) (A) and the maximum value of dynamic storage modulus (G0max) (B) versus reaction time
for the enzyme-induced gelation of 7.9 wt % aqueous solution of ABA-1 at pH 4.67. The values of Tsolꢀgel and G0max were obtained from oscillatory shear
experiments, which were performed in a heating ramp mode using a frequency of 1 Hz, a strain amplitude of 0.2%, and a heating rate of 3 °C/min.
Figure 11. Plot of solꢀgel transition temperature (Tsolꢀgel) (A) and the maximum value of dynamic storage modulus (G0max) (B) versus reaction time
for the enzyme-induced gelation experiment of a 7.9 wt % aqueous solution of ABA-2 at pH = 4.41. The values of Tsolꢀgel and G0max were obtained from
oscillatory shear experiments, which were performed in a heating ramp mode using a fixed frequency of 1 Hz, a strain amplitude of 0.2%, and a heating
rate of 3 °C/min.
Figure 9 shows the scattering intensity at scattering angle of 90°
and apparent hydrodynamic size, obtained from CONTIN
analysis, as a function of temperature. Below 17 °C, the scattering
intensity was low and the apparent hydrodynamic size was small
(<10 nm), indicating that the polymer dissolved molecularly in
the buffer. When the temperature was raised above 16 °C, the
scattering intensity began to increase and above 25 °C the
hydrodynamic size was around 60 nm. The critical micellization
temperature (CMT) determined from the plot of scattering
intensity vs temperature was 17 °C. The thermo-induced
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micellization was reversible; lowering the temperature dissociated
the micelles. Differently, there was very little change in the
scattering intensity of 0.02 wt % aqueous solution of ABA-1 in
the same temperature range.38 Clearly, in the gelation experi-
ment, the enzymatic cleavage of hydrophilic phosphate groups
made the outer blocks less hydrophilic and decreased the LCST.
When the LCST became lower than the experimental tempera-
ture (37 °C), the triblock copolymer began to self-assemble into
micelles and formed a three-dimensional network gel with the
PEO blocks forming bridges. Note that the Tsolꢀgel of the final
sample was 33 °C, higher than the CMT at the same pH (17 °C).
This is understandable because the gelation requires the forma-
tion of a three-dimensional network with a sufficient mechanical
strength, while the CMT is the temperature at which the
thermosensitive P(DEGEA-co-OHBA) blocks begin to self-as-
semble to form micelles in a dilute aqueous solution.
copolymer at the same pH (4.41) was 13 °C,38 4 °C lower than
that of the polymer formed from ABA-1.
’ CONCLUSIONS
By incorporating phosphate groups into thermosensitive
blocks, we synthesized two thermo- and enzyme-responsive hydro-
philic ABA triblock copolymers, P(DEGEA-co-OPBA)-b-PEO-b-
P(DEGEA-co-OPBA), via ATRP of DEGEA and BPBA from a
difunctional PEO macroinitiator and subsequent removal of tert-
butyl groups of BPBA units. A model study using a random
copolymer P(DEGEA-co-OPBA) showed that the time for the
0.5 wt % solution to turn cloudy in the presence of acid phos-
phatase at 37 °C decreased with the decrease of pH from pH 5.48
to 4.39 and leveled off when the pH was further lowed to 4.23.
Therefore, we chose pH 4.4 to conduct the enzyme-induced
gelation experiments. As expected, a 7.9 wt % aqueous solution of
ABA-1 in the presence of acid phosphatase at 37 °C turned into a
gel. Rheological measurements showed that the Tsolꢀgel de-
creased with the increase of reaction time and reached 37.2 °C
at 390 min. The frequency sweep study of the final sample indicated
that 28.5% of polymer chains formed effective network strands in
the gel at 40 °C. 1H and 31P NMR spectroscopy analysis showed
that the degree of the cleavage of phosphate groups was high. The
enzymatic dephosphorylation of the triblock copolymer yielded
hydrophobic 4-hydroxybutyl groups in the thermosensitive outer
blocks and decreased the LCST from above to below the experi-
mental temperature (37 °C), resulting in the formation of a
3-dimentional network gel. This was confirmed by a DLS study
of the dephosphorylated triblock copolymer showing that the
CMT was 17 °C. In contrast, the gelation at pH = 4.67 took a
much longer time, which was in line with the effect of pH on the
rate of enzymatic cleavage of phosphate groups in a random
copolymer. A similar but slightly faster gelation behavior was
observed in the study of ABA-2 that had a higher molecular
weight and a slightly greater OPBA content. Although the gelation
in the present work took >5 h for both triblock copolymers, we
believe that it can be improved, for example, by selecting a
thermosensitive water-soluble polymer with a lower LCST, tuning
the phosphate content and the block length of thermosensitive
blocks, using a larger amount of enzyme, etc. Since the enzyme-
catalyzed reactions are highly specific and efficient, the method
reported in this article can be used to design biologically induced
micellar gels for potential applications in biomedical areas.
Enzyme-Induced Gelation of 7.9 wt % Aqueous Solution
of ABA-1 at pH = 4.67. As shown in Figure 4, the dephospor-
ylation of P(DEGEA-co-OPBA) (R-1) by acid phosphatase was
heavily affected by the solution pH. For example, the clouding
time at pH = 4.70 (tclouding = 41.4 min) was 5.4 times that at pH =
4.39 (tclouding = 7.6 min), though the difference in pH values was
only 0.31 pH unit. To gain an insight into the effect of pH on the
gelation, we carried out an experiment at a slightly higher pH
value (4.67) while other conditions remained the same. The
gelation was significantly slower (Figure 10A); it took 1560 min
for the Tsolꢀgel to decrease to 37.5 °C,38 while at pH 4.40 the
Tsolꢀgel reached 37.2 °C in 390 min, which was 4 times faster.
Interestingly, the G0max at the same or similar stage of the gelation
process was not heavily dependent on the solution pH. For
example, for the aliquot with Tsolꢀgel = 37.5 °C, the G0max was
542 Pa (Figure 10B), essentially identical to that of the aliquot
with Tsolꢀgel of 37.2 °C at pH 4.40 (G0max = 524 Pa, Figure 7B).
In addition, the highest value of G0max observed in this gelation
experiment (717 Pa) was comparable to that at pH = 4.40 (821 Pa).
Enzyme-Induced Gelation of 7.9 wt % Aqueous Solution
of ABA-2 at pH = 4.41. We also studied the gelation of a 7.9 wt %
solution of ABA-2 in a 50 mM citrate buffer induced by acid
phosphatase under the same conditions (pH = 4.41 and tem-
perature =37 °C). The amount of acid phosphatase with respect
to the mass of the thermosensitive blocks of ABA-2 was also
maintained at the same level, that is, 7.5 to 100. Note that ABA-2
had a slightly higher molecular weight (the Mn,SEC of its precursor
ABA-2-P = 58.9 kDa, PDI = 1.11) and a slightly higher OPBA
content (see Table 1). Same as ABA-1, the 7.9 wt % aqueous
solution of ABA-2 turned i0nto a micellar gel.38 Figure 11 shows
the plots of Tsolꢀgel and G max versus reaction time. Compared
with ABA-1, the gelation was slightly faster; the Tsolꢀgel de-
creased to 35.8 °C after 331 min. In addition, the Tsolꢀgel of the
final sample was lower, 28.5 °C, and the highest value of G0max
(1036 Pa) was slightly higher (821 Pa for ABA-1). This is likely
because the thermosensitive blocks were longer (DPtotal = 232 in
comparison to 171 of ABA-1) and the OPBA content was slightly
higher. 1H and 31P NMR spectroscopy analysis showed that the
degree of the cleavage of phosphate groups was similar to that of
ABA-1 at the end of the gelation experiment.38 Thus, one can
imagine that after dephosporylation the thermosensitive blocks
of the triblock copolymer were slightly more hydrophobic due to
the slightly higher content of hydrophobic 4-hydroxybutyl acrylate
units. This was confirmed by a dynamic light scattering study,
which showed that the CMT of the dephosphorylated triblock
’ ASSOCIATED CONTENT
Supporting Information. SEC of ABA-2-P and 1H NMR
S
b
spectra of ABA-2-P, ABA-2, and dephosphorylated triblock
copolymer from ABA-2, 31P NMR spectra of ABA-2-P, ABA-
2, and dephosphorylated polymer from ABA-2, SEC trace of R-
1-P and 1H NMR spectra of R-1-P and R-1, 31P NMR spectra of
1
R-1-P and R-1, H NMR spectra of R-1 and the polymers
isolated from the 0.5 wt % aqueous solutions of R-1 with pH
values of 4.23 and 5.48 in the presence of acid phosphatase at the
clouding times, additional rheological data for enzymatic gelation
experiment of ABA-1 at pH 4.40, DLS data for 0.02 wt % solu-
tion of ABA-1 with pH of 4.40, rheological data from gelation
experiment of ABA-1 at pH 4.67, rheological data from gelation
experiment of ABA-2, and DLS data for the dephosphorylated
polymer from ABA-2. This material is available free of charge via
the Internet at http://pubs.acs.org.
5774
dx.doi.org/10.1021/ma200991d |Macromolecules 2011, 44, 5764–5775

Macromolecules
ARTICLE
’ AUTHOR INFORMATION
(22) Lodish, H.; Berk, A.; Matsudaira, P.; Kaiser, C. A.; Krieger, M.;
Scott, M. P.; Zipursky, L.; Darnell, J. Molecular Cell Biology, 5th ed.;
Freeman: New York, 2003.
(23) Yang, Z.; Xu, B. Adv. Mater. 2006, 18, 3043–3046.
(24) Yang, Z.; Liagn, G.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2006,
128, 3038–3043.
Corresponding Author
*E-mail: zhao@ion.chem.utk.edu.
(25) Yang, Z.; Liang, G.; Xu, B. Acc. Chem. Res. 2008, 41, 315–326.
(26) Ehrbar, M.; Rizzi, S. C.; Schoenmakers, R. G.; Miguel, B. S.;
Hubbell, J. A.; Weber, F. E.; Lutolf, M. P. Biomacromolecules 2007,
8, 3000–3007.
’ ACKNOWLEDGMENT
This work was supported by the National Science Foundation
through Award DMR-0906913.
(27) Aimetti, A. A.; Tibbitt, M. W.; Anseth, K. S. Biomacromolecules
2009, 10, 1484–1489.
(28) Hu, B.-H.; Messersmith, P. B. J. Am. Chem. Soc. 2003, 125,
14298–14299.
’ REFERENCES
(29) Thornton, P. D.; Mart, R. J.; Ulijn, R. V. Adv. Mater. 2007,
19, 1252–1256.
(30) Yang, Z.; Gu, H. W.; Fu, D.; Guo, P.; Lam, J. K.; Xu, B. Adv.
Mater. 2004, 16, 1440–1444.
(31) Sperinde, J. J.; Griffith, L. G. Macromolecules 1997, 30,
5255–5264.
(32) Sanborn, T. J.; Messersmith, P. B.; Barron, A. E. Biomaterials
2002, 23, 2703–2710.
(33) Jin, R.; Hiemstra, C.; Zhong, Z.; Feijen, J. Biomaterials 2007,
28, 2791–2800.
(34) Park, K. M.; Shin, Y. M.; Joung, Y. K.; Shin, H.; Park, K. D.
Biomacromolecules 2010, 11, 706–712.
(35) Amir, R.; Zhong, S.; Pochan, D. J.; Hawker, C. J. J. Am. Chem.
Soc. 2009, 131, 13949–13951.
(1) Wichterle, O.; Lim, D. Nature 1960, 185, 117–118.
(2) Okano, T., Ed. Biorelated Polymers and Gels; Academic Press:
Boston, MA, 1998.
(3) He, C. L.; Kim, S. W.; Lee, D. S. J. Controlled Release 2008,
127, 189–207.
(4) Jeong, B.; Kim, S. W.; Bae, Y. H. Adv. Drug Delivery Rev. 2002,
54, 37–51.
(5) Joo, M. K.; Park, M. H.; Choi, B. G.; Jeong, B. J. Mater. Chem.
2009, 19, 5891–5905.
(6) Yu, L.; Ding, J. Chem. Soc. Rev. 2008, 37, 1473–1481.
(7) Cushing, M. C.; Anseth, K. S. Science 2007, 316, 1133–1134.
(8) Burdick, J. A.; Anseth, K. S. Biomaterials 2002, 23, 4315–4323.
(9) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science
2009, 324, 59–63.
(36) Jin, N. X.; Woodcock, J. W.; Xue., C. M.; O’Lenick, T. G.; Jiang,
X. G.; Jin, S.; Dadmun, M. D.; Zhao, B. Macromolecules 2011, 44,
3556–3566.
(10) Hamley, I. W. Block Copolymers in Solution: Fundamentals and
Applications; John Wiley & Sons: Chichester, U.K., 2005.
(11) Hamley, I. W. Philos. Trans. R. Soc. London A 2001, 359,
1017–1044.
(12) Jeong, B. M.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997,
388, 860–862.
(13) Kirkland, S. E.; Hensarling, R. M.; McConaught, S. D.; Guo, Y.;
Jarrett, W. L.; McCormick, C. L. Biomacromolecules 2008, 9, 481–486.
(14) Woodcock, J. W.; Wright, R. A. E.; Jiang, X. G.; O’Lenick, T. G.;
Zhao, B. Soft Matter 2010, 6, 3325–3336.
(15) (a) Li, C.; Tang, Y.; Armes, S. P.; Morris, C. J.; Rose, S. F.; Lloyd,
A. W.; Lewis, A. L. Biomacromolecules 2005, 6, 994–999. (b) Madsen, J.;
Armes, S. P.; Lewis, A. L. Macromolecules 2006, 39, 7455–7457. (c) Fechler,
N.; Badi, N.; Schade, K.; Pfeifer, S.; Lutz, J.-F. Macromolecules 2009, 42,
33–36.
(37) (a) Aoshima, S.; Kanaoka, S. Adv. Polym. Sci. 2008, 210,
169–208. (b) Han, S.; Hagiwara, M.; Ishizone, T. Macromolecules
2003, 36, 8312–8319. (c) Lutz, J.-F.; Hoth, A. Macromolecules 2006,
39, 893–896. (d) Zhao, B.; Li, D. J.; Hua, F. J.; Green, D. R. Macro-
molecules 2005, 38, 9509–9517. (e) Jiang, X. G.; Lavender, C. A.;
Woodcock, J. W.; Zhao, B. Macromolecules 2008, 41, 2632–2643.
(f) Jiang, X. G.; Zhao, B. Macromolecules 2008, 41, 9366–9375.
(g) O’Lenick, T. G.; Jiang, X. M.; Zhao, B. Polymer 2009, 50, 4363–4371.
(h) Jiang, X. M.; Wang, B. B.; Li, C. Y.; Zhao, B. J. Polym. Sci., Part A:
Polym. Chem. 2009, 47, 2853–2870. (i) Jiang, X. G.; Jin, S.; Zhong, Q. X.;
Dadmun, M. D.; Zhao, B. Macromolecules 2009, 42, 8468–8476.
(j) Qiao, Z.-Y.; Du, F.-S.; Zhang, R.; Liang, D.-H.; Li, Z.-C. Macro-
molecules 2010, 43, 6485–6494. (k) Horton, J. M.; Bai, Z. F.; Jiang, X. M.;
Li, D. J.; Lodge, T. P.; Zhao, B. Langmuir 2011, 27, 2019–2027.
(38) The data can be found in the Supporting Information.
(39) (a) Yin, X.; Hoffman, A. S.; Stayton, P. S. Biomacromolecules
2006, 7, 1381–1385. (b) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W.
Macromolecules 1993, 26, 2496–2500. (c) Urry, D. W. J. Phys. Chem. B
1997, 101, 11007–11028.
(16) Mortensen, K.; Brown, W.; Jørgensen, E. Macromolecules 1994,
27, 5654–5666.
(17) (a) Hvidt, S.; Jørgensen, E. B.; Schillꢀen, K.; Brown, W. J. Phys.
Chem. 1994, 98, 12320–12328. (b) Mortensen, K.; Brown, W.; Norden,
B. Phys. Rev. Lett. 1992, 68, 2340–2343. (c) Wanka, G.; Hoffmann, H.;
Ulbricht, W. Macromolecules 1994, 27, 4145–4159.
(18) (a) Li, C.; Madsen, J.; Armes, S. P.; Lewis, A. L. Angew. Chem.,
Int. Ed. 2006, 45, 3510–3513.(b) Sun, K. H.; Sohn, Y. S.; Jeong, B.
Biomacromolecules 2006, 7, 2871ꢀ2848.(b) (c) Vogt, A. P.; Sumerlin,
B. S. Soft Mater 2009, 5, 2347–2351.
(40) (a) Chen, Y.; Jakoncic, J.; Carpino, N.; Nassar, N. Biochemistry
2009, 48, 1681–1690. (b) Van Etten, R. L.; Waymack, P. P. Arch.
Biochem. Biophys. 1991, 288, 634–645.
(41) (a) He, Y. Y.; Boswell, P. G.; B€uhlmann, P.; Lodge, T. P. J. Phys.
Chem. B. 2007, 111, 4645–4652. (b) He, Y. Y.; Lodge, T. P. Macro-
molecules 2008, 41, 167–174.
(42) Noro, A.; Matshushita, Y.; Lodge, T. P. Macromolecules 2009,
42, 5802–5810.
(43) (a) Ferry, J. D. Viscoelastic Properties of Polymers, 3rd ed.; Wiley:
New York, 1980. (b) Larson, R. G.; Sridhar, T.; Leal, L. G.; McKinley,
G. H.; Likhtman, A. E.; McLeish, T. C. B. J. Pheol. 2003, 47, 809–818.
(c) Yoshida, T.; Kanaoka, S.; Watanabe, H.; Aoshima, A. J. Polym. Sci.,
Part A: Polym. Chem. 2005, 43, 2712–2722.
(19) (a) Determan, M. D.; Cox, J. P.; Seifert, S.; Thiyagarajan, P.;
Mallapragada, S. K. Polymer 2005, 46, 6933–6946. (b) Determan, M. D.;
Guo, L.; Thiyagarajan, P.; Mallapragada, S. K. Langmuir 2006, 22,
1469–1473. (c) Shim, W. S.; Yoo, J. S.; Bae, Y. H.; Lee, D. S. Bio-
macromolecules 2005, 6, 2930–2934. (d) Shim, W. S.; Kim, S. W.; Lee,
D. S. Biomacromolecules 2006, 7, 1935–1941. (e) Park, S. Y.; Lee, Y.; Bae,
K. H.; Ahn, C. H.; Park, T. G. Macromol. Rapid Commun. 2007, 28,
1172–1176. (f) Suh, J. M.; Bae, S. J.; Jeong, B. Adv. Mater. 2005,
17, 118–120.
(20) (a) Shim, W. S.; Kim, J. H.; Park, H.; Kim, K.; Kwon, I. C.; Lee,
D. S. Biomaterials 2006, 27, 5178–5185. (b) Dayananda, K.; He, C. L.;
Park, D. K.; Park, T. G.; Lee, D. S. Polymer 2008, 49, 4968–4973.
(21) (a) O’Lenick, T. G.; Jiang, X. G.; Zhao, B. Langmuir 2010,
26, 8787–8796. (b) O’Lenick, T. G.; Jin, N. X.; Woodcock, J. W.; Zhao,
B. J. Phys. Chem. B. 2011, 115, 2870–2881.
5775
dx.doi.org/10.1021/ma200991d |Macromolecules 2011, 44, 5764–5775