D-Glucosamine-based supramolecular hydrogels to improve wound healing

Article abstract of DOI:10.1039/b616563j

A simple supramolecular hydrogel based on d-glucosamine, a naturally occurring aminosaccharide, promises new biomaterials for applications such as wound healing. The Royal Society of Chemistry.

Full text of DOI:10.1039/b616563j

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D-Glucosamine-based supramolecular hydrogels to improve wound
healing
Zhimou Yang,a Gaolin Liang,ab Manlung Ma,a A. Sunny Abbah,c W. William Luc and Bing Xu*ab
Received (in Cambridge, UK) 13th November 2006, Accepted 23rd November 2006
First published as an Advance Article on the web 8th December 2006
DOI: 10.1039/b616563j
with good biocompatibility. More importantly, the resulting
hydrogel assists wound healing and prevents the formation of
scars on a mouse model. The result of this work also supports the
notion that the self-assembly of bioactive molecules8,11 to form
networks of nanofibers in the hydrogel may offer a useful and
effective way to generate biomaterials.
A simple supramolecular hydrogel based on D-glucosamine, a
naturally occurring aminosaccharide, promises new biomater-
ials for applications such as wound healing.
Glucosamine, a naturally occurring compound found in healthy
cartilage, serves as a normal constituent of glycoaminoglycans in
cartilage matrix and synovial fluid in the form of glucosamine
sulfate, which strengthens cartilage and aids the synthesis of
glycosaminoglycan. Glucosamine acts as one of the components in
a widely used pain management for osteoarthritis (OA) patients
and achieves moderate effectiveness.1 Glucosamine also plays a
role in the process of wound healing,2 which has led to the
successful demonstration that the dendrimer of glucosamine
prevents the formation of scar tissue in a clinically relevant rabbit
model.3 Apparently, the dendrimer of glucosamine inhibits Toll-
like receptor 4 (TLR4) to achieve defined immuno-modulatory
and antiangiogenic effects for synergistically preventing the
formation of scar tissue.3 The biological importance of glucosa-
mine, the success of the design and application of polyvalent
glucosamine,3 and the successful generation and applications of
low molecular weight gelators based on carbohydrates4,5 encou-
rage us to incorporate glucosamine into supramolecular hydro-
gelators as the starting point towards self-assembled polyvalency
of D-glucosamine for wound healing and other biomedical
applications.
Despite intense research interest in glucosamine6 and the
increased efforts on supramolecular gelators or self-assembled
nanofibers,4,7–9 the use of glucosamine as a building block to
generate supramolecular hydrogels remains unexplored, except
that Hamilton et al. suggested that the conformational rigidity of
sugars plays an important role in hydrogelation via directing the
intermolecular hydrogen-bond networks.7 Moreover, the poly-
meric hydrogels that incorporate glucosamine or aminosugars
exhibit increased adhesion with neural tissue of the host, improved
vascularization, and enhanced infiltration of non-neuronal cells of
the host.10 This observation suggests that glucosamine may exert
similar beneficial effects on the supramolecular hydrogels and
render them a new type of biomaterials for applications in
biomedicine.
Scheme 1 illustrates the structures of the two hydrogelators,
which consist of D-glucosamine, L- or D-phenylalanine, and a
naphthalene group. L-phenylalanine or D-phenylalanine reacts
with N-hydroxy succinimide (NHS) activated ester of 2-(naphtha-
len-2-yloxy)acetic acid to afford (S)-2-(2-(naphthalen-2-yl)aceta-
mido)-3-phenylpropanoic acid (3) and (R)-2-(2-(naphthalen-2-
yl)acetamido)-3-phenylpropanoic acid (4), respectively. Then,
NHS assisted coupling between 3 and D-glucosamine gives pure
compound of 1 in 66% yield after HPLC, and the coupling
between 4 and D-glucosamine affords pure compound of 2 in 63%
yield after HPLC. Both 1 and 2 are effective hydrogelators.
Typically, after 2 mg of 1 are suspended in 1.0 mL of water, the
increase of the temperature to y80 uC gives a clear solution.
Cooling the solution to room temperature leads to a slightly
opaque hydrogel (Gel I, Fig. 1A). A similar procedure affords the
hydrogel of 2 (Gel II, Fig. 1B). The pH values of Gels I and II are
around 7, and the hydrogels are stable at room temperature for
several months. We also synthesized Nap-D-glucosamine and
Nap-L-Phe-L-Phe-D-glucosamine, which fail to form supramole-
cular hydrogels. This result indicates that the balance between
hydrophobicity and hydrophilicity is important for a molecular
hydrogelator.
Fig. 1C and 1D show the rheological data of Gels I and II.
Using the mode of dynamic strain sweep at the frequency of
10 rad s21, we determined the optimal conditions for the
measurements of dynamic frequency sweep. As shown in Fig. 1C,
the values of G9 and G0 remain constant from 0.1 to about 0.7%
strain for Gel I and from 0.1 to 1.0% strain for Gel II. Both
samples’ values of G9 are larger than values of G0, indicating that
both samples are viscoelastic. Although the value of G9 of Gel I is
larger than that of Gel II, the range of plateau of Gel II is wider
than that of Gel I, suggesting that Gel I is slightly more
viscoelastic, but Gel II is more tolerant of external force. Based on
We found that the attachment of appropriate hydrophobic
groups to the glucosamine affords supramolecular hydrogelators
aDepartment of Chemistry, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China.
E-mail: chbingxu@ust.hk; Fax: +852 2358 1594; Tel: +852 2358 7351
bCenter of Cancer Research, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China
cDepartment of Orthopedics and Traumatology, The University of Hong
Kong, Hong Kong, China
Scheme 1 The chemical structures of the hydrogelators, Nap-L-Phe-D-
glucosamine (1) and Nap-D-Phe-D-glucosamine (2).
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L-phenylalanine has a stronger tendency to aggregate to form
larger bundles and a more crosslinked network.
The circular dichroism (CD) and fluorescence spectra of the
hydrogels also help further understanding of the molecular
arrangements in Gels I and II. As shown in Fig. 1G, the peak at
191 nm and the trough at 205 nm in Gel I result from exciton
splitting of the peptide p–p* transition, while the peak at 222 nm is
due to the peptide n–p* transition. In Gel II, the peptide p–p* and
n–p* transition bands appear at 195 nm and 212 nm, respectively.
The peaks at around 218 nm indicate unordered conformations of
peptide bonds of both compounds (1 and 2) in their gel phases.
These CD signals (below 240 nm) share common features with the
CD of the b-sheet of a polypeptide,12 suggesting that the self-
assembly of the hydrogelators leads to a b-sheet like super-
structure. We also observed a broad positive peak centered at
about 272 nm (np* of aromatic parts) and a broad negative peak
centered at about 315 nm (pp* of aromatic parts). The CD signals
of Gels I and II exhibit similar shapes, suggesting that they were
mainly induced by the D-glucosamine.
Fig. 1H shows the emission spectra of both 1 and 2 in solution
and gel phases. Both compounds exhibit broad peaks centered at
340 nm in their corresponding solution phase. In their gel phases,
the peaks show slightly red shifts (to 347 nm for Gel I and to
343 nm for Gel II). These small red shifts indicate the lack of
efficient p–p stacking of naphthalene groups of both compounds
in their gel phases. The observation of a slightly bigger red shift
and a higher shoulder peak at 375 nm in Gel I than in Gel II
correlates well with the results obtained from rheological
measurements (higher elasticity or bigger G9 value for Gel I) and
TEM images (more entangled fibers in Gel I).
Fig. 1 Optical images of (A) Gel I and (B) Gel II, (C) strain and (D)
frequency dependence of dynamic storage moduli (G9) and loss moduli
(G0) of the hydrogels, TEM images of (E) Gel I and (F) Gel II, (G) the
circular dichroism (CD) of the hydrogels, and (H) the emission spectra of 1
and 2 in solution and in the hydrogels.
After characterizing the physiochemical properties of the
hydrogels, we evaluated their biocompatibility, one of the major
requirements for the application of the hydrogels. The cytotoxicity
assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT) indicates that 73.8% and 79.0% of HeLa cells survive
in 100 mM of 1 and 2 at 24 h, respectively. Based on this result, we
chose Gel II to test its ability to reduce formation of scar tissue at
the wound site on the mice wound model13 since 2 is more
biocompatible than 1. Subsequently, we tested whether the
hydrogel would improve the wound healing using the following
protocol:{ Six Balb/C mice, aged 6 weeks, were randomly divided
into two groups viz: treatment and control. A cut (7 mm in length,
2 mm in width, and 2 mm in depth) was made on the middorsal
skin of each mouse. After 30 seconds, 1 mL (2 mg of 2 in PBS) of
Gel II was applied on the cut (a liquid bandage was also used to fix
the hydrogel). For the negative control, only a liquid bandage was
applied on the wounds. No clinical, hematological or biochemical
(including blood glucose) toxicity was observed, and there were no
local or systemic bacterial, viral, or fungal infections in either of the
two groups treated over 18 days. On inspection, the mice treated
with Gel II exhibited a much faster wound healing process and
smaller scars than those of the control group at day 6 (Fig. 2 A &
B). Histological examination of the skins at the wound site also
shows that higher density of fibroblasts (scar tissues) is presented in
the skin of the mice in the control group. A large amount of
keratinocytes migrate to the extracellular matrix (ECM) near the
scar tissue (Fig. 2C), which indicates that on day 6, the wound on
the untreated mice is at the re-epithelialization phase, one of the
five typical phases of wound healing.14 In contrast, there is
the above results, we measured the dynamic frequency sweep of
both hydrogels at the strain of 0.4%. The values of their storage
moduli (G9) exceed those of their loss moduli (G0) by a factor of 10
(for Gel I) and 1.5 (for Gel II),10 indicating that these two samples
are viscoelastic and behave like a typical hydrogel. For Gel I, the
value of G9 exhibits weak dependence on frequency (from 0.1 to
100 rad s21) at the stress above 1000 Pa; for Gel II, its value of G9
changes from about 200 Pa at low frequency (0.1 rad s21) to more
than 1000 Pa at high frequency (100 rad s21). This observation
indicates that the matrices of Gel I have a good tolerance to the
change of external force.
To study the microstructure of Gels I and II, we obtained the
transmission electron micrograph (TEM) images of the hydrogels.
As shown in Fig. 1E, the irregular small ribbons form large
bundles and tangle with each other in Gel I. We also observed a
small amount of helical fibers with a width ranging from 27 to
55 nm in Gel I. For Gel II (Fig. 1F), small rigid ribbons with a
width of 35–50 nm form well-distributed matrices. The sizes of the
ribbons in Gel II are more uniform than those in Gel I. The
density of nanostructures in Gel I is higher than that in Gel II,
which accounts for a slightly larger value of G9 of Gel I than that
of Gel II. The different morphologies in the two gels are likely a
result of their different structures because the concentration of 1 or
2 is the same in their corresponding hydrogels and the only
difference is the configuration of phenylalanine (L- for 1 and D-
for 2). According to TEM images, compound 1 with an
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1 D. O. Clegg, D. J. Reda, C. L. Harris, M. A. Klein, J. R. O’Dell,
M. M. Hooper, J. D. Bradley, C. O. Bingham, M. H. Weisman,
C. G. Jackson, N. E. Lane, J. J. Cush, L. W. Moreland,
H. R. Schumacher, C. V. Oddis, F. Wolfe, J. A. Olitor, D. E. Yocum,
T. J. Schnitzer, D. E. Furst, A. D. Sawitzke, H. Shi, K. D. Brandt,
R. W. Moskowitz and H. J. Williams, N. Engl. J. Med., 2006, 354, 795.
2 M. Fukasawa, S. M. Bryant, H. Orita, J. D. Campeau and
G. S. DiZerega, J. Surg. Res., 1989, 46, 166; M. Lehto and
M. Jarvinen, Eur. Surg. Res., 1985, 17, 179; R. J. Schmidt,
O. Spyratou and T. D. Turner, J. Pharm. Pharmacol., 1989, 41, 781.
3 S. Shaunak, S. Thomas, E. Gianasi, A. Godwin, E. Jones, I. Teo,
K. Mireskandari, P. Luthert, R. Duncan, S. Patterson, P. Khaw and
S. Brocchini, Nat. Biotechnol., 2004, 22, 977.
4 S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato and
I. Hamachi, Nat. Mater., 2004, 3, 58.
5 S. Kiyonaka, K. Sugiyasu, S. Shinkai and I. Hamachi, J. Am. Chem.
Soc., 2002, 124, 10954; I. Yoshimura, Y. Miyahara, N. Kasagi,
H. Yamane, A. Ojida and I. Hamachi, J. Am. Chem. Soc., 2004, 126,
12204.
6 C. R. Cooper and T. D. James, Chem. Commun., 1997, 1419;
T. E. McAlindon, M. P. LaValley, J. P. Gulin and D. T. Felson,
JAMA, J. Am. Med. Assoc., 2000, 283, 1469; F. Nicotra, Top. Curr.
Chem., 1997, 187, 55; E. Saxon, S. J. Luchansky, H. C. Hang, C. Yu,
S. C. Lee and C. R. Bertozzi, J. Am. Chem. Soc., 2002, 124, 14893.
7 L. A. Estroff and A. D. Hamilton, Chem. Rev., 2004, 104, 1201.
8 G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. Harrington,
J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352; S. G. Zhang,
T. C. Holmes, C. M. Dipersio, R. O. Hynes, X. Su and A. Rich,
Biomaterials, 1995, 16, 1385.
9 H. A. Behanna, J. J. J. M. Donners, A. C. Gordon and S. I. Stupp,
J. Am. Chem. Soc., 2005, 127, 1193; L. A. Estroff and A. D. Hamilton,
Angew. Chem., Int. Ed., 2000, 39, 3447; M. A. Kostiainen, J. G. Hardy
and D. K. Smith, Angew. Chem., Int. Ed., 2005, 44, 2556; P. Terech and
R. G. Weiss, Chem. Rev., 1997, 97, 3133; B. G. Xing, C. W. Yu,
K. H. Chow, P. L. Ho, D. G. Fu and B. Xu, J. Am. Chem. Soc., 2002,
124, 14846; Z. M. Yang, H. W. Gu, D. G. Fu, P. Gao, K. J. K. Lam
and B. Xu, Adv. Mater., 2004, 16, 1440; Z. M. Yang, H. W. Gu,
Y. Zhang, L. Wang and B. Xu, Chem. Commun., 2004, 208; Z. M. Yang,
G. L. Liang, L. Wang and B. Xu, J. Am. Chem. Soc., 2006, 128, 3038;
Z. M. Yang and B. Xu, Chem. Commun., 2004, 2424; Z. M. Yang,
K. M. Xu, L. Wang, H. W. Gu, H. Wei, M. J. Zhang and B. Xu, Chem.
Commun., 2005, 4414; Y. Zhang, H. Gu, Z. Yang and B. Xu, J. Am.
Chem. Soc., 2003, 125, 13680; Y. Zhang, Z. M. Yang, F. Yuan,
H. W. Gu, P. Gao and B. Xu, J. Am. Chem. Soc., 2004, 126, 15028;
T. C. Holmes, S. de Lacalle, X. Su, G. S. Liu, A. Rich and S. G. Zhang,
Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 6728; A. Aggeli, I. A. Nyrkova,
M. Bell, R. Harding, L. Carrick, T. C. B. McLeish, A. N. Semenov and
N. Boden, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 11857; S. Bhuniya
and B. H. Kim, Chem. Commun., 2006, 1842; S. Bhuniya, S. M. Park
and B. H. Kim, Org. Lett., 2005, 7, 1741; A. M. Bieser and J. C. Tiller,
Chem. Commun., 2005, 3942; M. Suzuki, Y. Nakajima, T. Sato,
H. Shirai and K. Hanabusa, Chem. Commun., 2006, 377; V. Jayawarna,
M. Ali, T. A. Jowitt, A. E. Miller, A. Saiani, J. E. Gough and R. V. Ulijn,
Adv. Mater., 2006, 18, 611; M. de Loos, B. L. Feringa and J. H. van
Esch, Eur. J. Org. Chem., 2005, 3615; M. Venanzi, E. Gatto,
G. Bocchinfuso, A. Palleschi, L. Stella, C. Baldini, F. Formaggio and
C. Toniolo, J. Phys. Chem. B, 2006, 110, 22834, ASAP; M. Suzuki,
M. Nanbu, M. Yumoto, H. Shirai and K. Hanabusa, New J. Chem.,
2005, 29, 1439.
Fig. 2 Gross appearance (A, B), histological cross-section images (C, D)
and enlarged images (E, F) of the dorsal skins of Balb/C mice on day 6
after wounding. (A, C, and E) negative control and (B, D, and F) Gel II-
treated immediately after the incision was made. Histological specimens
were embedded in paraffin wax and stained with hematoxylin and eosin. a,
scar tissue; b, extracellular matrix (ECM); c, keratinocytes.
minimal formation of scar tissue at the wound site of the Gel II-
treated mice on day 6. A large amount of ECM forms between the
fibroblasts and the keratinocytes, which indicates the Gel II treated
mice are at the later matrix deposition phase of wound healing
(Fig. 2D). These results are consistent with the appearance of the
skin of the wound site after healing.
In summary, based on a biologically important aminosaccharide
derivative, we successfully synthesized two novel small molecule
hydrogelators, which form biocompatible and stable hydrogels.
The mice with wounds on their back recovered more rapidly when
one of the hydrogels was applied than those without the treatment.
This result indicates that the strategy reported in this paper
promises a new way to develop candidates for wound healing.
Further work will focus on studying the detailed relationship
between self-assembly of the glucosamine-based hydrogelators and
the beneficial effects of the hydrogels for other biomedical
applications.
10 G. W. Plant, S. Woerly and A. R. Harvey, Exp. Neurol., 1997, 143, 287.
11 B. G. Xing, P. L. Ho, C. W. Yu, K. H. Chow, H. W. Gu and B. Xu,
Chem. Commun., 2003, 2224.
BX acknowledges financial support from both the RGC (Hong
Kong) and EHIA (HKUST).
12 N. Sreerama and R. W. Woody, in Circular Dichroism: Principles and
Applications, ed. N. Berova, K. Nakanishi and R. W. Woody, John
Wiley & Sons, New York, 2000, pp. 601.
13 T. Hirobe, Development, 1988, 102, 567.
14 C. Amendt, A. Mann, P. Schirmacher and M. Blessing, J. Cell Sci.,
2002, 115, 2189; N. T. Bennett and G. S. Schultz, Am. J. Surg., 1993,
165, 728.
Notes and references
{ All animal procedures were approved by a local animal ethics committee.
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Chem. Commun., 2007, 843–845 | 845