Supramolecular hydrogels with reverse thermal gelation properties from (Oligo)tyrosine containing block copolymers

Article abstract of DOI:10.1021/bm301629f

Novel block copolymers comprising poly(ethylene glycol) (PEG) and an oligo(tyrosine) block were synthesized in different compositions by N-carboxyanhydride (NCA) polymerization. It was shown that PEG2000-Tyr ₆ undergoes thermoresponsive hydrogelation at a low concentration range of 0.25-3.0 wt % within a temperature range of 25-50 C. Cryogenic transmission electron microscopy (Cryo-TEM) revealed a continuous network of fibers throughout the hydrogel sample, even at concentrations as low as 0.25 wt %. Circular dichroism (CD) results suggest that better packing of the β-sheet tyrosine block at increasing temperature induces the reverse thermogelation. A preliminary assessment of the potential of the hydrogel for in vitro application confirmed the hydrogel is not cytotoxic, is biodegradable, and produced a sustained release of a small-molecule drug.

Full text of DOI:10.1021/bm301629f

Article
pubs.acs.org/Biomac
Supramolecular Hydrogels with Reverse Thermal Gelation Properties
from (Oligo)tyrosine Containing Block Copolymers
†
‡
‡
§
∥
∥
Jin Huang, Conn L. Hastings, Garry P. Duffy, Helena M. Kelly, Jaclyn Raeburn, Dave J. Adams,
,†
and Andreas Heise*
†
School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
‡
§
Department of Anatomy and School of Pharmacy, The Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2,
Ireland
∥
Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69 7ZD, United Kingdom
*
S Supporting Information
ABSTRACT: Novel block copolymers comprising poly(ethylene glycol) (PEG) and an
oligo(tyrosine) block were synthesized in different compositions by N-carboxyanhydride
(
NCA) polymerization. It was shown that PEG2000-Tyr6 undergoes thermoresponsive
hydrogelation at a low concentration range of 0.25−3.0 wt % within a temperature range of
5−50 °C. Cryogenic transmission electron microscopy (Cryo-TEM) revealed a continuous
2
network of fibers throughout the hydrogel sample, even at concentrations as low as 0.25 wt %.
Circular dichroism (CD) results suggest that better packing of the β-sheet tyrosine block at
increasing temperature induces the reverse thermogelation. A preliminary assessment of the
potential of the hydrogel for in vitro application confirmed the hydrogel is not cytotoxic, is
biodegradable, and produced a sustained release of a small-molecule drug.
INTRODUCTION
hydrogel matrix through noncovalent interactions such as
■
hydrogen bonding, electrostatic interactions, π-stacking, and
Hydrogels are critical materials in biomedical science. Their
high water content and permeability, allied to structural
integrity, similar to the extracellular matrix, means that
hydrogels are ideal building blocks in regenerative medicine
5
hydrophobic interactions. A prominent example is the peptide
amphiphiles introduced by Stupp consisting of a hydrophobic6
chain segment attached to a hydrophilic amino acid sequence.
This design promotes supramolecular self-assembly into three-
dimensional networks of fibrils in aqueous solution, stabilized
through peptide interactions at a low concentration. While this
design concept is very efficient, the self-assembly process is
highly sensitive to the length and sequence of the amino acid
block and relies on tedious multistep peptide and organic
1
and as therapeutic delivery systems. Despite the immense
progress made in the development of hydrogels, their first
clinical applications and the large and diverse number of
materials disclosed in the academic and patent literature, there
is still a high demand for the development of new hydrogels
with material properties that better match application demands.
Of particular interest are reverse thermoresponsive gels, that is,
solutions, which transform into a gel upon temperature
7
synthesis.
Utilizing a polymerization approach would potentially
simplify the synthetic efforts, however, at the expense of
monodispersity and the oligopeptide sequence. Synthetic
polypeptides can readily be prepared by the ring-opening8
polymerization of amino acid N-carboxyanhydrides (NCA).
Recent advances demonstrate the feasibility of NCA polymer-
ization for the synthesis of complex polymer structures capable
2
increase. In particular, thermoresponsive hydrogels are highly
amenable to the localized delivery of small-molecule drugs and
other therapeutic molecules due to their tendency to maintain a
liquid state at room temperature, thereby enabling a minimally
invasive administration via syringing and subsequent ability to
rapidly form a robust gel bolus upon heating to physiological
temperature, thereby enforcing drug cohesion and facilitating
sustained release at the intended site of action. Such systems
can potentially achieve local efficacy for extended periods,
requiring the administration of smaller doses, thereby reducing
the manifestation of off-target effects associated with systemic
9
of self-assembly, some of which are reported to form
hydrogels. For example, Deming employed NCA polymer-
izations for the synthesis of hydrogel forming diblock
10
copolypeptide amphiphiles. These materials contain water-
soluble polyelectrolyte blocks such as poly(L-lysine) or poly(L-
3
administration of drugs.
A promising class of materials is that of synthetic hydrogel4s
obtained by self-assembly of amphiphilic polypeptides.
Amphiphilic peptides can self-assemble into the supramolecular
Received: October 22, 2012
Revised: November 26, 2012
©
XXXX American Chemical Society
A
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glutamate) and an α-helical poly(L-leucine) hydrophobic block.
The gelation is driven by the stiff α-helical conformation of the
hydrophobic domain and the electrostatic repulsion from the
polyelectrolyte segments. However, hydrogelation was only
achieved when both the hydrophobic and hydrophilic segments
had a sufficiently high molecular weight. Jeong reported a series
of amphiphilic hybrid block copolymers obtained by ring-
O-Benzyl-L-tyrosine (Tyr-NCA) and Phenylalanine (Phe-NCA). α-
Pinene (15 g, 110.3 mmol) and O-benzyl-L-tyrosine (10.0 g, 36.9
mmol) were dissolved in 80 mL of anhydrous ethyl acetate in a three-
neck round-bottomed flask. The mixture was stirred and heated to
reflux with a slow flow of nitrogen. Then, a solution of triphosgene
(5.5 g, 18.4 mmol) in anhydrous ethyl acetate (40 mL) was added
dropwise. Two-thirds of the solution was added within 1 h after which
the reaction was left at reflux for another hour. The rest of the
triphosgene solution was added until the reaction mixture became
clear. Subsequently, around 60 mL of the solvent was removed under
pressure and 120 mL of n-heptane was added slowly to precipitate
NCA. After filtration, the solid was recrystallized from ethyl acetate
11
opening polymerization of alanine or phenylalanine NCAs.
These materials self-assemble into nanostructures such as
micelles in aqueous solutions and further transition into
hydrogels at high polymer concentration in the range of 3.0−
and n-heptane (1:2) four times until the NCA was recovered as an off-
14 wt % through intermicellar aggregation when the temper-
1
white crystal. Yield: 8.2 g (75%). H NMR (400 MHz, CDCl , δ,
3
ature increases. Very recently, Chen also reported micelle-
assembled thermosensitive hydrogels based on poly(L-
glutamate)s bearing different hydrophobic side groups via
NCA polymerization. Depending on the nature of the side
chains, the polymers underwent sol−gel transitions with
increasing temperature up to 62 °C at high polymer
ppm): 2.95 (dd, J = 14.13, 8.36 Hz, 1H, -CH-CH -), 3.21 (dd, J =
2
1
4.13, 4.10 Hz, 1H, -CH-CH -), 4.49 (dd, J = 4.10, 8.36 Hz, 1H, -NH-
2
CH-), 5.04 (s, 2H, -CH -O-), 5.96 (s, 1H, -NH-CH-), 6.95 (d, J = 8.61
2
1
3
Hz, 2H, ArH), 7.10 (d, J = 8.61 Hz, 2H, ArH), 7.39 (m, 5H, ArH).
NMR (400 MHz, CDCl , δ, ppm): 37.1 (-CH-CH -), 58.9 (-CH-CH
, 70.0 (-O-CH -), 115.6 (Ar), 125.9 (Ar), 127.5 (Ar), 128.1 (Ar),
C
-
3
2
2
)
2
12
128.7 (Ar), 130.2 (Ar), 136.6 (Ar), 151.5 (Ar), 158.5 (-O-C(O)-CH-),
concentration (typically 9.0 wt %).
1
68.6 (-C(O)-NH-).
We were interested to investigate if the advantages of NCA
polymerization for the synthesis of amphiphilic polypeptides
could be married with efficient hydrogelation at low
concentration and thermoresponsive behavior. In this concept,
a single amino acid must be able to display multiple modes of
interaction, triggering self-assembly into the hydrogel matrix
through noncovalent interactions. We hypothesized that
tyrosine is a promising amino acid candidate. Despite its
polar phenol group in the side chain, tyrosine residues are
relatively hydrophobic when the pH of the solution is lower
The same procedure was applied to the synthesis of phenylalanine
1
NCA. Yield: 5.3 g (45%). H NMR (400 MHz, CDCl , δ, ppm): 2.99
3
(
dd, J = 14.16, 8.59 Hz, 1H, -CH-CH -), 3.30 (dd, J = 14.16, 4.06 Hz,
2
1H, -CH-CH -), 4.54 (dd, J = 4.06, 8.59 Hz, 1H, -NH-CH-), 6.03
2
1
3
(s,1H, -NH-CH-), 7.20 (m, 2H, ArH), 7.35 (m, 5H, ArH). C NMR
(400 MHz, CDCl , δ, ppm): 38.0 (-CH-CH -), 58.9 (-CH-CH -),
28.2 (Ar), 129.3 (Ar), 129.4 (Ar), 134.0 (Ar), 151.8 (-O-C(O)-CH-),
68.7 (-C(O)-NH).
3
2
2
1
1
General Procedure for the Synthesis of PEG-b-poly(O-benzyl-L-
tyrosine). O-Benzyl-L-tyrosine NCA (715 mg, 2.4 mmol) was
dissolved in 30 mL of dry DMF in a Schlenk tube. A solution of α-
13
than the pK of the phenol. In addition to the ability to form
a
methoxy-ω-amino poly(ethylene glycol) (M = 2000 g/mol, PDI 1.03,
w
stable β-sheet assemblies through a main chain amide bond
driven hydrogen bonding, it has been shown that hydrogen
bonding from the tyrosine −OH group contributed favorably to
960 mg, 0.48 mmol) in 10 mL of dry DMF was added after the NCA
had dissolved. The reaction was left to stir at room temperature for 4
days in a dry nitrogen atmosphere until NCA was completely
consumed (as monitored by FTIR). The reaction mixture was
precipitated into an excess diethylether, filtered, and dried under
1
4
protein stability. Because one of the two lone electron pairs is
partially delocalized within the aromatic ring, it can act either as
1
15
vacuum as a pale yellow solid (yield 80%). H NMR of PEG2000-b-
hydrogen bonding donor or acceptor. Baker and Hubbard
revealed that Tyr-OH groups form more hydrogen bonds to
water molecules than intramolecular hydrogen bonds; although,
in proteins, the tyrosine phenolic hydroxyl group generally
forms a single intramolecular hydrogen bond with a main-chain
6
poly(O-benzyl-L-tyrosine) (400 MHz, TFA-d with CDCl , δ, ppm):
6
3
2
.66−3.03 (br m, 12H), 3.25−4.07 (br m, 176H), 4.5−5.17 (br m,
1
8H), 6.70−7.44 (br m, 54H).
PEG-b-poly(L-tyrosine). PEG-b-poly(O-benzyl-L-tyrosine) (1 g) was
dissolved in 10.0 mL of hexafluoroisopropanol (HFIP) with 2 mL of
trifluoroacetic acid (TFA). A 6-fold excess with respect to O-benzyl-L-
tyrosine of 33% of HBr in acetic acid (1.6 mL) was added. After 16 h,
the mixture was added dropwise into diethyl ether. The polymer was
filtered and redissolved in deionized water and dialyzed against water
for 7 days. The solution was lyophilized as a white fluffy powder (yield
16
carbonyl oxygen or a side chain carboxyl group. If desired,
17
tyrosine can also be enzymatically cross-linked for hydrogel
1
8
stabilization.
To explore if the unique features of tyrosine can be utilized
to trigger supramolecular hydrogel formation at low concen-
tration, we designed a range of PEG conjugated tyrosine-based
amphiphiles by simple NCA polymerization. The hydrogelation
profile is highly sensitive to the polymer composition and the
materials display rare reverse thermogelation. We determine the
applicability of this polymer to form an injectable drug delivery
vehicle and undertake a preliminary appraisal of the
biodegradability and toxicity of the formulation in cultured
cells. The release of a model compound, desferrioxamine
1
6
5
5%). H NMR of PEG2000-b-poly(L-tyrosine) (400 MHz, TFA-d ,
6
δ, ppm): 2.62−3.26 (br s, 12H), 3.30−4.23 (br m, 176H), 4.58−4.97
(br s, 6H), 6.70−7.27 (br m, 31H).
Hydrogel Preparation and Sol−Gel Transition. Hydrogel solutions
were prepared by dissolving freeze-dried polypeptide samples in
deionized water at the desired concentrations. The homogeneous
solutions were obtained by sonication at 20 °C until the solutions
became clear. The formation of the gel was determined via vial
19
inverting method. Each sample at a given concentration was
dissolved into distilled water in 2 mL vials. After equilibration at
room temperature overnight, the vials containing samples were
immersed in a water bath equilibrated at each given temperature for
(DFO), a small-molecule pro-angiogenic, from the thermores-
ponsive gel was investigated.
1
5 min. The sol−gel transition was determined after inverting the vial.
If no flow was observed within 1 min, the sample was regarded as a gel.
For the temperature-dependent measurements, the temperature was
raised in 1 °C steps (the precision of the sol−gel transition
temperature was ±1 °C). Each temperature data point represents an
average of three measurements. The gelation time as a function of
block copolymer concentration at the physiologically relevant
temperature of 37 °C was measured in a 37 °C water bath. The
EXPERIMENTAL SECTION
■
Materials. All chemicals were purchased from Sigma-Aldrich and
used as received unless otherwise noted. O-Benzyl-L-tyrosine and
phenylalanine were supplied by Bachem. Anhydrous DMF, ethyl
acetate, and THF were used directly from the bottle under an inert
and dry atmosphere.
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gelpoint was determined via vial inverting method every 15 min in the
initial 3 h. Thereafter, the gelation was checked every 30 min. The
duration from the time point the polymer solution was put into the
water bath to the time point the gelation was observed was defined as
the gelation time. Each gelation time data point was determined from
an average of three measurements.
Hydrogel Degradation Profile. Degradation of the hydrogel was
assessed by measuring the weight loss every 24 h for nine days in
triplicate. A 1 mL aliquot of deionized water containing 1.0 and 2.0 wt
Anton Paar Physica MCR101 rheometer. For the oscillatory shear
measurements, a sandblasted parallel top plate with a 25 mm diameter
and 1.0 mm gap distance were used. Evaporation of water from the
hydrogel was minimized by covering the sides of the plate with low
viscosity mineral oil. The measurements of storage modulus (G′) and
loss modulus (G″) with gelation were made as a function of time at a
frequency of 1.59 Hz (10 rad/s) and at a constant strain of 2% as the
temperature increased from 25 to 60 °C by a heating rate of 1.0 °C/
min. A frequency sweep (1−100 Hz) was performed on the recovered
gel at a constant strain of 2%. The morphology images of hydrogel
were obtained by using a Hitachi S3400n SEM instrument. The
preparation of samples for SEM analysis involved placing a drop of
hydrogel on the thin carbon-coated film. The hydrogel was subjected
to shock-freezing by liquid nitrogen, followed by lyophilization for 2 h.
It was then submitted to a SEM scan after gold-coating for 2 min.
Cryogenic−transmission electron microscopy (Cryo-TEM): Sample
preparation was carried out using a CryoPlunge 3 unit (Gatan
Instruments) employing a double blot technique. A total of 3 μL of
sample was pipetted onto a plasma etched (15 s) 400 mesh holey
carbon grid (Agar Scientific) held in the plunge chamber at
approximately 90% humidity. The samples were blotted from both
sides for 0.5, 0.8, or 1.0 s, dependent on sample viscosity. The samples
were then plunged into liquid ethane at a temperature of −170 °C.
The grids were blotted to remove excess ethane then transferred under
liquid nitrogen to the cryo TEM specimen holder (Gatan 626 cryo
holder) at −170 °C. Samples were examined using a Jeol 2100 TEM
operated at 200 kV and imaged using a GatanUltrascan 4000 camera;
images captured using Digital Micrograph software (Gatan). High-
performance liquid chromatography (HPLC) for DFO release studies
was performed on an Agilent 1120 Compact LC with a Phenomenex
Gemini 5u C18 column, mobile phase acetonitrile/phosphate buffer
(10/90% v/v), containing 2 mol ethylenediaminetetraacetic acid
%
block copolymer, respectively, was placed in a vial and left in a water
bath at 37 °C for 30 min to allow gelation to occur. A 2 mL aliquot of
prewarmed Krebs buffer solution was gently added into each vial,
taking care not to disturb the hydrogels. The vials were then left in the
water bath for the duration of the degradation study. Every 24 h, the
Krebs buffer solution was removed along with any gel debris that had
accumulated. Vials were then weighed to assess the change in weight
of the hydrogels over the duration of the study. In the enzymatic
degradation experiment, the enzymes were doped into the hydrogel by
dissolving the enzymes in the 5 mg/mL (∼0.5 wt %) polymer solution.
The activity of both enzymes in the final polymer solution is 28.5 unit/
mL. The hydrogelation was triggered by heating the solutions to 37 °C
in a water bath (around 4 h). The formation of the hydrogel was
determined by the vial inverting method.
Desferrioxamine (DFO) Release Experiment. Analysis of DFO
release from a 2.0 wt % (20 mg/mL) PEG2000-Tyr hydrogel was
6
20
undertaken as previously described. Hydrogels containing DFO of
1
00 μM concentrations were prepared. A total of 2 g of 20 mg/mL
polymer with 100 μM DFO was placed in each of three glass vials and
allowed to gel in a water bath for 2 h at 37 °C. A 1 mL aliquot of
phosphate buffer (pH 7.2) was added to each vial and the samples
were allowed to incubate at 37 °C while shaking at 75 rpm for the
duration of the release study (2 mL of buffer was used at 4 and 24 h to
ensure appropriate sink conditions in release media at early time
points). The phosphate buffer was completely removed and replaced
at 4 h, 24 h, day 3, day 5, and day 7 and frozen until analysis. All
studies were performed in triplicate. Samples were analyzed for DFO
content via HPLC.
1
13
(EDTA), pH adjusted to 6.5 and UV detection at 440 nm. H and
C
NMR spectra were recorded at room temperature with a Bruker
Avance 400 (400 MHz). CF COOD and CDCl were used as solvents
3
3
and signals were referred to the signal of residual protonated solvent
signals.
Hydrogel Biocompatibility. The biocompatibility of the hydrogel
was preliminarily assessed through the culture of cell monolayers on a
RESULTS AND DISCUSSION
preformed PEG2000-Tyr hydrogel substrate. Mesenchymal stem cells
■
6
were derived from bone marrow aspirates of Wistar rats (rMSCs)
under ethical approval. Cells were cultured in Dulbecco’s modified
Eagles medium (DMEM) supplemented with 10% fetal bovine serum
Poly(ethylene glycol-b-O-benzyl-L-tyrosine) (PEG-PBTyr)
block copolymers were synthesized by ring-opening polymer-
ization of O-benzyl-L-tyrosine NCA using α-methoxy-ω-amino
(
FBS), 1% L-glutamine, 2% penicillin/streptomycin, 1% nonessential
poly(ethylene glycol) (M = 2000 and 5000 g/mol) as the
w
amino acids, and 0.5% Glutamax. A total of 50000 rMSCs were seeded
on a preformed gel layer consisting of 100 μL of 2.0 wt % gel allowed
to set at 37 °C for 1 h in wells of a 24-well plate. Cell viability and
morphology was assessed qualitatively at 48 h, as previously
macroinitiator (Scheme 1). The ratio of PEG macroinitiator to
NCA was systematically varied to obtain block copolymers with
different block lengths ratios (Table 1). H NMR analysis
confirmed the very good agreement between the PEG to NCA
monomer feed ratio with the composition of the obtained block
copolymers, although, for PEG5000, a slight deviation was
1
21
described, through use of a Live/Dead stain (Molecular Probes),
which labels cells with calcein or ethidium homodimer to stain live
cells green and dead cells red. The proportion of live to dead cells and
the ability of cells to spread and attach to the gel substrate was assessed
qualitatively through visual inspection of stained cells at 48 h.
Methods. ATR-FTIR spectra were collected on a Perkin-Elmer
a
Scheme 1. Synthesis of PEG/Tyr Block Copolymers
−1
Spectrum 100 in the spectral region of 650−4000 cm and were
−1
obtained from 16 scans with a resolution of 2 cm . A background
measurement was taken before the sample was loaded onto the ATR
unit for measurements. CD data were collected on a Jasco J-810 CD
spectrometer (Japan Spectroscopic Corporation) with a path length of
0
.1 cm and a bandwidth of 1 nm. Three scans were conducted and
−1
averaged between 185 and 350 nm at a scanning rate of 20 nm min
with a resolution of 0.2 nm. The data were processed by subtracting
solvent as background. CD temperature-dependent measurements
were performed on a Jasco J-815 spectropolarimeter (Japan
Spectroscopic Corporation) with a PFD-425S/15 Peltier-type temper-
ature controller with a temperature range from 10 to 60 °C and a
temperature slope of 1 °C/min. The solution was allowed to
equilibrate for 10 min at each temperature prior to data collection.
The measurements were done between 185 and 350 nm in a 0.1 mm
quartz cell. Dynamic rheological experiments were measured using an
a
Reagents and conditions: (a) DMF, 0 °C; (b) HBr/acetic acid,
hexafluoroisopropanol/TFA, r.t.
C
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Table 1. Characteristics of Poly(ethylene glycol-b-L-tyrosine)
(
PEG-Tyr) Obtained by Macroinitiation of O-Benzyl-L-
tyrosine NCA from Amine-Functionalized PEG (PEG-NH2)
a
b
PEG-NH2
feed ratio PEG/NCA
composition block copolymer
PEG2000
PEG2000
PEG2000
PEG5000
PEG5000
PEG5000
PEG5000
1:5
PEG2000-Tyr6
PEG2000-Tyr10
PEG2000-Tyr14
PEG5000-Tyr9
PEG5000-Tyr13
PEG5000-Tyr16
PEG5000-Tyr23
1:10
1:15
1:10
1:15
1:20
1:30
a
Molar ratio of PEG macroinitiator to O-benzyl-L-tyrosine NCA.
b
1
Calculated from H NMR (CF COOD) using the integrated peak
3
area ratios of the PEG ethylene signals at 3.30−4.23 ppm and the C
α
signal of poly(L-tyrosine) at 4.58−4.97 ppm while using the aromatic
groups at 6.70−7.27 ppm and C signal at 2.62−3.26 ppm of poly(L-
β
tyrosine) as the internal standards.
observed at higher ratios. The subsequent quantitative
deprotection of the tyrosine benzyl ether was monitored by
1
H NMR spectroscopy by the disappearance of the benzyl ether
peaks (7.2−7.4 ppm) and afforded the poly(ethylene glycol-b-L-
tyrosine) (PEG-Tyr).
After deprotection of the PEG5000 series, PEG5000-Tyr23
was water insoluble, while all other block copolymers were
readily soluble but did not form hydrogels at room temper-
ature, even at polymer concentrations >120 mg/mL (12 wt %,
higher concentrations were not investigated). Of the PEG2000
series, both PEG2000-Tyr10 and PEG2000-Tyr14 were water
Figure 2. Hydrogelation temperature as a function of PEG2000-Tyr
6
insoluble. PEG2000-Tyr , on the other hand, formed trans-
concentration (top). Hydrogelation time as a function of PEG2000-
Tyr concentration at 37 °C (bottom).
6
parent hydrogels in deionized (DI) water at a range of
concentrations and temperatures. Cryogenic transition electron
microscopy (cryo-TEM) revealed a continuous network of
fibers throughout the hydrogel sample, even at concentrations
as low as 0.25 wt % (Figure 1). The fibers appear
6
%
and 39 °C at 1.0 wt %. It is remarkable that even at a
polymer concentration of 0.25 wt % stable hydrogels can be
observed upon heating to 50 °C. The second phase diagram in
Figure 2 depicts the gelation time as a function of block
copolymer concentration at the physiologically relevant
temperature of 37 °C. The data signify that the hydrogelation
time can be completely controlled by the polymer concen-
tration in water. Moreover, at concentrations >2.0 wt %,
hydrogelation occurs immediately at this temperature. The
thermogelation profile observed for PEG2000-Tyr is contrary
6
to most polypeptide aqueous systems that form a stable
hydrogel via hydrogen bonding or ionic interactions at a low
temperature and then undergo gel-to-sol transition (gel
2
2,23
melting) when the temperature increases.
Most relevant
Figure 1. (Left) Cryogenic transmission electron microscopy (Cryo-
in that respect is the example of telechelic Fmoc-protected
dityrosine end-capped PEG recently published by Hamley. Self-
assembled β-sheet fibril-based hydrogels from these materials
TEM) images of PEG2000-Tyr hydrogels at concentrations of 0.25
6
wt %; the scale bar represents 200 nm. (Right) Scanning electron
microscopy (SEM) images of PEG2000-Tyr hydrogel (0.5 wt %) after
6
24
lyophilization; the scale bar represents 20 μm.
exhibited a gel-to-sol transition with increasing temperature.
All these results suggest that there is a very delicate block
length ratio that needs to be met in this system for efficient
hydrogel formation to occur because the hydrogelation was not
interconnected by cross-link points. SEM images of samples
treated with liquid nitrogen followed by lyophilization to
preserve the hydrogel structure also confirm the three-
dimensional interconnected network structure (Figure 1).
Interestingly, the sol−gel phase diagram revealed a temper-
ature-dependent profile at low solid content. Figure 2 shows
observed for PEG2000-Tyr10 and PEG2000-Tyr . An obvious
14
explanation is the hydrophilic−hydrophobic balance between
the PEG and the tyrosine block that is determining for the
1
4,25
hydrogel formation.
Moreover, the self-assembly of the
that PEG2000-Tyr undergoes thermoresponsive gelation at a
PEG2000-Tyr in water can to a large extent be ascribed to the
6
6
concentration range of 0.25−3.0 wt % within a temperature
range of 25 to 50 °C. The sol-to-gel transition temperature
depends on the polymer concentration and increases with
decreasing polymer concentration, for example, 28 °C at 2.0 wt
β-sheet interaction in agreement with previously reported
26
peptide hydrogels. FTIR and CD spectra confirm that the
tyrosine block adopts a typical β-sheet conformation when the
hydrogel is formed as evident from the amide I band at 1623
D
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−1
cm and the minimum at 220 nm in the CD spectrum. The
hydrogels are stable up to pH 13, which coincides with the
complete deprotonation of the tyrosine hydroxy groups
The sensitivity of the hydrogelation to the block lengths
composition highlights the delicate balance of these inter-
actions.
(
Supporting Information). Further evidence for the importance
The viscoelastic properties of the gels were explored using
oscillatory rheology, in which the temporal evolution of storage
modulus (G′) and loss modulus (G″) is typically measured to
of the tyrosine phenolic groups in the current hydrogels was
obtained from block copolymers in which the tyrosine was
replaced by phenylalanine (Phe). Although the FTIR spectra of
both PEG2000-Phe and PEG2000-Phe confirmed a β-sheet
29
observe the gelation behavior. Figure 4 shows the dynamic
5
10
conformation, with a higher hydropathy index of 2.8 than Tyr
2
7
(
−1.3), neither of the polymers were water-soluble and could
form hydrogels. Apparently, the phenolic hydroxy groups
render the tyrosine block more polar and better soluble than
the phenylalanine block. This slight difference in structure
favorably tips the amphiphilic balance and affects the self-
assembly in aqueous solution. Furthermore, the partly polar
phenolic group could promote intramolecular hydrogen bonds
with water molecules or main-chain carbonyls and, thus,
16
contributes to the molecular interaction in the hydrogels.
While these results explain the hydrogel formation, they do
not rationalize the reverse thermoresponsive sol-to-gel
transition of PEG2000-Tyr . To investigate the conformational
6
changes of the block copolymer in aqueous solution, temper-
ature-dependent CD spectra were recorded. As revealed in
Figure 3, the negative band at 220 nm, corresponding to β-
Figure 4. Dynamic mechanical changes in the modulus of PEG2000-
Tyr at 2.0 and 1.0 wt % as a function of temperature.
6
mechanical changes in the modulus of PEG2000-Tyr6 at
different concentration of 2.0 and 1.0 wt % as a function of
temperature. The G′ of the polymer in both cases is an order of
magnitude higher than G″ as the temperature gradually
increases from 25 to 60 °C. This is indicative of the formation
of an elastic solid-like material as the temperature increases. At
higher polymer concentrations, higher values of G′ and G″ were
obtained, indicating a more rigid gel. G′ was found to be
insensitive to the frequency in a range of 1−100 rad/s and only
increase slightly with increasing frequency in all different
30
concentrations (Supporting Information).
The information obtained from the spectroscopic and
rheological analysis suggests that the hydrogel formation of
PEG2000-Tyr6 is collectively driven by several factors,
including amphiphilic balance between PEG and Tyr blocks,
hydrogen bonding by β-sheet conformation and phenolic
group, and possibly also aromatic interaction from the side
chains of poly(L-tyrosine). Better packing of the β-sheet
tyrosine block at increasing temperature induces a reverse
thermogelation. This is a desirable property for biomedical
hydrogels because, at room temperature, a liquid gel-precursor
solution could be administered by injection, possibly together
with drugs or growth factors, followed by in situ gelling at body
Figure 3. Circular dichroism spectra of PEG2000-Tyr6 aqueous
solution (0.25 mg/mL) as a function of temperature. Inset: Elipticity
change of PEG2000-Tyr (0.25 mg/mL) at 220 nm as a function of
6
temperature.
sheet conformation, gradually increased as the temperature
increased from 10 to 60 °C, in particular, above 30 °C. This
implies that the increment of β-sheet conformation at increased
temperature increases, thereby promoting better intermolecular
interaction and hydrogelation. We hypothesize that two
phenomena contribute to this behavior. The first is the
known dehydration of PEG at higher temperatures, causing
3
temperature, thus, avoiding surgical implantation. The
applicability of PEG2000-Tyr as biomedical hydrogels was
6
therefore assessed in preliminary experiments.
Biostability and resorbability under simulated physiological
condition are essential criteria in the assessment of biomedical
hydrogels. The stability of the hydrogel at block copolymer
concentrations of 2.0 and 1.0 wt % was measured in Krebs
buffer solution. Krebs buffer is a physiological buffer that, to
some degree, mimics the ionic composition, pH, and osmotic
behavior of tissue fluid and, so, is a useful medium to examine
the behavior of biomaterials in vitro. Figure 5 reveals a weight
loss of around 20% and 5% after nine days for the gel of 1.0 and
2.0 wt %, respectively. While both hydrogels remained quite
28
the PEG blocks to become more hydrophobic. This offsets
the hydrophobic/hydrophilic balance in the block copolymer
and decreases the molecular motion of the PEG block. On the
other hand, an increase in temperature increases the molecular
motion of the Tyr domain, promoting better phase packing and
also enhancing its molecular self-organization via hydrogen
bonding, as demonstrated by the stronger β-sheet conformation
at elevated temperatures. This process facilitates block
copolymer self-assembly and hydrogel network formation.
E
dx.doi.org/10.1021/bm301629f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Figure 5. Weight loss of PEG2000-Tyr hydrogel at 2.0 and 1.0 wt %
6
in Krebs buffer solution. Inset: Degradation of hydrogels in the
presence of (A) no enzyme, (B) chymotrypsin, and (C) trypsin.
Enzymes (28.5 unit/mL) were incorporated in the hydrogel at 0.5 wt
Figure 6. Cumulative release profile of DFO from 2.0 wt % hydrogel
containing 100 μM DFO at 37 °C over seven days (n = 3, mean +
standard deviation plotted).
%
in a 37 °C water bath; pictures taken after 7 days.
stable over the monitored time, the 2.0 wt % hydrogel displayed
higher stability and lower weight loss due to its higher
mechanical stability, as shown in Figure 4. This lack of
mechanical strength of the 1.0 wt % hydrogel could allow for
more uptake of water, resulting in more swelling, with
concomitant structural breakdown. Second, a weaker gel will
be more prone to mechanical disruption due to physical
manipulation associated with buffer changes. Both of these
factors together could account for the observed change in
weight. In a second set of experiments, enzymes were
encapsulated into the hydrogel (0.5 wt % block copolymer).
Chymotrypsin was chosen as a positive control because it
preferentially cleaves peptide amide bonds adjacent to an
aromatic amino acid such as tyrosine, tryptophan and
molecule drug confirmed that the PEG2000-Tyr hydrogel
6
could potentially function as an injectable drug depot, which
could exert sustained drug release and local efficacy for an
extended period.
Finally, the cytotoxicity of the hydrogel was assessed by
seeding rMSCs onto a preformed 2.0 wt % PEG2000-Tyr gel
6
layer and staining with a Live/Dead stain. No dead cells were
apparent and cells were adherent to the gel layer, suggesting
that the gel did not exert significant cytotoxicity after 48 h. At
4
8 h, the majority of cells appeared rounded, suggesting that
the cells were not presented with sufficient attachment sites to
enable spreading (Supporting Information).
3
1
phenylalanine. Trypsin was selected as negative control as it
cleaves peptide chains mainly at the carboxyl side of the amino
CONCLUSIONS
■
32
acids lysine or arginine. The enzymes of 28.5 unit/mL were
doped into the hydrogels by dissolving them in a 0.5 wt %
polymer solution and subsequently triggering hydrogelation by
heating to 37 °C. The hydrogel containing chymotrypsin
showed a gradual disintegration within 24 h and completely
broke into pieces after 7 days (inset of Figure 5). In contrast,
no visible change was observed for the hydrogels containing
trypsin or no enzyme in the same time period highlighting the
selective enzymatic degradation by chymotrypsin. This experi-
ment further shows that biomolecules can be incorporated
without compromising the hydrogel formation.
Novel block copolymers comprising a PEG and an oligo-
(tyrosine) block were synthesized in different compositions by
NCA polymerization. The ability to form hydrogels is delicately
dependent on the block length ratio with an optimum
composition of PEG2000-Tyr . This material undergoes
6
thermoresponsive gelation at a low concentration range of
0.25−3.0 wt % within a temperature range of 25 to 50 °C. The
hydrogel formation of PEG2000-Tyr is collectively driven by
6
several factors including amphiphilic balance between PEG and
Tyr blocks, hydrogen bonding by β-sheet conformation and
phenolic group. CD analysis confirms that better packing of the
β-sheet tyrosine blocks at increasing temperature induces the
reverse thermogelation. A preliminary assessment of bio-
compatibility in vitro indicated that the hydrogel is not
cytotoxic, is biodegradable, and produced a sustained release
of a small-molecule drug. Therefore, this formulation may have
potential as an injectable drug delivery vehicle.
We also explored the use of the hydrogel for the entrapment
and release of small drug molecules. A 2.0 wt % PEG2000-Tyr6
hydrogel was utilized to release the pharmacological pro-
20
angiogenic desferrioxamine (DFO) over a period of 7 days.
DFO was dissolved within the gel as it is a water-soluble drug.
Release of DFO from the hydrogel into phosphate buffer by
diffusion at 37 °C was measured via HPLC. As shown in Figure
6
, the gel produced a burst release within the first 24 h and then
ASSOCIATED CONTENT
■
a sustained, albeit decreasing, release over the period of seven
days. Interestingly, the gel released ∼20% of the total
encapsulated drug within seven days. However, the amount
of drug released is sufficient to exert a biological effect. The
remainder of the drug may have become degraded, within both
the gel and release buffer. The sustained release of the small-
*
S
Supporting Information
FTIR and CD spectra as a function of pH, frequency sweep,
20
and storage modulus data for PEG2000-Tyr hydrogels; cell
6
toxicity image. This material is available free of charge via the
Internet at http://pubs.acs.org.
F
dx.doi.org/10.1021/bm301629f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
2
012, 22, 6072. (f) Park, M. H.; Joo, M. K.; Choi, B. G.; Jeong, B. Acc.
AUTHOR INFORMATION
■
Chem. Res. 2012, 45, 42.
12) Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Zhuang, X.; Huang, Y.;
Chen, X. Biomacromolecules 2012, 13, 2053.
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Notes
(
The authors declare no competing financial interest.
(
8
(
ACKNOWLEDGMENTS
■
Financial support from the Science Foundation Ireland (SFI)
Principle Investigator Award 07/IN1/B1792 (J.H. and A.H.) is
gratefully acknowledged. A.H. is a SFI Stokes Senior Lecturer
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dx.doi.org/10.1021/bm301629f | Biomacromolecules XXXX, XXX, XXX−XXX