Carbohydrate-based crosslinking agents: Potential use in hydrogels

Article abstract of DOI:10.1002/pola.24892

A bis(methacrylamido) derivative of glucose has been used as a crosslinking agent for PHEMA sponges formed by polymerization-induced phase separation. Copyright

Full text of DOI:10.1002/pola.24892

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Carbohydrate-Based Crosslinking Agents: Potential Use in Hydrogels
Stefan M. Paterson,1 Jasper Clark,1 Keith A. Stubbs,1 Traian V. Chirila,2,3,4,5
Murray V. Baker1,2
1Chemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley,
Western Australia 6009, Australia
2Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101, Australia
3Faculty of Science and Technology, Queensland University of Technology, Brisbane, Queensland 4001, Australia
4Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, Queensland 4072, Australia
5Faculty of Health Sciences, University of Queensland, Herston, Queensland 4006, Australia
Correspondence to: M. V. Baker (E-mail: murray.baker@uwa.edu.au) or K. A. Stubbs (E-mail: keith.stubbs@uwa.edu.au)
Received 17 May 2011; accepted 16 July 2011; published online 17 August 2011
DOI: 10.1002/pola.24892
KEYWORDS: biomaterials; carbohydrates; macroporous polymers; poly(2-hydroxyethyl methacrylate); synthesis
carbohydrates represent another class of naturally occurring
biomacromolecules that has been exploited to render hydro-
gels biodegradable. The carbohydrates class is comprised of a
broad range of compounds, ranging from monosaccharides to
polysaccharides. Various polysaccharides (e.g., chitosan) have
been used in the formation of hydrogels,14,15 and have been
incorporated into polymer networks to form polymer-polysac-
charide hydrogel conjugates.16,17
INTRODUCTION Tissue engineering requires the use of poly-
mer scaffold biomaterials—made from either natural or syn-
thetic polymers—that are biocompatible, porous, and
biodegradable. One synthetic polymer in particular, poly(2-
hydroxyethyl methacrylate) (PHEMA), has an established his-
tory of use as a biomaterial in the field of medical devices1–5
but has also received attention as a potential scaffold material
for tissue engineering purposes.2,6–8
Although there are many reports on using polysaccharides
to form hydrogels, there are only a few reports on using mon-
osaccharide- to oligosaccharide-based crosslinking agents to
form hydrogel conjugates.18,19 This paucity of research in this
area is most likely a consequence of the difficulty of synthesiz-
ing specific carbohydrate-based compounds—the large number
of hydroxyl groups on a saccharide unit dictates the use of
extensive protection and deprotection chemistry when a specif-
ically functionalized carbohydrate is required. Nevertheless, the
differences between carbohydrates and peptides (e.g., glyco-
sidic linkages vs. peptide bonds) means that hydrogels and
other biomaterials involving cleavable carbohydrate-based
groups may have advantages (e.g. propensity for degradation
by different classes of enzymes, different rates of degradation)
over similar peptide-based materials in certain applications.
Of particular interest to us are carbohydrate-based crosslink-
ing agents, especially monosaccharide- to oligosaccharide-
based crosslinking agents that could be used as components of
PHEMA hydrogels. Although there have been reports on the use
of 2-hydroxyethyl methacrylate (HEMA) carbohydrate conju-
gates as monomers, and their corresponding polymers,20–25 to
the best of our knowledge, there are no reports of monosaccha-
ride- to oligosaccharide-based crosslinking agents. For this rea-
son, we have developed a synthetic route to a glucopyranose-
based crosslinking agent as a proof-of-concept investigation to
The wide-spread use of PHEMA materials has been attrib-
uted to its excellent biocompatibility2 and the fact that it can,
by polymerization-induced phase separation, be easily made in
macroporous forms having morphologies suitable for tissue
engineering applications.2,8–12 However, the use of PHEMA
hydrogels for tissue engineering purposes is limited because
PHEMA itself is not biodegradable. Fortunately, it has recently
been shown that incorporation of biodegradable elements,
namely peptide-based crosslinking agents, into PHEMA-based
networks renders PHEMA-based materials biodegradable.8,13
Peptide-based crosslinking agents impart biodegradability
because they can be cleaved enzymatically, enabling a network
polymer to fragment into soluble, linear degradation products.
Peptides have been targeted as crosslinking agents because:
they are relatively easy to synthesize; there are many enzymes
that can be used to cleave peptide-based crosslinking agents;
the degradation properties of peptides can be tuned by select-
ing, for example, peptide sequences that will be targeted by
particular enzymes;8 and as peptides are naturally occurring
biomacromolecules, cytotoxicity issues are unlikely for pep-
tide-based crosslinking agents. To widen the potential for bio-
degradable biomaterials to be used in tissue engineering
applications, there is ongoing research to develop new and
more specific ways to impart biodegradability. For this reason,
Additional Supporting Information may be found in the online version of this article.
C
V
2011 Wiley Periodicals, Inc.
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SCHEME 1 Synthesis of the carbohydrate-based crosslinking agent 1.
explore the use of monosaccharide-based crosslinking agents in
PHEMA-based hydrogels. This proof-of-concept study opens the
way for more elaborate carbohydrate-based crosslinkers to be
used in the preparation of materials for tissue engineering. Poly-
mers that use carbohydrate-based crosslinkers as enzyme-cleav-
able units will allow researchers to design new biodegradable
materials that can be degraded by different classes of enzymes
compared to polymers incorporating well-established peptide-
based crosslinkers.
and characterization techniques are described in Supporting
Information.
RESULTS AND DISCUSSION
To explore the potential for carbohydrate-based crosslinking
agents to be used in the synthesis of PHEMA-based materials,
the crosslinker 1 was devised (Scheme 1). First, using a previ-
ously reported procedure,26 the glycoside
3 was prepared
using 2-bromoethanol and the pentaacetate 2. Treatment of 3
with sodium azide gave the azide 4 in excellent yield. With one
azide already installed at the C1-position, we turned our atten-
tion to the installation of the next azide at the C6-position. The
azide 4 was treated with catalytic sodium methoxide to yield
EXPERIMENTAL
The materials, details for preparation of the carbohy-
drate-based crosslinking agent, polymerization methods,
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typically greater than 80%. In addition, the synthesis is robust and
can be scaled easily to accommodate larger quantities. We note,
however, that care needed to be taken during the functionaliza-
tion of 12 with the methacryloyl groups to give 1, and premature
polymerization occurred if the reaction mixture was concentrated
and subjected to even moderate heat (>33 ꢀC) during isolation of
1. The yield of 1 was only moderate (31%), but none of the reac-
tion conditions used in Scheme 1 have been optimized.
An advantage of our route to 1 is that it includes the azides
5 and 11 as intermediates. In our synthesis, azido groups were
used as protecting groups, but rather than simply being pro-
tecting groups in synthetic intermediates, the azido groups can
also be exploited as functional groups to undergo 1,3-cycload-
dition with alkynes in ‘‘click’’ chemistry. Click reactions of 5
could be used to functionalize alkyne-containing polymers,27
and similar click chemistry could allow 11 to be used as a
crosslinking agent for alkyne-functionalized polymers, to form
polymer networks.27–29 Furthermore, the hydroxyl groups on 1,
5, and 11 can be functionalized with biologically relevant
groups, such as cell adhesion ligands.30
To test the ability of 1 to act as a crosslinking agent, PHEMA
sponges (formed by polymerization-induced phase separation)
that were crosslinked with
1 were compared to PHEMA
sponges crosslinked with tetraethylene glycol dimethacrylate
(TEGDMA). Samples crosslinked with 1 or TEGDMA appeared
white, which is expected for PHEMA sponges.8,12,31 Scanning
electron microscopy (SEM) analysis of PHEMA sponges cross-
linked with 1 revealed morphologies based on polymer drop-
lets about 5 lm in diameter and pores in the order of 20 lm
[Fig. 1(A)]. For PHEMA samples crosslinked with TEGDMA,
SEM images showed morphologies with droplets about 2–3
lm in diameter and narrow pores with dimensions in the order
of 10 lm [Fig. 1(B)]. This difference in the size of the polymer
droplets and pores is not unexpected. Polymerization-induced
phase separation relies on the growing polymer chains precipi-
tating out of the polymerization solution. Any changes to the
hydrophilicity of the growing polymer chains will alter the mo-
lecular weight at which the polymer chains precipitate.31,32 If
the growing polymer chains are more hydrophobic, the poly-
mer will precipitate out of the polymerization solution at a
lower molecular weight and form comparably smaller polymer
droplets. Likewise, if the growing polymer chain is more hydro-
philic, the polymer will stay in the polymerization solution lon-
ger to give higher molecular weight polymer chains, forming
larger polymer droplets. As 1 is more hydrophilic compared to
TEGDMA, mainly due to 1 being a triol, the growing polymer
chains should be more hydrophilic, delaying the onset of
phase separation and producing polymer droplets that are
larger when compared to the more hydrophobic TEGDMA.
Simple differences in hydrophilicity between TEGDMA and 1
are unlikely to fully account for the differences in polymer mor-
phology. Other factors, such as differences between the reac-
tivity ratios relative to HEMA of TEGDMA and 1 (which will be
influenced by hydrophilicity and other factors), may also con-
tribute to the differences in morphologies, and this possibility
warrants further investigation.
FIGURE 1 SEM images of PHEMA sponges crosslinked with (A)
the carbohydrate-based crosslinking agent 1 or (B) TEGDMA.
the tetrol 5, and selective protection of the primary alcohol in 5
using triphenylmethyl chloride gave the triol 6 in good overall
yield. The triol 6 was then converted to the triacetate 7, after
which the trityl ether protecting group was removed using
aqueous acid to give the alcohol 8 in good yield. Activation of
the alcohol 8 using methanesulfonyl chloride gave the sulfo-
nate 9, and subsequent treatment of 9 with sodium azide gave
the diazide 10. Removal of the acetyl protecting groups gave
the desired triol 11. The azido groups on 11 were then reduced
to amino groups using conventional hydrogenolysis condi-
tions, and the amino groups were functionalized with metha-
cryloyl moieties to afford the desired monosaccharide
crosslinker 1. Although the overall synthetic route involves
multiple steps, each step only involved the use of relatively
simple synthetic procedures, and the yield for each step was
CONCLUSIONS
We developed
a simple, multistep synthetic route to
glycosylpyranose-based crosslinking agent 1. The route to 1
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can easily be adapted to the synthesis of other crosslinking
agents suitable for click-type chemistry with alkynes. Cross-
linker 1 was successfully incorporated into PHEMA sponges by
copolymerization with HEMA under conditions that induce
phase separation. The resulting PHEMA sponges exhibited
morphologies based on polymer droplets that were larger that
those seen for otherwise similar sponges crosslinked with
TEGDMA. The difference in morphologies was attributed to the
differences in hydrophilicity of the glucopyranose-based cross-
linking agent 1 and TEGDMA. To the best of our knowledge,
this is the first report of a monosaccharide-based crosslinking
agent, and we believe that our synthetic route will be of benefit
to researchers interested in the field of hydrogel synthesis. In
addition, although the crosslinker 1 was not designed to be
enzymatically degradable, we believe that further investiga-
tions into the synthesis of disaccharide- to oligosaccharide-
based crosslinking agents that are enzymatically degradable
could lead to the development of new and exciting biodegrad-
able materials for tissue engineering purposes.
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