Enzyme-controllable delivery of nitric oxide from a molecular hydrogel

Article abstract of DOI:10.1039/c3cc45666h

A β-galactosidase-responsive molecular hydrogelator of a nitric oxide (NO) donor can release NO in a controllable manner to improve wound healing.

Full text of DOI:10.1039/c3cc45666h

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Enzyme-controllable delivery of nitric oxide from
a molecular hydrogel†
Cite this: Chem. Commun., 2013,
49, 9173
a
a
Jie Gao,z Wenting Zheng,z Jimin Zhang,a Di Guan,a Zhimou Yang,*a
Received 25th July 2013,
Accepted 5th August 2013
Deling Kongab and Qiang Zhao*a
DOI: 10.1039/c3cc45666h
www.rsc.org/chemcomm
A b-galactosidase-responsive molecular hydrogelator of a nitric oxide of many diseases, such as tumor inhibition and wound healing.
(NO) donor can release NO in a controllable manner to improve In this study, we combine a b-galactosidase-responsive caged
wound healing.
NO molecule and a self-assembling short peptide to produce a
molecular hydrogelator. The resulting hydrogel can sustainedly
NO, as a diatomic free radical produced in the human body, plays release NO molecule by the addition of b-galactosidase and
vital roles in cardiovascular, respiratory, nervous, and immune therefore assist the wound healing in mice models.
systems.1 It has been demonstrated to act as a functional platelet
Molecular hydrogels are formed by the self-assembly of low-
aggregation inhibitor, a neurotransmitter, and an antimicrobial molecular weight molecules.7 They have shown great potential
and antitumor agent.2 Though NO has been demonstrated to for the controlled release of bioactive molecules,8 imaging
be an important molecule, it is unstable under physiological important biological molecules,9 and regenerative medicine.10
conditions. What’s more, its concentration should be precisely In this study, we opt to combine a sugar caged NO donor (2 in
regulated for its normal and correct biological functions. Scheme 1) and a short peptide of Nap-FFGGG to generate a
Higher concentrations of NO than its normal biological levels hydrogelator that can form hydrogels for local delivery of NO.
will lead to undesirable outcomes such as toxicity to cells. The synthetic route for the designed gelator is shown in
Therefore, NO donors,3 such as organic compounds, metal Scheme 1. The Cu(I) catalyzed a click reaction between an
complexes, dendrimers, and micelles, have been developed to alkyne containing a short peptide derivative (1 in Scheme 1)
store and deliver NO in a controllable manner (e.g. light and an azide containing a caged NO donor leading to the
irradiation, pH and temperature change, enzymes).4,5 The formation of the possible gelator (3 in Scheme 1).
development of these NO donors helps to understand the
biological function of NO and may lead to novel therapeutic performance liquid chromatography, the gelation ability of 3
agents for the treatment of NO-involved diseases. was tested using the inverted tube method. A transparent
After the synthesis and purification using reverse phase high
Biological systems use enzyme and signal transduction hydrogel (inset image on the left bottom corner of Fig. 1A)
machinery to regulate the level of NO. Inspired by such principles could form within 2 minutes upon cooling the hot phosphate
in nature, caged NO molecules with enzyme-responsive properties buffered saline (PBS) solution (pH = 7.4) containing 0.5 wt% of
have been reported.5,6 The NO released from caged NO molecules 3 to ambient temperature (25 1C). The hydrogel was stable for at
can be controlled by using different concentrations of enzymes. least one month. A rheometer was used to characterize the
However, most of enzyme-responsive caged NO molecules are free mechanical properties of the hydrogel at 37 1C. Both values of
molecules that will diffuse easily to the whole body. Local delivery storage moduli (G0) and loss moduli (G00) were independent of
of enzyme-responsive caged NO molecules will be a more precise
way that may eliminate undesirable side effects in the treatment
a State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences,
Nankai University, Tianjin 300071, P. R. China. E-mail: yangzm@nankai.edu.cn,
qiangzhao@nankai.edu.cn
b Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical
Engineering, Chinese Academy of Medical Science, Tianjin 300192, P. R. China
† Electronic supplementary information (ESI) available: Synthesis and character-
ization, hydrogel preparation, HE staining, details experimental procedure in the
animal study. See DOI: 10.1039/c3cc45666h
Scheme 1 Synthetic route of the hydrogelator containing a caged NO donor.
Chem. Commun., 2013, 49, 9173--9175 9173
‡ The authors pay equal contributions to this work.
c
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system, such a molecular hydrogel possessed the ability of
continually releasing NO at controllable rates. This property
was vital in NO therapies because the effective therapeutic dose
of NO might vary greatly and needed to be precisely controlled.12
The MTT assay of NIH 3T3 mouse fibroblast cells indicated
that more than 90% of 3T3 cells were alive after being cultured
in the medium containing 3 at concentrations lower than 800 mM
for 24 hours (Fig. S-7, ESI†). These observations indicated the
compatibility of 3 with 3T3 cells at these concentrations. One of
the vital biomedical applications of NO is wound healing. NO is
believed to assist wound repair by enhancing re-epithelializiation,
increasing collagen synthesis, and promoting angiogenesis by
inducing vascular endothelial growth factor expression.13 The
wound healing promoting ability of our molecular hydrogels
was investigated in C57 mice and the results including areas of
wounds and density of small new-born blood vessels were
assessed.
Animals were separated into five groups and submitted to
excisional wounds on the dorsal surface on the first day. Since
gels of 3 would change to solutions gradually upon the treatment
with b-galactosidase and hydrogel could prevent contamination
of environmental microbe, we used a mixed component gel for
the evaluation. 20 mL of the mixed component hydrogel (0.6 wt%
of 3 (120 nmol) and 1.0 wt% of Nap-FF (432 nmol)) were applied
Fig. 1 TEM images of hydrogel containing 0.5 wt% of 3 0 h and (B) 24 h after
the addition of b-galactosidase (3000.0 U Lꢀ1) (insets: optical images of the gel
and solution). (C) The release profile of NO from gel containing 0.5 wt% of 3 with
the addition of different concentrations of b-galactosidase. For results using
higher concentrations of enzyme (20, 200, 2000 U Lꢀ1), please see Fig. S-6 (ESI†).
the frequency at the range of 0.1 to 100 rad sꢀ1 (Fig. S-5, ESI†). on the wound bed in the NO Gel + GAL and NO Gel groups.
The G0 value was about an order of magnitude greater than the During the first 4 days, b-galactosidase was added daily in the
G00 value (Fig. S-5, ESI†), indicating the formation of a true NO Gel + GAL group, while the wounds in the NO Gel group
hydrogel.11 We also used transmission electron microscopy were treated with only the mixed component gel without
(TEM) to reveal the micro-structure in the gel. Results in b-galactosidase. The wound in the Nap-FF group was topically
Fig. 1A indicated that the gel was constructed by entangled treated with 20 mL of the Nap-FF hydrogel (1.0 wt%) without 3
long nano-fibers with diameters of 30–80 nm.
for the same period. The other two control groups were treated
We then tested the enzyme-responsive property of the hydrogel with PBS only (PBS group) and caged NO donor (Free NO + GAL
and studied the NO release behavior from the gel. The enzyme group: PBS solution containing 0.2 wt% (120 nmol) of 2, with
b-galactosidase was used to remove the protective galactose daily b-galactosidase treatment as well), respectively. The
from the NO donor and trigger the release of NO from 3 wound area on the first day (Day 0) and seven days after
(Scheme S-3, ESI†). A gel to sol phase transition (inset image treatment (Day 7) was measured. The wound area at Day 0
on left bottom corner of Fig. 1B) was observed 24 hours after was set to be 100% and the wound area reduction at Day 7 was
the addition of b-galactosidase. TEM images showed that the calculated for all groups. As shown in Fig. 2B, the reduction of
original long fibers in the gel (longer than 2.5 mm, in Fig. 1A) wound area was 81.9, 64.6, 62.1, 60.6, and 57.9% for NO Gel +
changed to short fibers in the resulting solution (30 to 600 nm, GAL, NO Gel, Free NO + GAL, Nap-FF, and PBS groups,
in Fig. 1B). These observations clearly indicated the enzyme- respectively. The results clearly indicated that, compared with
responsiveness of the gel.
other groups, the wound areas were dramatically reduced in the
In order to evaluate the controllable NO releasing ability of NO Gel + GAL group (Fig. 2B), which indicated the importance
the hydrogel, 50 mL of PBS solution containing different of NO molecules in the wound healing. The wound area
amounts of b-galactosidase was placed on top of 50 mL of gel reduction was significantly greater in the NO Gel + GAL group
(0.5 wt% of 3). The amount of released NO in the upper solution (81.9%, Fig. 2A) than that in NO Gel group (17.3% greater than
was measured every 1 hour using a classic Griess reaction Nitric NO Gel group (P = 0.012)), indicating that the additional
oxide assay. The results of accumulation of released amounts of b-galactosidase could catalyze the release of NO at higher levels.
NO from the gel were determined (Fig. 1C and Fig. S-6, ESI†). As for the Free NO + GAL group, it exhibited a similar wound
During the 10 hours of experimental time, the proportion of healing effect on the PBS control group probably due to the fast
total released NO was tunable, ranging from 1.1% to 17.8% by clearance of 2 by rapid diffusion to the whole body of mice. These
varying the amount of enzyme added to the gel. The releasing results also indicated the importance of hydrogel formulation for
rate remained constant without the burst release phenomenon topical treatments.
during the whole experimental procedure. Meanwhile, no NO
We then investigated the effect of hydrogels on angiogenesis
molecule could be detected when PBS without enzyme was in wounded skin by measuring the expression of vWF in
added on top of the gel. These results clearly demonstrated the wound area (Fig. S-8 and S-10, ESI†) and the border area
that, by adjusting the concentration of b-galactosidase in the (Fig. S-9, ESI†). As shown in Fig. S-8 (ESI†), there were about
c
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of wound because it could promote angiogenesis in the wound
bed, thus accelerating the wound healing process. The production
of hydrogel formulation could minimize fast diffusion and
clearance of bioactive molecules. We therefore believed that
our hydrogel system had great potential for local controllable
delivery of NO for regenerative medicine and tissue engineering.
This work is supported by National Basic Research Program
of China (2011CB964903) and NSFC (31070856, 81220108015,
81171478, and 81000680).
Fig. 2 (A) Percentage of wound area left in different groups at Day 7 compared
to the original wound area (mean ꢂ SEM) at Day 0. (B) Photographs of wounds in
animals treated with PBS, NapFF (hydrogel containing 1.0 wt% NapFF), Free NO +
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Chem. Commun., 2013, 49, 9173--9175 9175