Biodegradation of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly{(2-hydroxyethyl methacrylate)-co-[poly(ethylene glycol) methyl ether methacrylate]} hydrogels containing peptide-based cross-linking agents

Article abstract of DOI:10.1021/bm100756c

PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]-peptide conjugate hydrogels [where PHEMA ) poly(2- hydroxyethyl methacrylate; PEGMA ) poly(ethylene glycol) methacrylate] were readily prepared via photoinitiated free-radical polymerization in water. The PHEMA-peptide hydrogels were opaque and had a heterogeneous morphology of interconnected polymer droplets, characteristic of polymers that separate from the aqueous phase during the polymerization experiment. The P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were transparent gels with a homogeneous morphology when formed in water, but when formed in aqueous NaCl solutions the P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were also opaque and exhibited the heterogeneous morphology of interconnected polymer droplets. When incubated in solutions containing activated papain, P[HEMA-co-(MeOPEGMA)]- peptide conjugates underwent degradation that was characterized by macroscopic changes to sample shape and size, sample weight, and microscopic structure. PHEMA-peptide conjugates did not undergo any significant degradation when incubated with papain, although ninhydrin-staining experiments suggested that some peptide cross-linker groups were cleaved during the incubation. The difference in degradation behavior of PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]- peptide conjugates is attributed to differences in aqueous solubility of PHEMA and P[HEMA-co-(MeO-PEGMA)].

Full text of DOI:10.1021/bm100756c

Biomacromolecules 2010, 11, 2949–2959
2949
Biodegradation of Poly(2-hydroxyethyl methacrylate) (PHEMA)
and Poly{(2-hydroxyethyl methacrylate)-co-[poly(ethylene
glycol) methyl ether methacrylate]} Hydrogels Containing
Peptide-Based Cross-Linking Agents
Ylenia S. Casadio,† David H. Brown,†,‡ Traian V. Chirila,§,|,⊥,# Heinz-Bernhard Kraatz,†,∇
and Murray V. Baker*,†,§,O
Chemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western
Australia, Crawley, W.A. 6009, Australia, Nanochemistry Research Institute, Department of Chemistry,
Curtin University of Technology, Kent St, Bentley, W.A. 6102, Australia, Queensland Eye Institute, 41
Annerley Road, South Brisbane, Queensland 4101, Australia, Faculty of Science and Technology,
Queensland University of Technology, Brisbane, Queensland 4001, Australia, Australian Institute for
Bioengineering and Nanotechnology, University of Queensland, St Lucia, Brisbane,
Queensland 4072, Australia, Faculty of Health Sciences, University of Queensland, Herston,
Queensland 4006, Australia, Department of Chemistry, The University of Western Ontario, Chemistry Building
1151 Richmond Street, London, Ontario, Canada N6A 5B7, and Department of Chemistry, College of
Science and Engineering, National Dong Hwa University, Hualien 974, Taiwan, ROC
Received July 7, 2010; Revised Manuscript Received September 14, 2010
PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]-peptide conjugate hydrogels [where PHEMA ) poly(2-
hydroxyethyl methacrylate; PEGMA ) poly(ethylene glycol) methacrylate] were readily prepared via photoinitiated
free-radical polymerization in water. The PHEMA-peptide hydrogels were opaque and had a heterogeneous
morphology of interconnected polymer droplets, characteristic of polymers that separate from the aqueous phase
during the polymerization experiment. The P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were transparent
gels with a homogeneous morphology when formed in water, but when formed in aqueous NaCl solutions the
P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were also opaque and exhibited the heterogeneous morphology
of interconnected polymer droplets. When incubated in solutions containing activated papain, P[HEMA-co-(MeO-
PEGMA)]-peptide conjugates underwent degradation that was characterized by macroscopic changes to sample
shape and size, sample weight, and microscopic structure. PHEMA-peptide conjugates did not undergo any
significant degradation when incubated with papain, although ninhydrin-staining experiments suggested that some
peptide cross-linker groups were cleaved during the incubation. The difference in degradation behavior of
PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]-peptide conjugates is attributed to differences in aqueous
solubility of PHEMA and P[HEMA-co-(MeO-PEGMA)].
MA) has proved to be a highly biocompatible hydrogel8 and
has been used in a multitude of biomedical applications, which
include soft contact lenses,9 an artificial cornea,10 a potential
substrate for artificial skin,11 rhinoplasty,12 drug delivery
systems,13–15 and bone composite materials.16 Thus, due to their
biocompatibility and hydrophilic nature, PHEMA hydrogels are
excellent candidates for use as tissue support scaffolds.
1. Introduction
An ideal scaffold for tissue engineering must possess three
key characteristics, namely biocompatibility, porosity, and
biodegradability. The scaffold should be compatible so as not
to elicit an unfavorable immune response and should have the
appropriate porosity to allow for cellular attachment and
proliferation.1–3 It is also advantageous for the scaffold to be
biodegradable, so that the need for surgical removal after tissue
regeneration has occurred is obviated.1,4,5
Poly(ethylene glycol) (PEG) is a complementary polymer that
has been investigated extensively for biomedical applications.
PEG is very hydrophilic, nonimmunogenic in nature and
generally considered to be a biocompatible polymer.17,18 PEG
has found use as a bioconjugate in drug delivery systems where
it is covalently attached to proteins/peptides and/or pharmaceu-
ticals in order to improve solubility in biological systems, as
well as to render the resulting conjugates nonimmunogenic.17,18
Hydrogel materials are rapidly gaining popularity as potential
scaffolds for tissue regeneration as they often display excellent
biocompatibility.6,7 Poly(2-hydroxyethyl methacrylate) (PHE-
* To whom correspondence should be addressed. Tel.: +61 8 6488 2576.
Fax: +61 8 6488 1005. E-mail: murray.baker@uwa.edu.au.
† The University of Western Australia.
In addition to being biocompatible, a general structural
requirement of the polymeric scaffolds used in tissue engineering
applications is an open-pore morphology, in which the pores
are larger than 10 µm in diameter,19–22 interconnected, and
uniform throughout the material. This morphology has been
proposed as the optimum to allow for cellular proliferation and
tissue development.1,5,7,19 PHEMA hydrogels with the ideal
‡ Curtin University of Technology.
§ Queensland Eye Institute.
| Queensland University of Technology.
⊥ Australian Institute for Bioengineering and Nanotechnology, University
of Queensland.
# Faculty of Health Sciences, University of Queensland.
∇ The University of Western Ontario.
O National Dong Hwa University.
10.1021/bm100756c
 2010 American Chemical Society
Published on Web 10/20/2010

2950 Biomacromolecules, Vol. 11, No. 11, 2010
Casadio et al.
porous morphology (interconnected pores of g10 µm diameter)
can be readily obtained via one-step photoinitiated polymeri-
zation-induced phase separation polymerization.23 In addition,
it has been shown that similar porous morphologies can be
produced for poly{(2-hydroxyethyl methacrylate)-co-[poly(eth-
ylene glycol) methyl ether methacrylate]} (P[HEMA-co-(MeO-
PEGMA)]) using a similar technique. This technique is advan-
tageous as it is rapid, uses water as the solvent, and produces
amaterialthatdisplaysaninterconnectedporousmorphology.19,23,24
hydrogels biodegradable. Khelfallah et al. prepared their hy-
drogels in a 1:1 water:ethanol solvent mixture. The addition of
ethanol and the high proportion of PEG in the polymerization
mixtures act to suppress the phase separation process, thus
resulting in nonporous hydrogels, and therefore the resulting
PHEMA-PEG materials, though biodegradable, would not be
suitable for tissue scaffolding applications.
In this paper, we describe the synthesis of a series of
dimethacrylate peptide-based cross-linking agents and the use
of these cross-linking agents in the preparation of PHEMA and
P[HEMA-co-(MeO-PEGMA)] hydrogels having porous mor-
phologies suitable for tissue engineering.10 The hydrogels, with
porosity on the micrometer scale, have morphologies based on
a network of interconnected polymer droplets that forms
naturally by phase separation during polymerization in aqueous
mixtures. Polymerization-induced phase separation is an attrac-
tive route to polymers for tissue engineering applications because
the porous microstructure is obtained without the need for the
use of poragens or leaching steps involving toxic solvents. The
cross-linking agents were designed to be susceptible to degrada-
tion by the enzyme papain to demonstrate proof-of-principle.
Papain shows a great deal of homology with mammalian
cysteine proteases such as the cathepsins36,37 which participate
in wound healing and cell migration processes.25,38 In addition,
we describe the in vitro degradation of PHEMA and P[HEMA-
co-(MeO-PEGMA)] hydrogels that have been cross-linked with
short peptide sequences.
The use of PHEMA and P[HEMA-co-(MeO-PEGMA)]
hydrogels as scaffolds for tissue regeneration is hampered by
the high stability of these networks in biological environments.
PHEMA and P[HEMA-co-(MeO-PEGMA)] are not biodegrad-
able and thus are confined to biomedical applications that are
semipermanent or permanent. Therefore, the aim of this work
was to prepare biodegradable scaffolds based on PHEMA and
P[HEMA-co-(MeO-PEGMA)] having the porous morphology
appropriate for implementation as scaffolds for tissue support.
PHEMA and P[HEMA-co-(MeO-PEGMA)] hydrogels dis-
playing the appropriate pore morphology are readily prepared
through the polymerization of 2-hydroxyethyl methacrylate
(HEMA) or HEMA and poly(ethylene glycol) methyl ether
methacrylate (MeO-PEGMA) in the presence of a dimethacry-
late cross-linking agent.23 A potential strategy to render these
hydrogels biodegradable is through the incorporation of a
biodegradable cross-linking agent. A cross-linking agent is
biodegradable if it contains bonds that are cleaved when placed
in a biological environment and such a suitable cross-linking
agent can be a molecule that contains hydrolytically labile bonds
or bonds that are susceptible to enzymatic hydrolysis, such as
esters and amides. The latter allows for greater control over
the degradation behavior by designing a cross-linking agent
specifically suited to a target enzyme. The target enzyme is
dictated by the intended implantation site of the scaffold and
the physiological wound healing process that occurs once the
scaffold is in place. Generally, the implantation of a biomaterial/
prosthesis into the body will cause local trauma and inflam-
mation in the tissue surrounding the implant. As a response,
migrating cells located in the extracellular matrix will secrete
enzymes that are capable of degrading and remodeling the
components of the extracellular matrix such as collagen.
Therefore, by designing a cross-linking agent that is degraded
by specific enzymes, one can fine-tune the degradation properties
of the implanted scaffold.25 Enzymatically labile cross-linking
agents that have been used to impart biodegradability to
nondegradable polymers include both proteins26–28 and peptides.
In particular, the use of cross-linking agents based on short
peptide sequences has yielded much success in both drug
delivery and tissue engineering and such cross-linking agents
have been used with a plethora of polymers including poly(N-
vinylpyrrolidone) (NVP),25 N-(2-hydroxypropyl)methacryl-
amide,29–31 poly(N-isopropylacrylamide),32 and poly(acrylam-
ide).33,34 Peptide-based cross-linking agents are advantageous
because the synthetic chemistry used to produce oligopeptides
is well understood, and the peptide sequence can easily be
tailored to the desired enzyme.
2. Materials and Methods
2.1. Synthesis of Peptide-Based Cross-Linking Agents. The
synthesis of the protected oligopeptides was carried out via conventional
carbodiimide-mediated coupling procedures.39 The orthogonally pro-
tected amino acid N-benzyloxycarbonyl-L-lysine methyl ester hydro-
chloride (lys(Z)-OMe.HCl) (2) was prepared according to literature
procedures40–43 and served as the starting material for the protected
oligopeptides. Boc-gly-L-leu-L-lys(Z)-OMe 7 was prepared in three steps
(65% overall yield). The protected oligopeptide 7 was then deprotected
and the resulting deprotected peptide was functionalized with meth-
acryloyl groups (MA, CH2dC(CH3)COs) to give MA-gly-L-leu-L-
lys(MA)-OH 4 (45% yield based on 7). Similarly, Boc-gly-L-phe-L-
leu-L-lys(Z)-OMe 8 was prepared in 5 steps (33% overall yield) and
converted into MA-gly-L-phe-L-leu-L-lys(MA)-OH 5 (60% yield based
on 8) and Boc-gly-L-phe-gly-L-leu-L-lys(Z)-OMe 9 was prepared in 7
steps (18% overall yield) and was converted into MA-gly-L-phe-gly-
L-leu-L-lys(MA)-OH 6 (64% yield based on 9). All oligopeptides were
characterized by 1H and 13C NMR spectroscopy and fast atom
bombardment mass spectrometry (FAB-MS) and cross-linking agents
were also characterized by microanalysis. Full details for synthesis and
characterization of peptide-based cross-linking agents 4-6 and the
protected oligopeptide precursors 7-9 are provided in the Supporting
Information.
2.2. Hydrolysis of Peptide-Based Cross-Linking Agents with
Papain. A reverse phase high performance liquid chromatography (RP-
HPLC) assay was used to monitor the cleavage reactions of each cross-
linking agent in the presence of papain. Many difficulties were
encountered when trying to purify the cross-linking agents by column
chromatography (see section 3.1). To avoid any unfavorable reactions
of the cross-linking agents on the RP-HPLC support, passivation of
the methacryloyl groups was necessary. Thus, the cross-linking agents
4, 5, and 6 were subjected to catalytic hydrogenation (H2/10% Pd on
C) to give the reduced products 4H4, 5H4, and 6H4 respectively (in
which the CdC bonds in the terminal methacryloyl groups are reduced),
in almost quantitative yields. Full details for the synthesis and
characterization of 4H4-6H4 are provided in the Supporting Informa-
tion.
During the course of this research, Khelfallah et al. reported
the preparation of biodegradable PHEMA-PEG hydrogels which
incorporated a dimethacrylate cross-linking agent consisting of
a gly-gly-L-leu tripeptide core, flanked by two PEG spacers.35
The cross-linked hydrogels were proteolytically degraded by
the enzyme subtilisin over a period of 50 days. This result was
encouraging as it indicated that peptide-based cross-linking
agents could be successfully used to render PHEMA-PEG

Biodegradation of PHEMA and P[HEMA-co-(MeO-PEGMA)]
Biomacromolecules, Vol. 11, No. 11, 2010 2951
Table 1. Formulations of PHEMA and P[HEMA-co-
(MeO-PEGMA)] Hydrogels I4-IIIT
2.4. Scanning Electron Microscopy (SEM). Cross sections (300-500
µm thick) of the hydrogels were obtained using a Vibratome 3000
instrument. Dehydration of the samples was achieved either by critical
point drying (Emitech K850) or by freeze-drying (Dynavac FD2) to
constant weight as previously described.23 After dehydration, the
samples were mounted on double-sided carbon tabs and coated with a
30 nm layer of carbon. The samples were imaged using a Zeiss 1555
VF-FESEM microscope at an accelerating voltage of 3 kV, with a
working distance of 6 mm and an aperture of 10 µm. Some of the
hydrogel samples were imaged using variable pressure (VP) SEM.45
These samples were prepared by critical point drying or freeze-drying
but they were not coated with carbon prior to imaging. The uncoated
samples were imaged using a Zeiss 1555 VF-FESEM microscope at
an accelerating voltage of 10 kV, a chamber pressure of 20 Pa, a
working distance of 12 mm and an aperture of 60 µm.
cross-linking
formulationa
H2O:HEMA, 80:20
agent
hydrogel
4
5
6
I4
I5
I6
TEGDMA
IT
1.3 M NaCl:HEMA:MeO-PEGMA, 80:20:6b
H2O:HEMA:MeO-PEGMA, 80:20:6
4
II4
II5
II6
IIT
III4
III5
III6
IIIT
5
6
TEGDMA
4
5
6
TEGDMA
a The hydrogels were prepared according to the weight ratio A:B:C,
where A ) parts by weight water, B ) part by weight HEMA, C ) part by
weight MeO-PEGMA. The concentration of initiator (DPAP) was 0.1 mol
% DPAP relative to HEMA and the concentration of the cross-linking agent
(4, 5, 6, or TEGDMA) was 1 mol % relative to HEMA. b 1.3 M NaCl solution
was used in place of water as a diluent.
2.5. Enzymatic Degradation of Peptide-Cross-Linked PHEMA
and P[HEMA-co-(MeO-PEGMA)] Hydrogels. The hydrogels were
carefully cut to provide cylindrical samples with approximate dimen-
sions of 10 mm (diameter) × 20 mm (length) for degradation
experiments. Each sample was freeze-dried (Dynavac FD2) to constant
weight and the initial dry weight (Wd0) was measured. The freeze-dried
hydrogels were then rehydrated by incubation (37 °C) in water
containing sodium azide (0.2 mg/mL) for at least one week. The water
was exchanged daily. The hydrated hydrogels were gently blotted with
a tissue to remove any excess water from the surface, and were then
weighed, then returned to the hydration solution. The blotting and
weighing process was repeated three times and an average weight was
determined to give the initial wet weight (Ww0). Under a blanket of
nitrogen, the hydrogel samples were then placed into flasks that
contained papain (1.0 mg/mL) in 10 mL degassed activation buffer
(5.6 mM L-cysteine, 1.1 mM Na2EDTA and 0.2 mg/mL sodium azide).
The flasks were incubated at 37 °C under nitrogen, and at various times
during the course of the degradation experiment samples were removed,
washed with water and placed in hydrogen peroxide solution (ca. 0.6%
in water) to deactivate the papain.44 The wet weight of each sample
(Wwi) was determined as described above for the initial wet weight
(Ww0). Each sample was then freeze-dried and its dry weight (Wdi) was
measured, after which the sample was discarded. Thus, for a particular
hydrogel, say II4, the degradation was monitored over time using 12-14
samples of II4, with the following calculations being done for each
sample:
RP-HPLC analysis of 4H4, 5H4, and 6H4 and the products of their
cleavage by papain was performed using an Agilent HPLC system
equipped with an Agilent UV/vis detector (λ ) 210 nm). Separation
was achieved using a Vydac 218TP54 C18 reverse phase column
equipped with a guard column. The mobile phase of the RP-HPLC
system was based on H2O with 0.1% TFA (solvent A) and acetonitrile
with 0.1% TFA (solvent B). Separation was achieved with a flow rate
of 1 mL/min using gradient elution starting at a ratio solvent A:solvent
B of 99:1, changing to 60:40 over 30 min.
The reduced products 4H4, 5H4, and 6H4 were dissolved in 0.1%
V/V aqueous TFA solution to give a final concentration of approximately
1 mg/mL. An aliquot of the activated papain solution (100 to 200 µL
of a 1 mg/mL solution in activation buffer containing 5.6 mM L-cysteine
and 1.1 mM Na2EDTA)44 was added to the substrate solution (typically
500 to 700 µL), and the reaction mixture was incubated at 37 °C for
30 min. Samples were analyzed by RP-HPLC before and after addition
of the activated papain solution. Control experiments were conducted
by incubating the oligopeptides 4H4, 5H4, and 6H4 with activation buffer
in the absence of papain.
2.3. Preparation of Peptide-Cross-Linked PHEMA and P[HEMA-
co-(MeO-PEGMA)] Hydrogels. Hydrogels were prepared via the
photopolymerization of HEMA and/or MeO-PEGMA in aqueous
solutions23 according to the three reagent formulations outlined in Table
1. For formulation I, the H2O:HEMA ratio was kept constant at 80:20
w/w. Formulations II and III were prepared using 80:20:6 parts by
weight ratio for H2O:HEMA:MeO-PEGMA. For formulation II, 1.3
M NaCl was used in place of water as the diluent in the polymerization
mixture to promote phase separation. The photoinitiator DPAP and the
relevant cross-linking agent (4, 5, or 6) were used at 0.1 and 1 mol %,
respectively, relative to HEMA. The procedure used for hydrogel II6
(Table 1) is provided as an example.
The pentapeptide cross-linking agent 6 (6.7 mg, 1.0 mmol) was added
to a solution of MeO-PEGMA (42 mg) and HEMA (120 µL, 130 mg,
1.0 mmol) in a 1.3 M NaCl (520 µL) in a quartz vial under nitrogen.
An ethanolic solution of DPAP (5 µL of a 26 mg/mL solution, 0.5
µmol) was then added, and the mixture was sonicated for approximately
5-8 min. Where necessary, a small amount of ethanol (∼20 to 50 µL)
was added to assist in the dissolution of the pentapeptide 6. The quartz
sample vial was then suspended in the center of a hollow quartz tube
positioned 10 cm from a Philips HPW125 Mercury Discharge lamp
(125 W) for 30 min. During the irradiation, a stream of compressed
air was passed through the quartz tube to keep the sample temperature
below 27 °C. When irradiation was complete, the resulting hydrogel
sample II6 was carefully removed and immersed in water. The water
was exchanged twice a day for a week to remove any unreacted HEMA
monomer. Samples were stored under water at all times.
(a) dry weight remaining (%) ) Wdi/Wd0 × 100, where Wd0 is the
initial dry weight of the sample and Wdi is the dry weight of the sample
at the time it was removed from the degradation medium;46
(b) wet weight change (%) ) (Wwi - Ww0)/Ww0 × 100, where Ww0
is the initial weight of the hydrated sample and Wwi is the weight of
the hydrated sample at the time it was removed from the degradation
medium.32
Each degradation study was accompanied by two controls, which
were (1) the hydrogel cross-linked with 1 mol % peptide-based cross-
linker and incubated at 37 °C in 10 mL degassed activation buffer
without papain and (2) the hydrogel cross-linked with 1 mol %
TEDGMA (nonbiodegradable) incubated at 37 °C in 10 mL degassed
activation buffer that included 1.0 mg/mL papain.
The control hydrogels were treated in the same manner as the
degradation samples but were only analyzed (% dry weight remaining,
% wet weight change) at the commencement and conclusion of each
degradation study.
3. Results and Discussion
3.1. Synthesis of Peptide-Based Cross-Linking Agents and
Evaluation of the Degradation Behavior. The peptide-based
cross-linking agents 4 - 6 were designed to be hydrolyzed by
papain, a cysteine protease that cleaves the internal peptide bond
adjacent to a hydrophobic residue (i.e., L-phe, L-leu, and
L-val).36,37,44 Three peptide-based cross-linking agents were

2952 Biomacromolecules, Vol. 11, No. 11, 2010
Scheme 1. Synthesis of Peptide-Based Crosslinking Agents
Casadio et al.
prepared by conventional carbodiimide-mediated coupling
procedures with a peptide sequence containing a internal
hydrophobic amino acid (Scheme 1).39 The orthogonally
protected L-lys(Z)-OMe residue 2 was coupled to Boc-L-leu 1
to give the Boc-protected dipeptide 3, which served as the
starting material for the subsequent protected oligopeptides. The
addition of an amino acid residue to a particular Boc-protected
peptide was achieved by removal of the Boc-group to expose
the N-terminal amine, followed by coupling the appropriate Boc-
protected amino acid to the N-terminal amine. The three
oligopeptides prepared in this manner, namely Boc-gly-L-leu-
L-lys(Z)-OMe (7), Boc-gly-L-phe-L-leu-L-lys(Z)-OMe (8), and
Boc-gly-L-phe-gly-L-leu-L-lys(Z)-OMe (9), were deprotected to
reveal the amine groups at the N-terminal glycine (RNH2) and
C-terminal lysine (εNH2). The dimethacryloyl derivatives were
then prepared via the carbodiimide-mediated coupling of

Biodegradation of PHEMA and P[HEMA-co-(MeO-PEGMA)]
Biomacromolecules, Vol. 11, No. 11, 2010 2953
Figure 2. PHEMA hydrogels cross-linked with 1 mol % pentapeptide
6: formulation I (H2O:HEMA 80:20), formulation II (H2O:HEMA:MeO-
PEGMA 80:20:6 where 1.3 M NaCl solution was used in the place of
H2O), and formulation III (H2O:HEMA:MeO-PEGMA 80:20:6). The
three samples were initially the same size (the initial size was
determined by the size of the vessel in which polymerization
occurred). The different sizes for the samples shown reflect their
different swelling abilities when soaked in water postpolymerization.
Figure 1. RP-HPLC chromatogram of (A) reduced pentapeptide 6H4
and (B) solution of 6H4 after incubation with papain for 30 min. The
peaks at retention times less than 6 min are not due to 6H4 or its
degradation products and were also seen in chromatograms of the
control samples.
indistinguishable from the chromatograms for the pure reduced
peptide cross-linker (Figure 1A), indicating that the peptide 6H4
was stable toward the activation buffer. Analogous results were
obtained for the experiments using the tripeptide 4H4 and the
tetrapeptide 5H4 (see the Supporting Information).
methacrylic acid to the amine groups to give methyl esters of
4OMe, 5OMe, and 6OMe. Attempts at purification of the
dimethacryloyl compounds using conventional silica column
chromatography failed to yield any pure product, a result that
was attributed to partial/premature polymerization on the
chromatography support. Thus, crude 4OMe, 5OMe and 6OMe
were not purified but were directly subjected to saponification.
Saponification served as a purification step, which obviated the
need for chromatography and its associated complications. The
unmasking of the carboxylic acid also rendered the cross-linking
agents more water-soluble. This is an important consideration
in the final design of the cross-linking agents, as they need to
be soluble in the aqueous reaction mixture used for the
preparation of the cross-linked sponges. In summary, three
peptide-based compounds, functionalized with terminal meth-
acryloyl groups were prepared, in yields ranging from 45 to
64% based on their respective protected oligopeptide precursors.
The proteolytic degradation of the oligopeptides by papain
was monitored by RP-HPLC. Due to the difficulty experienced
during purification of the cross-linking agents, the dimethacry-
loyl compounds were subjected to catalytic hydrogenation to
reduce the double bonds and thus passivate the methacryloyl
groups. The relative rates of hydrolysis of the oligopeptides 4,
5, and 6 were qualitatively tested by exposure of samples of
the reduced oligopeptides (1 mg/mL) to similar quantities of
papain in activation buffer solution. In these experiments, the
hydrolyses of 4H4, 5H4, and 6H4 were complete within ca. 6,
3, and 0.5 h, respectively. These results are consistent with
previous studies29,30 that found that the rate of enzymatic
hydrolysis is enhanced by increasing the length of the oligopep-
tide, but the peptide sequence may also important in determining
the relative rates of hydrolysis of 4H4-6H4. Typical initial and
final chromatograms for the hydrolysis of pentapeptide 6H4 are
illustrated in Figure 1 and indicate a Rt of 21.98 min for the
pure substrate (Figure 1A). After hydrolysis (Figure 1B) the
peak at 21.98 min is absent, and three new peaks at lower
retention times (i.e., 14.13, 11.27, and 9.03 min) are present.
These results are consistent with the complete hydrolysis of the
pentapeptide 6H4 to give a number of smaller, more polar
fragments. The chromatograms obtained for the control experi-
ment (pentapeptide 6H4 and activation buffer only) were
3.2. Preparation and Characterization of PHEMA and
P[HEMA-co-(MeO-PEGMA)] Containing Peptide-Based Cross-
Links. A series of PHEMA and P[HEMA-co-(MeO-PEGMA)]
hydrogels were prepared via the photoinitiated induced-induced
phase separation of HEMA and MeO-PEGMA with 1 mol %
(relative to HEMA) of the corresponding peptide-based cross-
linking agents 4, 5, and 6 using the formulations specified in
Table 1. The macroscopic appearance of the hydrogels depended
on the formulation used. The appearances of the hydrogels
prepared with the pentapeptide cross-linking agent 6 using three
different formulations (hydrogel I6, II6, and III6) are shown in
Figure 2. The appearances shown in Figure 2 are typical; that
is, all formulation I hydrogels I4, I5, I6, and IT have similar
macroscopic appearances, and so on.
The hydrogels prepared using formulation I (I4, I5, I6, and IT)
were obtained using a standard set of experimental parameters
(H2O:HEMA 80:20 wt/wt, 0.1 mol % DPAP, and 1 mol % cross-
linker) and were opaque hydrogel sponges. These sponges were
characterized by SEM, which required that the samples be
dehydrated prior to imaging. For some samples, dehydration was
accompanied by irreversible alteration of the structure of the
hydrogel, which resulted in artifacts in SEM images. Heterogenous
hydrogels (i.e., Formulation I and II) can be prepared both by
freeze-drying and critical point drying to give SEM images that
are indistinguishable.23 Homogenous hydrogels (i.e., Formulation
III) were prepared for SEM by freeze-drying because critical point
drying results in the shrinkage of the samples due to the lowered
Tg value of the P[HEMA-co-(MeO-PEGMA)] hydrogel.23 The
SEM images of I4 and I5 (Figure 3) and IT revealed spherical,
contiguous droplets, constituting a morphology typical of PHEMA
hydrogel sponges. In contrast, the SEM images of hydrogel I6
(Figure 3) revealed the presence of agglomerates of smaller polymer
droplets that were situated in and around continuous polymer folds.
The reduced polymer droplet size in I6 may be a consequence of
the greater hydrophobicity (lower solubility in water) of
the pentapeptide cross-linking agent 6 compared to 4 and
5. The relative solubility of the cross-linking agent added to the
polymerization mixture has an effect on the polymer morphology,24
by accelerating the phase separation process. The earlier onset of
phase separation results in the rapid precipitation of polymer from

2954 Biomacromolecules, Vol. 11, No. 11, 2010
Casadio et al.
Figure 3. SEM images of PHEMA and P[HEMA-co-(MeO-PEGMA)] hydrogels cross-linked with 1 mol % peptide-based cross-linking agent: I4,
II4, and III4 cross-linked with tripeptide 4; I5, II5, and III5 cross-linked with tetrapeptide 5; and I6, II6, and III6 cross-linked with pentapeptide 6. The
hydrogels were prepared according to formulation I (H2O:HEMA 80:20), formulation II (H2O:HEMA:MeO-PEGMA 80:20:6 where 1.3 M NaCl
solution was used in the place of H2O), or formulation III (H2O:HEMA:MeO-PEGMA 80:20:6). Hydrogels prepared according to formulations I
and II were dehydrated via critical point drying, and hydrogels prepared according to formulation III were freeze-dried (see text).
the polymerization mixture, ultimately leading to a reduced polymer
droplet size.24 Thus it appears that the more hydrophobic pen-
tapeptide 6 could be acting to accelerate the phase separation
process, ultimately leading to smaller polymer droplets.
and cross-linking agent. The initial dry weight (Wd0) and the
initial wet weight (Ww0) were determined for each sample. Once
these parameters had been determined, the hydrated samples
were incubated with deoxygenated buffered enzyme solution
containing activated papain (1 mg/mL) and sodium azide (0.2
mg/mL). At predetermined time intervals, samples were re-
moved from the solution, rinsed, and their final wet weight (Wwi)
and final dry weight (Wdi) were determined. The weight changes
seen for samples exposed to the degradation solutions for
different times were used to construct a time course for the
degradation of each hydrogel formulation in terms of the dry
weight of the hydrogel remaining after degradation and change
to weight of the wet (hydrated) hydrogel caused by degradation.
In addition, where possible, the hydrogel samples were imaged
using SEM techniques (both conventional and variable pressure)
to assess the effect of degradation on the microstructure of the
hydrogel, which is of utmost importance if the hydrogel is to
support cellular growth while undergoing degradation. The
results obtained from the degradation studies with papain for
each of the three formulations are now discussed.
3.3.1. Formulation I. Hydrogel sponges prepared using
formulation I (I4, I5, and I6) exhibited negligible weight change
when incubated with papain. Hydrogels cross-linked with the
tetrapeptide 5 (I5) and pentapeptide 6 (I6) were incubated for
35 days. Analysis of the SEM images obtained from the samples
postincubation indicated some minor differences in their
morphological features compared to those of samples incubated
in buffer that did not contain papain (Figure 4). Similarly,
hydrogels cross-linked with the tripeptide 4 (I4) displayed
negligible weight loss, even after an extended period of
incubation with papain (120 days). However, SEM images of
the incubated I4 sample revealed a significant change in the
original polymer droplet morphology, whereby the sample
The hydrogels prepared using formulation II (II4, II5, II6,
and IIT) were obtained by the copolymerization of HEMA and
MeO-PEGMA in a NaCl solution. The introduction of MeO-
PEGMA enhances the hydrophilicity of the hydrogel and the
addition of NaCl to the polymerization mixture ensures that the
hydrogel forms with the appropriate spongy morphology (ag-
glomerated polymer droplets, Figure 3) by promoting phase
separation.23,47 These hydrogels varied from opaque to almost
translucent and they exhibited a greater swelling capacity
compared to their Formulation I counterparts (Figure 2).
The hydrogels prepared using formulation III (III4, III5, III6,
and IIIT) were obtained by the copolymerization of HEMA and
MeO-PEGMA in the absence of NaCl, were clear to transparent
in appearance, and also exhibited an increased swelling capacity
relative to the hydrogels prepared with Formulation I. The
transparency of these samples suggests that they are homoge-
neous gels that have not undergone polymerization-induced
phase separation. The SEM images of the Formulation III
hydrogels revealed an interesting cellular or “honeycomb”
structure, with the width of the cells ranging from 15 to 25 µm
(Figure 3; III4, III5, and III6), but this structure is most likely
an artifact of the sample drying process undertaken before SEM.
The addition of MeO-PEGMA acts to suppress phase separation,
and thus the interconnected polymer droplet microstructure is
not observed.23
3.3. In Vitro Degradation of PHEMA and P[HEMA-co-
(MeO-PEGMA)] Hydrogels Prepared with Peptide-Based
Cross-Linking Agents. In vitro degradation experiments each
used a batch of samples (typically 12) from the same formulation

Biodegradation of PHEMA and P[HEMA-co-(MeO-PEGMA)]
Biomacromolecules, Vol. 11, No. 11, 2010 2955
Figure 5. Samples of PHEMA I4 and IT cross-linked with 1 mol % 4
and TEDGMA respectively. The samples were incubated with
activated papain or activation buffer in the absence of papain and
then treated with ninhydrin.
Figure 6. SEM images of PHEMA hydrogels II4. The hydrogels were
incubated with activated papain or activation buffer in the absence
of papain for 91 days.
Figure 4. SEM images of PHEMA hydrogels (formulation I) cross-
linked with 1 mol % cross-linking agent and incubated with activated
papain or activation buffer in the absence of papain. Hydrogel I4 was
incubated for a period of 120 days, and hydrogel I5 and I6 were
incubated for a period of 35 days.
activation buffer) remaining in the hydrogel. Overall these
results are consistent with papain being able to diffuse into the
I4 sponge and catalyze the hydrolysis of the peptide-based cross-
linking agent, a result which is of fundamental importance for
potential of such hydrogels as biodegradable tissue engineering
scaffolds.
Since the ninhydrin test suggests that the peptide cross-links
in the hydrogels are cleaved by papain, we suggest that the lack
of weight loss during the degradation of the sponges prepared
using formulation I is likely due to poor solubility of the linear
PHEMA chains (which would be released by cleavage of
peptide-based cross-links). This result highlights the need to
enhance the hydrophilicity of the linear PHEMA chains, thereby
increasing their solubility in water.
3.3.2. Formulation II. Hydrogel sponges prepared using
formulation II and cross-linked with the tripeptide 4 (II4)
exhibited negligible weight loss over an incubation period of
91 days. SEM images of the incubated hydrogels (Figure 6, +
papain) showed that the samples no longer displayed the
characteristic polymer droplet morphology, however, but ap-
peared to be somewhat “mottled”, indicating that the polymer
droplet structure had collapsed during incubation with papain,
consistent with the results obtained for samples prepared using
formulation I. The SEM images of the control sample (hydrogel
cross-linked with 1 mol % tripeptide incubated in activation
buffer containing no papain) displayed an unaffected polymer
droplet morphology (Figure 6, - papain). These results suggest
that papain was able to hydrolyze the peptide cross-linked
hydrogel, but, again, the lack of any significant weight loss
indicated that either the polymer chains released when peptide
links were cleaved were still not sufficiently water-soluble to
diffuse away from the bulk hydrogel, or that papain was not
hydrolyzing sufficient numbers of peptide cross-links to fully
release linear PHEMA fragments.
appeared to exhibit a microstructure based on “collapsed”
polymer droplets together with small groupings of polymer
droplets located throughout the sample. SEM images of the I4
sample incubated in the absence of papain revealed a relatively
unaffected polymer droplet morphology (Figure 4).
The changes to the hydrogel morphology of I4 caused by
incubation with papain suggested that there was some form of
degradation of the material by papain despite the constant weight
of the sample. A weight loss will only be observed if the
fragments that are being hydrolyzed, either from the surface or
from the bulk hydrogel, are water-soluble and thus able to
diffuse away from the hydrogel. Since only very short PHEMA
chains (i.e., e20 repeat units) are water-soluble,48 and free-
radical polymerization of PHEMA (in the absence of cross-
linking agent) typically produces molecular weight fragments
ranging from 10 to 1000 kDa,49 the absence of any weight loss,
despite the evidence for some degradation, is perhaps not
surprising. Another possibility is that papain was not hydrolyzing
sufficient numbers of peptide cross-links to fully release linear
PHEMA fragments.
To confirm that papain was cleaving the peptide cross-links,
a colorimetric assay based on ninhydrin was developed to test
for hydrolyzed peptide bonds within the polymer network.
Figure 5 illustrates a series of hydrogels prepared according to
Formulation I using either 1 mol % tripeptide cosslinking agent
or 1 mol % nondegradable cross-linking agent TEGDMA (I4
and IT respectively). PHEMA discs were incubated with
activated papain or in buffer solution in the absence of papain
(Figure 5). The enzyme and buffer solutions were exchanged
every day for 8 days, after which time, the hydrogel samples
were washed with water and soaked in a dilute ethanolic solution
of ninhydrin. Gentle heating of the samples treated with
ninhydrin resulted in an intense blue coloration of only one
sample, I4 which had been incubated with papain, indicating
the presence of free amines within this sample.50 The remaining
samples all displayed only a pale blue tint, presumably resulting
from traces of residual enzyme or L-cysteine (used in the
Formulation II hydrogels cross-linked with tetra- and pen-
tapeptide (II5, II6 respectively) displayed significant weight loss
during incubation with papain over a period of days. The
degradation of hydrogels II5 and II6 is illustrated in Figure 7
by plots of dry weight remaining (%)46 and wet weight change
(%).32 Hydrogels II5 and II6 lost approximately 80% of their

2956 Biomacromolecules, Vol. 11, No. 11, 2010
Casadio et al.
Figure 7. Degradation of P[HEMA-co-(MeO-PEGMA)] hydrogels (formulation II). Photograph of P[HEMA-co-(MeO-PEGMA)] cross-linked with
1 mol % pentapeptide 6 (hydrogel II6) undergoing degradation over 24 h. Degradation plots for P[HEMA-co-(MeO-PEGMA)] hydrogels cross-
linked with 1 mol % tetrapeptide 5 (hydrogel II5) and pentapeptide 6 (hydrogel II6).
7), presumably due to an increased ability for the samples to
swell as cross-links are cleaved. We expect that when the
amount of polymer remaining in the sample (as indicated by
the % dry mass remaining) is greatly reduced, the small amount
of polymer remaining would be insufficient to maintain a large
volume of absorbed water, and so the wet weight should decline.
This effect is clearly reflected in the wet weight change for II5
(Figure 7); the wet weight for II5 samples drops precipitously
when the dry mass remaining falls below 40%. We note that as
degradation proceeds, measurement of sample wet weights
becomes increasingly difficult and prone to error (the samples
become increasingly fragile and the test involves blotting the
samples to remove excess water). Although the increase in wet
weight of the samples at the beginning of the degradation
experiments is clear, care should be taken not to overinterpret
the wet weight data at the later stages of the degradation
experiments.
Figure 7 also shows photographs of a series of samples of
P[HEMA-co-(MeO-PEGMA)] cross-linked with 1 mol % pen-
tapeptide 6 (hydrogel II6), at different times during the course
of the degradation. The general size and shape of the hydrogel
samples is roughly maintained up until approximately 12 h of
Figure 8. SEM images of P[HEMA-co-(MeO-PEGMA)] hydrogels
incubation, after which time the samples begin to collapse and
cross-linked with (a-e) 1 mol % pentapeptide cross-linking agent 6
loose their original forms. The samples of II6 hydrogels change
from opaque to become increasingly translucent as the degrada-
tion proceeds, to the point at which the sample removed after
24 h is almost transparent, indicating possible changes in the
hydrogel microstructure. It is noteworthy that similar changes,
but over a longer period of time, were observed for the II5
hydrogels.
(hydrogel II6) and (f) 1 mol % TEDGMA (hydrogel IIT). The samples
were incubated with activated papain for (a-d) 1 h, (e) 4 h, or (f) 90 h.
SEM images b and c are magnifications of regions within the white
rectangles in images a and b respectively.
dry weight over a period of 92 and 30 h, respectively. These
results are consistent with those obtained for the RP-HPLC
assays carried out on the modified peptide substrates 5H4 and
6H4, which indicated that papain catalyzed the hydrolysis of
the pentapeptide 6H4 at a faster rate than for the tetrapeptide
5H4. The weights of the wet (hydrated) samples of II5 and II6
initially increased sharply (by approximately 60-80%, Figure
SEM images of hydrogels II5 and II6 incubated with papain
displayed very similar morphological features. Figure 8 shows
SEM images that display the degradation of hydrogel II6 after
1 h and show that the polymer droplet morphology is still present
in some areas but the hydrogel appears to be pitted with large

Biodegradation of PHEMA and P[HEMA-co-(MeO-PEGMA)]
Biomacromolecules, Vol. 11, No. 11, 2010 2957
sample as the degradation proceeds. Hydrogel IIT, lacking the
biodegradable cross-links, maintained its polymer droplet
morphology when incubated with papain (Figure 8f).
SEM images of the II6 samples after 12 and 24 h of
incubation show that the original polymer droplet morphology
is no longer present (Figure 9). Difficulties were encountered
when preparing the degraded hydrogels for SEM imaging as
they collapsed noticeably during critical point drying, a result
that may be attributed to the hydrogel having a low Tg value,23
or an increased solubility of the degraded hydrogel in acetone
(critical point drying transitional fluid) used during the drying
procedure. The degraded samples were therefore dehydrated via
freeze-drying and imaged under variable pressure (VP) condi-
tions.45 Although not an ideal dehydration method, the difficul-
ties encountered in the preparation of the samples and the
collapsed microstructure displayed in the micrographs (Figure
9) clearly indicate that the original polymer droplet morphology
is no longer present and the samples are in an advanced stage
of degradation. Analogous results were obtained for the
degradation of samples of the hydrogel II5.
In control experiments (in which hydrogel samples cross-
linked with peptide-based cross-linkers were incubated in buffer
solution without added papain, or hydrogel samples cross-linked
with TEGDMA were incubated with buffer containing papain)
the change to the samples’ dry and hydrated weights were
negligible (g96% dry weight remaining and change to wet
weight of samples e7% after incubation; Table 2). There was
no change to the macroscopic appearance of the control samples
during incubation, and SEM images of the control samples (e.g.,
Figure 8f) indicated that the polymer droplet morphology was
unaffected by the incubation conditions. These results show that
under the experimental conditions, the control hydrogels remain
intact (i.e., they do not lose weight and do not absorb water
during the incubation) and thus little to no degradation of the
control samples occurred. The weight change profiles and SEM
images of the sponges incorporating the tetra- and pentapeptide
Figure 9. SEM images of P[HEMA-co-(MeO-PEGMA)] hydrogels
(formulation II) cross-linked with 1 mol % pentapeptide cross-linking
agent 6 (hydrogel II6). The hydrogels were incubated with activated
papain for (a) 12 h and (b) 24 h. The degraded samples were freeze-
dried and imaged using VP-SEM.
Table 2. Effect of Incubation on Sample Weight for Formulation II
Hydrogels
incubation
conditions
dry weight
remaining (%)
wet weight
change (%)
hydrogel
II5
activation buffer
without papain
activation buffer
without papain
activation buffer with
activated papain
97
99
2
1
7
II6
IIT
101
cavities (Figure 8a-c). SEM images of the interior of the
cavities indicate that they are densely occupied with polymer
droplets and that the spherical droplets appear to be “melting”
in the walls of the newly formed cavities to form a smoother,
more continuous structure (Figure 8c,d). The SEM image of
the hydrogel after 4 h (Figure 8e) shows that the number of
cavities has increased over time with a corresponding increase
in the overall proportion of the smooth areas throughout the
sample, indicating a significant degree of microstructural change
during the degradation. The increased homogeneity and loss of
the polymer droplet morphology of the samples, as observed
by SEM, is consistent with the increased transparency of the
Figure 10. Degradation of P[HEMA-co-(MeO-PEGMA)] hydrogels (formulation III). Photographs of P[HEMA-co-(MeO-PEGMA)] cross-linked
with 1 mol % pentapeptide 6 (hydrogel III6) undergoing degradation over 6.75 h. Degradation plots for P[HEMA-co-(MeO-PEGMA)] hydrogels
cross-linked with 1 mol % tetrapeptide 5 (hydrogel III5) and pentapeptide 6 (hydrogel III6).

2958 Biomacromolecules, Vol. 11, No. 11, 2010
Casadio et al.
Table 3. Effect of Incubation on Sample Weight for Formulation
III Hydrogels
indicating that polymerization had induced phase separation.
The remaining hydrogels formed in the absence of phase
separation during polymerization. Interestingly, subtle changes
in the peptide cross-linking agents had significant effects on
the polymer microstructure. The effects are related to the
hydrophobicities of the various cross-linking agentssthe more
hydophobic cross-linker 6 appeared to encourage earlier onset
of phase separation, with consequent smaller polymer droplet
size.
incubation
conditions
dry weight
remaining (%)
wet weight
change (%)
hydrogel
III5
activation buffer
without papain
activation buffer
without papain
activation buffer with
activated papain
98
98
12
32
4
III6
IIIT
100
When the HEMA and P[HEMA-co-(MeO-PEGMA)] hydro-
gels were incubated with papain their degradation behavior was
dependent on both the formulation of the hydrogel and the nature
of the peptide-based cross-linking agent. Hydrogels prepared
according to formulation I (without MeO-PEGMA) did not
display any significant weight loss, whereas hydrogels prepared
according to formulations II and III (both including MeO-
PEGMA) degraded over time when incubated with papain. The
enhanced degradability of the hydrogels incorporating PEG is
presumably due to the increased solubility of MeO-PEGMA-
PHEMA chains compared to simple PHEMA chains.
Hydrogels incorporating the pentapeptide cross-linking agent
6 generally degraded at a faster rate than the those incorporating
the tetrapeptide cross-linking agent 5, while hydrogels containing
the tripeptide cross-linking agent 4 showed little degradation,
suggesting that the time frame of degradation can be controlled
by the length of the peptide-based cross-linking agent. This result
is not too surprising given the steric congestion that undoubtedly
exists for shorter peptide linkers that will reduce the efficiency
of protease degradation of the peptide linker. While our results
suggest that longer linkers are cleaved more rapidly than shorter
linkers, more experiments are necessary to define the relationship
between hydrolysis rate and the length and peptide sequence
of the cross-linker. One of the next steps will be to incorporate
peptide targets that will be affected by specific proteases.
cross-linkers, together with the results obtained for the control
samples, show that the hydrogels prepared according to
formulation II can be degraded by papain. The hydrogels
prepared using Formulation II typically display a polymer
droplet morphology, which is consistent with their classification
as sponges.24 The porous nature of the sponges allows for the
diffusion of the enzymatic solution throughout the hydrogel
sample. The SEM images show that the droplet morphology is
lost during degradation, although the samples themselves remain
largely intact on the macroscopic scale. These results suggest
that degradation is occurring throughout the hydrogel structures
and not just at the surfaces of the constituent polymer droplets.
If degradation were instead occurring via a surface mechanism,
it would be expected that degradation would be characterized
by decreasing size of polymer droplets in SEM images, rather
than a complete loss of the droplet morphology.51
3.3.3. Formulation III. The degradation of the P[HEMA-co-
(MeO-PEGMA)] hydrogels prepared with the tetrapeptide 5 and
pentapeptide 6 cross-linking agents (hydrogels III5 and III6
respectively) in the absence of NaCl are illustrated in Figure
10. Upon incubation with papain, both of the hydrogels were
fully degraded. Complete dissolution of the pentapeptide cross-
linked hydrogel occurred within 8 h, while the tetrapeptide cross-
linked hydrogel dissolved over a 48 h period. The relative rates
of degradation of the samples are consistent with results
mentioned above, where the pentapeptide 6H4 and the pen-
tapeptide-cross-linked hydrogel II6 exhibited faster degradation
kinetics than their tetrapeptide analogues.
Acknowledgment. The authors acknowledge the Australian
Research Council for funding (to M.V.B., T.V.C., and H.-B.K.)
and an Australian Postgraduate Award (to Y.S.C.). We are
grateful for a Jean Rogerson Supplementary Scholarship (to
Y.S.C.). D.H.B. thanks Curtin University of Technology for a
Research and Teaching Fellowship; M.V.B. thanks the National
Science Council of Taiwan for a Visiting Professorship; H.-
B.K. thanks NSERC for funding and The University of Western
Australia for a Gledden Fellowship. The authors acknowledge
the facilities and scientific and technical assistance of the
Australian Microscopy and Microanalysis Research Facility at
the Centre of Microscopy, Characterization and Analysis, The
University of Western Australia, a facility funded by The
University of Western Australia, the Australian Government,
and the state Government of Western Australia.
While the dry weight of the sample decreased monotonically
during the degradation experiment, the wet weight of the sample
showed a rapid increase at the start of the degradation experi-
ment followed by a decline as the sample degraded further. This
increase may be a consequence of the cleavage of cross-links
allowing polymer chains to mover further apart, so that the
materials can absorb more water and swell. An alternative
possibility is that the increase in wet weight is consequence of
morphological changes to the samples (Tg below 36 °C23) that
occur during incubation at 37 °C (the optimal temperature for
papain).
The weight study carried out for the control samples III5-IIIT
(Table 3) indicated that there was negligible loss of dry weight
(e2%) during incubation. There were significant changes to the
samples’ wet weights (indicated by percentage wet weight gains
during incubation of ca. 4 to 32%). It may be that these weight
gains are also a consequence of morphological changes to the
samples (Tg ca. 36 °C) that occur during incubation at 37 °C
(the optimal temperature for papain).
Supporting Information Available. Full details for synthesis
and characterization of peptide-based cross-linking agents 4-6,
the protected oligopeptide precursors 7-9, and the reduced
cross-linking agents 4H4-6H4 and chromatograms for studies
of degradation of 4H4 and 5H4. This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
Conclusions
(1) Cima, L. G.; Vacanti, J. P.; Vacanti, C.; Ingbar, D.; Mooney, D.;
Langer, R. J. Biomech. Eng.-T. ASME. 1991, 113, 143–151.
(2) Scaffolding in Tissue Engineering, 1st ed.; Ma, P. X., Elisseeff, J.,
Eds.; CRC Taylor and Francis: Boca Raton, FL, 2006.
(3) Park, H.; Cannizzaro, C.; Vunjak-Novakovic, G.; Langer, R.; Vacanti,
PHEMA-peptide and [PHEMA-co-(MeO-PEGMA)]-pep-
tide conjugate hydrogels were readily prepared via photoinitiated
free-radical polymerization in water. Five of these hydrogels
displayed an interconnected polymer droplet morphology,
C. A.; Farokhzad, O. C. Tissue Eng. 2007, 13, 1867–1877.

Biodegradation of PHEMA and P[HEMA-co-(MeO-PEGMA)]
Biomacromolecules, Vol. 11, No. 11, 2010 2959
(4) Atzet, S.; Curtin, S.; Trinh, P.; Bryant, S.; Ratner, B. Biomacromol-
ecules 2008, 9, 3370–3377.
(29) Ulbrich, K.; Strohalm, J.; Kopecˇek, J. Biomaterials 1982, 3, 150–
154.
ˇ
(5) Vacanti, J. P.; Morse, M. A.; Saltzman, W. M.; Domb, A. J.; Perez-
Atayde, A.; Langer, R. J. Pediatr. Surg. 1988, 23, 3–9.
(6) Wichterle, O.; Lim, D. Nature 1960, 185, 117–118.
(7) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869–1879.
(8) Khutoryanskaya, O. V.; Mayeva, Z. A.; Mun, G. A.; Khutoryanskiy,
V. V. Biomacromolecules 2008, 9, 3353–3361.
(9) Lai, Y. C.; Wilson, A. C.; Zantos, S. Z. In Kirk-Othmer Encyclopedia
of Chemical Technology; Howe-Grant, M., Ed.; John Wiley and Sons,
Inc.: New York, 1993; Vol. 7, pp 192-218.
(10) Chirila, T. V. Biomaterials 2001, 22, 3311–3317.
(11) Young, C.-D.; Wu, J.-R.; Tsou, T.-L. Biomaterials 1998, 19, 1745–
1752.
(12) Voldrˇich, Z.; Toma´nek, Z.; Vac´ık, J.; Kopecˇek, J. J. Biomed. Mater.
Res. 1975, 9, 675–685.
(13) Dziubla, T. D.; Torjman, M. C.; Joseph, J. I.; Murphy-Tatum, M.;
Lowman, A. M. Biomaterials 2001, 22, 2893–2899.
(14) Hsiue, G.-H.; Guu, J.-A.; Cheng, C.-C. Biomaterials 2001, 22, 1763–
1769.
(15) Lou, X.; Munro, S.; Wang, S. Biomaterials 2004, 25, 5071–5080.
(16) Song, J.; Saiz, E.; Bertozzi, C. R. J. Eur. Ceram. Soc. 2003, 23, 2905–
2919.
(17) Harris, J. M. In Poly(ethylene glycol) chemistry: biotechnical and
biomedical applications; Harris, J. M., Ed.; Plenum Press: New York,
1992; pp 1-14.
(18) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M.
Chem. ReV. 1999, 99, 3181–3198.
(30) Subr, V.; Duncan, R.; Kopecˇek, J. J. Biomater. Sci., Polym. Ed. 1990,
1, 261–278.
(31) Duncan, R.; Hume, I. C.; Yardley, H. J.; Flanagan, P. A.; Ulbrich,
K.; Subr, V.; Strohalm, J. J. Controlled Release 1991, 16, 121–136.
(32) Kim, S.; Healy, K. E. Biomacromolecules 2003, 4, 1214–1223.
(33) Plunkett, K. N.; Berkowski, K. L.; Moore, J. S. Biomacromolecules
2005, 6, 632–637.
(34) Moss, J. A.; Stokols, S.; Hixon, M. S.; Ashley, F. T.; Chang, J. Y.;
Janda, K. D. Biomacromolecules 2006, 7, 1011–1016.
(35) Khelfallah, N. S.; Decher, G.; Me´sini, P. J. Macromol. Rapid Commun.
2006, 27, 1004–1008.
(36) Turk, V.; Turk, B.; Turk, D. EMBO J. 2001, 20, 4629–4633.
(37) Turk, D.; Guncˇar, G.; Podobnik, M.; Turk, B. Biol. Chem. 1998, 379,
137–147.
(38) Lo¨ser, R.; Schilling, K.; Dimmig, E.; Gu¨tschow, M. J. Med. Chem.
2005, 48, 7688–7707.
(39) Bodanszky, M. Peptide Chemistry: A Practical Textbook, 2nd ed.;
Springer-Verlag: Berlin, 1988.
(40) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis, 2nd
ed.; Springer-Verlag: Berlin, 1994.
(41) Balajthy, Z. Org. Prep. Proced. Int. 1991, 23, 375–376.
(42) Carter, H. E.; Frank, R. L.; Johnston, H. W. Org. Synth. Coll. Vol. II
1943, 13–16.
(43) Slotin, L. A.; Lauren, D. R.; Williams, R. E. Can. J. Chem. 1977, 55,
4257–4266.
(44) Glazer, A. N.; Smith, E. L. In The Enzymes; Boyer, P. D., Ed.;
Academic Press: New York, 1971; Vol. III p501-545.
(45) Egerton, R. E. Physical Principles of Electron Microscopy. An
Introduction to TEM, SEM, and AEM; Springer: New York, 2007.
(46) Guan, J.; Fujimoto, K. L.; Sacks, M. S.; Wagner, W. R. Biomaterials
2005, 26, 3961–3971.
(47) Liu, Q.; Hedberg, E. L.; Liu, Z.; Bahulekar, R.; Meszlenyi, R. K.;
Mikos, A. G. Biomaterials 2000, 21, 2163–2169.
(48) Weaver, J. V. M.; Bannister, I.; Robinson, K. L.; Bories-Azeau, X.;
Armes, S. P.; Smallridge, M.; McKenna, P. Macromolecules 2004,
37, 2395–2403.
(19) Chirila, T. V.; Constable, I. J.; Crawford, G. J.; Vijayasekaran, S.;
Thompson, D. E.; Chen, Y.-C.; Fletcher, W. A.; Griffin, B. J.
Biomaterials 1993, 14, 26–38.
(20) Sharkawy, A. A.; Klitzman, B.; Truskey, G. A.; Reichert, W. M.
J. Biomed. Mater. Res., Part A 1997, 37, 401–412.
(21) Sharkawy, A. A.; Klitzman, B.; Truskey, G. A.; Reichert, W. M.
J. Biomed. Mater. Res., Part A 1998, 40, 586–597.
(22) Sharkawy, A. A.; Klitzman, B.; Truskey, G. A.; Reichert, W. M.
J. Biomed. Mater. Res., Part A 1998, 40, 598–605.
(23) Baker, M. V.; Brown, D. H.; Casadio, Y. S.; Chirila, T. V. Polymer
2009, 50, 5918–5927.
(24) Chirila, T. V.; Chen, Y.-C.; Griffin, B. J.; Constable, I. J. Polym. Int.
1993, 32, 221–232.
(25) West, J. L.; Hubbell, J. A. Macromolecules 1999, 32, 241–244.
(26) Park, K. Biomaterials 1988, 9, 435–441.
(49) Casadio, Y. S. Biodegradable PHEMA-based Biomaterials, PhD
Thesis, The University of Western Australia: Crawley, Australia, 2008.
(50) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595–598.
(27) Shalaby, W. S. W.; Park, K. Pharm. Res. 1990, 7, 816–823.
(28) Shalaby, W. S. W.; Blevins, W. E.; Park, K. J. Controlled Release
1992, 19, 131–144.
(51) Go¨pferich, A. Biomaterials 1996, 17, 103–114.
BM100756C