International Journal of Biological Macromolecules 208 (2022) 149–158

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Injectable hyaluronic acid hydrogel encapsulated with Si-based NiO

nanoflower by visible light cross-linking: Its antibacterial applications
Kihak Gwon a, b, Jong-Deok Park c, Seonhwa Lee a, Won Il Choi d, Youngmin Hwang e,
Munemasa Mori e, Jong-Sung Yu c, *, Do Nam Lee a, *
a
Ingenium College of Liberal Arts (Chemistry), Kwangwoon University, Seoul 01897, Republic of Korea
b
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
c
Department of Energy Science and Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Republic of Korea
d
Center for Convergence Bioceramic Materials, Convergence R&D Division, Korea Institute of Ceramic Engineering and Technology, 202, Osongsaengmyeong 1-ro,
Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 28160, Republic of Korea
e
Columbia Center for Human Development (CCHD), Pulmonary Allergy & Critical Care Medicine, Department of Medicine, Columbia University Irving Medical Center,
New York, NY 10032, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Bacterial infections have become a severe threat to human health and antibiotics have been developed to treat
Antibacterial nanoflower them. However, extensive use of antibiotics has led to multidrug-resistant bacteria and reduction of their ther­
Photocrosslinking hydrogel apeutic effects. An efficient solution may be localized application of antibiotics using a drug delivery system. For
Biodegradable polymer
clinical application, they need to be biodegradable and should offer a prolonged antibacterial effect. In this
study, a new injectable and visible-light-crosslinked hyaluronic acid (HA) hydrogel loaded with silicon (Si)-based
nickel oxide (NiO) nanoflowers (Si@NiO) as an antibacterial scaffold was developed. Si@NiO nanoflowers were
synthesized using chemical bath deposition before encapsulating them in the HA hydrogel under a mild visible-
light-crosslinking conditions to generate a Si@NiO-hydrogel. Si@NiO synthesis was confirmed using scanning
electron microscopy, transmission electron microscopy, and powder X-ray diffraction. As-prepared Si@NiO-
hydrogel exhibited enhanced mechanical properties compared to a control bare hydrogel sample. Moreover,
Si@NiO-hydrogel exhibits excellent antibacterial properties against three bacterial strains (P. aeruginosa,
K. pneumoniae, and methicillin-resistant Staphylococcus aureus (>99.9% bactericidal rate)) and negligible cyto­
toxicity toward mouse embryonic fibroblasts. Therefore, Si@NiO-hydrogel has the potential for use in tissue
engineering and biomedical applications owing to its injectability, visible-light crosslink ability, degradability,
biosafety, and superior antibacterial property.

1. Introduction
Bacterial infections constitute one of the greatest global public
healthcare challenges today [1–4]. In particular, with the rapid devel­
opment of biomaterials and medical products such as wound dressings,
catheters, joint implants, and contact lenses, healthcare-associated in­
fections (HAIs) have been considered as severe problems by clinicians
[2]. Current treatments for bacterial infections mainly rely on various
antibiotics; however, using antibiotics has led to serious multidrug
resistance in bacteria and rapid reduction in therapeutic effects [5].
Therefore, there is a demand for an efficient and safe drug delivery
system that can reduce the risk of bacterial drug resistance, control and
prolong drug release, and regulate the toxicity of antibacterial drugs [6].
In recent years, various metals and metal oxides, such as silver (Ag) [7],
gold (Au) [8], copper (Cu) [9], copper oxide (CuO) [10], zinc oxide
(ZnO) [11], titanium dioxide (TiO2) [12], and nickel oxide (NiO) [13],
have been used in antibacterial applications owing to their high surface/
volume ratio and positive surface charge [14]. More recently, Si-based
nanocomposites have received considerable attention owing to their
desirable characteristics, such as a high theoretical specific surface area
and easy tuning of size and shape. In particular, the wrapping of metal
nanoparticles on these nano-supports has led to developing core-shell
and yolk-shell structures that have been trialed as new technologies
for energy-related and medicinal applications [15–17]. Because of its

* Corresponding authors at: Ingenium College of Liberal Arts (Chemistry), Kwangwoon University, Seoul 01897, Republic of Korea.
E-mail addresses: jsyu@dgist.ac.kr (J.-S. Yu), donamlee@hanmail.net (D.N. Lee).

https://doi.org/10.1016/j.ijbiomac.2022.03.051
Received 25 September 2021; Received in revised form 3 March 2022; Accepted 9 March 2022
Available online 16 March 2022
0141-8130/© 2022 Published by Elsevier B.V.
K. Gwon et al.
high surface area, the nanoflower-like core-shell structure provides
better antibacterial activity than the spherical or rod structure [18,19].
However, these materials can inhibit and kill not only pathogenic mi­
crobes but also normal cells in the human body, which limits the po­
tential applications of these materials [6]. Thus, there is a demand for
potent delivery systems that can not only reduce the side effects but also
increase the efficacy of the drug by releasing it near the target site.
Hydrogels, which comprise large amounts of water and a crosslinked
polymer network, are an appealing material for drug delivery systems.
Hydrogels provide physical and biological similarities to the extracel­
lular matrix of various tissues and have high water content, excellent
biocompatibility, and the capability to easily encapsulate hydrophilic
drugs [20–24]. Various natural and synthetic polymers are considered to
form promising hydrogel scaffolds. Natural polymers such as collagen,
chitosan, dextran, alginate, and hyaluronic acid (HA) have been widely
used as the backbone polymer [25]. Among them, HA has been the most
extensively studied because it has been well established as a biocom­
patible and biodegradable polymer that has been approved by the
United States Food and Drug Administration and has long been used for
the development of tissue engineering scaffolds and controlled drug
delivery systems [26,27]. If hydrogels can be made to be injectable, they
can be simply applied to the target site without an invasive surgical
procedure [28]. In particular, injectable hydrogels with in situ gel-
forming capability in response to chemical, temperature, pH, or light
stimuli can be applied to the target region without requiring invasive
surgery [28]. Although injectable hydrogels have several advantages,
they also have certain drawbacks. Most injectable hydrogels, for
example, require a significant gelation period, which may negatively
affect performance and lead to pain and infection risk for the patient.
The usage of photocrosslinkable hydrogels can help solve some of these
issues. These photocrosslinkable hydrogels can be quickly cured in
desired locations under physiological conditions in a spatiotemporally
controlled manner while avoiding obvious pH or temperature changes in
biological tissue, making them ideal for in situ injectable hydrogels for
wound treatment or drug delivery systems [29–32].
Ultraviolet (UV) light is a common light source for photocrosslinking
[33]. However, exposure to UV light can lead to cell damage and impair
cellular function. Moreover, the commercially available UV photoinitiator
Irgacure 2959 has low water solubility [34,35]. In contrast, the visible-
light photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate
(LAP) has higher water solubility, permits cell encapsulation at lower
photoinitiator concentrations, and initiates photopolymerization at a
longer light wavelength (405 nm), facilitating more efficient polymeri­
zation than Irgacure 2959 [36]. Moreover, visible light is expected to
cause less damage to cells and penetrate tissues at higher depths with
lower energy compared to UV light [37,38]. These favorable character­
istics make visible light suitable for developing in situ injectable materials
to be used for minimally invasive procedures. Yang et al. developed an
antimicrobial dressing agent based on a visible light-cured methacrylated
collagen hydrogel containing a betacyclodextrin/triclosan and demon­
strated its wound healing capacity [39]. Annabi et al. investigated visible-
light crosslinkable antimicrobial hydrogels to treat peri-implant diseases.
This antimicrobial hydrogel was engineered by incorporating a cationic
antimicrobial peptide into a photocrosslinkable gelatin methacryloyl
hydrogels and exhibited antibacterial properties against Porphyromonas
gingivalis, a Gram-negative bacterium involved in the pathogenesis of peri-
implant diseases [40]. A survey of the literature indicates that only a few
studies have investigated metal oxide encapsulated photocrosslinked
antibacterial hydrogels [41].
This study was performed to: (1) synthesize antibacterial Si@NiO
nanoflowers, (2) fabricate an antibacterial nanoflower-encapsulated
hydrogel (Si@NiO-hydrogel) via a visible-light-photocrosslinking
approach, and (3) verify the chemical and mechanical properties
Si@NiO-hydrogel as well as its antibacterial property and in vitro
biocompatibility to develop new injectable antibacterial hydrogels.
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2. Materials and methods
2.1. Materials
Sodium hyaluronate (HA, Mw: 40 kDa) was purchased from
Bloomage Freda Biopharm Co., Ltd. (Jinan, China). 4-Arm poly(ethylene
glycol) thiol (PEG4SH; Mw: 10 kDa) was obtained from Sunbio Inc.
(Anyang, Korea). Methacrylic anhydride (MA), deuterium oxide (D2O),
hyaluronidase (HAse) from bovine testes, ethyl alcohol (200 proof,
≥99.5%), plasma-synthesized silicon nanoparticles (20 mg; particle size:
50–100 nm; purity: 99%), nickel(II) acetate (Ni(CH3COO)2⋅4H2O),
ammonium hydroxide solution (NH3⋅H2O) and potassium perox­
odisulfate (K2S2O8) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). LAP was obtained from Cellink Life Sciences (Boston, MA, USA). A
dialysis membrane was obtained from Spectrum Laboratories (Rancho
Dominguez, CA, USA). Polydimethylsiloxane (PDMS, SYLGARD 184
Silicone Elastomer Kit) was purchased from Dow Corning (Midland, MI,
USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum
(FBS), penicillin/streptomycin, and Collagen I (rat tail) were purchased
from Gibco (Grand Island, NY, USA). An MTS Assay Kit (Cell Prolifera­
tion) was purchased from Abcam (Cambridge, MA, USA). A disposable
plastic syringe, mouse embryonic fibroblasts (MEFs), and a LIVE/DEAD
assay kit were purchased from ThermoFisher Scientific (Waltham, MA,
USA).
2.2. Preparation of Si-based Ni composite nanoflowers (Si@NiO)
Si@NiO was synthesized using chemical bath deposition (CBD) as
reported previously [42]. Plasma-synthesized silicon nanoparticles (20
mg) were dispersed in a beaker with a mixture of water (4 mL) and
ethanol (2 mL) and sonicated for 2 h (Solution 1). Ni(CH3COO)2⋅4H2O
(0.25 g) and potassium peroxodisulfate (0.05 g) were dissolved in
another beaker containing water (4 mL) and ethanol (2 mL) (Solution 2).
Solution 1 was mixed with Solution 2 under magnetic stirring at 200 rpm
(Solution 3). NH3⋅H2O (28–37%, 0.2 mL) was diluted in deionized water
(10 mL), and the diluted solution was added dropwise (1 drop⋅s− 1) to
Solution 3 by stirring. The yellowish solution slowly turned dark after
several minutes. The reaction continued for 30 min following NH3⋅H2O
addition, and the precipitates were washed with a large amount of
deionized water to remove other adsorbed ions (SO42− , K+, Ni2+,
CH3COO− , etc.). Brown Si@NiOOH nanoflowers were obtained, which
were dried in an oven at 60 ◦ C for 6 h. Dried Si@NiOOH was trans­
formed to NiO-coated Si (Si@NiO) by heating the former in a tube
furnace under increasing temperature up to 350 ◦ C at a heating rate of
2 ◦ C⋅min− 1 with Ar flow using a temperature control program before
natural cooling to ambient temperature [42].
2.3. Preparation of injectable Si@NiO-hydrogels
2.3.1. Step 1: HA-MA synthesis
To realize photopolymerization, a modified HA was designed by
introducing photo reactive groups and synthesized as previously re­
ported [43]. Briefly, sodium HA (200 mg) was dissolved in deionized
water (20 mL). After dissolution, a five-fold molar excess of MA was
slowly added. The reaction was conducted in an ice bath for 12 h, and
the pH value was maintained between 8 and 10 by adding 1 N or 5 N
NaOH. The final product (HA-MA) was precipitated in a five-fold excess
amount of cold absolute ethanol. After centrifugation at 5000 rpm for 5
min at 4 ◦ C, the supernatant was removed, and the precipitant was
redissolved in deionized water (20 mL). To remove any unreacted re­
agents, the solution was put into a dialysis bag (Mw cut-off: 3.5 kDa) and
dialyzed for 3 days. The resultant solution was freeze-dried (Sentry 2.0,
SP Scientific, Warminster, PA, USA) at − 80 ◦ C and 100 mTorr for 3 days
and stored at − 20 ◦ C for later use. The degree of methacrylate was
characterized using 1H nuclear magnetic resonance (NMR) spectroscopy
in D2O using 400 MHz spectrometers (JEOL RESONANCE ECZ 400S,
K. Gwon et al.
JEOL, Tokyo, Japan).
2.3.2. Step 2: Preparation of Si@NiO-mixed polymer precursor solution
To fabricate antibacterial Si@NiO-hydrogels, precursor solutions for
photocrosslinking were prepared as follows. First, PEG4SH and HA-MA
(thiol/acrylate in a 1:1 M ratio) were sequentially dissolved in 0.01 M
phosphate-buffered saline (PBS; pH: 7.4) to make 3%, 5%, or 7% (w/v)
solution. Another solution of LAP (100 mM) was also prepared in
distilled water. Next, LAP was added to this polymer solution with a final
concentration of 5 mM, and the pH of the polymer solution was adjusted
to 8.0 using 1 N NaOH or HCl and filtered through a 0.2 μm sterile filter
to sterilize the precursor solution [44]. Before crosslinking, Si@NiO
nanoflowers (0.2% (w/v)) were further mixed with the above polymer
solution. Then, the mixed solution was loaded into a plastic syringe and
was made to flow through a 25-gauge needle.
2.3.3. Step 3: Fabrication of photocrosslinkable and injectable Si@NiO-
hydrogels
To create a cylindrical PDMS well, a PDMS base was mixed with a
curing agent in a 10:1 ratio, and the mixture was poured into 15 cm Petri
dishes. After all the air bubbles were removed by placing the dishes in a
vacuum desiccator for 30 min, PDMS was baked in an 80 ◦ C convection
oven for 2 h. PDMS was peeled from the Petri dishes and then punched
using a stainless-steel punch (diameter: 1.2 cm). The PDMS pieces were
bonded to a glass slide after oxygen plasma treatment (G-500 Plasma
Cleaning System, Yield Engineering Systems, Livermore, CA, USA). The
Si@NiO-mixed polymer solution was injected into a 1.2 cm cylindrical
PDMS well. The well was then exposed to blue light by employing an
OmniCure S2000 Spot UV Curing System (Excelitas Technologies,
Waltham, MA, USA) using a 400–500 nm band-pass filter at 200
mW⋅cm− 2 for 1 min. For clarity, the Si@NiO-embedded hydrogel and
the hydrogel without Si@NiO are denoted as Si@NiO-hydrogel and the
control hydrogel, respectively.
2.4. Characterization of Si@NiO
The morphologies of the Si nanoparticles and Si@NiO nanoflowers
were characterized using scanning electron microscopy (SEM; Hitachi
SU8230, Hitachi High-Tec, Tokyo, Japan) at an acceleration voltage of
10 kV and transmission electron microscopy (TEM; Tecnai G2 F20 TWIN
TMP, FEI, Hillsboro, OR, USA) at 120 kV.
2.5. Characterization of Si@NiO-hydrogels
Powder X-ray diffraction (PXRD) patterns of Si@NiO, Si@NiO-
hydrogel, and the control hydrogel were recorded using a diffractom­
eter with Cu Kα radiation and a Ni filter operated at 40 kV and 30 mA
(SmartLab, Rigaku, Tokyo, Japan). The X-ray tube of the diffractometer
was operated at 40 kV and 30 mA with a scan rate of 4◦ /min and an
interval of 0.02◦ [42]. Fourier-transform infrared spectroscopy (FT-IR)
spectra were recorded on a Bio-Rad FTS 135 spectrometer (Hercules,
CA, USA) with KBr pellets. The thermal properties were evaluated using
thermogravimetric analysis (TGA; TG 209 F3 Tarsus, NETZSCH, Selb,
Germany). For rheological measurements, Si@NiO-hydrogels and the
control hydrogel were prepared in a cylinder-shaped PDMS mold
(diameter: 1.2 cm, depth: 1 mm) via the photocrosslinking process. The
formed hydrogels were removed from the mold and made to undergo
swelling with 0.01 M PBS (pH: 7.4) for 2 days at 37 ◦ C on an orbital
shaker. A frequency sweep test (from 0.01 to 100 rad⋅s− 1) was per­
formed using a rheometer (Discovery HR 10, TA Instruments, New
Castle, DE, USA) at a constant strain of 1% at 25 ◦ C. Both the hydrogel
samples were imaged before and after the swelling process using optical
microscopy (Stemi DV4 Stereomicroscope, Carl Zeiss, NJ, USA). The
swelling properties of Si@NiO-hydrogels and the control hydrogel were
investigated to determine the water sorption capacities. Fully swollen
cylindrical hydrogels were taken out from the PBS solution, gently
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International Journal of Biological Macromolecules 208 (2022) 149–158
blotted using filter paper to remove the excess surface water, and
immediately weighed (this weight is denoted as Ws). Then, the hydro­
gels were frozen at − 80 ◦ C before being made to undergo lyophilization
for 24 h. Next, their weights in the dried state were measured (this
weight is denoted as Wd). The equilibrium swelling ratio (ESR) was
calculated as the ratio of the mass value of the samples after swelling to
the mass value of the dried samples after lyophilization (Eq. (1)):
Ws
ESR = (1)
Wd
The morphological properties of the freeze-dried Si@NiO-hydrogels
and control hydrogel were analyzed using SEM. Before the SEM analysis,
the lyophilized hydrogels were coated with a gold‑palladium (Au–Pd)
alloy for 30 s. For the in vitro degradation studies, 5% (w/v) photo­
crosslinked hydrogels were incubated for 48 h in 0.01 M PBS (1 mL, pH:
7.4) for swelling. Fully swollen cylindrical hydrogels were taken out
from the PBS solution, the residual moisture on the hydrogel surface was
drained using filter paper, and the hydrogels were individually weighed
(this weight is denoted as Wi). Then, the samples were soaked in a PBS
solution (1 mL) containing 10–500 IU⋅mL− 1 HAse in an oscillator (25 ◦ C,
80 rpm) [43]. The enzyme solutions were refreshed at predetermined
points to ensure that the enzyme activity was sustained. At the pre­
determined interval, the residual hydrogels were taken out from the
enzyme solution, the residual moisture was carefully removed, and the
hydrogels were weighed (this weight is denoted as Wt). The weight
remaining ratio (WRR) was defined according to Eq. 2:
Wt
WRR (%) = × 100 (2)
Wi
where Wi and Wt are the weights of the samples before and after
degradation for time t, respectively.
2.6. Assessment of metal ion release
To assess whether Si@NiO or Si@NiO-hydrogel could release metal
ions in tryptic soy agar plates, Si@NiO or Si@NiO-hydrogel (400 μg) was
put in PBS (1 mL) and stirred for 6–48 h at 25 ◦ C [45]. This solution was
centrifuged at 25 ◦ C and 5000 rpm for 5 min, and the supernatant was
separated from the reaction tube. Metal ions released in the samples
were measured using inductively coupled plasma optical emission
spectrometry (ICP-OES; iCAP 7400 ICP-OES Duo, ThermoFisher Scien­
tific, Waltham, MA, USA). The concentration of the metal ions released
into the medium is reported in parts per million (ppm).
2.7. Antibacterial activity assessment
The antimicrobial activities of the control hydrogel, Si@NiO, and
Si@NiO-hydrogel against three strains of bacteria: P. aeruginosa (ATCC
15442), K. pneumoniae (ATCC 4352), and methicillin-resistant Staphy­
lococcus aureus (MRSA; ATCC 33591) were determined in terms of the
minimum bactericidal concentration (MBC) that killed over 99.9% of
the bacteria, following a previously published method [45–47]. To
measure the antibacterial activity of Si@NiO-hydrogel against the three
bacterial strains, three hydrogel sheets (2 mg⋅mL− 1, 5 × 5 ± 0.2 cm2)
and stomacher film for blank were prepared and tested. A control
hydrogel (without Si@NiO) was also prepared using the same method
for comparison. The surface of the test sample was dried after it was
wiped 2–3 times using an ethanol-soaked gauze. The platelets of the
precultured test bacteria with a concentration of 1–4 × 105 colony-
forming units (CFU)⋅mL− 1 were routinely inoculated. Each test sample
was placed in a Petri dish with the test side facing up. Subsequently, an
aliquot (0.2 mL) of the test solution was inoculated onto each test sample
using a pipette. The dropped test bacterium was covered using a film,
and the film was pressed lightly to spread the test bacterium. The test
sample inoculated with the test strain and the Petri dish containing the
K. Gwon et al.
Fig. 1. Synthesis and characterization of Si@NiO nanoflowers. (A) Schematic representation of Si@NiO prepared using silicon (Si) nanoparticles and Ni(II) acetate
(Ni(CH3COO)2). (B) SEM (top row) and TEM (bottom row) images of Si (left) and Si@NiO (right).
control hydrogel were incubated for 24 h at 37 ◦ C. Immediately after
inoculation, the uncoated test samples with the test strain were sepa­
rated using sterilized tweezers while ensuring that the respective solu­
tions did not flow, and a soyabean casein digest lecithin polysorbate
medium (10 mL) was added to wash off the test bacteria. The washing
solution was used to quickly measure the viable cell count. Next, the
washing solution (1 mL) was added to a test tube containing physio­
logical saline (9 mL) and mixed thoroughly. In this procedure, the
washing solution was diluted stepwise, and the diluted solution (0.1 mL)
was plated onto three nutrient agar plates and incubated for 24 h at
37 ◦ C. All the experiments were performed in triplicate. The bactericidal
rate was calculated using Eq. 3: [45–47].
(A − B)
Bactericidal rate (%) = × 100
A
where A represents the CFUs of the bacteria cultured in the presence of
stomacher film and B represents those of the bacteria cultured in the
presence of the control hydrogel, Si@NiO, or Si@NiO-hydrogel.
2.8. In vitro cytotoxicity assays
The cytotoxicities of Si@NiO and Si@NiO-hydrogels were evaluated
using a direct contact method, as described previously [46–48]. Briefly,
a Si@NiO-mixed polymer solution (2 mg⋅mL− 1) was injected into a cy­
lindrical PDMS mold (diameter: 1.2 cm, depth: 1 mm), and hydrogels
were formed in the same manner. Meanwhile, a fibroblast monolayer
was formed on a glass slide coated with Collagen I (10 μg⋅mL− 1) at a
density of 5 × 104 cells⋅cm− 1 and incubated for 3 h. Then, non-adhered
cells were rinsed with DMEM (supplemented with 10% (v/v) FBS, 200
IU⋅mL− 1 penicillin, and 200 μg⋅mL− 1 streptomycin), transferred to a 12-
well plate, and cultured in the cell culture medium at 37 ◦ C in a hu­
midified incubator containing 5% CO2. The following day, the prepared
International Journal of Biological Macromolecules 208 (2022) 149–158
Si@NiO-hydrogel and control hydrogel were carefully placed on the cell
monolayer or Si@NiO (0.2% (w/v)) was carefully mixed on the cell
monolayer, and then, incubation was conducted for either 1 day or 3
days. The cells were analyzed using a live/dead assay (ThermoFisher
Scientific, Waltham, MA, USA), in which the live cells were visualized in
green fluorescence and the dead cells in red. In brief, the MEF cells were
rinsed with PBS and then incubated in PBS containing 4 μM calcein-AM
and 2 μM ethidium homodimer-1 (EtBr-1) for 30 min. After PBS
washing, the stained cells were observed using inverted fluorescence
microscopy (IX83, Olympus, Center Valley, PA, USA). Cell viability was
quantified by calculating the ratio of the live cells to the total number of
cells.
In addition, the cell viability was quantitatively analyzed using an
(3) MTS assay, which is like an MTT assay and measures the metabolic rates
of cells. For this purpose, Si@NiO-hydrogel and hydrogel or Si@NiO
were incubated in a cell culture medium for 24 h to obtain the extract
solutions, as described in previous reports [45,46] and the cells were
seeded in a 24-well plate at a density of 5 × 104 cells per well. After
incubation at 37 ◦ C for 24 h, the culture medium in each well was
replaced with DMEM containing the extract solution (200 μL). After
incubation for 24 h, the DMEM-containing extract solution was carefully
removed; then, an MTS cell proliferation assay kit solution (20 μL) and a
fresh medium (200 μL) were added into each well. After incubation for
an additional 4 h, the absorbance at a wavelength of 490 nm was
measured using a microplate reader (Synergy H1, BioTek, Winooski, VT,
USA). The number of proliferated cells was quantified using a standard
curve of cells.
2.9. Statistical analysis
All the data were expressed as means ± standard deviation using at
least triplicate samples. Statistically significant differences were
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K. Gwon et al. International Journal of Biological Macromolecules 208 (2022) 149–158

Fig. 2. Fabrication of Si@NiO-loaded, injectable, and visible-light-crosslinked HA hydrogel (Si@NiO-hydrogel). (A) Absorption spectra of 5 mM LAP. (B) PEG4SH
mixed with methacrylated HA in presence of LAP. Si@NiO was combined with a polymer precursor solution. (C) Si@NiO-mixed polymer solution transferred to
syringe and subcutaneously injected into PDMS mold. The hydrogel was formed by visible-light-mediated thiol-ene photopolymerization between HA-MA and
PEG4SH with LAP as the photoinitiator.

Fig. 3. Characterization of Si@NiO-hydrogel. (A) PXRD patterns of control hydrogel, Si@NiO, and Si@NiO-hydrogel. (B) FT-IR spectrum, (C) TGA profiles, and (D)
SEM images of control hydrogel and Si@NiO-hydrogel.

153
K. Gwon et al.
Fig. 4. Mechanical properties of Si@NiO-hydrogel. (A) Storage modulus (swollen state) and (B) ESR for various concentrations of control hydrogel and Si@NiO-
hydrogel. (C) Photographs of control hydrogel and Si@NiO-hydrogel before and after swelling for 48 h. Scale bar: 5 mm.
evaluated using a t-test, and statistical significances were (*) p < 0.05,
(**) p < 0.01, and (***) p < 0.001.
3. Results and discussion
3.1. Characterization of Si@NiO nanoflowers
Si@NiO nanoflowers were prepared using modified versions of re­
ported methods, as shown schematically in Fig. 1A [42]. During the CBD
process, Si nanoparticles were encapsulated in porous flowerlike NiOOH
shells (i.e., Si@NiOOH), and Si@NiOOH was then reduced to porous
flowerlike NiO shell by mild heating to 350 ◦ C with Ar flow, yielding
Si@NiO. SEM and TEM images showed that the Si nanoparticles had a
spherical shape with an average size of 100 nm, and Si@NiO exhibited a
flowerlike core-shell structure (Fig. 1B), where the silicon core was
uniformly covered by a thin nanoflower-like NiO shell. The flowerlike
morphology was stable and well maintained even after further annealing
processes. As indicated by previously reported results [42], the porous
shell layers had a uniform thickness of 10–30 nm, as observed using TEM
(Fig. 1B, bottom).
3.2. Synthesis and characterization of HA-MA
In this study, we used HA as the backbone polymer because it has
been well established as a biocompatible and biodegradable polymer,
[26,43,49–51]. Moreover, HA can be easily modified using various
functional groups by utilizing the abundantly available hydroxyl and
carboxylic acid groups [51]. First, to modify the photopolymerizable
group to the HA, the primary hydroxyl group of HA was converted to a
methacryl group via a transesterification reaction (Fig. S1A). The NMR
spectrum of HA-MA showed that the peaks at 6.1 and 5.6 ppm were
assigned to the vinyl groups of MA, indicating that the HA-MA synthesis
succeeded. Based on a comparison of the areas of the methyl peaks of the
N-acetyl group in HA at 1.9 ppm and the methacrylate peaks of MA at
5.6 and 6.1 ppm, the degree of methacrylation was 50% (Fig. S1B) [43].
3.3. Fabrication and characterization of Si@NiO-hydrogel
To create photocrosslinked hydrogels, both UV-light and visible-light
sources are commonly used to initiate photopolymerization [33,52].
However, UV light can lead to cell damage and can impair cellular
function. In addition, the commercially available UV photoinitiator
Irgacure 2959 has a low water solubility of less than 0.5 wt% [34,35].
Thus, we used LAP as a photoinitiator owing to its good water solubility,
cytocompatibility, and visible-light initiation properties [36] Fig. 2A
shows, LAP exhibited absorption at 405 nm; thus, LAP allowed photo­
crosslinking under more cell friendly conditions than other UV photo­
initiators (e.g., Irgacure 2959) [53]. Si@NiO-hydrogels were prepared
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International Journal of Biological Macromolecules 208 (2022) 149–158
via visible-light-initiated photopolymerization between PEG4SH and
HA-MA with LAP as the photoinitiator (Fig. 2B and C). Upon visible-light
exposure (400–500 nm), LAP, which exhibited absorption at 405 nm,
was excited to abstract hydrogen from PEG4SH, thus forming thiyl
radicals. These radicals attacked methacryl groups on HA-MA to form
carbon radicals. These radicals either propagated through other meth­
acryl moieties in HA-MA or further abstracted hydrogen from other thiol
moieties on PEG4SH, generating photocrosslinked Si@NiO-hydrogels
(Fig. 2C).
To characterize the compositions, the PXRD patterns and FT-IR
spectra of the control hydrogel, Si@NiO, and Si@NiO-hydrogel were
analyzed. As shown in Fig. 3A, for Si@NiO, high-intensity diffraction
peaks were observed at 2θ = 28.3◦ , 47.2◦ , 55.9◦ , 75.7◦ , and 87.9◦ cor­
responding to Si and additional peaks were observed at 2θ = 37.1◦ ,
43.3◦ , and 62.6◦ corresponding to the coated NiO. The hydrogel was
observed to show major PXRD peaks at 2θ = 31.7◦ and 45.4◦ and a few
minor peaks and these observations are consistent with the data in a
previous report [42]. Si@NiO-hydrogel showed similar patterns with
hydrogel and a few additional peaks corresponding to Si@NiO were
observed. These PXRD patterns presents that Si@NiO is well incorpo­
rated in the hydrogel.
FT-IR spectral analysis was performed for the control hydrogel and
Si@NiO-hydrogels. Fig. 3B shows the O–H stretching (3428 cm− 1),
C–H stretching, pyranose ring (2892 cm− 1), C– – O stretching, NH-Ac
− 1 − 1
(1644 cm ), and C N stretching (1473 cm ) in the control hydro­
–
gel sample; these observations are consistent with the data in a previous
report [54]. Si@NiO-hydrogel showed similar characteristic peaks to the
control hydrogel, however, peak assigned to Ni–O at near 420 cm− 1
was not observed due to overlapping with C–H stretching band and its
low concentration.
Thermal stabilities of the control hydrogel and Si@NiO-hydrogel
were investigated using TGA, and the results are shown in Fig. 3C.
The control hydrogel and Si@NiO-hydrogel lost approximately 8.3%
and 14.4% of their weight, respectively, up to 200 ◦ C due to the loss of
absorbed water. The TGA thermogram of control hydrogel and Si@NiO-
hydrogel showed that thermal decomposition of hydrogel occurred in
one major step, and their initial decomposition began at 210 ◦ C and
ended to 350 ◦ C, with an increase in weight loss to 65.8% and 60.0%,
respectively. Finally, their weight loss at 800 ◦ C was 84.7% and 69.6%,
respectively. These observations agreed with the findings of other pre­
vious studies [55].
To investigate the surface topography of the control hydrogel and
Si@NiO-hydrogel, fully swollen hydrogels in deionized water were
lyophilized and characterized using SEM-EDS. As shown in Fig. 3D, all
the hydrogels had porous and irregular network structures, and nano­
sized Si@NiO was well encapsulated in the hydrogel network (Fig. 3D,
right). Moreover, analysis by SEM-EDS was carried out to obtain the
morphologies and chemical compositions of the control hydrogel and
K. Gwon et al.
Fig. 5. Degradation property of Si@NiO-hydrogels. Enzymatic degradability of
control hydrogel and Si@NiO-hydrogels at HAse concentrations of 10, 50, 100,
and 500 IU⋅mL− 1.
the Si@NiO-hydrogel. More specifically, Na, S, O and C were evenly
spread throughout both networks, and the additional elements of Ni, Si
and P were also found in the EDS spectrum of Si@NiO-hydrogel
Fig. 6. Antibacterial property. (A) Representative images of bacteria grown
under different conditions after incubation for 24 h compared to control
hydrogel: top to bottom: P. aeruginosa, K. pneumoniae, and MRSA; left to right:
blank, hydrogel, Si@NiO, and Si@NiO-hydrogel. (B) Antibacterial efficiency of
hydrogel, Si@NiO, and Si@NiO-hydrogel toward P. aeruginosa, K. pneumoniae,
and MRSA.
155
International Journal of Biological Macromolecules 208 (2022) 149–158
(Fig. S2).
The storage moduli of the control hydrogel and Si@NiO-hydrogel
were determined to evaluate their mechanical properties, which are
mainly determined by the crosslinking density of the polymer networks
and can strongly influence the hydrogel functions [43,56]. A higher
precursor concentration increases the crosslinking density, which results
in a higher modulus and lower ESR of the obtained hydrogel [35,43].
Our results showed that after swelling in PBS, Si@NiO-hydrogels
exhibited an increased storage modulus from 0.47 ± 0.15 kPa to 3.88
± 0.51 kPa and a decreased ESR from 39.6 ± 3.6 to 22.4 ± 0.8 when the
total polymer concentrations were increased from 3% (w/v) to 7% (w/
v). Moreover, the results for the control hydrogel were compared. As
expected, an increase in the total polymer concentration from 3% to 7%
(w/v) led to a notable increase in the storage modulus from 0.14 ± 0.04
kPa to 2.38 ± 0.29 kPa and a decrease in the ESR from 44.5 ± 3.6 to
26.9 ± 0.8 (Fig. 4A and B). To investigate the Si@NiO encapsulation
effect on the mechanical properties of the hydrogel, we compared the
mechanical properties of Si@NiO-hydrogel and the control hydrogel at
the same polymer concentrations. When Si@NiO nanoflowers were
embedded, the storage moduli increased to 3.3, 1.3, and 1.6 times for
the 3% (w/v), 5% (w/v), and 7% (w/v) Si@NiO-hydrogels, respectively,
compared to the storage modulus of the control hydrogel. Incorporating
Si@NiO in the hydrogel are supposed to induce additional interactions
with nickel ion with hyaluronic acid through -Ni-OCO- or -Ni-NH-CO-
coordination [57].
This may cause an improvement in the intermolecular interactions
and hydrogel stiffness, as well as a decrease in the ESR and pore sizes. In
addition, both the control hydrogel and Si@NiO-hydrogel were imaged
before and after swelling. As shown in Fig. 4C, the increase in the
diameter of the control hydrogel was greater than Si@NiO-hydrogel
after swelling. This may be caused by an improvement in the intermo­
lecular crosslinking density of Si@NiO-hydrogel compared to that of the
control bare hydrogel because of the coordination of Ni(II) ions or oxide
ions on the nanoflower surface of the hydrogel. The Si@NiO nano­
flowers were evenly dispersed in the hydrogel, whereas the control
hydrogel was transparent. In addition, the encapsulated Si@NiO was
maintained in the hydrogel even after extended (48 h) incubation in
PBS. These data confirm that the Si@NiO nanoflowers are well encap­
sulated in the hydrogel during the photocrosslinking process and that
the Si@NiO nanoflowers are stable in the hydrogel.
3.4. Degradation property
HA-based scaffolds have become promising candidates for tissue
engineering and drug delivery [26,27,50,51]. For potential in vivo ap­
plications, implanted materials (sutures, gauzes, or hydrogels) should be
biodegradable so a secondary surgery is not required to remove the
implant [58]. Thus, we evaluated the biodegradability of the control
hydrogel and Si@NiO-hydrogels against HAse owing to their promising
therapeutic and potential in vivo applications. Si@NiO-hydrogels and
the control hydrogel were placed in 0.01 M PBS or HAse solutions
(10–500 IU⋅mL− 1), and the resulting percentage weight loss was moni­
tored. Fig. 5 and Fig. S3 show the in vitro degradation behaviors. Upon
exposure to HAse, each hydrogel showed obvious degradation behavior
but a different degradation rate. First, enzymatic degradation trends
were affected significantly by both the HAse concentration (10–500
IU⋅mL− 1) and Si@NiO incorporation in the hydrogel. For example, when
the Si@NiO-hydrogels were placed in solutions containing 10, 50, 100,
and 500 IU⋅mL− 1 HAse, the degradation rates were 1.2%, 1.4%, 1.5%,
and 2.2% of weight loss per day, respectively, and for the control
hydrogel, the corresponding degradation rates were 3.0%, 5.8%, 6.8%,
and 8.2% of weight loss per day, respectively. Both Si@NiO-hydrogels
and the control hydrogel incubated with PBS underwent negligible
weight loss.
We also compared Si@NiO-hydrogels and the control hydrogel at a
fixed HAse concentration of 500 IU⋅mL− 1 for 12 days. In 12 days, the
K. Gwon et al.
Fig. 7. Cytotoxicity test for Si@NiO-hydrogel. (A) Live/dead staining images of MEFs after contact with control hydrogel, Si@NiO, or Si@NiO-hydrogel for day 1 and
day 3. Positive control (blank): cells cultured with no contact. Negative control: cells contacted with EtOH. (B) In vitro cytotoxicity of extract solution of control
hydrogel, Si@NiO, or Si@NiO-hydrogel toward MEFs. Scale bar: 200 μm.
control hydrogel degraded completely, whereas the Si@NiO-hydrogels
degraded by less than 25% (Fig. S3). The degradation rate of Si@NiO-
hydrogels was slower than the control hydrogel, thus the Si@NiO
incorporation affected the sensitivity of the hydrogel to enzymatic
degradation. Incorporating Si@NiO in the hydrogel increased the
intermolecular interaction between nickel ion and hyaluronic acid via
coordination reaction. This may cause the enhanced hydrogel stiffness,
as well as reduced the enzymatic degradation rate [57].
3.5. Ion release test
The degradation of Si@NiO and Si@NiO-hydrogel was tested in 400
μg⋅mL− 1
of PBS at 25 ◦ C for 6, 12, 24, and 48 h. The quantity of Ni ions
released from the samples was measured using ICP-OES. As shown in
Fig. S4, the concentration of Ni ions released from Si@NiO only
increased from 8.01 ppm at 6 h to 18.04 ppm after 48 h, indicating that
Si@NiO robustly maintained its flowerlike structure in PBS. Notably, the
amount of Ni(II) ions released from Si@NiO-hydrogel showed a 50-fold
decrease (0.36 ppm after 48 h) over that released by Si@NiO. Si@NiO,
immobilized on the inner polymer network, might have been slightly
inhibited by the direct contact with aqueous solutions, reducing the
amount of metal ion release. The amount of Ni(II) ions released from the
control hydrogel was negligible.
3.6. Antibacterial activity
To investigate the bioactivity of the antibacterial hydrogel, the
antibacterial activity of the control hydrogel, Si@NiO, and Si@NiO-
156
International Journal of Biological Macromolecules 208 (2022) 149–158
hydrogel was tested three times in the concentration range of 200
μg⋅mL− 1 to 2 mg⋅mL− 1, and the CFUs of P. aeruginosa, K. pneumoniae,
and MRSA were counted after incubation with Si nanoparticles at 37 ◦ C
for 24 h. The bactericidal rate for Si@NiO and Si@NiO-hydrogel was
99.9% for all three tested species at 200 μg⋅mL− 1 and 2 mg⋅mL− 1,
respectively (Fig. 6A and B and Table S1). In contrast, the control
hydrogel showed very low antibacterial activity against all the tested
bacteria at the same film size (5 × 5 cm2): P. aeruginosa (5.1%),
K. pneumoniae (11.9%), and MRSA (6.7%). This finding agrees with that
in previous reports: 1) HA itself has been previously shown to possess
bacteriostatic activities [59] and 2) high positive charges of metal-
coated nanoparticle surfaces can provide effective bactericidal activity
through the interaction with the negative charges of the bacterial cell
surface [45]. Therefore, at a low MBC of 200 μg⋅mL− 1, Si@NiO with two
positive charges showed bactericidal properties stronger than other
neutrally charged nanoparticles, such as nanosized Ag/Cu-graphene
[60]. Although the amount of Ni(II) ions released from Si@NiO-
hydrogel reduced compared to that released from Si@NiO owing to
the encapsulation of Si@NiO in the HA hydrogel, the bactericidal rate of
Si@NiO-hydrogel was 99.9% at a concentration of 2 mg⋅mL− 1. From
these results, we can speculate that the bactericidal process in Si@NiO-
hydrogel proceeds through direct contact with the cell membrane rather
than the inflow of antibacterial metal ions released from nanoflowers
into the cell, resulting in killing the bacteria.
3.7. Cytotoxicity
Finally, we studied the cell biocompatibility of Si@NiO-hydrogel
K. Gwon et al.
based on a direct contact method [38–40,61]. MEFs as a model cell line
have been used in in vitro studies due to their significant role in wound
healing [62,63]. To verify the toxicity of the control hydrogel, Si@NiO,
and Si@NiO-hydrogel, three test samples were prepared using the same
method. Furthermore, as an additional positive control (blank), an MEF
monolayer with no contact was prepared. As a negative control, an MEF
monolayer contact with a 10% EtOH solution was used. The MEF
viability was monitored using live/dead staining and a colorimetric MTS
assay. As shown in the fluorescence microscopy images (Fig. 7A), the
cell viabilities for the blank, Si@NiO, Si@NiO-hydrogel, and control
hydrogel samples exceeded 98% 1 day after culture, whereas with the
MEF monolayer, most cells died upon exposure to EtOH. It was found
that the cells gradually adhered, spread, and flattened on the surface
after seeding, before forming a confluent layer that survived for 3 days;
this was not observed in the EtOH group. These results indicate that
neither Si@NiO-hydrogel nor Si@NiO and the hydrogel alone were toxic
to the cells. Further quantification was conducted using an MTS assay
(Fig. 7B), wherein Si@NiO, the control hydrogel, and Si@NiO-hydrogel
extracted from cell culture medium solutions were serially diluted. For
example, 100% refers to the original extraction medium, and 25% in­
dicates a dilution of four times from the original extraction medium.
After the formation of the MEF monolayer, the culture medium was
changed to a medium with the desired concentration containing the
extract solution and was cultured. The obtained MTS results demon­
strated that Si@NiO-hydrogel had good cytocompatibility considering
that the MEF viability was greater than 95% in all the cases; however,
most MEFs died upon exposure to EtOH, confirming the low cytotoxicity
of the Si@NiO and Si@NiO-hydrogel extracts.
4. Conclusions
In this study, we designed an injectable and visible-light-mediated
hydrogel with an excellent antibacterial property. Si@NiO nano­
flowers were well encapsulated with high stability in the hydrogel
during the photocrosslinking process. The physical and chemical prop­
erties of Si@NiO-hydrogel were characterized using PXRD, FT-IR, TGA,
SEM, and ICP-OES. The mechanical properties of Si@NiO-hydrogel (i.e.,
rheology and swelling behavior) could be modulated by varying the
polymer precursor solution. Furthermore, the Si@NiO-hydrogel
exhibited an excellent antibacterial property (>99.9% bactericidal
rate) against three strains of bacteria (P. aeruginosa, K. pneumoniae, and
MRSA) at a concentration of 2 mg⋅mL− 1 and displayed negligible cyto­
toxicity toward MEFs. Using visible light may help in circumventing the
potential biosafety issues associated with toxic initiators or UV light. The
injectability and biodegradability of Si@NiO-hydrogel make it a prom­
ising platform for tissue engineering and biomedical applications.
However, further work, including in vitro cell migration and in vivo
wound closure assay, is required to promote the future use of Si@NiO-
hydrogel as a new generation of powerful and efficient antibacterial
hydrogels as a wound dressing with wide-ranging applications.
CRediT authorship contribution statement
Kihak Gwon: Conceptualization, Methodology, Investigation, Formal
analysis, Data curation, Funding acquisition, Writing – original draft.
Jong-Deok Park: Methodology, Investigation, Data curation, Writing –
review & editing. Seonhwa Lee: Methodology, Investigation, Formal
analysis, Data curation, Writing – review & editing. Won Il Choi: Data
curation, Writing – review & editing. Youngmin Hwang: Investigation,
Methodology, Data curation, Writing – review & editing. Munemasa
Mori: Methodology, Data curation, Writing – review & editing. Jong-
Sung Yu: Funding acquisition, Resources, Project administration, Su­
pervision, Writing – review & editing. Do Nam Lee: Conceptualization,
Funding acquisition, Resources, Project administration, Supervision,
Writing – review & editing.
157
International Journal of Biological Macromolecules 208 (2022) 149–158
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
No data was used for the research described in the article.
Acknowledgments
This work was supported by National Research Foundation of Korea
(NRF) grants funded by the Korean government (2021R1A2C1004285,
2017R1A6A3A11030955, and 2019R1A2C2086770). The present
research was conducted supported by a research grant provided by
Kwangwoon University in 2021. The authors want to thank the KBSIs at
Daegu and Busan and the CCRF at the DGIST for their assistance with the
SEM, TEM, and PXRD measurements.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2022.03.051.
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