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The preparation and biocompatible evaluation

of injectable dual crosslinking hyaluronic acid
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7, 4413 hydrogels as cytoprotective agents

Wanxu Cao,a Junhui Sui,a Mengcheng Ma,a Yang Xu,a Weimin Lin, b
Yafang Chen,a Yi Man,b Yong Sun, *a Yujiang Fan a and Xingdong Zhanga

Received 29th April 2019,
Accepted 2nd June 2019
DOI: 10.1039/c9tb00839j
rsc.li/materials-b
Injectable hydrogels attract a great deal of attention due to their in situ formation in vivo, which
minimizes the risk of major trauma during the surgical procedure as compared to traditional hydrogel
scaffolds. Click chemistry is widely used in the preparation of injectable hydrogels, although some of
them require photoinitiators or catalysts that may be cytotoxic and impair the proliferation of
encapsulated cells. In the present study, an injectable dual crosslinking hydrogel was designed by a
thiol–ene click reaction and conversion between sulfhydryl and disulfide bonds at a neutral pH. The
sulfhydryl group and acrylate group were successfully grafted onto HA and characterized using FT-IR
1
and H-NMR. The gelation time, morphology, injection force, swelling, degradation, mechanical
properties and drug release behavior of the hydrogel varied with the molecular weight of the hydrogel
(Mw = 0.1, 1.0, 2.0 MDa). MTT, FDA/PI staining and scanning electron microscopy (SEM) showed that the
encapsulated L929 proliferated well in different molecular weight hydrogels. It was also observed that in
a low molecular weight (0.1 MDa) hydrogel system, the cells were better distributed and secreted more
extracellular matrix. Subcutaneous injection in mice also demonstrated that hydrogels could support the
proliferation of encapsulated L929 cells in vivo, and no significant infiltration of peripheral cells into the
interior of the material was observed. These results indicate that this injectable hyaluronan-based
hydrogel is an excellent cell protectant and exhibits good biocompatibility, with potential for biomedical
applications such as cell delivery and postoperative anti-adhesion.

1. Introduction biochemical and mechanical properties could also improve cell

Direct injection delivery of cell suspensions has been intensively
used in the domains of clinical application due to its simple
operation and low invasiveness. However, such an approach
might bring about low cell survival and retention due to cell
shearing during injection, hypoxic microenvironments with
limited nutrient supply and dynamic mechanical stress in the
recipient tissue, and may even generate unsatisfactory therapeutic
efficiency. Compared with individual cell injection, biomaterials
could serve as 3D tissue engineering scaffolds for cell delivery,
which could protect cells from unwanted injection shearing,
provide cell growth spatial structural support, and improve
therapeutic efficiency.1,2 In addition, biomaterials with optimized
a
National Engineering Research Center for Biomaterials, Sichuan University,
29 Wangjiang Road, Chengdu 610064, P. R. China.
E-mail: sunyong8702@scu.edu.cn; Fax: +86-28-85417654; Tel: +86-28-85417654
b
Diseases and Department of Oral Implantology, Department of Oral Implantology,
West China Hospital of Stomatology, Sichuan University, Chengdu, 610041,
P. R. China
survival and retention, and promote cell-mediated tissue matrix
production, vascularization, and integration with endogenous
tissues.3,4 Tissue engineering scaffolds based on biomaterials
are mainly independent of implantable pre-fabricated and inject-
able in situ consolidation forming technology.5 The implantable
scaffolds require invasive surgery for transplantation and initiate
the risk of external trauma during the implantation procedure. In
addition, the pre-fabricated process might influence uniform
cell distribution in most individuals, and even cause a certain
amount of cell death. However, injectable scaffolds, as an
emerging strategy for therapeutic drugs and cells delivery, have
been widely explored for biomedical applications.6 They are self-
adaptable in matching complex defect tissue models under
physiological conditions, they could guarantee intensive mixing
between injectable biomaterials and encapsulate cells or drugs,
minimize the surgical trauma, and facilitate wound healing via a
minimally invasive manner to reduce patient pain and medical
expenses.7–10
Biocompatibility is one of the major issues associated with
implanted materials.11 Synthetic hydrogels are rich in chemical

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modification sites and are capable of providing better mechanical
properties. However, some chemical reactions involving the cross-
linking of hydrogels interfere with the biological processes of cells,
and the degradation products of certain scaffolds exhibit varying
degrees of cytotoxicity, which may lead to a sharp or long-term
decline in cell survival.12,13 Naturally derived hydrogels, such as
collagen and Matrigel, have excellent cell compatibility and bio-
degradability but they may cause inflammatory responses associated
with their immunogenicity.14 Hyaluronic acid (HA) derivatives as
injectable scaffolds have received widespread attention because of
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their excellent biocompatibility, moisture capacity, viscoelasticity,
lubricity and non-immunogenicity.15,16 Various chemical and
physical modification methods have been applied to obtain
hyaluronan-based hydrogels,17,18 such as enzymatically cross-
linking,16 photo polymerization,19 disulfide bond formation20
and click chemistry-mediated hydrogels21 for chemical cross-
linking, together with freeze–thaw-induced22 for physical cross-
linking. Click chemistry has been intensively demonstrated in
cross-linking hydrogel networks due to their significant features
such as high reaction speed, high selectivity, and mild reaction
conditions.23,24 Several click reactions have been greatly expanded
in the drug delivery and tissue engineering field including azide–
alkyne cycloadditions,25 thiol–ene/yne conjugation reactions,26
Diels–Alder and inverse electron demand Diels–Alder cyclo-
additions,27,28 strain-promoted azide–alkyne cycloadditions,29
tetrazine–norbornene reactions,30 and so on. Nevertheless, some
of these click reactions require transition-metal catalysts, UV light
irradiation, or complex of precursor synthetic approaches, which
complicate the experimental operations and restrict the applications.
Compared to other traditional cross-linking methods of fabricating
hyaluronic acid hydrogel, the Michael addition reaction conditions
of thiol–ene are relatively mild, and no initiator or cross-linking
agents are required, which guarantee the biocompatibility of the
injectable scaffolds.31–33 Moreover, hyaluronic acid lacks cell
adhesion sites and thus, it is difficult for cells to obtain good
adhesion; as such, hyaluronan-based biomaterials are widely
used for postoperative anti-adhesion, as cytoprotective agents
and carriers, and in other biomedical applications.34–36
In previous studies, Huang et al. designed a pH-responsive
hydrogel via a spontaneous amino–yne click reaction,37 which
had more serious cytotoxicity in high concentration. Further-
more, Segura et al. used acrylated hyaluronic acid and MMP
peptide to build an injectable hydrogel, but the strength of the
hydrogel was weak and its range of application is limited to brain
repair.38 Herein, we have designed an in situ-formed injectable
dual crosslinking hydrogel via a thiol–ene click reaction and
conversion between sulfhydryl and disulfide bonds. The use
of click chemistry ensured that the hydrogel could be formed in
a relatively short period of time, while the dual crosslinking
structure endowed the gel with more excellent stability and
intelligent responsiveness to cell proliferation. Acrylated hyaluronic
acid (HA-Ac) and thiolated hyaluronic acid (HA-SH) were synthe-
sized and characterized by 1H-NMR and FT-IR. Also, the effects of
molecular weight and concentrations of the polymer precursors on
the gelation time, morphology, injection force, swelling properties,
degradation rates, mechanical properties and drug release behavior
4414 | J. Mater. Chem. B, 2019, 7, 4413--4423
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Journal of Materials Chemistry B
were investigated in this study. Finally, the biocompatibility of
these hydrogels and the influence of molecular weight on
cytoprotective were studied in vitro and in vivo.
2. Materials and methods
2.1. Materials
Hyaluronic acid (HA, cosmetic grade, Mw = 0.1, 1.0, 2.0 MDa)
was obtained from Bloomage Freda Biopharm Corporation
(Shandong, China). 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide
hydrochloride (EDCI, 99%), N-hydroxysuccinimide (NHS, 99%),
sodium chloride (NaCl, 99%), ethylenediaminetetraacetic acid
(EDTA, 99%), cysteamine hydrochloride (CSAHCl, 99%), adipic
acid dihydrazide (ADH, 99%), N-acryloxysuccinimide (NHS-Ac),
dithiothreitol (DTT, 99%) and 4-(2-hydroxyethyl)-1-piperazine
ethanesulfonic acid (HEPES) were purchased from Best-Reagent
Corporation (Chengdu, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-
diphenyl-2-H-tetrazolium bromide (MTT, 98%), DMEM medium
(Hyclone) and phosphate buffered saline (PBS, 0.0067 M) were
purchased from Thermo Fisher Scientific Corporation (USA). Fetal
bovine serum (FBS, Gibco, South America origin) was bought from
Life Technologies Corporation (USA). All other chemicals were
used as-received unless otherwise specified.
2.2. Fabrication of the HA-SH–Ac-HA hydrogel
2.2.1. Preparation and characterization of hyaluronic acid
derivatives (HA-SH, HA-ADH, and HA-Ac). Thiolated hyaluronic
acid (HA-SH) was synthesized through the following process as
a representative example. Firstly, HA (1 mmol) and NHS
(2 mmol) were dissolved thoroughly in 80 mL of deionized
water under stirring at room temperature. Then, EDCI (3 mmol)
in solid form was added into the mixture solution and reacted
for 2 h to activate the carboxylic group of HA. Subsequently,
CSAHCl (3 mmol) was dissolved in 10 mL of deionized water
and added to the mixture solution. The pH of the mixture
solution was adjusted to 4.75 by adding 1.0 M NaOH or 1.0 M
HCl during the reaction process. After stirring for 24 h, the
solution mixture was transferred to the dialysis tubing (8000–
14 000 MWCO) and dialyzed exhaustively against dilute HCl
solution (pH 3.5) containing NaCl (concentration gradient from
100 mM to 0) for 72 h. The acidified solution was then
lyophilized to yield HA-SH as a spongy solid. The chemical
structures of HA and HA-SH were determined by 1H-NMR
(400 MHz, Bruker AMX-400, USA) using D2O as a solvent and
FT-IR (Nicolet 6700, USA).
Acrylated hyaluronic acid (HA-Ac) was prepared using a two-
step synthesis through the following process. Firstly, HA was
activated to get the HA-NHS solution as described above. This
was then added into 50 mL of ADH (10 mmol) deionized
solution, and the pH value of the mixture solution was kept at
4.75. After 24 h, the solution was transferred to the dialysis tubing
(8000–14 000 MWCO) and dialyzed against NaCl solution
(concentration gradient from 100 mM to 0) for 72 h. The purified
intermediate HA-ADH was lyophilized and stored at 20 1C. Next,
HA-ADH (1 mmol) was reacted with NHS-Ac (5 mmol) in HEPES
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buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.2) for
12 h at room temperature, and then dialyzed for 72 h under the
same conditions. The solution was then lyophilized to yield HA-Ac as
a spongy solid. The chemical structures of HA-ADH and HA-Ac were
determined by FT-IR and 1H-NMR using D2O as a solvent.
2.2.2. The fabrication of HA-SH–Ac-HA hydrogel. The
mixed solution of HA-SH and HA-Ac can rapidly crosslink under
mild conditions by the Michael addition reaction between thiol
groups and double bonds, while the free sulfhydryl groups
can also undergo oxidation reactions to form disulfide bonds
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by self-crosslinking. Equal volumes of lyophilized HA-SH and
HA-Ac polymer (Mw = 0.1, 1.0, 2.0 MDa) were dissolved in water
at pH 3.5 and 7, respectively, and then mixed to form a
homogeneous acidic solution. To fabricate the hydrogel, the
pH value of the mixture solution was adjusted to 7.4 to get a
neutral precursor solution by adding 1.0 M NaOH. Afterwards,
the freshly prepared neutral precursor solution was immediately
injected into the self-made annular molds (diameter 8.5 mm,
height 3.0 mm) to acquire the HA-SH–Ac-HA hydrogel.
2.3. Characterization of the HA-SH–Ac-HA hydrogel
2.3.1. The gelation time, morphology and injection force.
Rheology measurements were performed to analyze the accurate
gelation time using a TA Discovery DHR-2 rheometer with
parallel-plate geometry (40 mm) and a 1.0 mm gap. The dynamic
strain sweep was operated with constant ramp strain of 10% and
frequency of 1 Hz at time sweep in 250 s. Meanwhile, an integrated
temperature controller was used to maintain the temperature at
37 1C. To investigate the influence of the molecular weight of HA
(Mw = 0.1, 1.0, 2.0 MDa) and polymer concentration (10 mg mL1,
20 mg mL1) on the gelation time of the hydrogel, a series of
HA-SH–Ac-HA solutions were prepared and the gelation time
was recorded. The gross view of each sample was investigated
using a digital camera. Subsequently, the hydrogel was rinsed
three times with deionized H2O and immersed in liquid nitro-
gen immediately before lyophilization. The cross-section of the
hydrogel was further sputter-coated with a gold layer to observe
the morphology and internal structure by scanning electron
microscopy (SEM, HITACHI S-800, Japan). The pore size of the
hydrogel was analysed by ImageJ software (1.51j8, National
Institutes of Health) based on SEM images. The injection force
was measured using an electromechanical universal testing
machine (Shimadzu Autograph AGS-X, Japan) with a 30 mm min1
crosshead displacement up to a maximum load of 500 N. A 1 ml
syringe (needle gauge: 26 G (0.45 mm); 7 mm diameter and 146 mm
in length) fitted with a cannula of 5 mm inner diameter and 75 mm
in length was applied. Each sample was measured in five replicates.
2.3.2. The swelling properties. Six groups of hydrogel
solution (pH = 7.4) were immediately injected into self-made
annular molds (diameter 8.5 mm, height 3.0 mm) to form
hydrogels at 37 1C. The hydrogels were freeze-dried and weighed
(W0) and then immersed into 10 mL PBS buffer (pH = 7.4) and
placed in a constant temperature shaker (ZHWY-2012C, Shanghai
Zhicheng, China) at 90 rpm and 37 1C. To calculate the swelling
rate, the hydrogels were weighed (W1) at a certain time interval
until swelling equilibrium was reached. Every sample was
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measured in three replicates. The swelling rate of the hydrogel
was calculated by the following formula:
Swelling ratio = (W1  W0)/W0
2.3.3. The degradation performance. The six groups of
prepared hydrogels were weighed (W0) as mentioned above
and then the degradation performances were measured using
two methods. On the one hand, they were immersed in PBS
solution containing hyaluronidase (50 mg mL1) and placed in a
constant temperature shaker at 90 rpm at 37 1C. On the other
hand, they were immersed in PBS solution containing DTT
(0.1 mM) and placed in a constant temperature shaker at 90 rpm at
37 1C. To calculate the weight loss percentage, the hydrogels were
taken out at a certain time point, washed in distilled water and
weighed again (Wr). Every sample was measured in three replicates.
The degradation behavior of the hydrogels was expressed as the
percentage of weight loss and was calculated as follows:
The percentage of the remaining hydrogel weight = Wr/W0  100%
2.3.4. Dynamic mechanical analysis (DMA). Hydrogel samples
of six groups were prepared as mentioned above for mechanical
analysis. The storage modulus (G0 ) and loss modulus (G00 ) of the
hydrogels were measured using a Dynamic Mechanical Analyzer
(DMA, TA-Q800, USA) instrument in the multi-frequency mode
(amplitude of 40 mm, a preload force of 0.002 N and a force track
of 105%) with a fixed frequency of 1–10 Hz at room temperature.
Every sample was measured in three replicates.
2.3.5. The simulation of albumen release behavior in the
hydrogel in vitro. Hydrogel samples of six groups were prepared
as mentioned above for in vitro simulation of the controlled
release behavior. BSA albumen (1 mg) was added to a certain
concentration of acidic HA-SH solution, shaken evenly with a
vortex shaker, and then an equal volume of HA-Ac solution was
added. After forming a homogeneous solution, the pH of the gel
was adjusted by adding 1.0 M NaOH. After lyophilization, six dry
hydrogels loaded with BSA were obtained at the final concentration.
Six dry BSA-loaded hydrogels were immersed in 5 mL of PBS buffer
in a 37 1C water bath to study the in vitro release behavior of the
BSA. At the set time point, 0.1 mL of the release solution was taken
while the same amount of PBS solution was added to the release
system. Next, 5 mL of Coomassie Brilliant Blue G250 solution was
added to 0.1 mL of the release solution, the absorbance was
measured at 595 nm using an ultraviolet spectrophotometer, and
set in parallel three times to calculate the cumulative release of BSA
according to the standard absorption curve. The cumulative release
of BSA (Mt) was calculated as follows:
The cumulative release of BSA (Mt) = ct  5
+ (c1 + c2 + c3 +    + ct1)  0.1
The percentage of the cumulative release of BSA (CR) was
calculated as follows:
The percentage of the cumulative release of BSA (CR)
= Mt/M0  100%
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where Mt is the cumulative release of BSA at time t, ct is the
concentration of BSA released in the medium at time t, and M0
is the initial negative initial loading of BSA in the drug-loaded
hydrogel. Every sample was measured in three replicates.
2.4. Cytotoxicity, cell viability and proliferation in vitro
2.4.1. Cytotoxicity in vitro. MTT experiments were performed
to evaluate the cytotoxicity of the hydrogel precursor polymers
(HA-SH, HA-ADH, HA-Ac) on mice fibroblast cells (L929) and human
breast cancer cells (MCF-7). L929 cells and MCF-7 cells were cultured
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using standard procedures in DMEM (containing 1% penicillin–
streptomycin and 10% FBS) and subsequently suspended and
placed in a 96-well plate with a concentration of 2000 cells per well
for 48 h in an incubator (37 1C, 5% CO2). Meanwhile, the hydrogel
precursor polymers with different molecular weights (Mw = 0.1,
1.0, 2.0 MDa) were dissolved in PBS and diluted with cell
culture medium to gradient concentrations from 4 mg mL1 to
2500 mg mL1. Afterwards, the cell culture medium was removed
from the wells and replaced with hydrogel precursor polymer
solutions. After 48 h, the MTT assay was carried out. Finally, the
absorbance was measured using a Thermo Scientific Varioskan
Flash Multiscan Spectrum at the wavelength of 490 nm.
2.4.2. Fabrication of 3D cell-hydrogel constructs. For cell
encapsulation, spongy HA-SH and HA-Ac solids were soaked in
75% ethanol for 5 h, and then the HA-SH and HA-Ac polymers
were dried overnight on a clean bench. Thereafter, the dried
HA-SH and HA-Ac were dissolved in serum-free DMEM medium,
and 1.0 M NaOH and 1.0 M HCl solutions (0.22 mm filter to sterilize)
were prepared to adjust the pH value to 7.4. L929 suspension
(100 mL) was immediately added to the polymer solution to
obtain the mixture solution with a cell concentration of 2 
106 cells per mL. The freshly prepared mixture solution was
immediately injected into a cylindrical mold (8.5 mm in diameter
and 3.0 mm in length). After several minutes, the hydrogels were
detached from the mold and immersed into DMEM medium
containing 1% penicillin–streptomycin and 10% FBS on 6-well
tissue culture plate at 37 1C in 5% CO2 (MCO-15AC, SANYO, Japan).
2.4.3. Cell viability and proliferation. After the cell-hydrogel
constructs were cultured for 1, 4, 7, 14, 21 and 28 days in vitro,
respectively, the constructs were removed. Live/dead assays were
used to observe the cell viability and distribution of encapsulated
cells. The cell-hydrogel constructs were immersed in PBS solution
containing 1 mg mL1 of fluorescein diacetate (FDA) (live, green) and
1 mg mL1 of propidium iodide (PI) (dead, red) for 2 min to stain the
cells, and then washed with PBS solution again. The viability and
distribution of L929 in hydrogels were observed on a Leica TCS SP5
confocal laser scanning microscope (CLSM). The proliferation of
L929 cells was quantified by MTT assays. After 4, 7, 14, 21 and
28 days of cultivation, the proliferative capacity of L929 was
measured. The optical absorbance value of the solution in each
well was measured at 490 nm using a Multiscan Spectrum
(Varioskan Flash, Thermo Fisher Scientific, USA). Each sample
was replicated three times.
2.4.4. Cell morphology and cytoskeletal F-actin staining.
The morphology of L929 encapsulated in hydrogels was observed
using a scanning electron microscope (SEM). The samples were
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Journal of Materials Chemistry B
fixed in 2.5% glutaraldehyde solution for 2.5 h at room temperature.
They were subsequently dehydrated against a graded series of
alcohol (20%, 40%, 60%, 80%, 100%, 100%, 100%) for 15 minutes,
and dried by critical point drying for 2 hours, and then cryo-
fractured in liquid nitrogen to observe the internal structure by
SEM. Cytoskeletal F-actin stain was performed by using rhodamine–
phalloidin (red, Ex = 496 nm, Em = 516 nm) and DAPI (blue, Ex =
364 nm, Em = 454 nm) after incubation for 4, 7, 14 days. More
specifically, the cell-hydrogel constructs were immersed in PBS to
remove the culture medium, and then were treated with 4.0%
formaldehyde to fix cells. Subsequently, the cell membranes were
ruptured by 0.5% Triton X-100 solution, and the cytoskeletal F-actin
of cells was stained by rhodamine–phalloidin for 40 minutes, next,
the DAPI solution was used to stain cell nucleus for 1 minute. The
fluorescence pictures of L929 cells were taken by CLSM.
2.5. Biocompatibility studies in vivo
All animal studies were approved by the Sichuan University
Medical Ethics Committee. All animal procedures were performed
in accordance with the guidelines for the care and use of
Laboratory Animals of Sichuan University. Female BALB/c mice,
aged 5–6 weeks, were supplied by the Experimental Animal
Center in Chengdu Dashuo Corporation (Chengdu, China).
They were fed ad libitum in a standard animal facility with
12 h light–dark cycles and kept at a constant temperature of
20–22 1C and a relative humidity of 50–60%. The sterilized
precursor polymer solution was prepared by the method as
described above. Subsequently, 0.15 mL sterilized precursor
mixture solutions (Mw = 0.1, 1.0, 2.0 MDa, n = 3 per time point)
were subcutaneously injected separately into the back of the
female BALB/c mice using a syringe with a 26 gauge needle. On
the 7th, 14th, 21st, and 28th days, the mice were euthanized,
and the hydrogels were removed and recorded with a digital
camera, and were promptly stained with FDA/PI and then
observed by CLSM to evaluate the cell viability in vivo as
mentioned in the experimental procedure in Section 2.4.3. For
histopathological evaluation, the freshly acquired hydrogels
were washed twice with PBS solution, and then fixed by 4%
paraformaldehyde solution for 48 h at 4 1C. The hydrogel pieces
were embedded in optimal cutting temperature compound (OCT,
Tissue-Tek) and were then sliced into a thickness of 5–10 mm. These
samples were stained by hematoxylin–eosin (H&E) and DAPI.
2.6. Statistical analysis
The statistically significant differences among groups were
verified using Student’s t-test. Differences were considered
significant if P o 0.05 (*), P o 0.01 (**) or P o 0.001 (***)
and were marked accordingly in the figures.
3. Results and discussion
3.1. Synthesis and characterization of HA-SH/HA-ADH/HA-Ac
Chan et al. believed that the sequence of reaction efficiency was
propylmaleimide 4 diethyl fumarate 4 diethyl maleate 4
dimethylacrylamide 4 acrylonitrile 4 ethyl crotonate 4 ethyl
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Journal of Materials Chemistry B
cinnamate 4 ethyl methacrylate.39 Besides, Morgan et al. studied
the influence of the molecular structure reaction of thiol–ene by
experimental means. It was believed that the reaction rate was
mainly determined by the addition rate of sulfhydryl and double
bond, and the decrease in the charge density of the CQC double
bond resulted in the lower reaction efficiency.40 According to the
above theory, the designed Michael addition reaction in this
paper had a relatively high reaction efficiency. The synthesis
protocol for the hyaluronic acid derivatives is shown in Fig. 1A. At
first, the carboxylic group of HA was reacted with NHS to prepare
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the HA-NHS active ester using EDCI as the condensing agent.
Then, HA-NHS was reacted with ADH/CSAHCl to synthesize
the HA-ADH/HA-SH polymer. Subsequently, to obtain the pure
product, HA-SH raw product was dialyzed exhaustively against
dilute HCl aqueous solution (pH = 3.5) which prevented the thiol
groups from reacting with each other to form the disulfide bond,
and the addition of NaCl could decrease the viscosity of the
solution. The HA-ADH was prepared by a carbodiimide-mediated
coupling reaction between HA and ADH, and was dialyzed against
100 mM NaCl solution to acquire a pure product. HA-Ac was
prepared via the nucleophilic substitution of NHS-Ac with HA-ADH
in HEPES buffer, and the product was purified as HA-Ac as
above. The reaction efficiency of the thiol–ene click reaction was
significantly affected by the different double bond activities on
different molecular structures.
As shown in Fig. 1D, HA-ADH has a broad association peak
at 3300–3400 cm1. As a derivative of HA, an amino group was
added to the HA-ADH chain, and the amino group has an
absorption peak at 3300–3400 cm1. However, the hydroxyl group
on the chain also has an absorption peak at 3200–3650 cm1, so
that a broad association peak appeared, which was as a result of
the superposition of the stretching vibration peaks of the amino
Fig. 1 The synthesis protocol (A), degree of substitution (B), 1H-NMR (C), and FT-IR (D) of the hyaluronic acid derivatives (HA-SH, HA-ADH and HA-Ac)
are presented.
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group and the hydroxyl group. Comparatively, HA-ADH had an
absorption peak at 1650 cm1, which was due to the stretching
vibration of –CQN, while two absorption peaks at 1607 cm1 and
1560 cm1 were attributed to the stretching vibration of –CQO.
At 1660 cm1, HA-Ac had an absorption peak, which was due to
the stretching vibration of the double bond. The characterization
of thiolated hyaluronic acid was conducted as previously
reported.20,41 In the HA-SH spectrum, a sharp peak appeared at
1665 cm1 as compared with HA, showing the existence of the
amide I band. Due to the presence of the III band, peak splitting
occurred at 1260 cm1. The above results confirmed the success-
ful modification of ADH, Ac and SH with hyaluronic acid.
The 1H-NMR spectrum was used to confirm the existence of
the conjugated thiol groups, and the typical spectra of native
HA and thiolated HA are shown in Fig. 1C. Compared with the
spectrum of native HA, it was obviously seen that new resonant
peaks of HA-SH appeared at 2.8 ppm, which represented the
methylene protons of the –CH2CH2SH.42 The degree of sub-
stitution could be calculated by taking the ratio of peaks at
2.8 ppm to the singlet peak of the acetyl methyl protons in HA
(1.88 ppm). The 1H-NMR spectrum of HA-ADH showed new
resonant peaks at 1.6 ppm and 2.3 ppm, representing the eight
hydrogens of the methylene groups on the ADH. The degree of
substitution could be calculated by taking the ratio of peaks at
1.6 ppm and 2.3 ppm to the singlet peak of the acetyl methyl
protons in HA (1.88 ppm).43 The 1H-NMR spectrum of HA-Ac
showed new resonant peaks at 5.65 ppm and 6.2 ppm, representing
the cis and trans acrylate hydrogens, respectively. The degree of
substitution was calculated by taking the ratio of peaks at 5.65 and
6.2 ppm to the singlet peak of the acetyl methyl protons in HA
(1.88 ppm);44 the higher the molecular weight of HA was, the
lower the degree of substitution was (Fig. 1B). The possible
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reason is that the high viscosity for high molecular weight HA
could result in lower liquidity,45 which might decrease the
opportunity for the reaction substrates to touch each other
and cause lower reaction activity.
3.2. Preparation and characterization of the dual crosslinking
hydrogels
The fast thiol/acrylate Michael addition reaction contributed to
the establishment of the primary network. The disulfide bonding
network as a secondary network could be slowly and spontaneously
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formed by the oxidation of thiol groups. Meanwhile, the secondary
network further stabilized and strengthened the primary network.
3.2.1. Gelation time. In the rheological test, the intersec-
tion of the storage modulus and the loss modulus indicates the
gelation time at the molecular level as shown in Fig. 2A and B.
The molecular weight and solution concentration of hyaluronic
acid both quite significantly affect the gelation time, but the
concentration exerted a greater impact on the gelation time.
Specifically, at a molecular weight of 2.0 MDa, the gelation can
be quickly completed at a minimum of 10 s; with the decrease in
molecular weight, the gelation time gradually increased to about 2
minutes. Compared with the lower concentration, increasing the
precursor solution concentration to 20 mg mL1 enhanced the
probability of the Michael addition reaction between reactive
functional groups per unit volume, which accelerated the
formation of 3D networks.
3.2.2. Morphology. The macroscopic pictures and internal
network structures of the hydrogels with different molecular
weights and concentrations of the hydrogels are shown in
Fig. 2B and D. It can be seen that all the hydrogels are transparent.
Pore size is an important parameter of three-dimensional porous
scaffolds; it has a great impact on material exchange inside and
outside the scaffold, affecting cell proliferation and regulating cell
physiology.46 A typical uniform sponge-like porous structure with a
pore size of 5 mm to 30 mm was observed. With the decrease in
molecular weight and concentration, the porosity of the hydrogel
network increased as shown in Fig. 2E.
3.2.3. Injection force. The HA-Ac and HA-SH were dis-
solved in deionized water and their final concentrations were
adjusted to 20 mg mL1 and 10 mg mL1, respectively by
mixing. The result (Fig. 2C) showed that the injection force of
20 mg mL1 (8–13 N) was significantly higher than that of
10 mg mL1 (2–4 N), while the molecular weight had little effect
on the injection force. One possible reason was that the increase
in hyaluronic acid concentration resulted in the increase in
covalent cross-linking sites in the dual crosslinking hydrogels.
3.2.4. Swelling and degradation. As shown in Fig. 3C, all
the hydrogels reached the equilibrium of swelling in about
10 hours, and the maximal value of the swelling ratio was up to
24 (0.1 MDa, 10 mg mL1). Generally speaking, the lower the
concentration and molecular weight of hyaluronic acid were,
the greater the equilibrium swelling rate of the gel was. With
the molecular weight increasing, the single molecular chains
formed many hydrogen bonds, they were more likely to become
entangled, and at the same time, the cross-linking points were
densely added, resulting in a decrease in the swelling ratio.
4418 | J. Mater. Chem. B, 2019, 7, 4413--4423
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Journal of Materials Chemistry B
Meanwhile, the increase in the hyaluronic acid concentration
ensured greater hydrogen bond content.
The in vitro degradation profile of hydrogels is shown in
Fig. 3A and B. With the molecular weight and concentration
increasing, the degradation rate gradually decreased in PBS
solution containing hyaluronidase. This was due to the fact that
the glycosidic bonds in the molecular structure of hyaluronic
acid hydrogel with low molecular weight were more likely to be
exposed and then be degraded. It was noteworthy that the early
degradation rate (from 0 h to 40 h) was slightly higher than that
of the late stage. However, in PBS solution containing DTT, only
the disulfide bonds in the hydrogel were degraded. In Fig. 3B,
the hydrogels degraded faster before 8 h, and then their masses
gradually stabilized. This phenomenon was due to the rapid
collapse of the secondary structure constructed by the S–S bond
in the first 8 h hydrogel under the action of DTT. After the DTT
completely degraded the S–S bond, the six groups of hydrogels
were equivalent to degradation in PBS buffer alone; this process
was very slow and required a long degradation time.
3.2.5. DMA. The storage modulus (G 0 ) and loss modulus
(G00 ) of hydrogels represent the energy stored or consumed per
unit of strain, respectively. As shown in Fig. 3D and E, with the same
concentration of hydrogels, the higher the molecular weight, the
greater the storage modulus, and the storage modulus (G0 ) slightly
increased with increasing frequency. When the concentration of
hyaluronic acid increased from 10 mg mL1 to 20 mg mL1, the
storage modulus increased by about twofold. On the other hand, the
loss modulus of the hydrogel progressively declined along with
increasing frequency. This trend showed that the hydrogels could
increase the storage modulus (G0 ) and reduce the loss modulus (G00 )
when subjected to periodic higher frequency shock.
3.2.6. The simulation of albumen release behavior in the
hydrogel in vitro. Fig. 3F shows the release profile of BSA in six
groups of hydrogels at 37 1C in PBS buffer. It can be seen from
the figure that in the PBS buffer solution the BSA was released
faster in the first 48 hours in the six groups of hydrogels, and the
cumulative release percentage of BSA in the high concentration
group (20 mg mL1) of hydrogels was about 40%; for hydrogels in
the low concentration group (10 mg mL1), the cumulative
release percentage of BSA was up to 75%. After 48 hours, the
release rate of BSA from six groups hydrogels was slowed down. The
cumulative release percentage of BSA in the high concentration
group (20 mg mL1) hydrogel was about 60%, while in the low
concentration group (10 mg mL1), the hydrogel was about 83% in
the 240 hours. The hydrogel from the high concentration group
(20 mg mL1) had a lower release rate than the low concentration
group (10 mg mL1) because of its crosslink density. The higher
crosslink density of the hydrogel and the smaller average molecular
distance between adjacent cross-linking points prevented the diffu-
sion of BSA molecules from the hydrogel. However, even in the
lowest cross-linking hydrogel group D (0.1 MDa, 10 mg mL1), BSA
was not completely released in 240 hours, probably because a partial
Michael addition reaction occurs between the amino on the BSA
and the CQC double bond on the HA-Ac during BSA loading,
which allows the BSA bond in the gel network. These results
indicate that the injectable hydrogel has a good sustained
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Fig. 2 Rheology measurements (A), gelation time and gross view of each sample (B), injection force (C), microstructure (D) and mean pore size (E) of
hyaluronic acid hydrogels are presented; scale bar: 10 mm. Error bars represent standard deviation (N = 5) in (C) and standard error of mean in (E).
(*P o 0.05, **P o 0.01, ***P o 0.001).

release of BSA in PBS solution, which could be applied in long-
term controlled release medium and protective agents for
proteins or cells, and effectively maintain their structure and
function. The above results indicate that the interior structure,
gelation time, injection force, swelling and degradation properties,
mechanical modulus and drug release behavior of dual crosslinking
hyaluronan-based hydrogels were precisely controlled by adjusting
the concentrations and molecular weights of HA-Ac and HA-SH.
These features facilitate the application of injectable dual cross-
linking HA-Ac–SH-HA hydrogels as scaffold materials for cell, drug
and bioactive substance delivery.
3.3. Cell viability and proliferation in vitro
In order to explore the cytotoxicity of the prepared dual cross-
linking hydrogels, normal cells (L929) and cancer cells (MCF-7)
were co-cultured with low-concentration aqueous solutions of

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Fig. 3 The in vitro simulated degradation behavior under different environmental response conditions with hyaluronidase (A) and DTT (B). The swelling
ratio (C), storage modulus (D), loss modulus (E), and simulation of albumen release behaviour (F) of dual crosslinking hyaluronic acid hydrogels with
different molecular weights.

hydrogel precursor polymers (HA-SH, HA-Ac, HA-ADH) in 96- concentration increasing (from 4 to 2500 mg mL1), but the
well plates, respectively. The results of the MTT assay showed viability of all materials was higher than 80% against L929 cells
that the cell activity presented a slight decline with solution (Fig. 4A) and MCF-7 cells (Fig. 4B), indicating good biocompatibility.

Fig. 4 Biocompatibility of hydrogel precursor polymers (HA-SH, HA-ADH, HA-Ac) against L929 (A) and MCF-7 (B). Live/dead staining of cell-hydrogel constructs
after 1 d, 4 d, 7 d, 14 d, 21 d and 28 d of culture are presented, where the green stain represents the live cells and the red stain represents dead cells (C). The
proliferative capacity of the L929 in hydrogel (D). The scale bar: 100 mm. Error bars represent standard deviation (N = 3) in (D). (*P o 0.05, **P o 0.01, ***P o 0.001).

4420 | J. Mater. Chem. B, 2019, 7, 4413--4423 This journal is © The Royal Society of Chemistry 2019

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Furthermore, FDA/PI staining and MTT assays were applied
to observe the cell proliferation profiles in hydrogels. As shown
in Fig. 4C, all the L929 cells were homogeneously distributed in
HA-Ac–SH-HA hydrogels with different molecular weights, and
had a prominent effect of agglomeration proliferation with the
extension of co-culture time. Interestingly, the HA-Ac–SH-HA
hydrogels with high molecular weights displayed more remark-
able cell agglomeration as compared to that of low molecular
weights, just as shown in Fig. 4C (2.0 MDa, 28 d). This
was consistent with the conclusion of other similar studies.20
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Also, the cell displayed a circular morphology, probably because
hyaluronan-based materials lack cell adhesion sites, which inhibit
the stretching of encapsulated cells. In Fig. 4D, the quantitative
analysis of cell proliferation was determined by MTT assay. It
could be seen that the number of cells in all groups increased with
time, which indicated that the hydrogels could effectively support
the proliferation of the encapsulated cells.
To further observe the morphology of the cells and the
secretion of the matrix, SEM and cytoskeletal staining were
performed. The results of SEM and phalloidin/DAPI staining
showed that almost all cells exhibited spherical growth.
The SEM (Fig. 5A) showed the abundant extracellular matrix
that was secreted by the encapsulated cells with increased
incubation time, which was mainly distributed on the surface of
Fig. 5 Cell morphologies of cell-hydrogel constructs after 1 d, 4 d, 7 d, 14 d, 21 d and 28 d of culture are presented (A); scale bar: 10 mm. Cytoskeletal
F-actin staining cell-hydrogel constructs after 4 d, 7 d and 14 d of culture are presented (B); scale bar: 25 mm.
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the cells and the scaffold. Similarly, the agglomeration proliferation
phenomenon was also detected. In addition, the phalloidin/DAPI
staining (Fig. 5B) could visually confirm the morphology of the
cytoskeleton and nucleus. These phenomena were consistent
with the previous results.
3.4. Biocompatibility studies in vivo
The injection force of the 20 mg mL1 gel precursor solution
was obviously higher than that of the 10 mg mL1 gel precursor
solution, which would inconvenience the actual injection operation
due to a reduction in the actual operation time. Meanwhile, the
higher the concentration of the gel precursor solution, the
faster the gelation speed. Therefore, the final concentration
of the 10 mg mL1 was selected for injection. At 1, 2, 3 and 4
weeks after injection, no obvious swelling phenomenon was
detected on the back of the mice. After euthanizing the mice, these
hydrogels were removed and presented excellent morphology
(Fig. 6A). FDA/PI staining was performed to observe the cell
viability by CLSM. As shown in Fig. 6B, compared with blank
hydrogel groups (1.0 MDa as control), a large number of living
cells with agglomerating growth were observed, and the cell
density was obviously increased after 4 weeks of culture.
H&E (Fig. 7A) staining results provided a histological
overview of the tissue response at the site of the injection.
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Fig. 6 Observations of the subcutaneous injection of hydrogel in BALB/c mice (A), and live/dead staining of cell-hydrogel constructs after 7 d, 14 d, 21 d and 28 d
of subcutaneous injection are presented, where the green stain represents the live cells and the red stain represents dead cells (B). Scale bar: 500 mm and 100 mm.

Fig. 7 HE staining (A) and DAPI staining (B) of cell-hydrogel constructs with different molecular weights after 7 d, 14 d, 21 d and 28 d of subcutaneous
injection; scale bar: 500 mm.

These L929 cells encapsulated in hydrogels were distributed
uniformly after subcutaneous injection. An orderly proliferation
phenomenon was obvious 4 weeks after injection. Relatively few or
no cells were noticed in the blank hydrogel group from 1 week to 4
weeks. DAPI (Fig. 7B) staining was adopted to confirm this
observation. On prolonging the time to 4 weeks, the intensity of
the internal fluorescence staining of the hydrogels gradually
increased, implying that the encapsulated cells were continuously
proliferating over time. Likewise, the blank hydrogel group only
showed a faint fluorescence. These results indicated that injectable
dual crosslinking HA-Ac–SH-HA hydrogel was of ideal biocompat-
ibility and could support cell proliferation. It should be noted that
rather high levels of infiltrating inflammatory cells were found at
early time points (at days 7 and 14) and gradually decreased with
extending the time to 28 days; they were mainly localized at the
boundary between surrounding tissue and the hydrogel. The
infiltration of inflammatory cells was hardly observed in the
inner areas of the hydrogel, suggesting that the hydrogel did not
induce a local inflammatory reaction in the long-term implantation
process. Therefore, the injectable dual crosslinking HA-Ac–SH-HA
hydrogel could rapidly form a 3D structure in situ while minimizing
invasion and allowing it to remain in the injection site for long
periods of time. Thus, the in situ formed HA-Ac–SH-HA hydrogel has
the potential for applications in postoperative anti-adhesion and
other applications that delaminate tissue interfaces.
4. Conclusions
The injectable dual crosslinking HA-Ac–SH-HA hydrogel was
prepared by a fast thiol/acrylate Michael addition reaction as
the primary network, and the spontaneous oxidation reaction
of thiol groups as the secondary network. The interior structure,
gelation time, injection force, swelling and degradation properties,
mechanical modulus and drug release behavior of the dual cross-
linking hyaluronan-based hydrogel were precisely controlled by
adjusting the concentrations and molecular weights of HA-Ac and
HA-SH. The research results in vivo and in vitro indicate that the

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Journal of Materials Chemistry B
HA-Ac–SH-HA hydrogel can effectively support and protect the
agglomeration proliferation of the encapsulated L929 cells and
present wonderful biocompatibility. It also has the ability to prevent
cell adhesion and infiltration, which opens up huge possibilities
and great application potential as a novel surgical anti-adhering
material, cytoprotective agent and bioinert tissue filler.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This work was sponsored by National Key Technology R&D Plan
(Grant No. 2018YFC1105900), National Natural Science Foundation
of China (51573113), Sichuan Provincial Key Technology R&D
Program (2016SZ0003, 2019YFS0007), Young Elite Scientists
Sponsorship Program by CAST (2017QNRC001), and Sichuan
university Innovation Spark Project (2018SCUH0089).
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