Improved method for synthesis of cysteine modified hyaluronic acid for in situ hydrogel formation

Article abstract of DOI:10.1039/c5cc02367j

We developed a new strategy for the functionalization of hyaluronic acid by chemical modification of its C-6 hydroxyl groups through an ether bond to obtain a cysteine-hyaluronic acid conjugate. This conjugate is suitable to prepare injectable and in situ formed hydrogels cross-linked by native chemical ligation and Michael addition under mild conditions.

Full text of DOI:10.1039/c5cc02367j

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Improved method for synthesis of cysteine
modified hyaluronic acid for in situ hydrogel
formation†
Cite this:DOI: 10.1039/c5cc02367j
Received 21st March 2015,
Accepted 7th May 2015
Xin Zhang,a Pengcheng Sun,a Lingzi Huangshan,a Bi-Huang Hu*a and
Phillip B. Messersmith*b
DOI: 10.1039/c5cc02367j
www.rsc.org/chemcomm
We developed a new strategy for the functionalization of hyaluronic bioactive surfaces,11 drug delivery,12 and minimally invasive
acid by chemical modification of its C-6 hydroxyl groups through in situ formation of hydrogels under physiological conditions.13
an ether bond to obtain a cysteine–hyaluronic acid conjugate. This Such applications are facilitated by modification strategies
conjugate is suitable to prepare injectable and in situ formed that permit chemical functionalization while preserving the
hydrogels cross-linked by native chemical ligation and Michael biological activity of HA.14
addition under mild conditions.
Strategies for modification of HA often target the carboxylic
acid of the glucuronic acid and primary hydroxyl groups of the
Hyaluronic acid (HA) is a non-sulfated anionic linear poly- biomacromolecule.15 The carboxyl groups have been commonly
saccharide consisting of b-(1, 4) linked D-glucuronic acid-b-(1, 3)- modified by amidation and esterification using carbodiimide-
N-acetyl-D-glucosamine disaccharide repeats. HA has a molecular mediated chemistry. However, derivatization of HA in this
weight ranging from 100 kDa to 8 MDa and is ubiquitous in the manner may form N-acylurea-type EDC adduct,16 which causes
human body, especially in the synovia of joints, the corpus the resulting HA conjugate to be immunogenic.17 Furthermore,
vitreum of the eyes and the dermis of the skin.1 As an essential the biological functions of HA are considered to be mediated
signalling biomacromolecule, HA is located in the extracellular through specific ionic interactions between HA carboxylates
matrix (ECM), at the cell surface and inside the cell, where it is and cationic amino acids of the cell surface receptor CD44, and
involved in many cellular functions such as water homeostasis, five or more HA disaccharide units in length have been shown
cell migration, proliferation and adhesion.2
to be necessary for optimal CD44 binding.18
HA has a short half-life of only about 1 to 2 days in tissue,
Alternative HA modification strategies to target the hydroxyl
limiting its applications as a biocompatible, biodegradable and groups of HA have employed divinyl sulfone, diglycidyl ether,
non-immunogenic polymer.3 Chemical modification and cross- glutaraldehyde, and ester linked glycidyl methacrylate, or methacrylic
linking of HA has been used to achieve longer residence time anhydride.4,5,15a,19 For the preparation of thiolated HA, a previous
in vivo while maintaining biocompatibility and viscoelastic report described an HA derivatized with free thiol groups (HASH)
properties of the naturally occurring biomolecule.4 Modified HA through ether bond linkage by reaction of the hydroxyl groups of
has been used in various physical forms – viscoelastic solutions, HA with ethylene sulphide.20 Unfortunately, the reported HASH
soft or stiff hydrogels, macroporous and fibrillar sponges, nano- was unable to form cross-linked HA hydrogels via disulfide bonds,
particulate fluids and flexible sheets.5 A wide range of biomedical or through bivalent thiol-reactive cross-linkers, indicating that
applications utilizing HA derivatives have been developed, such employing this strategy for HA gel formation was rather challenging.
as viscosupplementation,6 cell differentiation,7 tissue sealants,8
In the interest of developing cysteine derivatized HA (Cys–HA)
tissue augmentation,9 ophthalmic surgery,10 engineering of for chemo-selective gel formation under mild conditions,21 here
we report a new strategy for the preparation of Cys–HA useful for
in situ formation of hydrogels by native chemical ligation. The
method preserves the free carboxylic acids by conjugating the
cysteine through a stable ether linkage via the HA hydroxyl
groups (Scheme 1). Starting with commercially available L-cysteine
hydrochloride 1, compound 3 was prepared in two steps,22 followed
by activation of the carboxylic acid with N-hydroxysuccinimide
(HOSu) to afford the active ester 4. Cystamine is an unstable
a Key Laboratory of Tropical Biological Resources of Ministry of Education,
College of Oceanography, Hainan University, Haikou, Hainan Province, 570228,
China. E-mail: bhhu@hainu.edu.cn; Tel: +86 0898 66289567
b Departments of Bioengineering and Materials Science and Engineering,
University of California, Berkeley, CA, 94720-1760, USA.
E-mail: philm@berkeley.edu; Tel: +1 510 643 9631
† Electronic supplementary information (ESI) available: Detailed experimental
procedures, chemical and molecular weight characterization and hydrogel for-
mation, as well as oscillatory rheology of hydrogels. See DOI: 10.1039/c5cc02367j liquid and is generally handled as the dihydrochloride salt,
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(0.1 M NaOH solution) could not afford the expected modified
HA, as indicated by the absence of the 1HNMR signal of the Boc
protecting group. A number of reaction conditions for this reaction,
including mixtures of alkaline acetonitrile or THF as well as use of
HA–TBA salt in DMSO after TBA salt conversion by ion exchange
chromatography, were explored without success. Finally, we dis-
covered that introduction of tetrabutylammonium hydroxide
(TBAH) into the reaction mixture of compound 7 with HA enabled
the synthesis of compound 8, characterized by 1HNMR data of the
conjugate, and verified by Ellman test for detection of free thiol
group24 as well as ninhydrin test for free amino group after removal
of protecting groups on the Cys moiety. Finally, Cys–HA conjugate 9
was obtained by cleavage of Boc protecting group of compound 8
with TFA, and followed by treatment with ethanol/H2O (1 : 1) to free
the Cys, monitored by 1HNMR analysis and Ellman test.
Measurement of the thiol content and the degree of modifi-
cation (DS, HA disaccharide unit %), indicated that the DS was
up to 3.3%. The modification sites on HA were proposed to be
6-position as illustrated, based on the hydroxyl group at 6-position
being a primary alcohol and much less sterically and spatially
hindered than the secondary alcohols at the 2- and 3- positions.25
The molecular weight (Mw) of Cys–HA conjugate 9 was determined
by gel permeation chromatography (GPC) to be 132 kDa (ESI†),
indicating that the molecular weight was only minimally reduced
compared to the HA starting material (143 kDa) during the
chemical modifications shown in Scheme 1.
To confirm the functionality of modified Cys–HA conjugate
9 and its potential application for in situ hydrogel formation, we
explored preparation of hydrogels of 9 cross-linked by native
chemical ligation (NCL),21a and Michael addition26 under mild
conditions using this HA conjugate. In the case of NCL, a thioester
can react with an N-terminal-Cys to form a new amide bond under
mild conditions (Scheme 2).27 Advantages of NCL include chemo-
selectivity (only an N-terminal-Cys is reactive), high efficiency,
mild aqueous reaction conditions, and regeneration of free thiol
side chain of the N-terminal Cys. NCL has been used to form
covalently cross-linked hydrogels,21a allowing further biofunction-
alization of the hydrogel network with bioactive compounds for
tailoring the biological properties of in situ formed hydrogels.21d
We found that mixing aqueous solutions of Cys–HA con-
jugate 9 and poly(ethylene glycol) (PEG) thioester 10 resulted
Scheme 1 Synthesis of Cys–HA conjugate 9.
scarcely soluble in organic solvents. Therefore, compound 4 in hydrogel formation (Scheme 3). Gel formation time could be
as an active ester can facilitate the reaction with cystamine significantly affected by buffer pH, conjugate concentration,
dihydrochloride in aqueous solution to generate compound 5 polymer architecture, reactant stoichiometry, and reaction
without an extra step for the conversion of the salt to the free temperature. Control experiments with 20 mM tricarboxyethyl-
amine. Selective reduction of the disulfide of compound 5 with phosphine (TCEP) in water to reduce intermolecular disulfide
NaBH4 yielded compound 6 with free thiol group,23 a nucleo- bonds, and with 100 mM 2-mercaptoethanol (MCE) to cleave
phile that is highly reactive with epoxides under mild conditions. the disulfide bonds and thioester bond by thiol exchange in
Addition of compound 6 to one epoxide group of 1,4-butanediol
diglycidyl ether (BDDE) produced compound 7 containing an
epoxide group, which was then used to modify HA at its alcohol
groups through an ether bond linkage.
The hydroxyl groups in HA are nucleophiles, which can react
with epoxides in a ring-opening process to form ether bonds
at high pH conditions (usually above 11). However, we found
Scheme 2 Chemistry of native chemical ligation (NCL).
that the reaction of the epoxide 7 with HA in alkaline solution
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Fig. 1 Oscillatory rheology of the hydrogel formed by native chemical ligation
of Cys–HA conjugate 9 (5% w/v) and 4-armed PEG-EMP thioester 10 (0.5% w/v)
in phosphate buffer solution (pH 7.5) at 25 1C with the molar ratio of cysteine and
thioester 2 : 1. Storage (G0) and storage (G00) modulus vs. time are shown.
Scheme 3 The hydrogel formed by Cys–HA conjugate 9 and 4-arm-
PEG-EMP thioester 10 by native chemical ligation.
water, were performed to confirm the formation of amide cross-
linked HA–PEG hydrogels. The persistence of hydrogels formed
from Cys–HA conjugate 9 and PEG thioester 10 in the presence
of TCEP and MCE confirmed that the gels formed mainly by
cross-linking through NCL rather than intermolecular disulfide
of Cys–HA conjugate, or thioester exchange.
Furthermore, the hydrogel formation process and visco-
elastic behavior of the hydrogels formed by NCL of Cys–HA
conjugate 9 and PEG thioester 10, were investigated by oscilla-
Fig. 2 Oscillatory rheology of the hydrogel formed by Michael addition of
Cys–HA conjugate 9 (5% w/v) and 4-armed PEG-ACLT conjugate (0.5% w/v)
in phosphate buffer solution (pH 7.5) at 25 1C with the molar ratio of cysteine
and acrylate 2 : 1. Storage (G0) and storage (G00) modulus vs. time are shown.
tory rheology using optimized conditions for measurement of a chemically modified HA in future biomedical applications where
G0/G00 crossover point.28 The time-dependent changes in storage interactions between HA and biomolecules (e.g. CD44) may be
modulus (G0) and loss modulus (G00) for the hydrogel composi- important. We demonstrated that the Cys–HA conjugate is reactive
tion tested were characteristic of elastic hydrogel formation with the polymers containing thiol-reactive groups and can be used
(Fig. 1), as indicated by a low initial G0 (G0 o G00), and a G0/G00 to prepare hydrogels cross-linked by native chemical ligation
crossover point representing a theoretical gelling point, followed and Michael addition under mild conditions, respectively. Inject-
by rapid increase in G0 to a plateau value. Subsequent frequency able and in situ formed HA hydrogels could be useful in tissue
and strain sweep experiments conducted after G0 reached a engineering, tissue repair, and drug delivery in a minimally invasive
plateau indicated the storage modulus was frequency and strain way. The modified HA conjugate could be used for further bio-
independent as expected for a covalently cross-linked hydrogel.29 functionalization with bioactive molecules and engineering of
To illustrate the versatile nature of Cys–HA conjugate 9, hydrogels bioactive surfaces through the thiol and amino groups. The
cross-linked by Michael addition26 were prepared under mild con- method of HA modification reported here may also be applicable
ditions by mixing Cys–HA conjugate 9 and 4-armed PEG–acrylate to the modification of other natural polysaccharides.
(ACLT) conjugate. It was found that hydrogels formed by Michael
This research was supported by the National Natural Science
addition of Cys–HA conjugate 9 and 4-armed PEG–ACLT conjugate Foundation of China (No. 21074027), Hainan University Starting
reached the gelling point more rapidly (Fig. 2) compared to those Grant, and grants from the National Institutes of Health (R01
formed by NCL under the same conditions (8 min vs. 20 min).
In summary, we developed a new strategy for the function-
alization of hyaluronic acid by chemical modification of alcohol
groups through a stable ether bond to obtain a cysteine–hyaluronic
acid conjugate. The method preserves the free carboxylic acid of
the disaccharide repeat unit of HA, therefore facilitating the use of
DE021215 and R01 DE021104).
Notes and references
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