Synthesis and characterization of enzymatically degradable PEG-based peptide-containing hydrogels

Article abstract of DOI:10.1002/mabi.200900295

Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG) ₂K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and microrheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, timecourse analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability. (Figure Presented)

Full text of DOI:10.1002/mabi.200900295

Full Paper
Synthesis and Characterization of
Enzymatically Degradable PEG-Based Peptide-
Containing Hydrogelsa
ˇ
Jiyuan Yang, Michael T. Jacobsen, Huaizhong Pan, Jindrich Kopecˇek*
Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG
differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides: [alkyne]-
GFLGK-[alkyne] and ([alkyne]-GFLG)2K. The physical properties of in situ formed hydrogels
were examined. The hydrogels were highly elastic as determined by rheological and micro-
rheological studies. Swelling degree and enzy-
matic degradation by papain were dependent
on the molecular weight of the PEG, but not the
peptide. For PEG8800-based hydrogels, time-
course analysis of degradation showed that the
molecular weight of the soluble fraction quickly
reached the PEG precursor value. These findings
may guide future design of hydrogels with con-
trollable mechanical properties and enzymatic
degradability.
Introduction
chemistry for the formation of reproducible, reversible 3D
networks with precisely defined structures, and biodegrad-
ability. Living radical polymerization has been used for the
control of the molecular weight distribution of the primary
chains of the network;[4,5] copper catalyzed[6,7] or copper-
free[8] Huisgen cycloaddition of azides and alkynes (click
chemistry) and Michael addition[9] have been used to
control crosslinking and/or attachment of biologically
active molecules.
Hydrogels were the first biomaterials designed for clinical
use.[1] They have found applications in numerous areas,
including tissue engineering, drug delivery, sensing,
diagnostics, and microfluidics.[2,3]
Several factors need to be considered when designing
biocompatible hydrogel biomaterials including controlled
The combination of synthetic polymers with sequences/
chains of natural polymers is a promising route for the
design and synthesis of enzymatically degradable hybrid
polymers/hydrogels.[2,10] To render a hybrid polymer/
hydrogel degradable, the structure of the degradable
sequences should match the active site of respective
enzyme(s); oligopeptide sequences have been frequently
used as degradable crosslinks in hydrogels. Hydrogels,
containing oligopeptide crosslinks susceptible to chymo-
trypsin-catalyzed hydrolysis were synthesized by cross-
linking N-(2-hydroxypropyl)methacrylamide (HPMA)
copolymers containing reactive side-chains (terminated
J. Kopeˇcek, J. Yang, M. T. Jacobsen, H. Pan
Departments of Pharmaceutics and Pharmaceutical Chemistry/
CCCD, 20 S 2030 E, BPRB 205B, University of Utah, Salt Lake City,
Utah 84112, USA
Fax: (þ801) 581 7848; E-mail: jindrich.kopecek@utah.edu
J. Kopeˇcek
Department of Bioengineering, University of Utah, Salt Lake City,
Utah 84112-9452, USA
a : Supporting information for this article is available at the bottom
of the article’s abstract page, which can be accessed from the
journal’s homepage at http://www.mbs-journal.de, or from the
author.
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DOI: 10.1002/mabi.200900295
445

J. Yang, M. T. Jacobsen, H. Pan, J. Kopecˇek
Canada). Side-chain protected Fmoc-amino acids, 2-chlorotrityl
resin, and 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HATU) were purchased from AAPPTec
(Louisville, KY). Piperidine (PIP; 99.5þ%, Biotech grade), triisopro-
pylsilane (TIS; 99%), papain (EC 3.4.22.2), glutathione, 5-hexynoic
acid, Bz-Phe-Val-Arg-NAp (chromogenic substrate), iodoacetic acid
sodium salt, CuBr, L-ascorbic acid, and O-phthaladehyde (OPA)/
3-mercaptopropionic acid (MPA) were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Ethyldiisopropylamine (DIPEA; 99%)
was from Alfa Aesar (Ward Hill, MA, USA). Diethyl ether and
dichloromethane (DCM) were purchased from Mallinckrodt Baker
(Phillipsburg, NJ, USA). Trifluoroacetic acid (TFA; 99%) was
purchased from Acros Organics (Morris Plains, NJ, USA). Surfactant
free fluorescent yellow–green amidine modified polystyrene (PS)
beads were from Interfacial Dynamics Corp. (Portland, OR).
in p-nitrophenoxy groups) with oligopeptide-containing
diamines.[10] Thedegradabilityofhydrogelswasdependent
on the length and detailed structure of the oligopeptide
sequence and on the equilibrium degree of swelling
(network density); the higher the degree of swelling, the
faster the rate of degradation. The degree of swelling also
has an impact on surface versus bulk degradation of the
hydrogel. If the enzyme cannot diffuse into the hydrogel
interior, only surface degradation takes place.[11] Similar
HPMA-based hydrogels degradable by cathepsin B, a
lysosomal thiol proteinase, have also been evaluated.[12]
In further experiments, HPMA-based hydrogels with
degradable crosslinks were shown to release FITC-dextran
and daunomycin (covalently bound via oligopeptide
spacer) during incubation with a mixture of lysosomal
enzymes (Tritosomes) or chymotrypsin.[13] Recently, Plun-
kett et al. studied acrylamide-based hydrogels containing
oligopeptides degradable by chymotrypsin.[14]
Synthesis of Enzyme-Degradable Peptides with
Dialkyne Modification
Thefunctionalized peptides, flankedby alkynegroups atbothends,
were synthesized using solid-phase methodology and manual
Fmoc/tBu strategy on 2-chlorotrityl resin. As an example, the
synthesis of GFLG1 is shown in Scheme 1. Fmoc-Lys(ivDde)ꢀOH
was employed as the first amino acid residue to introduce two free
amine groups. The protecting groups (Fmoc- and ivDde-) were
removed selectively; one was used to introduce alkyne group into
the C-terminus of the peptide, whereas the other functioned in
constructing the peptide sequence. Incorporation of alkyne at the
N-terminus was accomplished by acylation of the peptide with
5-hexynoic acid using the standard coupling protocol (ꢀCOOH/
DIPEA/HATU in DMF). The resin was then washed sequentially
with 3 ꢁ DMF, 3 ꢁ DCM, and 3 ꢁ MeOH, and dried under vacuum.
The resulting resin-bound peptides were cleaved from the resin,
and side-chains were deprotected using TFA/H2O/TIS (95:2.5:2.5)
cocktail. Crude peptides were purified by RP-HPLC (Agilent
Technologies 1100, semipreparative Zorbax 300SB-C18 column,
Poly(ethylene glycol) (PEG) is a hydrophilic polymer that
has been used in several clinical applications. PEG-based
hydrogelshavebeenextensivelystudied.Hubbell’slaboratory
used multiarm-PEG and Michael-type addition to synthesize
extracellular matrix mimicking hydrogels degradable by cell-
excretedmatrixmetalloproteinases.[9,15] PEG-basedhydrogels
containing Schiff base linkages were designed for low
molecular weight (doxorubicin) drug delivery.[16] Azide-
alkyne click chemistry was also used for the synthesis of
PEG-based hydrogels.[6,17] Hawker and coworkers[6] synthe-
sized hydrogels by reacting a tetraazide-modified tetraethy-
lene glycol with telechelic alkyne terminated PEGs of varying
molecular weight. Yang and coworkers employed 4-arm
alkyne terminated PEG and crosslinked it with a series of RGD
containing dialkyne-peptides. However, both alkyne groups
were attached to the N-terminal lysine. Consequently, the
side-chains were degradable, but the hydrogel crosslinked
backbone remained stable.[17]
˚
250 mm ꢁ 9.4 mm, 300 A pore size, 5 mm particle size, flow rate
2.5 mL ꢂ minꢀ1) employing a gradient from 50 to 100% B over
30 min,wherebufferAwas0.1%TFAinwaterandbufferB0.1%TFA
in 90:10 v/v methanol/water. The purity of peptides GFLG1 and
GFLG2 was verified by analytical RP-HPLC (Figure S1 and S2 of
Supporting Information). Peptide structures were ascertained by
MALDI-TOF mass spectrometry (Voyager-DE STR Biospectrometry
Workstation, Perseptive Biosystems) (Figure S3 and S4 of Support-
ing Information). For GFLG2, Fmoc-Lys(Fmoc)-OH was used as the
first amino acid to incorporate a branched structure after removal
of Fmoc-group. The end-modification was realized by acylation of
the peptide with 5-hexynoic acid. The molecular structures of the
two peptides are shown in Table 1.
In this study we synthesized two 4-arm azide-terminated
PEGs differing in molecular weight (2 100 and 8 800) and
two alkyne-terminated peptides, [alkyne]-GFLGK-[alkyne]
(GFLG1) and ([alkyne]-GFLG)2K (GFLG2). The hydrogels were
synthesized by Huisgen cycloaddition (click chemistry) of PEG
azides with peptide alkynes and contained enzymatically
degradablecrosslinks. Thekineticsofthecrosslinkingreaction
was monitored by dynamic rheology. The hydrogels were
characterized by the equilibrium degree of swelling, their
morphology, and bulk and micro-rheology. The relationship
betweenthestructureofthehydrogelsandtheirdegradability
by papain, a thiol proteinase, was also evaluated.
Synthesis of Azido-Terminated Poly(ethylene glycol)
(PEG)
Experimental Part
Thesyntheticprocedureconsistedoftwosteps(Scheme2A):4-arm-
PEG-OH (PEG-4OH) was first transformed into an active inter-
mediate(mesylate-terminatedPEG), followedbyreplacementwith
azido functional group. A typical procedure for synthesis of 4-arm
PEG-N3 with Mn 8 800 g ꢂ molꢀ1 (PEG-4N3 8 800) is briefly described.
Materials
Four-armPEGterminatedwithhydroxygroups(PEG-4OH2 100and
PEG-4OH 8 800) was purchased from Polymer Source (Montreal,
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Synthesis and Characterization of Enzymatically Degradable . . .
ether. A fine powder was obtained. The
yield was 82.5%. The product was
confirmed by 13C NMR (CDCl3) and used
without further purification.
PEG-4N3 with Mn ꢃ 2 100 g ꢂ molꢀ1
(PEG-4N3 2 100) was synthesized using
a similar procedure.
Preparation of Hybrid
Hydrogels via Click Reaction
All hydrogels were prepared by the
reaction of 4-arm azide-terminated
PEGs described above with dialkyne-
flanked peptides in the presence
Scheme 1. Synthesis of alkyne-modified peptide GFLG1.
of CuBr/L-ascorbic acid in DMF
(Scheme 2B). For example, to prepare
hybrid hydrogel PEG8800-GFLG1, PEG-
In a 25 mL dry round-bottom flask, PEG-4OH 8 800 (0.7 g, [OH]
0.32 mmol) was dissolved in 4.5 mL anhydrous DCM, then kept in
an ice-ethanol bath (ꢀ10 to ꢀ15 8C). Triethylamine (0.88 mL,
6.4 mmol) was dropwise added to the flask under stirring before
addition of 0.5 mL (6.4 mmol, 20ꢁ) methanesulfonyl chloride. The
reaction was allowed to proceed at 0 8C for 3 h and then at room
temperature for another 16 h. Precipitation was observed, and the
color of the system gradually changed from colorless to yellow and
then to a dark, tea-like color. At the end of reaction 20 mL DCM was
added, and the precipitate was removed by filtration. The filtrate
was washed sequentially with 1 N HCl, saturated NaCl solution, 1 N
NaOH, and saturated NaCl solution. The organic phase was dried
over MgSO4, and solvent was removed by evaporation. A yellowish
powder (0.56 g) was obtained.
4N3 8 800 (20 mg, [azide] 9 mmol), and GFLG1 (2.83 mg, [alkyne]
9 mmol) were dissolved in deoxygenated DMF (60 mL) in a 1 mL
Eppendorf tube. L-Ascorbic acid solution (20 mL containing 0.35 mg
L-ascorbic acid, 0.25ꢁ relative to alkyne and azide groups) was
addedtothistube,andthesystemwasbubbledwithnitrogen.CuBr
solution (100 mL containing 0.56 mg CuBr, 0.5ꢁ relative to alkyne
and azide groups) was added just prior to transfer of the mixture to
themold,andthereactionwascarriedoutatroomtemperature.Gel
formation was examined by vial tilting method.
Rheology
Gelation process was dynamically monitored by time sweep
rheology using a TA-Instruments 550 Advanced Rheometer (New
Castle, DE). Immediately after CuBr addition, sample was loaded on
a Peltier plate underneath 20 mm diameter geometry with gap
distance 103 mm, at 25 8C. Time sweeps were performed at fixed 1%
strain amplitude and 6 rad ꢂ sꢀ1 frequency until the curve achieved
plateau, with datacollection every10–30 s. Following timesweeps,
frequency sweep rheology, at fixed 1% strain amplitude over a
Part of this intermediate (0.3 g, [OSO2CH3] 0.132 mmol) was
dissolved in 5 mL DMF, and then NaN3 (0.43 g, 50ꢁ) was added. The
reaction was undertaken at 80 8C for 20 h. DMF was removed by
evaporation under reduced pressure. The resulting intermediate
was redissolved in 30 mL DCM. Undissolved solid was removed by
filtration. The filtrate was washed with saturated NaCl, dried over
MgSO4, and then condensed and precipitated into cold diethyl
Table 1. Enzymatically degradable peptides with dialkyne modification.
Peptide
Molecular structure
MR
GFLG1
708.38
GFLG2
1 082.58
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447

J. Yang, M. T. Jacobsen, H. Pan, J. Kopecˇek
until constant weight. Hydrogel equili-
brium (gravimetric) swelling ratio was
calculated by (Ww – Wd)/Wd, where Ww
is the weight of fully swollen species
and Wd is the weight of dry species.
Kinetics of hydrogel swelling (volu-
metric) was performed in water and
monitored under optical microscope
(Nikon E800) using 2ꢁ objective. Gel
radii were measured using ImagePro
Plus software (Media Cybernetics,
Bethesda, MD, USA). Assuming isotro-
pic swelling, volume swelling ratio, Q,
was calculatedby (rt/r0)3, wherert is the
radius of swelling gel at different time
points, and r0 is the radius of the dried
gel.
Scanning Electron Microscope
(SEM)
Scheme 2. (A) Synthesis of 4-arm azido-terminated PEG. (B) Synthesis of degradable hydrogels
Water-swollen hydrogel samples, pre-
pared as described above, were shock-
frozen by immersion into liquid nitro-
by click reaction of 4-arm PEG-N3 with dialkyne modified peptide.
frequency range from 0.1 to 100 rad ꢂ sꢀ1, was performed to
determine hydrogel rheological properties: storage, G0, and loss, G00,
moduli.
gen for 10 min, then lyophilized overnight. Freeze-dried samples
weremountedonSEMaluminumstubsandsputteredwithgoldfor
40 s to obtain a conductive surface. Observation of the hydrogel
morphology was carried out on a SEM (Hitachi S-2460N SEM) at an
accelerating voltage of 10 kV and appropriate magnifications.
Microrheology
Particle-tracking microrheological measurements were performed
as previously described by us[18–20] and by others.[21,22] Surfactant
free fluorescent yellow–green amidine modified PS beads (Inter-
facial Dynamics Corp., Portland, OR) (1 mm in diameter) were
dispersed into the click reaction solution at 0.5% w/v final
concentration. After gel formation (minimally 24 h after mixing
of reagents), gel slices were mounted on slides underneath No. 1.5
coverslips and then visualized on a Nikon Eclipse E800 microscope
equipped with a 100ꢁ oil-immersion objective. The samples were
focused in a manner that avoided wall effects. The Brownian
motion of the PS beads for each sample was analyzed by IDL
software (Research Systems Inc., Boulder, CO) based on 3 000
consecutive images captured by a CCD camera (Dage-MTI, DC330)
and recorded at intervals of 33 ms using StreamPix software
(Norpix, Montreal, Canada). The mean square displacement (MSD)
of tracer particles was determined using algorithms previously
developed.[23]
Enzyme-Catalyzed Degradation of Hybrid Hydrogels
Hydrogel degradation was performed in McIlvaine’s buffer
(50mmol ꢂ Lꢀ1 citrate/0.1M phosphate) at pH 6.0, 37 8C, using papain
as model enzyme. Papain, at concentration of 0.05 mg ꢂ mLꢀ1
(determined by UV at 280 nm, with absorbance of 25 at 1%[24]), was
reduced with 5 ꢁ 10ꢀ3 M glutathione for 5 min. The enzyme activity
was confirmed usingBz-PheValArg-NAp as chromogenic substrate.
A seriesof dried hydrogels(4–10 mg)were pre-swollen in theabove
buffer before addition of enzyme solution (3 mg ꢂ hydrogel ꢂ mLꢀ1
enzyme solution). At predetermined time points after addition of
enzyme, hydrogelsamples were removedfrom the medium and re-
immersed in a large volume of water to remove salts before wet
mass weighing and drying. The medium was treated with enzyme
inhibitor solution (3 ꢁ 10ꢀ3
M iodoacetic acid sodium salt in
McIlvaine’s buffer, 10 mL) to stop the hydrogel degradation. Then,
the medium was analyzed with size-exclusion chromatography on
an AKTA FPLC system (Pharmacia) equipped with miniDAWN
TREOS and OptilabEX detectors (Wyatt Technology, Santa Babara,
CA, USA) using a Superose12 HR/10/30 column with PBS (pH 7) as
mobile phase. The average molecular weight of the degraded
products was determined by light scattering, UV, RI and calculated
by ASTRA software. The peptide content in remaining hydrogels
wasdeterminedbyaminoacidanalysis(seebelow).Theweightloss
(WL) was calculated as
Hydrogel Swelling
Hydrogelsretrievedfromthereactionmoldswerefirstimmersedin
DMF.Toremoveorganicsolventandcoppersalts, hydrogelsamples
were incubated in a series of DMF/H2O solutions containing
10 mmol ꢂ Lꢀ1 EDTA (75:25, 50:50, 25:75, 0:100 v/v; each step lasted
at least 12 h), and finally swollen in pure H2O until no weight changes
were detectable. Hydrogels were then dried at room temperature
WL ¼ Wt=W0 ꢁ 100%
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Synthesis and Characterization of Enzymatically Degradable . . .
where Wt is the dry weight at selected time point and W0 is the dry
a second copy of the degradable sequence (GFLG2). The
identities of the peptides were ascertained by MALDI-TOF
mass spectra, indicating that the alkyne groups were stable
under peptide synthesis/cleavage conditions. The yields of
both peptides were in the range of 70–90%. Thus, the
synthetic approach provided an efficient method for the
insertion of alkyne groups into peptide sequences.
weight of original gel.
Amino Acid Analysis
Hydrogelsamples(2–3 mg)atselectedtimepointswerehydrolyzed
with 0.5 mL of 6 N HCl in sealed ampoules at 160 8C for 3 h. Then,
the samples were dried under vacuum (with the protection of
NaOH pellet). The hydrolysates were dissolved in 1 mL of distilled
water. Pre-column OPA/MPA derivatization followed by RP-HPLC
analysis (Agilent Technologies 1100, Eclipse XDB-C8 column,
4.6 mm ꢁ 150 mm, with Ex 229 nm and Em 450 nm) was used.
The glycine (G) and L-phenylalanine (F) contents in the hydrogels
were determined.
Hydrogel Formation
The degradable PEG hydrogels were formed by click reac-
tion between 4-arm azido-terminated PEG and dialkyne-
modified peptides, GFLG1 or GFLG2. The reaction
conditions, such as the molar ratio of azide/alkyne and
the catalyst system, were selected based on preliminary
experiments (results not shown). The gelation was
performed at equimolar azide/alkyne ratio at room
temperature, using DMF as solvent and 0.5 equivalent of
copper bromide/0.25 equivalent of L-ascorbic acid as the
catalyst system. We observed that the PEG concentration
significantly affects the gelation process. To optimize
gelation conditions, three different PEG concentrations
(from 2.5 to 10 wt.-%) were tested (Table 2). No hydrogel
formation was observed at the lowest polymer concentra-
tion of 2.5%. As expected, higher concentration of polymer
in the reaction mixture shortened the time needed for gel
formation and increased the reaction yield (Table 2).
Consequently, in further experiments, a polymer concen-
tration of 10 wt.-% was used in agreement with similar
reactions described in the literature.[8,17]. Moreover, the
occurrence of gelation was determined more accurately
fromrheologicalcurvesasthecrossoverofstoragemodulus,
G0, and loss modulus, G00.
Results and Discussion
Synthesis of 4-Arm Azido-Terminated Poly(ethylene
glycol)s (PEGs) and Peptides with Dialkyne
Modification
The synthesis of tetraazido-functionalized PEG (PEG-4N3)
followed a published procedure[25] with slight modifica-
tions. As shown in Scheme 2A, the activation of 4-arm
hydroxy-terminatedPEG(Mn ¼ 2 100and8 800)withmesyl
chloride, followed by nucleophilic substitution with
sodium azide produced PEG-4N3. The presence of azido
groups was evidenced by the methylene resonance in
13C NMR spectrum (Figure 1). The presence of the peak at
d 50.9 ppm (CH2N3) and the absence of the peak at
d 61.7 ppm (CH2OH) in the product demonstrated that the
conversion to azido groups was nearly 100%.
Theinclusionofalkynegroupsatbothendsofdegradable
peptides, GFLG1 and GFLG2, was achieved via standard
Fmoc/tBu solid-phase peptide synthesis, and by incorpora-
tion of lysine (K) residue that created a branched structure
for the introduction of either an alkyne group (for GFLG1) or
The effect of peptide structure on the gelation process
was also examined. There were no significant differences in
gelationtimeforpeptidesGFLG1andGFLG2, buttheyieldof
hydrogel was slightly lower when the longer sequence
(GFLG2) was used (Table 2, gels 4
and 6). As described by Liu,[17] the
lengths of the peptide crosslinkers
may influence the gelation pro-
cess. Alkyne groups in GFLG1 and
GFLG2possessthesamereactivity;
thus, there were no differences in
initial gelation time. However,
generally, peptides have a high
persistence length;[26] once the
viscosity of the reaction mixture
increases, the longer GFLG2 has a
lower mobility thus leading to
incomplete reaction compared to
the shorter GFLG1.
Poly(ethylene glycol) (PEG)
Figure 1. 13C NMR spectrum (CDCl3) of PEG-4N3 8 800.
length has an opposite effect
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449

J. Yang, M. T. Jacobsen, H. Pan, J. Kopecˇek
Table 2. Synthesis of PEG-based hybrid hydrogels by click reactions.a)
No.
Dialkyne peptide
PEG Conc, w/v
%
Gelation time
Yieldb)
%
PEG-4N3 Mn
g ꢂ molꢀ1
1
2
3
4
5
6
8 800
8 800
8 800
8 800
2 100
2 100
GFLG1
GFLG1
GFLG1
GFLG2
GFLG1
GFLG2
2.5
5
no gel
h
–
66.9
94.6
58.1
56.8
45.1
10
10
10
10
12 minc)
9 minc)
9 minc)
ꢃl0 minc)
a)Experimental conditions: [azide]0/[alkyne]0/[CuBr]0/[L-ascorbic acid]0 ¼ 1:1:0.5:0.25; rt in DMF; b)Yield ¼ (weight of dried gel)/(initial
weight of PEG-4N3 þ initial weight of dialkyne peptide); c)Gelation time was obtained from dynamic time-sweep rheology experiments.
compared to peptide length. The yield of the hydrogel for
thePEG2100waslower thanthat forPEG8800(Table2). One
could hypothesize that the intramolecular reaction of
peptide alkynes with the same PEG molecule is more
probable with PEG2100 due to the closer vicinity of the
polymer arm termini.
mainly degree of gelation—in numerous soft materials,
ranging from primarily viscous to primarily elastic[18–20]
Briefly, particle-tracking microrheology is based on the
Brownian motion of beads embedded within materials;
these motions can be tracked to calculate MSDs, which
relate to the material’s viscoelastic properties. Some
advantages of microrheology techniques include wide
frequency range, local environmental probing, very small
volumes, and cost efficiency,[28] due to few requirements of
fluorescent microscope and specific software (see Experi-
mental Part). On a log–log MSD plot (MSD vs. lag time), a
primarily viscous liquid yields a slope of 1, viscoelastic
between 0 and 1, and elastic of 0.[18,29] We obtained MSD
plots (Figure 2G) with slopes near 0, which indicated elastic
hydrogels and supported our bulk rheology findings.
Figure 2H qualitatively demonstrates hydrogel forma-
tion. Top image shows a gel that was formed during the
time-sweeprheology;here, thehydrogelwasremovedfrom
the plate immediately after rheology. Bottom image
shows a hydrogel formed using a plastic syringe mold.
This particular gel was used for enzymatic degradation
studies (see discussion in later section).
Physical Characterization of Hydrogels
Hydrogel formation was monitored by bulk dynamic
rheology experiments. Under constant strain and fre-
quency, G0 and G00 were measured immediately after
addition of catalyst CuBr (Figure 2A–C). Onset of gelation
was observed at ꢃ10 min (insets in Figure 2A–C) and a
plateau was reached in a range of 70–150 min.
Following time-sweep experiments of hydrogel forma-
tion, frequency-sweeps were performed to more thor-
oughly characterize the rheological properties of the
hydrogels (Figure 2D–F). Hydrogels showed highly elastic
orgel-likecharacter, withG0 values2–3ordersofmagnitude
greaterthanG00. Inourstudy, G0 valueswerearound6 000 Pa
when using PEG8800 and a 1:1 azide/alkyne ratio at
10% w/v, which mirrored values in click reaction work
from Anseth’s group, where they observed mean final
elastic (storage) modulus of 5 160 Pa when using PEG10000
and a 1:1 azide/acetylene ratio at 13.5% w/v.[27]
A significant increase in G0 was observed when PEG2100
(Figure 2C and F) was used compared to PEG8800(Figure 2A,
B, D, and E). This finding suggests that the PEG chain length
strongly affects the mechanical properties of hydrogels,
whereas the peptide composition (GFLG1 or GFLG2) had a
minoreffect(GFLG2 didreachplateaufasterthantheGFLG1
peptide: 70 min with GFLG2, compared to 120 or 150 min
with GFLG1).
The swelling degree of hydrogels was measured both
gravimetrically and volumetrically, and consistent data
were obtained. Figure 3A shows the equilibrium swelling
ratios that were calculated by comparing the weights of the
dry and water-swollen hydrogels; time-course studies of
volumetric swelling ratios were performed to assess the
kinetics of hydrogel swelling (Figure 3B). In both experi-
ments, PEG8800GFLG1 showed significantly higher degree
of swelling than PEG2100GFLG1. Equilibrium swollen state
wasreachedinmorethan4 hwithPEG8800GFLG1, whereas
this state was reached in about 1 h with PEG2100GFLG1.
Both hydrogels, PEG8800GFLG1 and PEG2100GFLG1, were
prepared under same weight concentration (10 wt.-%). In
the same volume, the number of functional groups in
PEG2100GFLG1 is 4.5 times that of PEG8800GFLG1, which
In addition to classical bulk rheology, we also performed
particle-tracking microrheology. We have used this tech-
nique successfully to analyze rheological properties—
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Synthesis and Characterization of Enzymatically Degradable . . .
Figure 2. Hydrogel formation. (A–C) Time-sweep rheology of gel formation, as monitored by increase in G0, storage modulus. Gel points were
identified when G0 was equal to G00, the loss modulus, as shown in the insets. A, PEG8800GFLG1; B, PEG8800GFLG2; C, PEG2100GFLG1. (D–F)
Frequency sweep rheology of hydrogels. D, PEG8800GFLG1; E, PEG8800GFLG2; F, PEG2100GFLG1. (A–F) Rheology studies were performed ꢄ 3
times; open diamond represents G0, closed diamond G00. (G) Microrheology of hydrogels, monitored by MSD versus lag time. (H) Top,
hydrogel obtained following rheological characterization; bottom, hydrogel molded in plastic syringe, and used for enzymatic degradation
studies.
Figure 3. Hydrogel swelling. (A) Equilibrium degree of swelling (in water), which was measured by (Ww – Wd)/Wd, where Ww is the weight of
fully swollen species and Wd is the weight of dry species. Standard deviation bars are indicated (n ¼ 5). (B) Kinetics of swelling (in water), as
measured by Q, which was calculated by (rt/r0)3, where rt is the radius of gel at time point and r0 is the radius of the dried gel.
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J. Yang, M. T. Jacobsen, H. Pan, J. Kopecˇek
factors important for the formation of enzyme–(polymer)
substrate complexes are the steric hindrance of the
polymeric chain and the length of the oligopeptide
sequence.[10] In a three-dimensional hydrogel structure,
there is transport (diffusion) limitation for the enzyme to
reach the peptide substrate. However, peptides bound to
PEG chain termini accommodate into the active site of
chymotrypsin more easily than peptides bound to syn-
thetic polymer side chains.[32] Consequently, the PEG chain
may accommodate relatively easily into the distant
subsites of papain’s active site.
The WL of hydrogels during degradation is shown in
Figure 5. PEG8800 hydrogels degraded considerably faster
than those based on PEG2100, indicating that PEG chain
length highly affected the rate of degradation. This is
related to the differences in hydrogel properties: degree of
swelling and elastic modulus. PEG8800 hydrogels showed
higher degree of swelling and lower elastic moduli
compared to PEG2100 hydrogels. This finding is in good
agreement with published data on the chymotrypsin-
catalyzed surface degradation of HPMA-based hydrogels
containing oligopeptide crosslinks.[11] However, the struc-
ture of the crosslinks, GFLG1 versus GFLG2, did not have an
impactontherateofdegradation. Apparently, theenzyme–
substrate interactions are controlled by diffusion, and the
presence of two enzyme attachment modes for GFLG2
whencompared toGFLG1did not result indifferences inthe
rate of degradation.
Figure 4. Scanning electron microscope (SEM) imaging of hydro-
gels. Freeze-dried hydrogels were imaged at 200ꢁ and 800ꢁ
magnification.
leads to a higher crosslinking density, consequently
resulting in higher G0. This hypothesis was supported by
SEM images (Figure 4) in which condensed pore sizes were
observed in PEG2100GFLG1 compared to PEG8800GFLG1.
Similar observations of the relationship between polymer
length and swelling degree were also reported by Lutolf and
Hubbell.[26]
The difference in the polymer–water interaction para-
meter, x, between PEG8800 and PEG2100 based hydrogels
also contributes to the lower swelling degree of hydrogel
PEG2100GFLG1. As PEG is the only water-soluble compo-
nent, the higher the PEG content, the higher the swelling
degree. The weight ratio of PEG:peptide changes from 7 in
PEG8800GFLG1 to 1.5 in PEG2100GFLG1, leading to an
increase in x with concomitant decrease in swelling ability.
From these studies, we confirmed that the structure of
the polymer component (PEG 2 100 or 8 800) played an
importantroleindeterminingthephysicalpropertiesofthe
hydrogel.
The analysis of cleavage products may contribute to our
understanding of hydrogel degradation. Size-exclusion
chromatography was used to detect changes in the soluble
fraction of papain-catalyzed PEG8800GFLG1 hydrogel
degradation, at different time points (Figure 6). With the
exception of the initial time points, where higher molecular
weight fractions were detected (at elution volume ꢃ11 mL,
indicated by arrows), the molecular weight of the soluble
Hydrogel Degradation
Hydrogels crosslinked by GFLG1 and GFLG2 were incubated
with papain for enzymatic degradation. The time-course of
hydrogel degradation was characterized by three para-
meters: (i) hydrogel WL, (ii) size-exclusion chromatography
of the soluble fraction, and (iii) amino acid analysis of the
remaining hydrogel samples.
Papain (EC 3.4.22.2) is a thiol protease of wide specificity.
˚
It has a large active site which extends over about 25 A and
can be divided into seven subsites, each accommodating
one amino acid residue.[30,31] The subsite S2 specifically
interactswithphenylalanine(F)(nomenclaturefrom[30]);in
substrates containing ꢀFꢀXꢀYꢀ, the peptide bond
between amino acid residues X and Y is cleaved. Other
Figure 5. Hydrogel WL. During degradation catalyzed by
0.05 mg ꢂ mLꢀ1 papain in citrate-phosphate buffer (pH 6.0) at
37 8C, the hydrogel WL was determined from WL ¼
Wt/W0 ꢁ 100% where Wt is dry weight at selected time point
and W0 is dry weight of original gel.
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Synthesis and Characterization of Enzymatically Degradable . . .
ratio in the hydrogel fraction at 0, 3, and 16 h of degradation
was 2.1, 1.8, and 1.3, respectively (Figure 7). This indicates
that the peptide cleavage occurs at more than one site.
Conclusion
PEG-based peptide-containing hydrogels were synthesized
by click chemistry and shown to have elastic rheological
properties, along with relatively high degrees of network
swelling. Hydrogels based on PEG8800 showed lower
elastic moduli, higher degree of swelling, and larger pore
sizes compared to PEG2100, which is likely a result of the
higher hydrophobicity of the PEG2100 hydrogel, and due to
the higher peptide:PEG ratio and higher crosslinking
density. Using a papain enzyme degradation assay, we
observed significant differences in hydrogel degradability
between PEG 2 100 and 8 800-based hydrogels. Time-course
analysis of degradation showed that the molecular weight
of the soluble fraction quickly reached that of the PEG
precursor. Finally, we have introduced in this work a ‘‘click
hydrogel’’ system and shown how specific changes in
composition can tune the hydrogel’s mechanical properties
and sensitivity to enzymatic degradation.
Figure 6. Size-exclusion chromatography of soluble fractions.
Soluble fractions were obtained during degradation of hydrogel
PEG8800GFLG1 with 0.05 mg ꢂ mLꢀ1 papain in citrate-phosphate
buffer (pH 6.0) at 37 8C. Inset shows initial molecular weight of
PEG-4N3 8 800 polymer, for comparison.
fraction quickly reached the initial value of the PEG
precursor.
TheimportanceofFinpositionP2 ofthesubstrateandthe
significance of at least four amino acid residues in the
oligopeptide substrate (to achieve high rate of degradation)
have been previously demonstrated to be important in the
papain-catalyzed cleavage of branched HPMA copolymer
with oligopeptide crosslinks.[33]
Acknowledgements: This research was supported in part by NIH
grant EB 005288 and the University of Utah Research Foundation
TCP Program. We thank Dr. Pavla Kopecˇkova´ for valuable
discussion and the Dr. Patrick Kiser laboratory for assistance
and use of Rheometer.
Apparently, the crosslinks in PEG hydrogels might
preferentially align with F in the S2 subsite; consequently,
the first cleavage would occur between L and G. However,
the wide specificity of papain suggests that other sites of
initial cleavage are also possible. The changes in peptide
content in the hydrogel PEG2100GFLG1 during papain
degradation were analyzed by amino acid analysis. The G/F
Received: August 15, 2009; Revised: December 1, 2009; Published
online: February 9, 2010; DOI: 10.1002/mabi.200900295
Keywords: biodegradable; click chemistry; hydrogels; peptide
crosslinks; poly(ethylene glycol)
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