Orthogonal enzymatic reactions to control supramolecular hydrogelations

Article abstract of DOI:10.1002/cjoc.201100082

Enzyme-responsive hydrogels have great potential in applications of controlled drug release, tissue engineering, etc. In this study, we reported on a supramolecular hydrogel that showed responses to two enzymes, phosphatase which was used to form the hydrogels and esterase which could trigger gel-sol phase transitions. The gelation process and visco-elasticity property of the resulting gel, morphology of the nanostructures in hydrogel, and peptide conformation in the self-assembled nanostructure were characterized by rheology, transmission electron microscope (TEM), and circular dichroism (CD), respectively. Potential application of the enzyme-responsive hydrogel in drug release was also demonstrated in this study. Though only one potential application of drug release was proved in this study, the responsive hydrogel system in this study might have potentials for the applications in fields of cell culture, controlled-drug release, etc. Copyright

Full text of DOI:10.1002/cjoc.201100082

FULL PAPER
DOI: 10.1002/cjoc.201100082
Orthogonal Enzymatic Reactions to Control
Supramolecular Hydrogelations
a
b
b
Chen, Guoqin (陈国钦)
Ren, Chunhua (任春华)
Wang, Ling (王玲)
,c,d
,e,f
Xu, Bing* (徐兵)
Yang, Zhimou* (杨志谋)
a
Department of Cardiology, Guangzhou Panyu Central Hospital, Guangzhou, Guangdong 511400, China
b
College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin,
00071, China
3
c
Department of Chemistry, The Hong Kong University of Science and Technology, Hongkong, China
d
Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02468, USA
e
Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tian-
jin, 300071, China
f
State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China
Enzyme-responsive hydrogels have great potential in applications of controlled drug release, tissue engineering,
etc. In this study, we reported on a supramolecular hydrogel that showed responses to two enzymes, phosphatase
which was used to form the hydrogels and esterase which could trigger gel-sol phase transitions. The gelation proc-
ess and visco-elasticity property of the resulting gel, morphology of the nanostructures in hydrogel, and peptide
conformation in the self-assembled nanostructure were characterized by rheology, transmission electron microscope
(
TEM), and circular dichroism (CD), respectively. Potential application of the enzyme-responsive hydrogel in drug
release was also demonstrated in this study. Though only one potential application of drug release was proved in
this study, the responsive hydrogel system in this study might have potentials for the applications in fields of cell
culture, controlled-drug release, etc.
Keywords self-assembly, hydrogel, enzyme, drug release
Introduction
drogelators is advantageous for biomedical applications
due to the highly efficient catalytic ability of enzymes in
As one of recently most investigated areas of mate-
rials research, supramolecular hydrogels has been de-
veloped into promising biomaterials for drug deliv-
mild conditions and the biocompatibility associated with
[17,20,39,40]
the enzymes.
Based on the successful demon-
strations of enzymatic formation and stage regulation of
[
1-6]
[7-12]
ery,
tissue engineering,
etc. due to their easily
[17,2,41,42]
supramolcular hydrogels,
we report here the
obtained biocompatibility and biodegradability that re-
sulted from non-covalent aggregation and self-assembly
of the hydrogelators.
regulation of supramolecular hydrogelations by a pair of
enzymes and its application in controlled drug release.
[13-16]
Recently, the development of
self-assembled oligopeptides as 3D scaffolds for tissue
engineering has stimulated the efforts to expand the ap-
plications of supramolecular hydrogels including the
development of novel biocompatible methods to form
Experimental
Materials
[
17-25]
Succinated 4-(hydroxymethyl)acethyl phenol NHS
activated ester was obtained from PracticaChem, LLC
the hydrogels.
Since the balance between hydro-
phobicity and hydrophilicity determines the ability of a
small molecule to form a hydrogel, the careful adjust-
ment of the balance would allow the control of
self-assembly of small molecules and the formations of
(
(
Tianjin). Alkali phosphatase was obtained from Takara
Dalian, D2250) Bio. Inc. Esterase was obtained from
Sigma-Aldrich. L-Tyrosine-COOBu-t, Fmoc-OSu, and
Fmoc-amino acids were obtained from GL Biochem
[26-29]
supramolecular hydrogels.
Although chemical or
(
Shanghai). All the other starting materials were ob-
physical perturbations (e.g., adjusting pH, temperature,
tained from Alfa. Commercially available reagents were
used without further purification, unless noted otherwise.
The solvents were dried according to regular protocols.
ionic strength, and ultrasonic agitation) are the usually
[2,30-38]
used methods to achieve that balance,
en-
zyme-responsive self-assembly and gelation of the hy-
*
E-mail: bxu@brandeis.edu.cn, yangzm@nankai.edu.cn; Tel.: 0086-022-23502875; Fax: 0086-022-23498775
Received June 30, 2011; accepted August 29, 2011; published online December 23, 2011.
Chin. J. Chem. 2012, 30, 53—58
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
53

Chen et al.
FULL PAPER
All other chemicals were reagent grade or better.
amino acid was loaded on the resin at the C-terminal
－
1
with the loading efficiency of about 0.6 mmol•g . 20%
piperidine in anhydrous N,N'-dimethylformamide (DMF)
was used during deprotection of Fmoc group. Then the
next Fmoc-protected amino acid was coupled to the free
amino group using O-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (HBTU) as the
coupling reagent. The growth of the peptide chain fol-
lowed the established Fmoc SPPS protocol. After the
last coupling step, excessive reagents were removed by
General procedure
1
H NMR spectra of the synthesized compounds were
characterized on a Bruker ARX 300 NMR spectrometer
using CDCl or DMSO-d as the solvent, and ESI-MS
3
6
spectrometric analysis were performed on the Thermo
Finnigan LCQ AD System. HPLC analyses were per-
formed on Waters 600E Multi-solvent Delivery System
using a C18 RP column with MeOH (0.1% of TFA) and
water (0.1% of TFA) as the eluents. Atomic force mi-
croscopy (AFM) was done on a Veeco multimode V
system with the tip of Veeco RTESP. Rheology test was
done on an AR 2000ex (TA instrument) system, 40 mm
parallel plates were used during the experiment at the
gap of 500 μm.
－
1
a single DMF wash for 5 min (5 mL•g of resin), fol-
lowed by 5 steps of washing using dichloromethane
－
1
(
DCM) for 2 min (5 mL•g of resin). The peptide de-
rivatives were cleaved from the resin by 1% of TFA in
－
1
DCM (5 mL•g of resin for t10 times). The mixtures
were combined and the solvents were removed under
vacuum. The crude product was purified using prepara-
tion reverse-phase HPLC to obtain compound 9. And
then compound 9 in DCM was treated with 25 equiv. of
TMSBr overnight to afford compound 10.
Syntheses and characterizations
Compound 1 was synthesized according to Scheme 1
and following the method reported in our previous pa-
[
40]
Preparation of title product 1 To a 5 mL of an-
hydrous DMF solution containing 84 mg of FFY(P) (10,
per. Preparation of compound 8 followed the method
in reference [40].
1
42.1 μmol) and 117 μL of diisopropylethylamine
Peptide synthesis The peptide of fFfFY(P) (10)
was firstly prepared by solid phase peptide synthesis
(
DIPEA) (710 μmol, 5 equiv.), 77.4 mg of NHS active
(
SPPS) using 2-chlorotrityl chloride resin and the cor-
ester (11, 213 μmol, 1.5 equiv.) was added. After the
responding N-Fmoc protected amino acids. The first
resulting reaction mixture being stirred at room tem-
Scheme 1
O
O
OH
OH
P
OEt
OEt
O
O
OH
O
ii
iii
i
O
N
H
O
N
O
H
H N
O
O
2
O
6
7
8
O
O
OEt
OEt
OH
OH
F
O
P
F
O
P
O
O
H
H
iv: spps
v
N
OH
N
OH
H N
N
H2N
N
2
H
H
O
O
O
O
9
F
10
F
O
O
O
OH
O
F
O
P
O
O
N
OH
O
O
11
O
O
O
O
H
O
O
O
N
OH
vi
N
H
N
H
O
O
1
F
3 2 2
Reagents and conditions: (i) Fmoc-OSu, NaHCO , acetone/H O; (ii) I , triethyl phosphite, pyridine, DCM; (iii) TFA, DCM; (iv) solid phase
peptide synthesis; (v) TMSBr, DCM; (vi) DIEA, DMF
54
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 53—58

Orthogonal Enzymatic Reactions to Control Supramolecular Hydrogelations
perature overnight, it was used for HPLC separation
7.4]. Compound 1 served as a substrate of both phos-
directly to obtain the title compound 1 in a yield of
phatase and esterase. And then its self-assembly behav-
ior was evaluated. We firstly added the phosphatase to a
PBS solution containing 0.2% of 1 (the optical image in
Figure 1A and the HPLC trace in Figure 1E). Similar to
1
7
0.2%. H NMR (DMSO-d
6
, 400 MHz) δ: 7.35—7.38
(
m, 2H), 7.23—7.28 (m, 2H), 7.18—7.21 (m, 3H),
6
4
.98—7.12 (m, 8H), 5.03 (s, 2H), 4.50—4.54 (m, 1H),
.43—4.45 (m, 2H), 2.97—3.04 (m, 2H), 2.88—2.92
[19,39,40]
our previous reports,
the cleavage of phosphate
group from 1 increased the hydrophobicity and the abil-
ity of self-assembly thus inducing the conversion of 1 to
2 and the formation of a supramolecular hydrogel (final
phosphatase concentration＝60 U/mL, gel I in Figure
(
2
m, 2H), 2.73—2.78 (m, 1H), 2.60—2.68 (m, 1H),
.41—2.45 (m, 2H), 2.31—2.34 (m, 2H), 2.26 (s, 3H);
+
MS m/z: 840.2 [(M+H) ]; HR-MS calcd for
C H F N O P [M ] 839.23, found 840.2375.
+
4
0
40
2
3
13
1
B). The HPLC result indicated that there was about
Hydrogel formation
97.1% of 1 being converted to 2. To test whether the
ester bond of 2 could be hydrolyzed by the enzyme of
esterase in the self-assembled stage, esterase was added
on the top of the gel. A gel-sol phase transition was ob-
served 2 h after the addition of esterase (40 U/mL, Fig-
ure 1C), which supported its potential applications for
controlled drug release, cell culture, etc. The HPLC re-
sult indicated that 93.5% of 2 had been converted to a
more hydrophilic 3, water soluble 5, and acetic acid at 6
h. Solution of 1 could not be changed to hydrogels by
the treatment of esterase (Figure 1D), suggesting that 2
was the only hydrogelator among compounds 1—4.
5
mg of compound 1 was dissolved in 0.50 mL of
PBS (pH＝8.0). 0.4 μL of alkaline phosphatase in 3.6
μL of PBS buffer was then added to above solution. Gel
I formed after the solution was kept at room temperature
(
20—25 ℃) for 2 min.
Results and Discussion
We firstly designed and synthesized the Ac-Ben-
Succ-fFfFY(P)-OH (1, Scheme 2) with a good solubility
[
solubility＞2.0% (w) in PBS buffer solution at pH＝
Scheme 2 Illustration for enzymatic conversions between hydrogelators and non-hydrogelators by phosphatase and esterase (only 2 can
form hydrogels, 1, 3, 4 and 5 are highly soluble in aqueous solutions)
F
F
O
O
O
Phosphatase
H
H
O
O
O
N
N
H
H
O
O
N
OH
N
N
H
O
O
N
OH
O
O
F
H
O
O
P
O
O
F
OH
OH
O
O
OH
1
2
Esterase
Esterase
F
F
O
O
O
Phosphatase
O
O
O
O
H
H
O
H
H
N
N
N
N
+
+
HO
N
OH
HO
N
OH
OH
H
H
O
O
F
O
O
5
O
OH
O
P
F
OH
OH
3
4
Chin. J. Chem. 2012, 30, 53—58
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
55

Chen et al.
FULL PAPER
Figure 1 Optical images of (A) a PBS sol. I containing 0.2% of 1, (B) gel I formed by treating solution in A with 60 U/mL of phos-
phatase, (C) sol. II formed by treating gel I with 40 U/mL of esterase, and (D) sol. III formed by treating sol. I with 40 U/mL of esterase,
and HPLC results of (E) sol. I, (F) gel I, (G) sol. II, and (H) sol. III.
To explore the kinetics of enzymatic hydrogelation, a
dynamic time sweep was applied to solution of 1 (0.2%)
with phosphatase (60 U/mL). As shown in Figure 2, the
hydrogelation happened right after the addition of the
enzyme, indicated by the results that the value of G'
Pascals and from about 0.3 to 8.5 Pascals, respectively,
suggesting that the enzyme kept converting 1 to 2 after
the gelation. The conversion was nearly finished 1 h
after the addition of phosphatase, indicated by both G'
and G'' reaching plateaus. The value of G' was more
than an order of magnitude greater than that of G'', in-
(
storage moduli and elasticity) was larger than that of
[
1,43]
G'' (loss moduli and viscocity). Both the values of G'
and G'' kept increasing from lower than 2 to about 100
dicating the formation of a true hydrogel.
Atomic force microscopy (AFM) was used to char-
acterize the morphology of microstructures in the gel.
As shown in Figure 3A, nanofibers with uniform size
(
25 nm) were observed and some of them were helical.
The fibers dispersed quite well and there were no in-
tense aggregations or large bundles of nanofiber being
observed. Circular dichroism (CD) was also used to
study the superstructures of self-assembled peptide de-
rivatives in the gel phase. As shown in Figure 3C, com-
pound 2 exhibited similar signals to collagen peptide,
which possessed a pronounced trough at about 202 nm
(ππ* transition) and the positive peak at about 224 nm.
This result suggested that compound 2 formed a triple
[44]
helix structure in gel I. The small positive peak at 190
nm and negative band at 208 nm suggested that small
amount of compound 2 exhibited the α-helical confor-
Figure 2 The dynamic time sweep of the PBS solution con-
taining 0.2% of 1 with 60 U/mL of phosphatase at the strain of
[44]
mation in the gels.
The results observed in the CD
2
% and the frequency of 2 rad/s.
measurement and the AFM image in Figure 3A sug-
56
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 53—58

Orthogonal Enzymatic Reactions to Control Supramolecular Hydrogelations
gested that the conformation of the gelator might be
able to transfer to the macroscopic self-assembled
structures.
by the addition of the esterase. The strategy of using
enzymes to regulate hydrogelations might be developed
into a powerful tool to generate promising biomaterials
for 3D cell culture, controlled drug release, etc.
Acknowledgement
This work was supported by National Natural Sci-
ence Foundation of China (No. 20974054) and Tianjin
MSTC (No. 11JCZDJC17200).
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Chin. J. Chem. 2012, 30, 53—58