Multi-responsive polypeptide hydrogels derived from: N -carboxyanhydride terpolymerizations for delivery of nonsteroidal anti-inflammatory drugs

Article abstract of DOI:10.1039/c7ob00931c

A polypeptide-based hydrogel system, when prepared from a diblock polymer with a ternary copolypeptide as one block, exhibited thermo-, mechano- and enzyme-responsive properties, which enabled the encapsulation of naproxen (Npx) during the sol-gel transition and its release in the gel state. Statistical terpolymerizations of l-alanine (Ala), glycine (Gly) and l-isoleucine (Ile) NCAs at a 1:1:1 feed ratio initiated by monomethoxy monoamino-terminated poly(ethylene glycol) afforded a series of methoxy poly(ethylene glycol)-block-poly(l-alanine-co-glycine-co-l-isoleucine) (mPEG-b-P(A-G-I)) block polymers. β-Sheets were the dominant secondary structures within the polypeptide segments, which facilitated a heat-induced sol-to-gel transition, resulting from the supramolecular assembly of β-sheets into nanofibrils. Deconstruction of the three-dimensional networks by mechanical force (sonication) triggered the reverse gel-to-sol transition. Certain enzymes could accelerate the breakdown of the hydrogel, as determined by in vitro gel weight loss profiles. The hydrogels were able to encapsulate and release Npx over 6 days, demonstrating the potential application of these polypeptide hydrogels as an injectable local delivery system for small molecule drugs.

Full text of DOI:10.1039/c7ob00931c

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Multi-responsive polypeptide hydrogels derived
from N-carboxyanhydride terpolymerizations for
delivery of nonsteroidal anti-inflammatory drugs†
Cite this: DOI: 10.1039/c7ob00931c
Jingwei Fan, Richen Li, Hai Wang, Xun He, Tan P. Nguyen, Rachel A. Letteri,
Jiong Zou and Karen L. Wooley
*
A
polypeptide-based hydrogel system, when prepared from a diblock polymer with a ternary
copolypeptide as one block, exhibited thermo-, mechano- and enzyme-responsive properties, which
enabled the encapsulation of naproxen (Npx) during the sol–gel transition and its release in the gel state.
Statistical terpolymerizations of L-alanine (Ala), glycine (Gly) and L-isoleucine (Ile) NCAs at a 1 : 1 : 1 feed
ratio initiated by monomethoxy monoamino-terminated poly(ethylene glycol) afforded a series of
methoxy poly(ethylene glycol)-block-poly(L-alanine-co-glycine-co-L-isoleucine) (mPEG-b-P(A-G-I))
block polymers. β-Sheets were the dominant secondary structures within the polypeptide segments,
which facilitated a heat-induced sol-to-gel transition, resulting from the supramolecular assembly of
β-sheets into nanofibrils. Deconstruction of the three-dimensional networks by mechanical force (soni-
cation) triggered the reverse gel-to-sol transition. Certain enzymes could accelerate the breakdown of
the hydrogel, as determined by in vitro gel weight loss profiles. The hydrogels were able to encapsulate
and release Npx over 6 days, demonstrating the potential application of these polypeptide hydrogels as an
injectable local delivery system for small molecule drugs.
Received 14th April 2017,
Accepted 25th May 2017
DOI: 10.1039/c7ob00931c
rsc.li/obc
Hydrogels have attracted significant attention in the past
decades for their potential applications as scaffolds for tissue
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been engineering, matrices for controlled drug release, and absor-
widely used in the clinical treatment of acute and chronic pain bents for aqueous pollutant capture and removal.7–11 They are
and inflammation by inhibiting cyclooxygenases (COXs).1 a class of materials comprising polymeric or low-molecular-
NSAIDs are usually ingested orally in high dosages.1,2 Despite weight crosslinked hydrogelators that entrap substantial
their wide and systemic usage, in such high dosages, NSAIDs amounts of water or aqueous solutions while maintaining a
can cause undesirable side effects, such as adverse gastrointes- distinct three-dimensional structure.12,13 The high water
tinal symptoms and cardiovascular risks. To minimize these content and tunable properties of these materials render them
side effects, NSAIDs can be incorporated in a drug delivery suitable synthetic mimics of soft tissue microenvironments, as
system, for direct injection into target sites.3,4 After injection well as promising media for localized storage and delivery of
of this drug-loaded complex, NSAIDs may be released in therapeutic agents.14,15 In addition, the controlled incorpor-
response to applied external stimuli or internal enzyme degra- ation of supramolecularly noncovalent interactions or chemi-
dation to circumvent systemic dosing and minimize undesir- cally covalent linkages into hydrogelators yields hydrogels that
able side effects.5,6 Therefore, it is expected to be clinically respond to various physical, chemical or biological
beneficial to develop
material into a localized delivery agent for the treatment of sional structures and transitions between the sol and gel states
a
stimuli-responsive biocompatible stimuli.16,17 Regulated reorganizations within the three-dimen-
inflammation, such as osteoarthritis.
provide tunable release rates of encapsulated small mole-
cules.18 Recently, Cao et al. reported the controlled release of
naproxen (Npx), one kind of NSAID, from a γ-ray-responsive
hydrogel, composed of a diselenide-containing polymer and
an Npx conjugated dipeptide, by UV irradiation.6 At present,
Npx-loaded hydrogels remain limited to primarily low-mole-
cular-weight hydrogelators,3,4 and few studies have been per-
formed on polymeric hydrogelators for NSAID delivery.
Departments of Chemistry, Chemical Engineering, Materials Science and
Engineering, and Laboratory for Synthetic-Biologic Interactions, Taxes A&M
University, P.O. BOX 30012, 3255 TAMU, College Station, TX, 77842, USA.
E-mail: wooley@chem.tamu.edu; Tel: +1 979-845-4077
†Electronic supplementary information (ESI) available: Fig. S1–S6. See DOI:
10.1039/c7ob00931c
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Among biodegradable polymers, polypeptides offer signifi- Instrumentation
cant advantages, including high biocompatibility, multiple
functionalities and tunable secondary structures.19–23 These 1H and 13C NMR spectra were recorded on a Varian Inova 300
materials have been widely used in the construction of supra- spectrometer interfaced to a UNIX computer using VnmrJ soft-
molecularly-assembled hydrogels.18,24–26 Their broad applica- ware. Chemical shifts were referenced to the solvent resonance
bility is attributed to the precisely-defined nano- and micro- signals. Attenuated total reflectance-Fourier transform infrared
structures derived from hierarchical assembly of polypeptides, spectroscopy (ATR-FTIR) spectra were recorded on an IR
and the variety of stimuli-responsive behaviors exhibited by Prestige 21 system (Shimadzu Corp.) and analysed using
natural and synthetic polypeptides.27–29 Secondary structures, IRsolution v. 1.40 software. Circular dichroism (CD) spectra
especially the α-helix and β-sheet conformations, can serve as were recorded on a Chirascan CD spectrometer from Applied
the driving forces to form well-defined fibrous structures, Photophysics, Ltd (Leatherhead, UK) equipped with a 150 watt
which further assemble into noncovalently crosslinked three- xenon arc lamp. The polymer solutions for CD measurements
dimensional networks that can encapsulate aqueous media to were prepared at the indicated concentration in nanopure
form hydrogels.24,30,31 Recently, Chen and co-workers reported water. CD spectra were acquired between 180 and 280 nm,
a
biocompatible polypeptide hydrogelator, poly(ethylene using a wavelength step of 1.0 nm, in a quartz cell with a path
glycol)-block-poly(γ-propargyl-L-glutamate) (PEG-b-PPLG), with length of 1.0 mm and analyzed using Pro-Data Version 5
a temperature-triggered sol-to-gel transition driven by the for- software.
mation of β-sheet structures.26 Herein, we report a thermo-,
Thermogravimetric analysis (TGA) was performed under
mechano-, and enzyme-responsive polypeptide-based hydroge- argon atmosphere using Mettler-Toledo model TGA/
lator, methoxy poly(ethylene glycol)-block-poly(L-alanine-co- SDTA851e (Mettler-Toledo, Inc., Columbus, OH), with
a
a
glycine-co-L-isoleucine) (mPEG-b-P(A-G-I)), synthesized by stat- heating rate of 10 °C min−1. Measurements were analysed
istical terpolymerization of α-amino acid N-carboxyanhydride using Mettler-Toledo STARe v. 7.01 software. Glass transition
(NCA) monomers of L-alanine, glycine and L-isoleucine. This temperatures (Tg) were measured by differential scanning
termonomer mixture was selected for statistical incorporation calorimetry (DSC) on a Mettler-Toredo DSC822®, with a
of matrix metalloproteinase (MMP) MMP-2 and 9-active G-IAG heating rate of 10 °C min−1. Measurements were analysed
cleavage sites.21,32 In contrast to many polypeptide hydrogela- using Mettler-Toledo STARe v. 7.01 software. The Tg was taken
tors that undergo gel-to-sol with increased temperature, a as the midpoint of the inflection tangent of the second
reverse thermal gelation profile and sonication-triggered heating scan.
gel-to-sol transition were observed for these materials.
Wide-angle X-ray scattering (WAXS) was performed on a
Detailed characterization studies and analyses were performed Bruker D8 Bragg Brentano X-ray powder diffractometer.
to investigate the driving force for the heat-induced hydrogela- Samples were placed in the sample holder of a two-circle
tion and to understand the correlation between nanostructural goniometer, in a radiation safety enclosure. The X-ray source
morphology and stimuli-responsive sol–gel transitions. The was a 2.2 kW Cu X-ray tube, maintained at an operating
encapsulation and release of Npx demonstrated the potential current of 40 kV and 40 mA. The standard Bragg–Brentano
application of these polypeptide hydrogels as a local delivery para-focusing mode was employed, with the X-ray source diver-
system to reduce side effects stemming from the systemic ging from a diffraction slit (1 mm) of the tube to irradiate the
delivery of Npx.
sample and then converging onto a position-sensitive X-ray
Detector (Lynx-Eye, Bruker-AXS). The two-circle 250 mm dia-
meter goniometer was computer-controlled with independent
step motors and optical encoders for the θ and 2θ circles with
a smallest angular step size of 0.0001° 2θ. Data collection was
automated using the COMMANDER program by employing a
Experimental section
Materials
Ethyl acetate, n-hexane, tetrahydrofuran (THF), diethyl ether, DQL file and analyzed in EVA software.
N,N-dimethylformamide (DMF, anhydrous), L-alanine, Transmission electron microscopy (TEM) was performed on
L-isoleucine, bis(trichloromethyl) carbonate, glycine, trichloro- a JEOL 1200 EX microscope operating at 100 kV, and micro-
methyl chloroformate, Proteinase K, Trypsin and naproxen graphs were recorded at calibrated magnifications using an
(Npx)
were
purchased
from
Sigma-Aldrich
(USA). SLA-15C CCD camera. The final pixel size was 0.42 nm per
Monomethoxy-monoamino-terminated poly(ethylene glycol) pixel. Samples for TEM measurements were prepared as
(mPEG45-NH2, Mn = 2000 g mol−1) was purchased from Rapp follows: 10 µL of the diluted solution (with a polymer concen-
Polymere (Germany). All chemicals were used without further tration of 1 mg mL−1) was deposited onto a carbon-coated
purification, unless otherwise noted. Nanopure water (18.2 copper grid, and after 2 min, the excess solution was quickly
MΩ cm) was acquired from a Milli-Q water filtration system, wicked away by a piece of filter paper. The samples were then
Millipore Corp. (St Charles, MO). Tris-HCl buffered saline and negatively stained with 1 wt% phosphotungstic acid (PTA)
phosphate buffered saline (PBS) were purchased from Sigma- aqueous solution. After 30 s, the excess staining solution was
Aldrich (USA) as dry powders, and dissolved in deionized water quickly wicked away by a piece of filter paper and the samples
(1.0 L) to obtain the desired buffer solutions.
were left to dry under vacuum overnight.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
Scanning electron microscopy (SEM) imaging was per- under N2 bubbling for 4.5 h. The reaction mixture was cooled
formed on a JOEL JSM 6400 SEM operated at an acceleration to room temperature and unreacted L-isoleucine was removed
voltage of 15 kV. The preparation of samples for SEM involved by filtration. The filtrate was concentrated and the resulting
placing 100 µL hydrogel on a carbon thin film. The gel was yellow oil was recrystallized three times from THF/n-hexane
then subjected to immediate freezing by liquid nitrogen, 2 : 1 (v/v), and dried under vacuum to obtain the product as
followed by lyophilisation for 3 days. The surface of the gel was white crystals (2.2 g, yield: quantitative). The product was
sputter-coated with gold for 3 min under argon before stored in a −20 °C freezer under N2 protection. 1H NMR
imaging.
(DMSO-D6, 300 MHz, ppm): δ 0.86 (t, 3H, J = 9.0 Hz,
Sonication-responsive experiments were performed with an CH3CHCH2CH3), 0.92 (d, 3H, J = 7.0 Hz, CH3CHCH2CH3), 1.28
ultrasonic homogenizer (maximum power, 150 W, 20 kHz, (m, 2H, CH3CHCH2CH3), 1.79 (s, 1H, CH3CHCH2), 4.39, (s, 1H,
Model 150 V/T, Biologics, Inc.) equipped with a micro tip with COCHNH), 9.09 (br, 1H, COCHNH). 13C NMR (DMSO-D6,
a diameter of 3.81 mm, employing a power outlet of 45 W at a 75 MHz, ppm): δ 11.3, 14.7, 23.8, 36.5, 61.7, 152.2, 170.8. FTIR
frequency of 20 kHz at room temperature.
(cm−1): 3500–3100, 2970, 2881, 1848, 1750, 1215, 1091, 1070,
939. HRMS: calculated [M − H]− for C7H11NO3: 156.0661,
found: 156.0665.
Experimental procedures
Synthesis of L-alanine N-carboxyanhydride (Ala NCA)
Synthesis of methoxy poly(ethylene glycol)-block-poly(L-
monomer. In a 250 mL three-necked round bottom flask alanine-co-glycine-co-L-isoleucine)
(mPEG-b-P(A-G-I))
by
equipped with a magnetic stir bar, condenser, and N2 inlet, statistical terpolymerization of NCA monomers initiated by
L-alanine (5.0 g, 56 mmol, 3 equiv.) and bis(trichloromethyl) mPEG-NH2. As a representative example, the synthesis of
carbonate (5.6 g, 19 mmol, 1 equiv.) were added and sus- mPEG45-b-P(A5-G7-I5) is described. In a 10 mL flame-dried
pended in 100 mL dry THF at 50 °C under N2 bubbling for 4 h. Schlenk flask equipped with a magnetic stir bar, mPEG45-NH2
The reaction mixture was cooled to room temperature and (80.0 mg, 0.04 mmol, 1 equiv.) was added into a solution of
unreacted L-alanine was removed by filtration. The filtrate was Ala NCA (23.0 mg, 0.2 mmol, 5 equiv.), Gly NCA (20.0 mg,
concentrated and the resulting brown oil material was further 0.2 mmol, 5 equiv.) and Ile NCA (31.4 mg, 0.2 mmol, 5 equiv.)
purified by recrystallization three times from THF/n-hexane dissolved in 6.4 mL anhydrous DMF solution. The reaction
2 : 1 (v/v), and dried under vacuum to obtain the product as mixture was stirred at 400 rpm under continuous N2 flow
white crystals (1.4 g, yield: 63%). The product was stored in a (100 mL min−1) at room temperature for 12 h. During the
−20 °C freezer under N2 protection. 1H NMR (DMSO-D6, polymerization, the Schlenk flask was capped with a rubber
300 MHz, ppm): δ 1.33 (d, 3H, J = 7.0 Hz, COCH(CH3)NH), 4.48 stopper with a needle outlet connected to a syringe barrel
(q, 1H, J = 7.0 Hz, COCH(CH3)NH), 9.00 (br, 1H, COCH(CH3) filled with drierite. Monomer consumption was monitored by
NH). 13C NMR (DMSO-D6, 75 MHz, ppm): δ 18.8, 52.8, 151.7, the disappearance of the NCA anhydride peak at 1788 cm−1
172.4. FTIR (cm−1): 3500–3100, 2838, 1840, 1760, 1280, 1139, using ATR-FTIR. The polymerization solution was precipitated
1023, 925. HRMS: calculated [M − H]− for C4H5NO3: 114.0191, into diethyl ether under vigorous stirring, and the polymer was
found: 114.0193.
Synthesis of glycine (Gly) NCA monomer. In a 250 mL three- temperature to yield a white powder (102.2 mg, yield: 80%). 1H
necked round bottom flask equipped with a magnetic stir bar, NMR (300 MHz, TFA-D, ppm): 0.70–1.10 (br, 15H,
isolated by centrifugation and dried under vacuum at room
δ
condenser, and N2 inlet, glycine (5.0 g, 67 mmol, 3 equiv.) was CH3CHCH2CH3; br, 15H, CH3CHCH2CH3), 1.46 (br, 10H,
suspended in 100 mL THF and heated to 50 °C. CH3CHCH2CH3; br, 15H, CH3CH), 1.60–2.10 (br, 5H,
Trichloromethyl chloroformate (4.4 g, 22 mmol, 1 equiv.) was CH3CHCH2CH3), 3.52 (s, 3H, CH3O), 3.85 (s, 180H, CH2CH2O),
added into the reaction by syringe and the reaction mixture 4.20 (br, 14H, NHCH2CO), 4.64 (br, 10H, NHCHCO). 13C NMR
was heated at reflux for another 4 h under N2 atmosphere. The (75 MHz, TFA-D, ppm): δ 12.9, 13.8, 15.8, 24.0, 36.9, 42.6, 50.1,
reaction mixture was filtered before cooling to room tempera- 57.5, 69.5, 70.7, 172.5, 174.8, 175.6. FTIR (cm−1): 3286, 2868,
ture. The filtrate was concentrated, recrystallized three times 1699, 1625, 1515, 1451, 1349, 1249, 1099, 948, 849, 696. DSC:
from THF/n-hexane 2 : 1 (v/v) and dried under vacuum to Tg = −17 °C, Tc = −15 °C, Tm = 30 °C, Tg = 126 °C. TGA in N2:
obtain the product as white crystals (1.5 g, yield: 67%). The 20–233 °C, 2% mass loss; 233–367 °C, 29% mass loss;
product was stored in a −20 °C freezer under N2 atmosphere. 367–430 °C, 46% mass loss; 430–500 °C, 3% mass loss; 20%
1H NMR (300 MHz, DMSO-D6, ppm): δ 4.18 (s, 2H, COCH2NH), mass remaining above 500 °C.
8.83 (br, 1H, COCH2NH). 13C NMR (75 MHz, DMSO-D6, ppm):
Determination of critical gelation temperature (Tgel
)
δ 46.3, 153.0, 169.4. FTIR (cm−1): 3500–3100, 2955, 1876, 1740,
1275, 1113, 1061, 925. HRMS: calculated [M − H]− for Polymer solutions were prepared by dissolving polypeptide
C3H3NO3: 100.0035, found: 100.0039. powders in nanopure water at the desired concentrations.
Synthesis of L-isoleucine (Ile) NCA monomer. In a 250 mL Homogeneous solutions were obtained by 2 min sonication
three-necked round bottom flask equipped with a magnetic with a probe sonicator at room temperature. Hydrogel for-
stir bar, condenser, and N2 inlet, L-isoleucine (5.0 g, 42 mmol, mation was determined using the test tube inversion
3 equiv.) and bis(trichloromethyl) carbonate (4.6 g, 14 mmol, 1 method.33 Briefly, each sample was dissolved in nanopure
equiv.) were added and suspended in 100 mL dry THF at 50 °C water at a predetermined concentration in a 2 mL vial. After
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
equilibrium at room temperature overnight, the vial was
immersed in an oil bath equilibrated at given temperatures for
Results and discussion
30 min, and the vial was then inverted. If no flow was observed Stemming from our general interest in the creation of bio-
after 5 min, the sample was regarded as a gel. For measure- degradable stimuli-responsive polymeric materials with simple
ment of the sol–gel transition temperature, the temperature molecular design and synthetic feasibility, especially for future
was raised at a step of 0.5 °C h−1. The sol–gel transition temp- biomedical applications, we began investigation into the
eratures were recorded as the average of three measurements.
syntheses and physical properties of hybrid diblock polymers,
having a statistical terpolypeptide as one of the block seg-
ments, with the expectation that the polypeptide would
respond to multiple external stimuli, including thermal, mech-
Hydrolytic and enzymatic degradation of polypeptide
hydrogels
Degradation of the polypeptide hydrogel was performed by anical and biological triggers. Recently, our group reported a
measuring the weight loss of the hydrogel at predetermined novel approach to tune the stimuli-responsive properties of
time points spanning 10 days after immersion of the hydrogel polypeptide gel systems via control of the secondary structures
in degradation media.24 An aliquot of polypeptide gelator solu- within the polypeptide segment, which was offered by vari-
tion (4 wt% in nanopure water, 0.3 mL) was placed in a 1.5 mL ation in the feedstock selection during the statistical copoly-
vial and maintained at 37 °C overnight for gel formation. Tris- merization of various N-carboxyanhydride (NCA) monomers.
HCl buffer (0.05 M, pH = 8.0, 0.2 wt% NaN3, 0.5 mL) was The last two decades have witnessed significant development
added gently into the vial without disturbing the gel. The vial of controlled ring-opening polymerization (ROP) of amino acid
was then maintained at 37 °C throughout the degradation NCAs as a convenient and efficient synthetic approach for the
study. Tris-HCl buffer solution, together with any accumulated preparation of well-defined polypeptide materials in large
gel debris, was removed at desired time points and the weight scales.35–41 In fact, NCA ROP can be conducted using normal
of the vial was recorded to assess the change of hydrogel Schlenk techniques, with the enhancement of polymerization
weight. The remaining weight percent of the hydrogels was cal- rate and retention of controlled features achieved through a
culated from the weight of the hydrogel at each time point rela- straightforward N2 flow method.40,42 Using this technique, the
tive to the weight measured before Tris-HCl buffer addition. In methoxy poly(ethylene glycol)-block-poly(L-alanine-co-glycine-
the enzymatic degradation study, the enzyme was first dis- co-L-isoleucine) (mPEG-b-P(A-G-I)) was synthesized by statistical
solved in Tris-HCl buffer solution (0.05 M, pH = 8.0, 0.2 wt% copolymerization of L-alanine (Ala), glycine (Gly) and
NaN3) with a concentration of 1 mg mL−1. The enzyme solu- L-isoleucine (Ile) NCAs, initiated by monomethoxy mono-
tion was stored at −20 °C and warmed to 37 °C immediately amino-terminated poly(ethylene glycol) (mPEG45-NH2, Mn
=
prior to addition into the hydrogel. Proteinase K and Trypsin 2000 g mol−1) as the macroinitiator under continuous N2 flow
were added at multiple time points over the duration of the (100 mL min−1), as shown in Scheme 1. A series of block
experiment, and each degradation study was repeated three polymers with polypeptide segments of various lengths was
times, with the remaining weight listed as the average of the synthesized by varying the ratio of NCA monomers to the
three measurements. Due to limited supply and high expense macroinitiator, while maintaining the ratio of three NCA
of MMP-2, the degradation study was conducted with a single monomers at 1 : 1 : 1 (Table 1). For each terpolymerization, the
initial time point for addition of MMP-2 and in a single run.
NCA monomers and macroinitiator were dissolved in anhy-
drous N,N-dimethylformamide (DMF) and the ROP was
allowed to proceed for 12 h at room temperature. Spontaneous
Naproxen (Npx) encapsulation and release
The loading of Npx was conducted by dissolving the block
polypeptides in PBS buffer solution (0.01 M, pH = 7.4, 0.3 mL)
containing 1 mg mL−1 of Npx. Each vial was held at 37 °C over-
night to facilitate hydrogel formation. PBS buffer solution (pH
= 7.4, 0.5 mL) was added to each vial and the samples were
incubated at 37 °C, shaking at 60 rpm during the release
study. At predetermined time points, the PBS buffer was
removed for analysis and replaced with fresh solutions
(0.5 mL). The release experiments were repeated three times
and the error bars shown are the standard deviations. The
concentration of the released Npx was determined as a func-
tion of time on a Shimadzu Prominence HPLC equipped with
an SPD-20 AV prominence UV-Vis detector set to 254 nm and a
Waters X Bridge C8 column (4.6 × 150 mm, 5 µm, 100 Å). The
system was operated at 40 °C in an eluent system composed of
55% 20 mM ammonium acetate buffer and 45% acetonitrile in
isocratic mode with a flow rate of 1 mL min−1 and a run time
of 15 min.34
Scheme 1 Synthetic
copolymer.
approach
for
mPEG-b-P(A-G-I)
diblock
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 Average degree of polymerization (DPn), number-averaged
molecular weight (Mn) and composition of the mPEG-b-P(A-G-I)
diblock copolymers synthesized with different monomer to initiator
ratios
ture (Tgel). The obtained hydrogels were stable and no gel-to-
sol transitions were observed when hydrogels were left undis-
turbed for at least 3 days at room temperature. The effects of
polymer concentration and composition on Tgel were deter-
mined by measurement of Tgel values for a series of block poly-
mers as a function of concentration, and the results are sum-
marized in Fig. 2. For each polymer, Tgel decreased as polymer
concentration increased, as seen from the sol–gel phase
diagram in Fig. 2. For example, the Tgel of polymer 1 decreased
from 84 °C for a 2 wt% solution to 22 °C for a 6 wt% solution.
b
Entry
M : Ia
DPn
Mn b (kDa)
Polymerb
1
2
3
4
5
15 : 1
30 : 1
50 : 1
80 : 1
100 : 1
17
30
43
63
72
3.3
4.2
5.1
6.4
6.9
mPEG45-b-poly(A5-G7-I5)
mPEG45-b-poly(A8-G13-I9)
mPEG45-b-poly(A15-G19-I9)
mPEG45-b-poly(A24-G29-I10
mPEG45-b-poly(A28-G34-I10
)
)
a All polymerizations were conducted using mPEG45-NH2 as the macro- For 6 wt% solutions, the Tgel value increased from 22 °C for
initiator at a concentration of 10 mg mL−1, and a molar ratio of
polymer 1, with a polypeptide block length of 17 repeat units,
Ala : Gly : Ile = 1 : 1 : 1. M : I indicated the ratio of the total monomer
(Ala + Gly + Ile) to initiator. b Calculated by 1H NMR spectroscopy.
to 32 °C for polymer 2, with a polypeptide block length of 30
repeat units, whereas the Tgel value decreased to 16 °C for
polymer 5, having the longest polypeptide block (DPn = 72).
Due to a Tgel value (35 °C) similar to the physiological tempera-
ture of the human body, we focused our efforts on hydrogels
formed from polymer 1 solutions (4 wt%) to investigate the
potential for application as a drug delivery system.
gelation in DMF was observed during each polymerization
within 8 h, and the targeted diblock polymers were obtained
as white powders by precipitation of the reaction mixture into
diethyl ether, followed by drying under vacuum.
In order to further understand the gelation mechanism, the
supramolecular structure of the 4 wt% polymer 1 hydrogel in
the solid state was investigated by attenuated total reflectance-
Fourier transform infrared spectroscopy (ATR-FTIR) and wide-
angle X-ray scattering (WAXS). The characteristic absorbance
bands of secondary structures were clearly observed in the
The molecular weight and composition of the block poly-
mers were determined by H NMR spectroscopy in deuterated
1
trifluoroacetic acid (TFA-D) to break the strong hydrogen
bonding interactions within the polypeptide segments in order
to solubilize the polymers (Fig. 1). Comparison of the intensi-
ties of the methylene proton resonances of the mPEG chains
at ca. 3.85 ppm (b in Fig. 1) with those of the Gly methylene
protons at ca. 4.20 ppm (e in Fig. 1), the Ile side chain methine
proton at ca. 1.85 ppm (g in Fig. 1), and the backbone methine
protons for both Ile and Ala at ca. 4.64 ppm (c + f in Fig. 1)
yielded the block lengths and compositions. For both Ala and
Gly, the calculated numbers of repeat units from 1H NMR
spectroscopy correlated linearly with the theoretical values,
while for Ile, the numbers of repeat units in the obtained poly-
mers plateaued in polymerizations conducted at Ile NCA
monomer to macroinitiator ratios above 10 (Fig. S1 in ESI†).
These results suggested higher reactivities of Gly and Ala NCA
monomers relative to the Ile NCA monomer.43–45
FTIR spectrum, especially in the amide
I
region
(1600–1700 cm−1) (Fig. 3). The absorbance bands at 1701 cm−1
and 1625 cm−1 (amide I), 1515 cm−1 (amide II) and 694 cm−1
(amide V), were attributed to β-sheet conformation.47 Further
quantitative analyses of secondary structure populations were
conducted by employing the secondary derivative of the absor-
bance as a function of wavenumber, and then using deconvo-
lution strategies to extract the relative peak intensities in the
heavily populated amide I region (Fig. S2 in ESI†).48–50 The
β-sheet contribution was calculated to be 63%, which was
similar to the β-sheet content (66%) for PEG45-b-PGly30 in the
The ability to form hydrogels of copolymers with different
polypeptide block lengths was characterized by test tube inver-
sion experiments.33,46 Polymer solutions were prepared at a
variety of concentrations in nanopure water at room tempera-
ture, and hydrogels formed upon heating the solutions until
gelation was observed, i.e., above the critical gelation tempera-
Fig. 2 Tgel of mPEG-b-P(A-G-I) hydrogels as a function of polymer
concentration.
Fig. 1 1H NMR spectrum of polymer 1 acquired in TFA-D.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 4 (a) CD spectra of polymer 1 in nanopure water (0.2 wt%) as a
function of temperature from 10 to 60 °C. (b) Estimation of β-sheet
content at each temperature from the CD spectra in the range of
190–240 nm with CDPro software using Program CONTILL and SDP48
as the reference sets.
Fig. 3 ATR-FTIR spectrum of polymer 1 in the solid state.
β-sheet content increased from 0% to 12%, suggesting gelation
was driven by temperature-triggered β-sheet formation.
Transmission electron microscopy (TEM) and scanning
electron microscopy (SEM) were utilized to study the nano-
and microstructure of the hydrogels from polymer 1. The TEM
sample was prepared from a 0.1 wt% polymer solution in
nanopure water and stained with 1 wt% phosphotungstic acid
(PTA) aqueous solution. The bright-field TEM image of the
hydrogels showed fibrillary nanostructures with an average
width of 13.3 nm (Fig. 5a). The narrow width distribution of
nanofibrils (standard deviation = 1.5 nm, Fig. S6 in ESI†),
obtained by counting 100 nanofibrils in the TEM image, indi-
cated that the hydrogels had a well-defined nanofibrillar mor-
phology. To visualize the microstructure of the hydrogels, SEM
samples were prepared by lyophilising the hydrogels with the
aim to maintain the gel networks. From the SEM image
(Fig. 5b), interconnected three-dimensional networks and
highly porous structures with various sizes were observed,
which could be potentially utilized to encapsulate and release
cargoes, such as therapeutic drugs or cells.
solid state reported previously.43 In addition, the WAXS pattern
of the dried hydrogels showed a d spacing of 4.6 Å with strong
signals from PEG crystallization reflection (Fig. S3 in ESI†),
consistent with the melting and crystallizing peaks in the DSC
traces of polymer 1 (Fig. S4 in ESI†). A d spacing of 4.6 Å has
also been observed in poly(L-alanylglycine)51 and the self-
assembled PEG-b-β-strand-peptides,52 and attributed to anti-
parallel β-sheet conformation. The results from qualitative and
quantitative FTIR analyses, together with the WAXS character-
ization, indicated the dominance of β-sheet structures in the
solid state of the hydrogels.
The strong absorption of water in the amide I region
(1600–1700 cm−1) complicates the analysis of the secondary
structure population of the hydrogels in the hydrated state.
However, the O-D bending of D2O appears at lower wavenum-
bers than that of H2O (Fig. S5a in ESI†), enabling in situ ana-
lysis of secondary structure.53 The hydrogel sample was pre-
pared by dissolving the polymer powder in D2O by sonication
and maintaining the polymer solution at 37 °C overnight prior
to analysis. With D2O as the background, the characteristic
β-sheet absorbance at 1625 cm−1 (amide I) was observed in the
FTIR spectrum (Fig. 6b and S5b in ESI†), indicating the domi-
nation of β-sheets in the secondary structure population in the
hydrated gels, which was in agreement with the results from
analyses conducted in the solid state.
Based on the results from NMR, ATR-FTIR, WAXS, CD, SEM
and TEM analyses, a mechanism for the hydrogelation of
mPEG-b-P(A-G-I) was proposed (Scheme 2). One-dimensional
stacking of the β-sheet-forming polypeptide segments afforded
well-defined fibrillary nanostructures, as observed spectrally
and microscopically. Using hydrogels from polymer 1 as an
To investigate the conformational changes of polymer 1 in
nanopure water, temperature-dependent circular dichroism
(CD) was employed to monitor the changes of the secondary
structure as a function of temperature. Polymer solutions pre-
pared at 0.2 wt% for CD measurements were placed in a
quartz cell with a path length of 1.0 mm and spectra were
acquired between 180 and 280 nm with a wavelength step of
1.0 nm. As revealed in Fig. 4a, the intensity of the negative
band at 220 nm increased gradually with temperature from 10
to 60 °C. The β-sheet contents at different temperatures were
estimated with CDPro software with Program CONTILL and
SDP48 as the reference sets, and the results are summarized in
Fig. 5 (a) TEM image of the hydrogel from polymer 1 and (b) SEM
Fig. 4b.54 As the temperature increased from 10 to 60 °C, the image of the lyophilized hydrogel from polymer 1.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 6 (a) Digital photographs showing reversible sol–gel transitions
triggered by thermal and sonication stimuli, respectively. (b) In situ FTIR
spectra in the gel state (red trace) and the sol state when sonication was
applied (black trace). (c) TEM image of the hydrogel in the sol state from
polymer 1 after sonication for 1 min.
Scheme 2 Proposed mechanism of the reversible sol–gel transitions
observed in the hydrogels from polymer 1, including the heat-induced
hydrogelation resulting from supramolecularly assembled nanofibrils
and sonication-triggered gel-to-sol transition.
example, the radius of gyration of mPEG with a molecular the copolymer after sonication, in situ FTIR of the hydrogels in
weight of 2000 Da can be estimated as 18 Å.52 The axial dis- the sol state was performed. As shown in Fig. 6b, the FTIR
tance between adjacent α-amino acids is about 3.5 Å, thus the spectrum of the sol state was almost identical to the spectrum
width of the polypeptide domain can be calculated to be 60 Å of the gel state, indicating that no significant change in the
in the conformation of β-sheet secondary structures. secondary structures occurred during the sonication treat-
Therefore, the diameter of the assembled nanofibrils can be ment, and that the dominant β-sheet contribution was main-
estimated to be about 13.2 nm, which is in agreement with the tained in the sol state. TEM was also employed to compare the
measured width (13.3 nm) of nanofibrils in the TEM image. difference of the nanoscale morphologies between the gel and
The hydrophilic mPEG segments are postulated to prevent the sol states, with the aim to further understand the sol–gel tran-
precipitation of the block polymer via increasing its aqueous sitions upon application of external stimuli. In the TEM image
solubility and maintaining the hydrogel networks by encapsu- of the hydrogel after sonicating for 1 min to produce a sol, the
lating water molecules. Heat-triggered gelation is attributed to well-defined nanofibrils observed in the gel state disappeared
the formation and strengthening of β-sheet secondary struc- (Fig. 5a), and short nanofibrils and spherical aggregates were
tures and the dehydration of PEG at elevated temperature to observed instead (Fig. 6c). Conversion of the longer nanofibrils
enhance the hydrophobic interactions. The entanglement of into short nanofibrils and spherical aggregates, which lacked
nanofibrils resulting from long range interactions, including long range connectivity, resulted in the breakdown of the
peptide–peptide interaction and PEG–PEG association, con- hydrogel networks.55 When the sol was heated to 37 °C for
structs the three-dimensional networks of this polypeptide over 1 h, reformation of the longer nanofibrils reconnected the
hydrogel system.55
hydrogel networks (Scheme 2).
The physical properties of the block polypeptide hydrogels,
Enzyme-responsive properties of synthetic polypeptide-
in particular stimuli-responsive properties, as well as the corre- based materials, including enzymatic degradation, are promis-
lation between nanostructure and physical properties were ing for biomedical applications. Therefore, in vitro gel weight
then investigated. As shown in the quantitative ATR-FTIR ana- loss profiles were obtained by measuring the weight loss of
lyses, the content of β-sheet secondary structure in the hydro- hydrogels formed from polymer 1 (4 wt%) at 37 °C.22,24,56 The
gels was calculated to be 63%, thus a mechano-triggered gel- enzymatic degradation medium was prepared by dissolving
to-sol transition was expected, based on previous studies.43,56 MMP-2, proteinase K or trypsin in Tris-HCl buffer (0.05 M,
The mechano-responsiveness of the hydrogels was tested by pH = 8.0, 0.2 wt% NaN3). The enzyme solution or Tris-HCl buffer
applying sonication to the hydrogel for 30 s and the gel-to-sol without enzymes (0.5 mL) was added gently into each vial and
transition was observed by the test tube inversion experiment. removed along with accumulated gel debris at the selected
This transition was reversible, with the hydrogel reforming time points. Measurements were performed in triplicate for
after maintaining the sol at Tgel (ca. 37 °C) for 1 h (Fig. 6a). proteinase K, trypsin and the control group (Tris-HCl buffer),
Due to the short time in the sonication experiment and the but only once for MMP-2, due to limited supply and high cost.
thermal stability of the hydrogels against heating up to 90 °C, According to the weight loss profiles (Fig. 7), trypsin, which
the mechanical force applied by the sonicator was proposed to cleaves peptide chains at lysine and arginine residues, had no
break the supramolecularly-assembled structures of hydrogels significant effect on the hydrogel degradation, resulting in a
formed from polymer 1, resulting in the deconstruction of the similar degradation profile as the control group. In contrast,
hydrogels. In order to investigate the secondary structures of the hydrogels treated with proteinase K, an aggressive and
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 8 Cumulative release of Npx from
a
hydrogel formed from
polymer (0.4 wt% in 0.01
1
M
PBS buffer, pH 7.4) containing
=
Fig. 7 Weight loss profiles of hydrogels formed from polymer 1 in the
presence and absence of enzymes. Measurements were repeated three
times for proteinase K, trypsin and control group, and error bars rep-
resent the standard deviations. A single measurement was performed
for MMP-2.
1 mg mL−1 Npx at 37 °C over 6 days. Measurements were repeated three
times and the error bars represented standard derivations.
system as a local drug delivery carrier to reduce the systematic
drug dosage and associated undesirable side effects.
broadly-active enzyme, exhibited much faster degradation, in
which over 90% weight loss was observed after 72 h. As
expected, MMP-2, which cleaves at G-IAG positions, resulted in
Conclusions
intermediate degradation kinetics. It is uncertain whether the In summary, a polypeptide hydrogel system prepared from
apparent plateauing of activity (after 48 h) for MMP-2 was due ternary sequences of L-alanine, glycine and L-isoleucine within
to a limited number of G-IAG sequences along the relatively a polypeptide segment of a PEG-peptide diblock structure
short polypeptide chains or to a loss in enzymatic activity over exhibited thermo-, mechano- and enzyme-responsive pro-
time. Further experiments would be necessary to sequence the perties, which enabled the encapsulation and release of Npx.
polymer and quantify the relative contributions from these The diblock polymer, mPEG-b-P(A-G-I), was facilely synthesized
effects, among others.
from the statistical ROP of Ala, Gly and Ile NCA monomers
The utilization of this hydrogel for the entrapment and initiated by mPEG-NH2. Reversible and repeatable sol–gel tran-
release of the small therapeutic drug, naproxen (Npx), was also sitions were observed in response to externally-applied stimuli,
investigated to explore the potential application of these with heat-induced sol-to-gel transitions and sonication-trig-
materials for local drug delivery. Npx has a low solubility in gered gel-to-sol transitions. The Tgel was largely dependent on
nanopure water in its protonated form, but it can be dissolved the polymer concentration, and the block length of the poly-
in PBS buffer (0.01 M, pH = 7.4) at concentrations up to 1 peptide segments, which could be tuned easily into the tar-
mg mL−1. Thus, Npx-loaded hydrogels were prepared by mixing geted temperature range, ca. 37 °C, to enable biomedical appli-
the weight fraction of polymer 1 with the Npx solution in PBS cation of these materials. Detailed characterization studies
buffer to yield a polymer concentration of 0.4 wt%, and trans- revealed that the hydrogelation was driven by the supramolecu-
parent hydrogels were generated after the solution was main- lar assembly of β-sheets into well-defined fiber-like nano-
tained at 37 °C overnight.6 The obtained hydrogels were stable, structures, which further entangled into a three-dimensional
and neither gel-to-sol transition nor precipitation of Npx was network through long range interactions. The mechanical
observed for at least one week at room temperature. The soni- force produced by sonication deconstructed the supramolecu-
cation-triggered gel-to-sol transition was also observed for the larly-assembled structures of the hydrogels, resulting in a gel-
Npx-loaded hydrogels upon sonication for 10 s. The release of to-sol transition. In addition, enzymes that are either broadly
Npx from mPEG-b-P(A-G-I) polymer 1 hydrogels was monitored aggressive or selective for G-IAG motifs were found to acceler-
by HPLC to measure the concentration of Npx in the surround- ate the breakdown of the polypeptide segments, resulting in
ing PBS buffer at 37 °C at predetermined time points over the reversion of the hydrogels into solutions. Npx was loaded
6 days. As shown in Fig. 8, a burst release of Npx up to 64%, into the hydrogels at room temperature in the sol state and
was observed within 24 h, followed by slower release over the encapsulated within the hydrogel after heating to the gelation
next 5 days, and reaching 80% at the end of the study. This temperature. The release of Npx from the hydrogels diffused
encapsulation and release of the small molecule drug into the surrounding environment, demonstrating the poten-
suggested the potential application of this polypeptide hydrogel tial application of this polypeptide-based hydrogel as a local
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
drug delivery system, desired to reduce the systemic dosage of 15 U. P. Shinde, H. J. Moon, D. Y. Ko, B. K. Jung and B. Jeong,
small molecule drugs with associated adverse side effects.
Biomacromolecules, 2015, 16, 1461–1469.
16 M. H. Park, M. K. Joo, B. G. Choi and B. Jeong, Acc. Chem.
Res., 2012, 45, 424–433.
17 E. A. Appel, J. del Barrio, X. J. Loh and O. A. Scherman,
Chem. Soc. Rev., 2012, 41, 6195–6214.
Acknowledgements
This work is financially supported by the National Heart, 18 Y. Chen, X. H. Pang and C. M. Dong, Adv. Funct. Mater.,
Lung and Blood Institute of the National Institutes of 2010, 20, 579–586.
Health as Program of Excellence in Nanotechnology 19 T. J. Deming, Nat. Mater., 2010, 9, 535–536.
(HHSN268201000046C), the National Science Foundation 20 A. Lalatsa, A. G. Schatzlein, M. Mazza, B. H. L. Thi and
(DMR-1105304, DMR-1309724, and DMR-1507429), and the I. F. Uchegbu, J. Controlled Release, 2012, 161, 523–536.
Welch Foundation through the W. T. Doherty-Welch Chair in 21 Y.-A. Lin, Y.-C. Ou, A. G. Cheetham and H. Cui,
Chemistry (Grant no. A-0001). The Microscopy & Imaging Biomacromolecules, 2014, 15, 1419–1427.
Center (MIC) at Texas A&M University is also gratefully 22 H. J. Moon, B. G. Choi, M. H. Park, M. K. Joo and B. Jeong,
acknowledged. The authors thank Professor Tadhg P. Begley at Biomacromolecules, 2011, 12, 1234–1242.
Texas A&M University for access to the circular dichroism 23 E. Y. Kang, B. Yeon, H. J. Moon and B. Jeong,
a
spectrometer.
Macromolecules, 2012, 45, 2007–2013.
24 J. Huang, C. L. Hastings, G. P. Duffy, H. M. Kelly,
J. Raeburn, D. J. Adams and A. Heise, Biomacromolecules,
2013, 14, 200–206.
25 A. P. Nowak, V. Breedveld, L. Pakstis, B. Ozbas,
D. J. Pine, D. Pochan and T. J. Deming, Nature, 2002,
417, 424–428.
References
1 F. E. Silverstein, G. Faich, J. L. Goldstein, L. S. Simon,
T. Pincus, A. Whelton, R. Makuch, G. Eisen, N. M. Agarwal,
W. F. Stenson, A. M. Burr, W. W. Zhao, J. D. Kent, 26 Y. Cheng, C. He, C. Xiao, J. Ding, H. Cui, X. Zhuang and
J. B. Lefkowith, K. M. Verburg and G. S. Geis, J. Am. Med.
Assoc., 2000, 284, 1247–1255.
2 R. S. Bresalier, R. S. Sandler, H. Quan, J. A. Bolognese,
X. Chen, Biomacromolecules, 2013, 14, 468–475.
27 J. Cao, P. Hu, L. Lu, B. A. Chan, B.-H. Luo and D. Zhang,
Polym. Chem., 2015, 6, 1226–1229.
B. Oxenius, K. Horgan, C. Lines, R. Riddell, D. Morton, 28 T. J. Deming, Chem. Rev., 2016, 116, 786–808.
A. Lanas, M. A. Konstam, J. A. Baron and 29 L. Merkel and N. Budisa, Org. Biomol. Chem., 2012, 10,
A. T. Investigators, N. Engl. J. Med., 2005, 352, 1092–1102.
3 J. Y. Li, Y. Kuang, Y. Gao, X. W. Du, J. F. Shi and B. Xu, 30 J. H. Hong, H. J. Lee and B. Jeong, ACS Appl. Mater.
J. Am. Chem. Soc., 2013, 135, 542–545. Interfaces, 2017, 9, 11568–11576.
4 J. Majumder, M. R. Das, J. Deb, S. S. Jana and P. Dastidar, 31 J. Bhattacharyya, I. Weitzhandler, S. B. Ho, J. R. McDaniel,
7241–7261.
Langmuir, 2013, 29, 10254–10263.
5 S. Bhuniya, Y. J. Seo and B. H. Kim, Tetrahedron Lett., 2006,
47, 7153–7156.
6 W. Cao, X. L. Zhang, X. M. Miao, Z. M. Yang and H. P. Xu,
Angew. Chem., Int. Ed., 2013, 52, 6233–6237.
7 L. A. Estroff and A. D. Hamilton, Chem. Rev., 2004, 104,
1201–1217.
8 S. Kiyonaka, K. Sugiyasu, S. Shinkai and I. Hamachi, J. Am.
Chem. Soc., 2002, 124, 10954–10955.
X. Li, L. Tang, J. Liu, M. Dewhirst and A. Chilkoti, Adv.
Funct. Mater., 2017, 27, 1605421.
32 B. E. Turk, L. L. Huang, E. T. Piro and L. C. Cantley, Nat.
Biotechnol., 2001, 19, 661–667.
33 L. Yu, H. Zhang and J. D. Ding, Angew. Chem., Int. Ed.,
2006, 45, 2232–2235.
34 J. Zou, F. Zhang, S. Zhang, S. F. Pollack, M. Elsabahy, J. Fan
and K. L. Wooley, Adv. Healthcare Mater., 2014, 3, 441–448.
35 T. Aliferis, H. Iatrou and N. Hadjichristidis,
Biomacromolecules, 2004, 5, 1653–1656.
9 Z. Zhou, Q. Yi, T. Xia, W. Yin, A. A. Kadi, J. Li and Y. Zhang,
Org. Biomol. Chem., 2017, 15, 2191–2198.
36 T. J. Deming, Nature, 1997, 390, 386–389.
10 X. Du, J. Zhou, J. Shi and B. Xu, Chem. Rev., 2015, 115, 37 I. Dimitrov and H. Schlaad, Chem. Commun., 2003, 2944–
13165–13307. 2945.
11 M. H. Park, Y. Yu, H. J. Moon, D. Y. Ko, H. S. Kim, H. Lee, 38 I. Conejos-Sanchez, A. Duro-Castano, A. Birke, M. Barz and
K. H. Ryu and B. Jeong, Adv. Healthcare Mater., 2014, 3,
1782–1791.
12 J. Kopecek and J. Yang, Angew. Chem., Int. Ed., 2012, 51,
7396–7417.
M. J. Vicent, Polym. Chem., 2013, 4, 3182–3186.
39 H. Lu and J. Cheng, J. Am. Chem. Soc., 2007, 129, 14114–
14115.
40 J. Zou, J. Fan, X. He, S. Zhang, H. Wang and K. L. Wooley,
Macromolecules, 2013, 46, 4223–4226.
13 S. C. Lange, J. Unsleber, P. Drucker, H.-J. Galla,
M. P. Waller and B. J. Ravoo, Org. Biomol. Chem., 2015, 13, 41 W. Zhao, Y. Gnanou and N. Hadjichristidis, Chem.
561–569. Commun., 2015, 51, 3663–3666.
14 P. M. Kharkar, K. L. Kiick and A. M. Kloxin, Chem. Soc. 42 J. Fan, R. Li, X. He, K. Seetho, F. Zhang, J. Zou and
Rev., 2013, 42, 7335–7372.
K. L. Wooley, Polym. Chem., 2014, 5, 3977–3981.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
43 J. Fan, J. Zou, X. He, F. Zhang, S. Zhang, J. E. Raymond and 50 J. K. Kauppinen, D. J. Moffatt, H. H. Mantsch and
K. L. Wooley, Chem. Sci., 2014, 5, 141–150. D. G. Cameron, Appl. Spectrosc., 1981, 35, 271–276.
44 A. Wamsley, B. Jasti, P. Phiasivongsa and X. L. Li, J. Polym. 51 A. Panitch, K. Matsuki, E. J. Cantor, S. J. Cooper,
Sci., Part A: Polym. Chem., 2004, 42, 317–325.
45 S. Hayashi, K. Ohkawa and H. Yamamoto, Macromol.
Biosci., 2006, 6, 228–240.
46 Y. Y. Choi, J. H. Jang, M. H. Park, B. G. Choi, B. Chi and
B. Jeong, J. Mater. Chem., 2010, 20, 3416–3421.
47 A. Aggeli, M. Bell, N. Boden, J. N. Keen, P. F. Knowles,
T. C. B. McLeish, M. Pitkeathly and S. E. Radford, Nature,
1997, 386, 259–262.
E. D. T. Atkins, M. J. Fournier, T. L. Mason and
D. A. Tirrell, Macromolecules, 1997, 30, 42–49.
52 A. Rosler, H. A. Klok, I. W. Hamley, V. Castelletto and
O. O. Mykhaylyk, Biomacromolecules, 2003, 4, 859–863.
53 E. H. Kim, M. K. Joo, K. H. Bahk, M. H. Park, B. Chi,
Y. M. Lee and B. Jeong, Biomacromolecules, 2009, 10, 2476–
2481.
54 N. J. Greenfield, Nat. Protoc., 2006, 1, 2876–2890.
48 V. K. Kotharangannagari, A. Sanchez-Ferrer, J. Ruokolainen 55 X. He, J. Fan and K. L. Wooley, Chem. – Asian J., 2016, 11,
and R. Mezzenga, Macromolecules, 2012, 45, 1982–
1990.
49 J. K. Kauppinen, D. J. Moffatt, H. H. Mantsch and
D. G. Cameron, Anal. Chem., 1981, 53, 1454–1457.
437–447.
56 X. He, J. Fan, F. Zhang, R. Li, K. A. Pollack, J. E. Raymond,
J. Zou and K. L. Wooley, J. Mater. Chem. B, 2014, 2, 8123–
8130.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017