Functionalization of hyaluronic acid with chemoselective groups via a disulfide-based protection strategy for in situ formation of mechanically stable hydrogels

Article abstract of DOI:10.1021/bm1007986

Functionalization of hyaluronic acid (HA) with chemoselective groups enables in situ (in vivo) formation of HA-based materials in minimally invasive injectable manner. Current methods of HA modification with such groups primarily rely on the use of a large excess of a reagent to introduce a unique reactive handle into HA and, therefore, are difficult to control. We have developed the new protective group strategy based on initial mild cleavage of a disulfide bond followed by elimination of the generated 2-thioethoxycarbonyl moiety ultimately affording free amine-type functionality, such as hydrazide, aminooxy, and carbazate. Specifically, new modifying homobifunctional reagents have been synthesized that contain a new divalent disulfide-based protecting group. Amidation of HA with these reagents gives rise to either one-end coupling product or to intra/intermolecular cross-linking of the HA chains. However, after subsequent treatment of the amidation reaction mixture with dithiothreitol (DTT), these cross-linkages are cleaved, ultimately exposing free amine-type groups. The same methodology was applied to graft serine residues to the HA backbone, which were subsequently oxidized into aldehyde groups. The strategy therefore encompasses a new approach for mild and highly controlled functionalization of HA with both nucleophilic and electrophilic chemoselective functionalities with the emphasis for the subsequent conjugation and in situ cross-linking. A series of new hydrogel materials were prepared by mixing the new HA-aldehyde derivative with different HA-nucleophile counterparts. Rheological properties of the formed hydrogels were determined and related to the structural characteristics of the gel networks. Human dermal fibroblasts remained viable while cultured with the hydrogels for 3 days, with no sign of cytotoxicity, suggesting that the gels described in this study are candidates for use as growth factors delivery vehicles for tissue engineering applications.

Full text of DOI:10.1021/bm1007986

Biomacromolecules 2010, 11, 2247–2254
2247
Functionalization of Hyaluronic Acid with Chemoselective
Groups via a Disulfide-Based Protection Strategy for In Situ
Formation of Mechanically Stable Hydrogels
Dmitri A. Ossipov,* Sonya Piskounova, Oommen P. Varghese, and Jo¨ns Hilborn
Polymer Chemistry, Material Chemistry Department, Uppsala University, S-75121 Uppsala, Sweden
Received March 5, 2010
Functionalization of hyaluronic acid (HA) with chemoselective groups enables in situ (in vivo) formation of
HA-based materials in minimally invasive injectable manner. Current methods of HA modification with such
groups primarily rely on the use of a large excess of a reagent to introduce a unique reactive handle into HA and,
therefore, are difficult to control. We have developed the new protective group strategy based on initial mild
cleavage of a disulfide bond followed by elimination of the generated 2-thioethoxycarbonyl moiety ultimately
affording free amine-type functionality, such as hydrazide, aminooxy, and carbazate. Specifically, new modifying
homobifunctional reagents have been synthesized that contain a new divalent disulfide-based protecting group.
Amidation of HA with these reagents gives rise to either one-end coupling product or to intra/intermolecular
cross-linking of the HA chains. However, after subsequent treatment of the amidation reaction mixture with
dithiothreitol (DTT), these cross-linkages are cleaved, ultimately exposing free amine-type groups. The same
methodology was applied to graft serine residues to the HA backbone, which were subsequently oxidized into
aldehyde groups. The strategy therefore encompasses a new approach for mild and highly controlled
functionalization of HA with both nucleophilic and electrophilic chemoselective functionalities with the emphasis
for the subsequent conjugation and in situ cross-linking. A series of new hydrogel materials were prepared by
mixing the new HA-aldehyde derivative with different HA-nucleophile counterparts. Rheological properties of
the formed hydrogels were determined and related to the structural characteristics of the gel networks. Human
dermal fibroblasts remained viable while cultured with the hydrogels for 3 days, with no sign of cytotoxicity,
suggesting that the gels described in this study are candidates for use as growth factors delivery vehicles for
tissue engineering applications.
Functionalization of HA with chemoselective groups is
advantageous for the preparation of glycoconjugates with
complex biomacromolecules and for the performance of chemi-
cally cross-linked hydrogel materials. Mild chemoselective
reactions, also referred to as click reactions,17,18 allow mild,
quick, and injectable immobilization of growth factors and cells
at physiological aqueous environment without formation of toxic
side products. This may significantly reduce cell death or lost
of bioactivity of the encapsulated growth factors in such
chemically formed HA hydrogel systems.8,9,11,12,14 HA func-
tionalization with chemoselective groups is, however, a chal-
lenging task due to multifunctionality and limited solubility of
HA in organic solvents. For example, modification of HA with
nucleophilic hydrazide groups19,20 has been accomplished by
reacting HA with homobifunctional adipic dihydrazide (ADH),
which can potentially act as a cross-linking agent at the stage
when only one of the two hydrazide groups is intended to be
coupled to the HA carboxylate. To avoid the concomitant cross-
linking during hydrazide functionalization, a large excess of
ADH was therefore applied. In spite of that, the cross-linking
effect of ADH was substantial even at high molar ratios of ADH
to polysaccharide carboxylate as well as when using low amount
of the condensing reagent, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC).21 Consequently, acrylation of the HA-
ADH derivative was utilized for the installation of an electro-
philic methacrylamide group.9,22 Haloacetate-derivatized HA
was prepared via direct acylation of HA hydroxyl in water using
a 10-fold excess of anhydride reagent due to a side reaction
with HA carboxylate groups and the formation of hydrolytically
Introduction
Hyaluronan (HA) is a major glucosaminoglycan component
of extracellular matrices (ECM) found in different mammalian
tissues. HA plays an important role in the structure and
organization of the ECM, regulation of cell adhesion, morpho-
genesis and modulation of inflammation.1,2 Native HA is highly
biocompatible, biodegradable, and nonimmunogenic which
makes it an ideal material for various biomedical applications,
such as for treatment of osteoarthritis,3 in ophthalmic surgery4
and wound healing.5 Chemical modifications of HA has allowed
preparation of HA-drug conjugates6 and various HA-based
nanocarriers7 which conferred even more extensive applications
of HA in drug delivery. Chemically cross-linked HA hydrogel
biomaterials provided control over degradation rates and me-
chanical stiffness of HA in ViVo. Matching the mechanical
properties of HA hydrogels to biological tissues together with
the use of HA hydrogels as carriers for growth factors8,9 and
cells9-11 resulted in successive regeneration of different tissues
including bone,9,12 cartilage,10,11 as well as in stimulation of
angiogenesis.8 Our group has recently developed cross-linkable
HA-bisphosphonate prodrug that are selectively taken up by cells
that produce the HA receptor CD44.13 We have also demon-
strated the hydrogel-mediated delivery of BMP-2 (bone mor-
phogenetic protein-2) to induce local bone formation14,15 as well
as the utility of guanidinium-modified HA hydrogel for gene
delivery.16
* To whom correspondence should be addressed. Tel.: +46-18-4717335.
Fax: +46-18-4713477. E-mail: dmitri.ossipov@mkem.uu.se.
10.1021/bm1007986
 2010 American Chemical Society
Published on Web 08/12/2010

2248 Biomacromolecules, Vol. 11, No. 9, 2010
Ossipov et al.
Scheme 1. Difunctional Symmetric Reagents 11-14 Protected with 2,2′-Dithiobis(ethoxycarbonyl) Group
unstable mixed anhydride.23 Partial oxidation of HA with
Results and Discussion
sodium periodate was also used to generate electrophilic
aldehyde groups in the oxidized open rings of the glucuronic
acid units.12 Periodate oxidation of HA requires, however,
prolonged reaction time due to extensive inter- and intramo-
lecular hydrogen bonding between hydroxyl and carboxylate
groups of HA which, as a side reaction, causes degradation of
HA backbone and reduction of the molecular weight by at least
an order of magnitude.24 A solution to controlled functional-
ization of HA with chemoselective groups would be temporal
blocking of the group of interest with a suitable protecting group.
The repertoire of protecting groups, the removal of which would
not be destructive for HA, is, however, quite limited because
both highly basic and acidic conditions degrade HA by cleavage
of the labile glycosidic bonds. The use of protection strategy
for functionalization of HA with chemoselective groups has been
limited so far to only the preparation of thiolated HA.25
Design of New Difunctional Reagents for Mild Incorpora-
tion of Chemoselective Nucleophilic Groups. We have elabo-
rated a synthetic method that allows one pot functionalization
of HA with various types of orthogonal groups. To accomplish
this, we have introduced a protecting group strategy that allows
removal of multiple protective groups under the same mild
reaction conditions eventually affording different types of
orthogonal click functionalities. A series of symmetrical di-
functional reagents 11-14 have been synthesized (Scheme 1)
that have a central divalent protecting group, 2,2′-dithio-
bis(ethoxycarbonyl) (Dtec), which links two identical molecules.
The synthesis of the reagent 2 (Scheme 1) presenting two
hydrazine molecules coupled together through the divalent Dtec
protecting group has been reported by us recently.13 The divalent
protecting group has been prepared from 2,2′-dithiodiethanol 1
by activation with 2 equiv of carbonyldiimidazole (CDI) and
used in situ for a subsequent carbamate coupling reaction with
2 equiv of an appropriate amine-type compound (hydrazide 3,
aminooxy compound 4, or serine derivative 5). This afforded
alcohol derivatives 6 and 7, as well as serine methyl ester 8.
Two hydroxyl groups of the condensation products 6 and 7 were
then extended with hydrazinocarbonyls by consecutive activation
with CDI and treatment with hydrazine in one pot to give the
corresponding reagents 11 and 12. In a similar way, N-(2-
hydroxyethoxy)phthalamide 9 was coupled to the reagent 2 to
obtain 10. The serine derivative 8 and pthalimido derivative 10
afforded corresponding hydrazide 13 and aminooxy derivative
14 upon treating with neat hydrazine.
Here we report the synthesis of a series of symmetrical
difunctional reagents that have a central divalent protecting
group, 2,2′-dithiobis(ethoxycarbonyl) (Dtec), which links two
identical molecules. These reagents allow mild and highly
controlled functionalization of HA with various types of
nucleophilic chemoselective groups, which is exemplified by
first time HA modification with aminooxy and carbazate groups.
In spite of the cross-linking effect of these reagents during the
first amidation coupling, they can be easily cleaved at the central
disulfide part afterward, ultimately liberating the masked nu-
cleophilic groups. The novelty with this approach is that no excess
of reagents is needed, no intermediate purification is needed, the
reaction yields are quantitative without side reactions, and it allows
the retention of the molecular weight of the HA. The new HA
derivatives were tested in preparation of hydrogel materials by
mixing with new electrophilic HA derivative containing aldehyde-
terminated side chains to produce a series of HA-based hydrogels
with tunable mechanical properties.
Grafting the Hyaluronic Acid with Side Chains
Terminated with Chemoselective Nucleophilic Groups. The
symmetrical linkers 11-14 have a terminal amine-type func-
tional group that can undergo an amide-type reaction with the
carboxylate residue of HA in aqueous solution. The self-
immolative central Dtec group of these linkers when treated
with external thiol generate unstable thiols that decompose
spontaneously, liberating ethylene episulfide, carbon dioxide,

Functionalization of Hyaluronic Acid
Biomacromolecules, Vol. 11, No. 9, 2010 2249
Scheme 2. Functionalization of HA with Nucleophilic Chemoselective Groups
and the corresponding free amine-type click functionality.26
Amidation of HA with bifunctional reagents 11-14 can give
rise to either a one-end coupling product or a two-end intra/
intermolecular cross-linking of HA chains. However, after
subsequent treatment of the reaction mixture with dithiothreitol
(DTT), these cross-linkages are cleaved, ultimately exposing
free amine-type groups (Scheme 2).
of the isolated HA derivative 15 (Figure 1b) showed only R, ꢀ,
γ, δ, and ε-methylene protons of the reagent 11 that are derived
from its synthetic progenitor 3 but not the protons corresponding
to the HSCH2- methylene of the thioethyl carbamate intermedi-
ate. This observation demonstrated that the thioethyl carbamate
residues were indeed unstable under the basic conditions applied
(pH 8.5), underwent the described in the literature26 fragmenta-
tion reaction, and ultimately liberated the hydrazide group.
When reagents 12, 13, and 14 were subjected for the EDC-
mediated coupling with HA, the thioethyl carbamate moiety was
1H NMR spectroscopy provided the evidence that DTT
treatment led to the formation of the unstable 2-thioethyl
carbamate side chains that underwent subsequent fragmentation
reaction (Figure 1). Thus, coupling of the known reagent 1925
to HA afforded the expected thiolated HA derivative with clearly
visible methylene protons of the HSCH2CH2CO- side chains at
2.73 and 2.58 ppm, respectively (Figure 1a). Conversely, when
the same procedure was applied using the reagent 11, 1H NMR
1
also not detected in the H NMR spectra of the corresponding
products (see Figure S1 of Supporting Information), demonstrat-
ing the generality of our protection strategy for different amine-
type functionalities. Thus, we were able to quantitatively
functionalize HA with a range of nucleophilic chemoselective
Figure 1. 1H NMR spectra in D2O of (a) thiolated HA and (b) hydrazide-modified HA 15. The region from 0.9 to 4.7 ppm for all spectra is
compared.

2250 Biomacromolecules, Vol. 11, No. 9, 2010
Ossipov et al.
Table 1. Reaction Conditions of HA Modification and Degrees of
Substitution (DS) of the Corresponding Products
Addition of the 150 mM solution of sodium periodate in water
to the 0.8 wt % aqueous solution of 17 at a 1:1 [HA
carboxylate]/[NaIO4] molar ratio led to the formation of a gel,
indicating a very fast reaction. The reaction was stopped after
5 min by the addition of a 10-fold excess of ethylene glycol
and the dilution of the mixture with water two times. The gel
dissolved gradually after stirring the diluted mixture overnight.
The oxidized HA product 20 (Figure 2) was isolated from the
dissolved mixture by dialysis against water two times followed
by lyophilization. The conversion of the serine side chains into
molar ratio
of HA/reagent/
EDC/HOBt
HA
derivative
DS
(%)
yield
(%)
reagent
15
16
17
18
11
12
13
14
1:0.15:0.15:1
1:0.15:0.3:1
1:0.15:0.3:1
1:0.15:0.4:1
8.9
7.5
6.0
6.5
95.8
91.2
97.9
98.9
1
aldehyde groups was proved by first examining the H NMR
groups including hydrazide (15), aminooxy (16), serine (17),
and carbazate (18) derivatives using low submolar amounts of
the new homobifunctional reagents and carbodiimide coupling
reagent (Table 1), unlike previously described adipic dihydrazide
(ADH) modification of HA, which normally uses a large excess
of ADH to avoid cross-linking.21
spectra of HA derivatives 17 and 20. Compared to native HA,
the spectrum of serine-modified HA 17 showed additional peaks
at 4.27 and 4.00 ppm that were assigned to the protons at the
carbon A (Figure 2). These signals, corresponding to the proton
peak 4 of the reagent 13, disappeared in the spectrum of the
oxidized product 20. Because aldehydes in water exist in
equilibrium with their hydrated forms as well as they can
potentially react with numerous hydroxyl groups of HA with
the reversible formation of hemiacetals, it should come as no
surprise that we did not observe a peak corresponding to the
aldehyde proton. Instead, we observed new resonances at 5.43
and 3.63 ppm that were attributed to the protons of the hydrate
and hemiacetal forms. The formation of reversible hemiacetal
cross-links could be responsible for the gel formation during
the oxidation of the HA-serine derivative. The fact that the gel
was dissolved when the excess of ethylene glycol was added
to the diluted reaction mixture confirms our assumption. The
neighboring electron-negative carbonyl group makes the serine-
derived aldehyde more reactive toward both alcohols and
amines. The amount of aldehyde groups in the obtained
derivative was quantified by reductive amination with tert-butyl
carbazate (TBC) and subsequent integration of the tert-butyl
peak at 1.4 ppm, which indicated 5% of aldehyde functional-
ization. It should be noted that the peak at 3.63 ppm disappeared
after the reaction with TBC, thus, corroborating its hemiacetal
nature.
Modification of Hyaluronic Acid with Aldehyde-Contain-
ing Side Chains. The use of modifying reagent 13 allowed also
facile grafting serine side chains via hydrazide linkages. The
1,2-aminoalcohol motif of the serine moieties is known to
participate in selective oxidation with sodium periodate into the
corresponding aldehyde. Periodate oxidation of 2-aminoalcohols
is a very fast reaction, reportedly 1000-fold faster than the
1,2-diols.27,28 Periodate, for example, effects quantitative oxida-
tion of N-terminal serine when present in only a low molar
excess over 2-aminoalcohol.29 On the other hand, the glycol
groups in the glucuronate residues of hyaluronan are resistant
to periodate oxidation, although identical structures in similar
polymers and environments are easily attacked.30 The reason
to that is the extensive inter- and intramolecular hydrogen
bonding between hydroxyl and carboxylate groups of HA, which
makes glucuronic hydroxyls at C2 and C3 less accessible for
-
IO4 anions. We hypothesized that the attachment of 2-ami-
noalcohols -CH(NH2)CH2OH as side chains to the HA polymer
backbone with the following quick periodate oxidation could
selectively convert serine side groups into aldehydes without
affecting the glycol groups of the HA backbone. To date, the
normal way of HA functionalization with aldehyde groups relies
on sodium periodate oxidation of the native hyaluronan for 2 h,
which is accompanied by glycoside bond cleavage and signifi-
cant reduction of the molecular weight by at least an order of
magnitude.24
Regioselectivity of periodate oxidation at the serine side
chains under the conditions applied was demonstrated by
performing oxidation of the native HA using the same mild
conditions (5 min reaction at room temperature, equimolar
amount of periodate). No oxidation of the glycol groups of the
Figure 2. 1H NMR spectra of the reagent 13 (up), HA-serine 17 (middle), and HA-aldehyde 20 (bottom) in D2O. The region from 0.9 to 6.0 ppm
for all spectra is compared.

Functionalization of Hyaluronic Acid
Biomacromolecules, Vol. 11, No. 9, 2010 2251
Table 2. Mechanical Properties of HA Hydrogels
a
b
gel composition
G′initial (Pa)
G′swollen Pa
swelling ratioc
15 + 20
1520 ( 40
2880 ( 60
2300 ( 60
765 ( 60
247 ( 6
259 ( 3
218 ( 9
dissolved
41.8 ( 0.9
40.3 ( 0.5
43.4 ( 2.1
dissolved
16 + 20
18 + 20
15 + HA-Alox
a G′initial: elastic modulus measured for the prepared gels after 3 h of
setting. b G′swollen: elastic modulus measured for the swollen gels after 8
days of incubation in PBS (pH 7.4). c Swelling ratios were determined
after 8 days of incubation in PBS (pH 7.4).
Figure 3. Human dermal fibroblast viability after incubation for 48 h
in the presence of either 0.1 or 0.6% HAs modified with nucleophilic
and electrophilic chemoselective groups. The left bar corresponds
to the untreated control. The columns represent the mean ( S.D.;
n ) 3.
HA glucuronate units was detected under such conditions as
was verified by a reaction of the periodate-treated native HA
with TBC. Hence, the approach presented here allows the
preparation of the HA derivative that is functionalized with
electrophilic aldehyde side groups without breaking the HA
polysaccharide chain.
prepared the HA-Alox with 10% of aldehyde content from 1.3
MDa HA by treatment with 1 mol equiv of sodium periodate
per HA repeating unit for 2 h. The 2% hydrogel formed from
HA-Alox and HA-hydrazide 15 was completely dissolved on
day 8 (Table 2). The stability of the hydrogel networks prepared
from the serine-derived HA-aldehyde 20 was confirmed by
monitoring the swelling ratios over the time of incubation in
PBS buffer (Figure 2S of Supporting Information). After 8 days,
the newly prepared gels had reached a plateau of swelling ratio
ranging from 40 to 45, while the swelling ratio for the (15 +
HA-Alox) hydrazone gel was steadily increasing ultimately
leading to the gel dissolution. This allows the design of gel
materials that are degraded by cell-directed endogenous pro-
cesses only and not by hydrolysis.
Degradation of Hyaluronic Acid-Based Hydrogels. The
hydrogels were completely degraded by 500 U/mL hyalu-
ronidase (Hase) within 24 h. The amount of Hase added to the
solution was much higher than that of physiological conditions.
These experiments clearly demonstrated that Hase can recognize
the cross-linked HA, proposing that the prepared gels would
be resorbable in vivo by native degradation.
In Situ Hydrogel Preparation Using New Hyaluronic
Acid Derivatives Modified with Chemoselective Groups.
After the new HA derivatives carrying different nucleophilic
chemoselective groups had been prepared, we examined them
for their ability to form hydrogel materials by mixing with the
new HA-aldehyde counterpart. This procedure is free of a low
molecular weight cross-linker, it utilizes chemoselective addition
cross-linking reactions that are rapid in aqueous solutions
without the formation of side products and, thus, can be applied
in vivo. Until the present, the only chemical gelling HA-based
materials utilizing an aldehyde as one of the cross-linking
reactants were hydrazone and imine cross-linked gels. The
hydrazone HA gels were, for example, formed from the HA-
ADH by reacting with either PEG dialdehyde31,32 or with HA
in which aldehyde groups were generated by sodium periodate
oxidation.12,24,33 Schiff’s base cross-linked hydrogels were
prepared by mixing of the periodate-oxidized HA with either
chitosan10 or gelatin.34 Labile imine bonds were also formed
by coupling of amine-modified HA with oxidized heparin.35
Here we sought to prepare new cross-linker free HA hydrogels
in which oxime or carbazone bonds serve as cross-linking points
to verify the influence of these cross-linkages on the hydrolytic
stability of the corresponding gels.
Hydrogels (2 wt % in PBS buffer) were formed by mixing
the nucleophilic HA derivatives with the aldehyde-derivatized
HA 20 at a 1:1 molar ratio of the cross-linkable functionalities
(Table 2). The gelation time was monitored using a test tube
inversion assay that showed hydrogel formation within half a
minute for all HA derivatives. Slight variations in mechanical
properties of the prepared hydrogels (G′initial) can reflect varia-
tions in degree of functionalization of the HA derivatives as
well as different hydrophobicity of the functional side chains.
However, after incubation in PBS buffer for 8 days, the gels
Cytotoxicity of the Modified HA Derivatives and the
Corresponding Hydrogels. Cytocompatibility of the nucleo-
philic HA derivatives was evaluated by culturing human dermal
fibroblasts (hDFns) for 48 h in the presence of the respective
HA macromolecules (0.1 or 0.6% w/v, Figure 3). For compari-
son, cell viability in presence of electrophilic aldehyde-
derivatized HA 20 was examined. MTT assay indicated that
HA derivatives modified with nucleophilic groups (15, 16, 18)
were less toxic than the aldehyde-modified HA 20 (Figure 3).
Higher toxicity of HA-aldehyde 20 might originate from higher
chemical reactivity of the aldehyde group that can potentially
react with amino groups of proteins. All the HA derivatives
showed dose-dependent cytotoxicity. It should be stressed,
however, that cells would not be exposed to the free functional
HA components for a long time in the case of in vivo injection
due to fast gelling kinetics. To confirm this, hDFn cells were
cultured for 3 days, while sharing medium with the hydrogels
via cell culture inserts. The corresponding gel-forming solutions
were premixed and immediately put in contact with the cell
culture medium. The results presented in Figure 4 clearly show
that sharing medium with the gels in all cases showed no
significant decrease in cell viability. It can therefore be
concluded that all potentially toxic components are efficiently
cross-linked in the hydrogels during formulation and therefore
could be prevented from exposure to endogenous cells while
injecting the gels in vivo.
became softer, as judged by a lower elastic modulus (G′swollen
Table 2).
;
After swelling in PBS buffer for 20 days, none of the HA-
derived hydrogels was hydrolytically degraded. In comparison,
previously described hydrogels made from hydrazide and
aldehyde-modified HA derivatives were highly deformable in
spite of much higher degrees of functionalization of the
corresponding HAs.12,24,33 The improved hydrolytic stability of
our hydrogel materials can be attributed to the use of the serine-
derived aldehyde component which should react with amines
forming more stable -CdN- adducts due to the resonance
stabilization with the adjacent carbonyl group. This assumption
was confirmed by replacing our HA-aldehyde with the well-
known HA derivative that is obtained by periodate oxidation
of the native HA, named here as HA-Alox.10,12,24,33,34 We have

2252 Biomacromolecules, Vol. 11, No. 9, 2010
Ossipov et al.
Disulfanediylbis(ethane-2,1-diyl) Bis(2-(6-((hydrazinecarbonyl)oxy)-
hexanoyl)hydrazinecarboxylate) 11. The compound 6 (2.34 g, 4.69
mmol) was coevaporated with dry pyridine two times and finally
dissolved in the same solvent (25 mL). CDI (1.83 g, 11.27 mmol) was
added to the solution and the reaction mixture was stirred at room
temperature for 2 h under an argon atmosphere. The reaction mixture
was then cooled to -5 °C on an ice/NaCl bath and hydrazine (455 µL,
9.39 mmol) was added to the mixture in three portions over 40 min
while maintaining the temperature at -5 °C. The mixture was stirred
at -5 °C for 1.5 h and then allowed to warm up to room temperature
for another 17 h. Pyridine was evaporated, and the residue was
coevaporated with toluene two times. The residue was redissolved in
a minimal amount of methanol and the concentrated methanolic solution
was poured into 200 mL of DCM, stirred for 15 min, and then kept for
1 h at -20 °C. The insoluble waxy product was collected by decantation
and dried under vacuo overnight. Yield: 2.11 g (3.43 mmol, 73%). 1H
NMR (CD3OD): 4.29 (4H, m, 2 × -OCH2CH2S-), 4.02 (4H, m, 2 ×
-CH2OC(O)NHNH2), 2.96 (4H, m, 2 × -OCH2CH2S-), 2.21 (4H, m, 2
× -CH2C(O)-, J ) 7.3 Hz), 1.64 (8H, m, 2 × -CH2CH2CH2-), 1.42
(4H, m, 2 × -CH2CH2CH2-). 13C NMR: 174.6, 174.3, 156.9, 64.8, 63.2,
37.1, 33.2, 28.5, 25.1, 24.8.
Figure 4. Human dermal fibroblast viability after incubation for 3 days
in the presence of 2% w/v gels that were formed by combination of
aldehyde-modified HA 20 with different nucleophile-modified HA
derivatives. The left bar corresponds to the untreated control. The
columns represent the mean ( S.D.; n ) 3.
Experimental Section
General. 1,1′-Carbonyldiimidazole (CDI), serine methyl eater
hydrochloride, ε-caprolactone, DL-dithiothreitol, hydrazine, N-hydroxy-
benzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC), and 2,2′-dithiobisethanol 1 were purchased from Aldrich
Chemical Co. Hyaluronic acid (HA) sodium salt of the molecular weight
130 kDa was purchased from Lifecore Biomedical. The reagents were
used as received. 2,2′-Dithiobis(ethanol-O-hydrazinocarbonyl) 1,13
2-aminooxyethanol 7,36 N-(2-hydroxyethyloxyphthalimide) 9,37 and
3,3′-dithiobis(propionic hydrazide) 1925 were synthesized according to
the literature procedures. All solvents were of analytical quality (p.a.)
and were dried over 4 Å molecular sieves. Dialysis membranes Spectra/
Por 6 (3500, g/mol cut off) were purchased from VWR International.
The NMR experiments (δ scale; J values are in Hz) were carried out
on Jeol JNM-ECP Series FT NMR system at a magnetic field strength
Disulfanediylbis(ethane-2,1-diyl) Bis(2-hydroxyethoxycarbamate)
7. The procedure was analogous to that for the preparation of 6. CDI
(712 mg, 4.4 mmol) was used for the activation of 2,2′-dithiobisethanol
1 (322 mg, 2.1 mmol) in 6 mL of DCM. 2-Aminooxyethanol 4 (339
mg, 4.4 mmol) and DMAP (25.5 mg, 0.21 mmol) in 12 mL of DMF
containing pyridine (0.36 mL, 4.4 mmol) were added to the solution
of activated 1. After 20 h, DMF was evaporated under vacuo. The
concentrate was coevaporated with toluene and the residue was triturated
with a mixture of DCM (200 mL) and diethyl ether (85 mL). The
1
precipitate (217 mg) was collected by filtration and analyzed by H
NMR. It consisted of the product 7 in a mixture with imidazole side
product. The filtrate was evaporated to dryness and the residue was
suspended in 20 mL of DCM. This gave first a turbid solution from
which the pure product 8 was then separated as a small liquid phase.
The small phase was isolated from the bulk DCM phase yielding 156.4
1
of 9.4 T, operating at 400 MHz for H.
1
mg of pure 7 (0.43 mmol, 20.7%). H NMR (CD3OD): 4.36 (4H, t, 2
6-Hydroxycaproic Acid Hydrazide 3. ε-Caprolactone (4.4 mL, 41.23
mmol) was dissolved in 40 mL of ethanol. Hydrazine (2 mL, 41.23
mmol) was added dropwise to the obtained solution under vigorous
stirring. The reaction mixture was heated to reflux for 20 h and then
cooled to room temperature. The obtained colorless crystals were filtered
× -OCH2CH2S-, J ) 6.4 Hz), 3.88 (4H, t, 2 × -NHOCH2-, J ) 4.8
Hz), 3.70 (4H, t, 2 × -CH2OH, J ) 4.8 Hz), 2.97 (4H, t, 2 × -CH2S-,
J ) 6.4 Hz).
5,14-Dioxo-3,6,13,16-tetraoxa-9,10-dithia-4,15-diazaoctadecane-1,18-
diyl Bis(hydrazinecarboxylate) 12. The procedure was analogous to
that for the preparation of 11. The compound 7 (160 mg, 0.44 mmol)
was activated with CDI (173 mg, 1.07 mmol) in dry pyridine (3 mL)
for 2 h. Hydrazine (65 µL, 1.33 mmol) was added to the activated 7.
After 21 h, pyridine was evaporated and the residue was coevaporated
with toluene two times. The residue was redissolved in a minimal
amount of methanol and the concentrated methanolic solution was
poured into 130 mL of DCM, stirred for 5 min, and then kept for 1.5 h
at -20 °C. The precipitate was collected by filtration and dried under
vacuo overnight. Yield: 78.5 mg (0.17 mmol, 38.6%). 1H NMR (D2O):
4.43-4.22 (8H, m, 2 × -OCH2CH2S- and 2 × -NHOCH2-), 4.12-3.96
(4H, m, 2 × -CH2OCONHNH2), 3.01-2.90 (4H, t, 2 × -CH2S-). 13C
NMR: 169.7, 169.5, 74.7, 74.2, 63.1, 37.0.
1
off and dried under vacuum. Yield: 3.87 g (26.45 mmol, 64.2%). H
NMR (D2O): 3.47 (2H, t, HOCH2-, J ) 6.6 Hz), 2.10 (2H, t,
-CH2CONHNH2, J ) 7.3 Hz), 1.52-1.39 (4H, m, -CH2CH2CH2-),
1.25-1.17 (2H, m, -CH2CH2CH2-).
Disulfanediylbis(ethane-2,1-diyl) Bis(2-(6-hydroxyhexanoyl)hydra-
zinecarboxylate) 6. 2,2′-Dithiobisethanol 1 (771 mg, 5 mmol) was
placed in a round-bottom flask that was degassed and then filled with
argon. A total of 15 mL of dichloromethane (DCM) was added to the
flask followed by CDI (1.7 g, 10.5 mmol) under stirring. Stirring was
continued for another 2 h under argon at room temperature. 6-Hy-
droxyhexanoic hydrazide 3 (1.46 g, 10 mmol) was dissolved in hot
DMF (15 mL) in a separate flask. After cooling to room temperature,
DMAP (61 mg, 0.5 mmol) followed by pyridine (0.81 mL, 10 mmol)
were added to the DMF solution. The solution of the CDI-activated
2,2′-dithiobisethanol was added dropwise to the solution of 6-hydroxy-
hexanoic hydrazide over 1 h at room temperature. The mixture was
stirred for 18 h and DMF was evaporated under vacuo. The concentrate
was coevaporated with toluene and the residue was triturated with a
mixture of DCM (200 mL) and diethyl ether (50 mL). The insoluble
waxy product was collected by decantation, and dried under vacuo
Dimethyl 2,15-Bis(hydroxymethyl)-4,13-dioxo-5,12-dioxa-8,9-dithia-
3,14-diazahexadecane-1,16-dioate 8. 2,2′-Dithiobisethanol 1 (500 mg,
3.24 mmol) was placed in a round-bottom flask that was degassed and
then filled with argon. A total of 16 mL of DCM was added to the
flask followed by CDI (1.31 g, 8.1 mmol) under stirring. Stirring was
continued for another 2 h under argon at room temperature. Serine
methyl ester hydrochloride 5 (1.26 g, 8.1 mmol) was added to the
reaction mixture followed by triethylamine (1.36 mL, 9.72 mmol). The
mixture was stirred for 18 h and evaporated to dryness. The crude
material was purified by silica gel flush column chromatography, eluting
with 0-6% ethanol in DCM. The product was eluted from the column
with 6% ethanol/DCM. Yield: 534 mg (1.2 mmol, 37.1%) for two
isolated stereoisomers. 1H NMR (CDCl3): 6.07 and 5.77 (2H, two broad
1
overnight. Yield: 2.34 g (4.69 mmol, 94%). H NMR (CD3OD): 4.35
(4H, t, 2 × -OCH2CH2S-, J ) 6.2 Hz), 3.54 (4H, t, 2 × -CH2OH, J )
6.4 Hz), 2.98 (4H, m, 2 × -OCH2CH2S-), 2.22 (4H, t, 2 × -CH2C(O)-,
J ) 7.3 Hz), 1.65 (4H, m, 2 × -CH2CH2OH), 1.55 (4H, m, 2 × -
CH2CH2C(O)-), 1.40 (4H, m, 2 × -CH2CH2CH2OH). 13C NMR: 157.0,
63.2, 61.4, 37.0, 33.4, 32.0, 25.1, 25.0.

Functionalization of Hyaluronic Acid
Biomacromolecules, Vol. 11, No. 9, 2010 2253
singlets, 2 × -NH-), 4.43 (4H, t, 4 × -SCH2CH2O-, J ) 6.2 Hz),
4.43-4.28 (2H, m, 2 × -CH<), 4.04-3.90 (4H, m, 2 × -CH2OH),
3.79 and 3.78 (4H, two singlets, 2 × -OCH3), 2.97 (4H, t, 2 × -CH2S-,
J ) 6.2 Hz). 13C NMR: 171.1, 156.1, 63.3, 63.2, 56.2, 52.9, 37.9.
followed by dialysis against dilute HCl, pH 3.5 two times, the solution
was lyophilized to give the corresponding nucleophile-modified HA
15, 16, 17, or 18. The incorporation of nucleophile-terminated side
chains was verified by 1H NMR. Specifically, the peaks corresponding
to the native HA protons, such as acetamide protons at 1.9 ppm, 2′-,
3′-, 4′-, 5′-, and 6′-protons of HA disaccharide unit at 3.2-4.0 ppm, as
well as anomeric 1′-protons at 4.4 ppm, were compared with newly
appeared peaks of the grafted side chains. Additionally, the presence
of free nucleophile-terminated side chains was confirmed spectropho-
tometrically by reaction with trinitrobenzene sulfonic acid (TNBS).38
Synthesis of Aldehyde-Modified HA 20. HA-serine derivative 17
(370 mg, 0.96 mmol of disaccharide units) was dissolved in 45 mL of
deionized water. A 0.15 M aqueous solution of sodium periodate (5.8
mL, 0.96 mmol) was added to the HA-serine solution, which caused
formation of a gel. The gel was diluted with 60 mL of water, which
led to the partial dissolution of the gel. After 5 min, ethylene glycol
(0.5 mL, 9.6 mmol) was added to the mixture. The mixture was stirred
in the dark overnight during which the gel was completely dissolved.
The reaction solution was dialyzed against pure water two times (Mw
cutoff ) 3500) and finally lyophilized. Yield: -347 mg (94.4%). The
amount of aldehyde groups was obtained by reaction with tert-butyl
carbazate (TBC) followed by reduction with NaBH3CN. Briefly, HA-
aldehyde (∼20 mg) was dissolved in 2 mL of water and to this solution
was added the 0.5 M aqueous solution of TBC (10-fold excess per
molar amount of sodium periodate that was used in the preparation of
HA-aldehyde derivative). The mixture was stirred for 1 h at room
temperature after which a 0.5 M aqueous solution of NaBH3CN
(equimolar amount to that of TBC) was added to the mixture. The
mixture was allowed to react for 24 h at room temperature. The TBC-
modified HA was recovered by dialysis in 3500 MW cut off
tubing against water twice. 1H NMR of the obtained product was
examined and the peak corresponding to the tert-butyl substituent
((CH3)3COCONHNH-, δ ) 1.38 ppm) was compared with the peak of
HA acetamide protons at 1.9 ppm. Degree of aldehyde functionalization
in 20 (∼5%) was calculated from the amount of reacted TBC reagent.
Hydrogel Formation and Characterization. HA hydrogels were
prepared in cylindrical glass vials (10 mm in diameter) from 2% w/v
precursor solutions listed in Table 2. In a typical gelation experiment,
HA-aldehyde and HA-nucleophile derivatives were dissolved separately
in water and then mixed by addition of the HA-nucleophile solution
(0.5 mL) to the HA-aldehyde solution (0.5 mL), followed by brief
vortexing for several seconds. Gelation time was determined by a flow
test as the point at which the mixture would no longer flow under the
force of gravity. The gels were left in the capped vials for another 4 h
to complete cross-linking reactions. The cylindrically shaped hydrogels
were then carefully transferred from the vials and applied to the bottom
plate of AR2000 rheometer (TA Instruments, Inc., U.K.). The mechan-
ical properties were measured at a frequency of 0.1 to 10 Hz at 25 °C
using an 8 mm aluminum plate geometry. The gap was adjusted starting
from the original sample height and compressing the sample to reach
a normal force of about 50 mN (gap sizes were between 7 and 8 mm).
Enzymatic Hydrogel Degradation. In 4 h after mixing of the
corresponding components, the hydrogels were swollen in PBS for 24 h
and the swelling buffer then was diluted with HAse solution such that
the final concentration of hyaluronidase was 500 U/mL. The hydrogels
were then stored in PBS buffer containing HAse at room temperature
for several days.
Disulfanediylbis(ethane-2,1-diyl) Bis((1-hydrazinyl-3-hydroxy-1-oxo-
propan-2-yl)carbamate) 13. Compound 8 (400 mg, 0.9 mmol) was
dissolved in methanol (9 mL). Hydrazine (135 µL, 2.7 mmol) was added
to the mixture, which was then stirred for 2 days at room temperature.
During the reaction, the product was precipitated, which was collected
by filtration. The solid white material was dried under vacuum. Yield:
191 mg (0.43 mmol, 47.8%). 1H NMR (D2O): 4.33 (4H, t, 2 ×
-SCH2CH2O-, J ) 5.5 Hz), 4.19 (2H, t, 2 × -CH<, J ) 5.1 Hz), 3.79
(4H, d, 2 × -CH2OH, J ) 5.1 Hz), 2.97 (4H, t, 2 × -CH2S-, J ) 5.5
Hz). 13C NMR: 171.3, 157.9, 63.4, 61.4, 56.0, 37.1.
2-Bis(2-((1,3-dioxoisoindolin-2-yl)oxy)ethyl) O′1,O1-(Disulfanediyl-
bis(ethane-2,1-diyl)) Bis(hydrazine-1,2-dicarboxylate) 10. N-(2-Hy-
droxyethyloxyphthalimide) 9 (904 mg, 4.36 mmol) was placed in a
round-bottom flask that was degassed and then filled with argon. A
total of 13 mL of DCM was added to the flask followed by CDI (745
g, 4.59 mmol) under stirring. Stirring was continued for another 3 h
under argon at room temperature. Dithiobis(ethanol O-hydrazinocar-
bonyl) 2 (512 mg, 1.9 mmol) was dissolved in hot DMF (13 mL) in a
separate flask. After cooling to room temperature, DMAP (61 mg, 0.5
mmol) followed by pyridine (0.81 mL, 10 mmol) were added to the
DMF solution. The combined solution of 2 and DMAP in DMF-pyridine
was added dropwise to the solution of CDI-activated 9 over a period
of 10 min at room temperature. The mixture was stirred for 20 h and
DMF was evaporated under vacuo. The concentrate was coevaporated
with toluene and the residue was worked-up with ethyl acetate-water.
The organic phase was separated, washed with water, and finally dried
over MgSO4. The solvent was evaporated, yielding yellow oily crude
material, which was purified by silica gel flush column chromatography
eluting with ethyl acetate-hexane 1:1 (v/v) mixture followed by pure
1
ethyl acetate. Yield: 387 mg (0.53 mmol, 53%). H NMR (CDCl3):
8.00-7.73 (8H, m, arom.), 4.59-4.36 (12H, m, 4 × -CH2OCONH-
and 2 × >NOCH2-), 3.08-2.95 (4H, t, 2 × -CH2S-).
2-Bis(2-(aminooxy)ethyl) O′1,O1-(Disulfanediylbis(ethane-2,1-diyl))
Bis(hydrazine-1,2-dicarboxylate) 14. Compound 10 (385 mg, 0.52
mmol) was dissolved in a mixture of ethanol and DCM (8 mL, 3:1
v/v). Hydrazine (150 µL, 3.14 mmol) was added to the mixture, which
was then stirred for 2 days at room temperature. The formed precipitate
was filtered off and the filtrate was evaporated to dryness. After
subsequent drying under vacuum, 113 mg of 12 was obtained as a white
solid. Yield: 0.24 mmol (45.4%). 1H NMR (D2O): 4.43-4.18 (8H, m,
4 × -CH2OCONH-), 3.96-3.80 (4H, m, 2 × -CH2ONH2), 3.05-2.89
(4H, t, 2 × -CH2S-).
Modification of HA with Side Chains Terminated with Chemose-
lective Nucleophilic Groups. Hyaluronan was dissolved in deionized
water at concentration 8 mg/mL. The reagent (11, 12, 13, or 14) was
added to the HA solution at the reagent/HA disaccharide molar ratio
specified in Table 1, and the obtained mixture was stirred until complete
dissolution of the reagent. N-Hydroxybenzotriazole (HOBt) was
separately dissolved in a 1:1 (v/v) mixture of acetonitrile-water at
concentration 0.2 M and added to the solution of HA. Molar ratio of
HOBt to HA disaccharide was 1 (see Table 1). The pH of the resultant
solution was adjusted to 4.7 after which the coupling reaction was
initiated by addition of solid EDC (see Table 1 for EDC/HA
disaccharide feeding ratio) to the reaction mixture. The mixture was
stirred overnight. The pH of the reaction solution was slightly increasing
during this time which evidenced the coupling of the reagents to HA
carboxylates. The reaction solution was basified to 8.5 with 1 M NaOH
and DTT was added to the solution. A 5-fold molar excess of DTT
was used relative to the applied reagent (11, 12, 13, or 14) to ensure
the cleavage of disulfide bond of the reagent. The mixture was again
stirred overnight, after which the solution was acidified to 3.5 with 1
M HCl and transferred to a dialysis tube (Mw cutoff ) 3500). After
exhaustive dialysis against dilute HCl (pH 3.5) containing 0.1 M NaCl,
Cell Culture. Human dermal fibroblasts (hDFns) were cultured in
complete DME/F12 medium (Dulbecco’s modified Eagle’s medium/
Nutrient Mixture F-12 Ham with L-glutamine, 15 mM HEPES, and sodium
bicarbonate (DME/F12) supplemented with 10% fetal bovine serum). Cells
were maintained at 37 °C in 5% CO2 and used at passages 4 and 5.
Cytotoxicity of the Individual Gel Components. hDFns were seeded
in 24-well plates at concentration 5 × 104 cells/well in 1 mL of complete
medium and grown at 37 °C, 5% CO2. After 24 h, the old medium
was replaced with fresh medium containing 0.1 or 0.6% of the HA
derivatives. The HA derivatives were dissolved directly in cell culture

2254 Biomacromolecules, Vol. 11, No. 9, 2010
Ossipov et al.
medium and then passed through 0.8 µm sterile filter before being added
to the cells. Fibroblasts grown in complete cell culture medium were
used as a control. Cells grown in plain cell culture medium were used
as a positive control. After 48 h of culturing, cytotoxicity of the
individual components was examined by performing thiazolyl blue
tetrazolium bromide (MTT) assay. MTT dye was dissolved in PBS at
a concentration of 5 mg/mL and passed through a 0.22 µm sterile filter.
A total of 100 µL of the sterile MTT solution was added to each well
and the plate was incubated at 37 °C, 5% CO2. After 4 h the medium
was carefully removed and the dark blue crystals were dissolved in 1
mL of DMSO. The absorbance was measured at 570 nm, and the results
were compared with that of the control wells to determine relative cell
viability.
Cytotoxicity of the Gels. The 2% w/v solutions of the corresponding
gel components were placed in two separate 1 mL syringes, the tips of
which were subsequently interconnected by means of a luer-lock
adapter. The solutions were mixed by passing the contents of the
syringes from one to another. After 20 passages, the hydrogels were
formed and extruded from the syringes into cell culture inserts (Falcon,
1.0 µm pore size). hDFns were seeded in 24-well plates at concentration
of 50000 cells/well in 1 mL complete medium and grown at 37 °C,
5% CO2. After 24 h, the medium was changed and the inserts with 0.2
mL of the hydrogels were added in contact with the medium. After 3
days of culturing, the cytotoxicity of hydrogels was evaluated by
performing an MTT assay analogously to the cytotoxicity analysis of
the individual gel components.
ratios of hydrogels as a function of incubation time in PBS
buffer (Figure S2). This material is available free of charge via
the Internet at http://pubs.acs.org.
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1
Supporting Information Available. H NMR spectra and
structures of the reagents 12 and 14 (Figure S1) and swelling
BM1007986