Title Page

ORIGINAL RESEARCH

Cu2WS4 Nanocrystals for Photocatalytic Inhibition of Staphylococcus

Aureus Biofilm Formation and Wound Management

Heng Dong1†, Kaili Yang2†, Yu Zhang1, Qiang Li1, Weijun Xiu2, Meng Ding1,
Jingyang Shan3, * and Yongbin Mou1, *
1. Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing,
China.
2. Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key
Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National
Synergetic Innovation Centre for Advanced Materials (SICAM), Nanjing University
of Posts and Telecommunications
3. Department of Neurology, Shenzhen Institute of Translational Medicine, The First
Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital,
Shenzhen 518000, China
†These authors contributed equally to this study.
*Corresponding author:
Jingyang Shan, Department of Neurology, Shenzhen Institute of Translational
Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second
People's Hospital, Shenzhen 518000, China. E-mail: jyshandr@163.com
Yongbin Mou, Nanjing Stomatological Hospital, Medical School of Nanjing
University, #30 Zhongyang Road, Nanjing 210008, China. E-mail:
yongbinmou@163.com

Author contributions: J.Y.S. and Y.B.M. conceived the concept of the study and
designed the experiments. H.D. and K.L.Y. performed the experiments analyzed the
experimental data and co-wrote the manuscript. Y.Z., Q.L. W.J.X., and M.D. helped
and gave valuable suggestions for experiments All the authors discussed, commented
and agreed on the paper.
Funding: This review was funded by the National Natural Sciences Foundation of
China (81371680), and Jiangsu Provincial Medical Talent (ZDRCC2016016), Key
Project of Research and Development Plan of Jiangsu Province (BE2020629), and
Development of Science and Technology of Nanjing (YKK19094).
Acknowledgments: This work was supported by the We also greatly appreciate the
support from Dr. Lihui Yuwen (Nanjing University of Posts and
Telecommunications).
Conflicts of Interest: The authors declare no conflict of interests.
Cu2WS4 Nanocrystals for Photocatalytic Inhibition of Staphylococcus

Aureus Biofilm Formation and Wound Management

Abstract:
Background: Bacteria biofilm related-wound infections threaten human health due to
the absence of efficient treatment. Therefore, developing novel strategy in wound
infection care is urgently needed.
Methods: Cu2WS4 nanocrystals (CWSNs) with cube-like shape were successfully
prepared via a microwave-assisted method. CWSNs, as the photocatalysis, were
firstly studied for the generation of reactive oxygen species (ROS) by using the
fluorescence spectroscopy. The antibacterial activity and biofilm inhibition ability of
CWSNs were determined in vitro by using Staphylococcus aureus (S. aureus) as the
model. Moreover, CWSNs gel were prepared and applied to treat S. aureus infected
wounds in mice. The toxicity of CWSNs were evaluated through in vitro cell and in
vivo animal experiments.
Results: Property studies demonstrated that CWSNs can catalyze the generation of
hydroxyl radicals (•OH) without addition of H2O2 by visible light, indicating their
photocatalytic ability. Moreover, in vitro experimental results showed that the CWSNs
could not only kill S. aureus, but also inhibited S. aureus biofilm formation. In vivo
animal results showed that the CWSNs gel achieved excellent antibacterial effect
against S. aureus infected wounds in mice and effectively promoted wound healing.
Furthermore, Toxicity tests showed that the CWSNs have the negligible toxicity in
vitro and in vivo.
Conclusion: This work may provide a potential antibacterial agent for the
photocatalytic inhibition of bacterial biofilms formation and the treatment of wound
infection.

Keywords: Cu2WS4 nanocrystals, Staphylococcus aureus, Visible light, Hydroxyl

radical, Biofilms
Introduction
Antibiotic-resistant bacteria have become a worldwide problem and a major
hidden danger that threatens global public health. Currently, the abuse of antibiotics is
a particularly serious problem, and it often leads to the emergence of drug-resistant
bacteria and even "superbacteria", such as Staphylococcus aureus (S. aureus)[1, 2].
Bacterial infections are very challenging to treat, as the action and penetration of
antibiotics are largely limited by the dormant lifestyle of bacteria and matrix of
extracellular polymeric substance (EPS) in bacterial biofilms [3, 4]. Bacterial biofilms
with an EPS matrix can resist host immune defenses and induce persistent
inflammation, thus allowing the bacteria to become highly resistant to traditional
antibiotics[5]. Ineffective treatments with antibiotics not only cause the rapid
emergence of drug-resistant S. aureus but could also result in the formation of S.
aureus biofilms[7]. Therefore, a new strategy to inhibit S. aureus biofilm formation
urgently needs to be developed.
With the advances in nanotechnology, nanomaterials have been considered for
antibacterial applications[8, 9]. To date, photocatalysts that utilize different parts of the
solar spectrum have been widely investigated. When photocatalytic nanomaterials
absorb visible-light and generate electron–hole pairs, electrons and holes can react
with water and dissolved oxygen separately to generate ROS. Compared to the normal
hydrogen electrode at pH 7, the potentials for ROS production at the hole side for
water oxidation are 1.1~1.9 V (H2O/OH•)[10]. Therefore, the bandgap of nanomaterials
is a key factor that affects the photocatalytic generation of ROS. Furthermore, many
nanomaterials, as photocatalysts, induce bactericidal effects through the
photocatalytic generation of ROS. TiO 2 is a prototypical transition metal that
functions as an oxidative photocatalyst; however, the common bulk phases of TiO 2
have a 3 eV bandgap, which limits their photoactivity to the ultraviolet region of the
spectrum[11]. Vertically aligned MoS2 nanofilms with a lower bandgap (~1.55 eV) can
harvest visible light to generate various ROS for killing bacteria [10]. Developing
antibacterial nanomaterials that possess characteristics, such as high antibacterial
activity, low toxicity, and photoresponsiveness, that are typical antibiotics is still a
great challenge.Cu2WS4 is a typical layered ternary chalcogenide and contains unique
physical and chemical properties that are conducive to various applications in
supercapacitors and hydrogen evolution[12, 13]. A pervious study demonstrated that the
Cu2WS4 catalyst has both enzyme-like (oxidase and peroxidase) properties and
selective bacteria-binding ability; thus, Cu 2WS4 can facilitate the production of ROS
to kill bacteria[14]. However, Cu2WS4 nanomaterials have shown broad-spectrum
antibacterial activity under visible light, and the antibacterial mechanism and
antibiofilm application of Cu2WS4 nanomaterials have not been fully explored.
In this study, we constructed Cu2WS4 nanocrystals (CWSNs) with a small cubic
size (~20 nm). The inhibitory ability of free bacteria (S. aureus) was evaluated, and in
vitro antibacterial experiments showed that the minimal inhibitory concentration
(MIC) of CWSNs was 16 μg/mL. In addition, the current study focused on the
interaction relationship between the photocatalytic performance and antibiofilm
properties of CWSNs. The S. aureus biofilms were reduced up to ~97% at 200 μg/mL
CWSNs under visible-light, while biofilms were only reduced up to ~46% at 200
μg/mL CWSNs in the dark. The therapeutic efficacy of S. aureus-infected wounds
was investigated in vivo, and CWSN gel can efficiently treat S. aureus infected
wounds and promote wound healing with good biocompatibility. We hope to develop
a highly efficient antibacterial nanomedicine for maximizing the treatment of bacterial
infection after photocatalytic CWSNs are administered under visible-light.

Materials and methods

Preparation and characterization of CWSNs

The CWSNs were prepared by using (NH4)2WS4, CuBr and 3-mercaptopropionic
acid according to the reported method [14]. The morphology of the CWSNs was
characterized by transmission electron microscopy (TEM, HT7700, Hitachi, Japan,
100 kV) and high resolution TEM (HRTEM, F200X, Talos, USA, 200 kV) with
energy dispersive spectrometry (EDS). X-ray diffraction (XRD) patterns were
recorded on a diffractometer (D8 Advance A25, Bruker, Germany) with Cu Kα
radiation. The bandgap of CWSNs was calculated by the obtained ultraviolet visible
near-infrared (UV–vis-NIR) absorption spectrum using UV–vis-NIR
spectrophotometer (UV-3600, Shimadzu, Japan). The valence band (VB) was
measured by ultraviolet photoelectron spectroscopy (UPS, Thermo, ESCALAB
250XI, USA).

Hydroxyl radical (•OH) detection

•OH can react with terephthalic acid (TA) to form 2-hydroxyl TA with
fluorescence. Two experimental groups with TA (5 mM) and CWSNs (100 μg/mL) +
TA were incubated at room temperature for 2 h. Afterward, 3 mL of the mixtures were
measured with a fluorescence spectrophotometer using 312 nm as the excitation
wavelength (RF-5301 PC, Shimadzu, Japan). (ref)

Bacterial culture
Luria-Bertani (LB) broth (ST156) and LB broth with agar (ST158) were
purchased from Beyotime Institute of Biotechnology (Shanghai, China). S. aureus
(ATCC25923) was revived and streaked on LB agar plates. The plate was incubated at
37°C for 12 h. A single colony of S. aureus was picked and used to inoculate 5 mL LB
overnight at 37°C under shaking at 220 rpm.

Antibacterial assay
The bacteria were washed and diluted to 2 × 10 6 colony-forming units
(CFUs)/mL by using ultrapure water. CWSNs at different concentrations (final
concentrations: 0.001, 0.01, 0.05, 0.1, and 1 μg/mL) were incubated with diluted S.
aureus (2 × 106 CFU/mL) at 37°C for 2 h. Ultrapure water was used as a control. The
mixtures were diluted 103 times with saline, and 100 μL was spread onto LB agar
plates. After incubation for 18 h at 37°C, the number of CFUs was counted. All assays
were performed as triplicates. Moreover, the bacteria were subjected to the reported
method and their morphology was characterized by scanning electron microscopy
(SEM, Hitachi S-4800, Japan)[15].

Bacterial regrowth assay (Assessment of bactericidal effect)

CWSNs (final concentrations: 0.05, 0.1, 0.5, 1, 2, and 5 μg/mL) were first
incubated with S. aureus (2 × 106 CFU/mL) at 37°C for 2 h, and the mixtures were
transferred into 1.5 mL tubes containing LB (1 mL). Then, 200 μL of the mixture was
transferred into a 96-well plate. Ultrapure water was used as a control. The 96-well
plate were incubated at 37°C. The bacterial optical density at a wavelength of 600 nm
was measured every hour by using a microplate spectrophotometer (PowerWave XS2,
BioTek, USA). All assays were performed as triplicates.

Minimal inhibitory concentration (MIC) assay

S. aureus was diluted to 2 × 107 CFU/mL by using LB. Briefly, 100 μL of
CWSNs at different concentrations (0.5, 1, 2, 4, 8, 16, 32, and 64 μg/mL) was placed
into each well of a 96-well plate, and then 100 μL of S. aureus suspension was added
to each well. The optical density (OD) at 600 nm of S. aureus suspensions was
measured at different times by using a microplate spectrophotometer. All assays were
performed as triplicates.

Assay on the inhibition of S. aureus biofilm formation

S. aureus was diluted to 2 × 107 CFU/mL by using LB with 1% glucose. Briefly,
100 μL of CWSNs at different concentrations (final concentration: 1, 10, 100, and 200
μg/mL) was placed into each well of a 96-well plate, and then 100 μL of S. aureus
suspension containing glucose was added into each well. The samples were incubated
at 37°C for 24 h. All assays were performed in triplicate.

Crystal violet staining assay

The crystal violet assay was used to quantificationally evaluate the total biofilm
biomass. The LB medium of all wells was aspirated, and the wells were washed two
times with saline. The biofilms were fixed with 2.5% glutaraldehyde for 30 min and
stained with 0.2% crystal violet at room temperature for 30 min. The excess stain of
each well was removed and washed with saline. Next, the crystal violet-stained
biofilms were quantified by adding 200 μL of 95% ethanol. The OD at 590 nm was
measured by using a microplate spectrophotometer. All assays were performed as
triplicates.

Morphology analysis of biofilms

S. aureus was diluted to 2 × 106 CFU/mL by using LB with 1% glucose. Briefly,
1 mL of CWSNs at different final concentrations (1, 10, 100, and 200 μg/mL) was
placed into confocal dishes, and then 1 mL of S. aureus suspension containing glucose
was added to each well. The samples were incubated at 37°C for 24 h. The excess
medium in confocal dishes was removed. The biofilms were stained with calcein-AM
at room temperature for 30 min and washed with saline to remove excessive stain. A
confocal laser scanning microscope was used to obtain 630 × 630 μm images (CLSM,
Olympus IX81).

Bacterial viability of biofilms

S. aureus suspensions (2 × 107 CFU/mL) with LB that contained 1% glucose
were incubated with 1 mL of CWSNs at different final concentrations (1, 10, 100, and
200 μg/mL) at 37°C for 24 h. Saline was used as a control. The LB medium of each
well was removed, and 200 μL saline was added to each well. Next, the bacterial
suspensions were transferred into 1.5 mL tubes containing 800 μL saline; and were
collected by ultrasonication for 5 min at room temperature. The mixtures were diluted
1~109 times with saline, and then 100 μL of the mixtures was spread onto LB agar
plates. After incubation for 18 h at 37°C, the number of CFUs was counted.
Triplicates were performed for all assays.
Preparation and detection of the gel antibiofilm potential in vitro
The gel (3% agar) was heated to 45°C, and saline or CWSNs were added to the gel.
Then, the mixture was cooled to room temperature to obtain the saline gel and CWSN
gel. S. aureus was diluted to 1 × 10 6 CFU/mL in agar plates with 1% glucose LB in
agar plates. Then, 50μL saline gel or CWSN gel was placed on agar plates and plates
were maintained and observed for 24 h at 37°C.

Mice and ethics statement

Specific pathogen-free female Balb/c mice (18-22 g) were purchased from the
Qinglong Mountain Company of China. All research procedures were approved by
the Animal Care and Use Committee of the Medical School of Nanjing University
and conformed to the National Institutes of Health Guide for Care and Use of
Laboratory Animals.

Treatment of wound infection

The animal models for infected wounds were generated with S. aureus and
Balb/c mice. After the dorsal hair was removed from the backs of mice and a circular
defect was created on the backs, all wounds were infected by using 100 μL of S.
aureus suspension (1 × 107 CFU/mL) within 24 h. The infected wounds were
established, and then the mice were randomly placed into two groups (n=6/group).
The infected mice were treated with saline gel (0.3% agar) and CWSN gel containing
0.3% agar (50 μg/mL CWSNs). The mice were sacrificed on the 4th day, and the
wound tissues were placed in saline by using ultrasonication to count the number of
CFUs in the wound. One hundred microliters of the obtained solutions was plated
onto LB agar plates and incubated at 37°C for 18 h. The small wound tissues were
harvested at therapeutic day 4 and fixed in 4% paraformaldehyde solution. Then the
tissues were paraffined, sectioned, and analyzed by hematoxylin-eosin (H&E) staining
and Masson’s trichrome staining. All samples were examined using a microscope
(Olympus IX-71).
Cytotoxicity assay
The cytotoxicity of CWSNs was evaluated using normal cells (Hok cell line, oral
epithelial cells of mice) by an lactate dehydrogenase (LDH cytotoxicity assay. A
LDH-cytotoxicity colorimetric assay kit was purchased from KeyGen (Nanjing,
China). Hok cells (104 cells/well) were added to 96-well plates. After 24 h, the
supernatant medium was removed and CWSN dispersions (200 μL) containing
different concentrations of CWSNs (25, 50, 100, and 200 μg/mL) were added to
wells. The wells supplemented with PBS were used as a low control (LC), and 1%
Triton X-100 was used as a high control (HC). After 24 h of coculture, 100 μL of
supernatant/well was removed, and then 100 μL of reaction solution/well was added.
After 30 min, a microplate reader was used to measure the OD495 nm. The viability
of cells was calculated according to the following formula: Cell viability = −(test
sample OD495 − LC OD495)/ (HC OD495 − test sample OD495) ×100% + 100%

Toxicity study of CWSNs in vivo

Ten Balb/c mice were randomly divided into two groups (5 mice/group) and an
i.v. injection with 200 μL PBS and 200 μL CWSNs (50 μg/mL), respectively, was
performed (ref). All mice were sacrificed on the 14th day to obtain the major organs
for H&E staining. The weights of the treated mice were recorded every two days.
No Statistical analysis mentioned?
Results
Preparation and characterization of CWSNs
The morphology of CWSNs is shown in Figure 1A and the nanoparticle size
distribution showed that the CWSNs have an average size of ~20 nm. The TEM
image showed that the CWSNs had cube-like appearances. The HRTEM image in
Figure 1B shows that the crystal lattice of the CWSNs had a distance of
approximately 0.495 nm. Furthermore, the high-angle annular dark field scanning
TEM (HAADF-STEM) image shows that the CWSNs had a clear morphology of the
CWSNs, and the corresponding EDS elemental mapping images show that the CWSN
have identical elemental distributions of Cu, W, and S (Figure 1C). EDX spectroscopy
also showed that the CWSN have identical elemental distributions of Cu, W, and S
(Figure 1D). The crystal structure of the CWSNs was characterized by XRD (Figure
1E). All results indicate that the CWSNs were successfully prepared.

Photocatalytic generation of •OH by CWSNs

Photocatalytic CWSNs can generate high levels of ROS (•OH) under visible-
light; thus, photocatalytic CWSNs have potential antibacterial applications (Figure
2A). As shown in Figure 2B, the bandgap extraction is 2.45 eV for the CWSNs. The
valence band (VB) was evaluated by using UPS. Figure 2C shows that the VB
position of the CWSNs is 4.51 eV. The conduction band (CB) of CWSNs and •OH
formation potential are shown in Figure 2D, which is specific that CWSNs are
suitable for generating •OH. Moreover, the generation of •OH was detected by using
TA through fluorescence characterization in water under visible-light. Figure 2E
shows that obvious fluorescence intensity was generated through the production of
•OH at a wavelength of 425 nm, indicating that the photocatalytic generation of OH•
by CWSNs had occurred.

Antibacterial action of CWSNs in vitro

The antibacterial activity of CWSNs was evaluated by using S. aureus, and the
bacteria were exposed to different concentrations of CWSNs in saline. Figure 3A
shows the concentration-dependent antibacterial activity, and 1 μg/mL CWSNs
achieved ~100% bacterial inactivation. The bacterial morphology was observed by
SEM (Figure 3B), as the SEM images showed that the S. aureus cells cultured in
saline (control) maintained a sphere-shaped morphology with intact and smooth cell
membranes, but those exposed to CWSNs became damaged and wrinkled. These
results show that CWSNs have a strong ability to kill free S. aureus. Next, bacterial
regrowth was studied after antibacterial experiments in LB broth for different times.
The S. aureus growth curves are shown in Figure 3C, and the results indicate that
bacterial regrowth was fully inhibited by 1 μg/mL CWSNs. To further study the
inhibition of bacterial growth, the bacteria were exposed to different concentrations of
CWSNs in LB broth. As shown in Figure 3D, bacterial growth was inhibited at a
CWSNs concentration of of ≥16 μg/mL, which indicates that the minimal inhibitory
concentration (MIC) of CWSNs was 16 μg/mL. CWSNs are encapsulated by LB, and
their catalytic activity is affected. Thus, the MIC of CWSNs was higher than the
antibacterial concentration in the bacterial regrowth testing.

Anti-biofilms potential of CWSNs

Previous results show that CWSNs can excite ROS to inhibit biofilm potential
under visible-light. The inactivation efficiency of S. aureus by CWSNs was studied
under visible-light and in the dark using a plate count method. The biofilm biomass of
was analyzed by crystal violet staining and is shown in Figure 4A. The biomass of
adhesion biofilms decreased with increasing concentrations of CWSNs, and biofilms
were reduced up to ~97% at 200 μg/mL CWSNs under visible-light. However,
biofilms were only reduced up to ~46% at 200 μg/mL CWSNs in the dark. To
investigate the antibiofilm potential of CWSNs under visible-light, we examined the
efficiency of CWSNs in inhibiting bacterial biofilm formation on the surface. CWSNs
at different concentrations were first added to each well of a 96-well plate, and then S.
aureus in LB with glucose was added to these wells. After incubation for 24 h under
static conditions, the biofilms obviously attached to the bare surface in the absence of
CWSNs, while few biofilms formed with CWSN concentrations of 100-200 μg/mL
(Figure 4B). CLSM images further showed that the amount of the adhered biofilm
was dose-dependent (Figure 4C), which is suitable for the crystal violet staining
results. As shown in Figure 4D, the CFU of S. aureus biofilms decreased with
increasing concentrations of CWSNs. Figure 4E shows that the viability of S. aureus
biofilms exposed to CWSNs (200 μg/mL) decreased by approximately 100%,
suggesting the excellent antibiofilm activity was excellent. All results indicate that
CWSNs can inhibit bacterial surface adhesion and biofilm formation.
CWSNs promoted the healing of infected wounds
To gain a better time of CWSNs action on the wound, we generated different
gels that were used to treat infected wounds (Figure 5A). Compared to saline gel
(control), the CWSN gel significantly inhibited the growth of bacterial biofilms on
agar plates (Figure 5B). To evaluate the tissue regeneration and antibacterial activity
of CWSNs in vivo, a wound infection model was established on the backs of BALB/c
mice (Figure 5C). The S. aureus-infected wounds were treated with saline gel and
CWSN gel. Photographs show that the wounds in the CWSN gel-treated groups were
smaller than those in the saline groups on the 3rd and 4th days (Figure 5D).
Obviously, the average wound area of CWSN gel-treated mice was 3.81 mm 2, which
was smaller than that of the saline gel-treated mice (average 6.98 mm 2) on 4th day,
indicating that CWSNs can promote healing of infected wounds (Figure 5E). The
H&E and Masson staining results are shown in Figure 5F. The length of the epithelial
gap in the CWSN gel-treated groups was smaller than that in the saline gel groups,
and the saline gel groups maintained obvious scabbing and abundant neutrophils
compared with that of the CWSN-treated groups. Moreover, the number of bacterial
CFUs from infected wound tissues was assessed by using the plate counting method
(Figure 5G). As shown in Figure 5H, the bacterial inactivation efficiency of the
CWSN gel-treated groups was ~ 2 log compared with that of the saline gel groups,
suggesting that CWSN gel can kill S. aureus in the infected wounds of mice. All
results indicate that CWSNs can efficiently treat S. aureus-infected wounds and
promote wound healing.

Biocompatibility of CWSNs
The toxicity evaluation is an essential factor for nanomaterials with potential
biomedical applications. The cytotoxicity of CWSNs was assessed through the LDH
cytotoxicity assay using Hok cells. As shown in Figure 6A, Hok cells remained >90%
viable in the presence of CWSNs at concentrations up to 200 μg/mL, indicating that
the CWSNs had a low toxicity. Furthermore, the long-term toxicity of CWSNs to
mice was also studied. As shown in Figure 6B, the body weights of the mice treated
with CWSNs steadily increased and were not obviously different from those of the
mice treated with saline. All mice were sacrificed on day 14, and the H&E staining
images of major organs from the mice after they obtained an i.v. injection of CWSNs
showed no noticeable damage or inflammatory lesions (Figure 6C). The results
indicate that CWSNs have excellent biosafety and future application potential.

Discussion
Bacteria usually exist as biofilms, and biofilm-associated infectious diseases,
such as chronic wound infections, pulmonary infections, endocarditis osteomyelitis,
musculoskeletal infections, dental periodontitis, peri-implantitis, or even nosocomial
infections, are an increasingly serious problems in the global medical community [16-19].
In the process of biofilm formation, bacteria attach to inert or biological surfaces and
become wrapped in self-produced extracellular polymeric substances (EPSs). EPSs
have many negative effects, such as facilitating bacterial aggregation and biofilm
cohesion, allowing cell–cell communication among the biofilm population and
providingas a source of nutrients. In addition, EPS also provides protection against
both mechanical stresses and antibiotic treatments [20]. Fighting bacterial biofilm-
associated infections is a major challenge with traditional therapies. Therefore, there
is an urgent need to develop novel treatment strategies that are effective at combating
bacterial biofilms.
Nanomedicine has shown great promise for the diagnosis and therapy of various
diseases[21, 22]
. In particular, nanomaterials have been extensively developed for the
treatment of bacterial biofilm infections[23, 24]
. These antibiofilm nanomedicine
technologies have many advantages; thus, an increasing number of antibiofilm agents
have been ingeniously designed to target EPSs from biofilms and to kill bacteria in
biofilms[25]. Recent studies have reported that novel photocatalytic antibiofilm agents
can possess photosensitive ability or enzyme-like catalytic activity, which could
disrupt the matrix of biofilms and damage the cell components of bacteria via the
catalytic generation of ROS[26, 27]
. Therefore, developing novel antibiofilm
photocatalytic nanomaterials is a promising treatment strategy for infectious diseases.
Ternary transitional metal sulfides possess a variety of intriguing chemical and
physical properties and can be used in many fields. We successfully prepared CWSN
nanocrystals by a microwave irradiation method. TEM results showed that CWSNs
were small (average 20 nm). The HRTEM image showed that the crystal lattice
distance of CWSN is approximately 0.495 nm, which can correspond to the (002)
plane of I-CWS[12]. The crystal structure of CWSNs was characterized by XRD, and
the diffraction peaks of CWSNs can be attributed to the I-CWS planes. A previous
study demonstrated that Cu2WS4 nanocrystals have oxidase- and peroxidase-like
activities that are involved in ROS production [14]. It has been reported that the UV–vis
diffuse reflectance spectrum suggested that the as-synthesized I-Cu 2WS4 submicron
crystallites had a direct bandgap of approximately 2.15 eV. The as-synthesized I-
Cu2WS4 submicron crystallites exhibited considerably high photocatalytic activity in
the reduction in aqueous Cr (VI) under visible-light (λ > 420 nm) irradiation [28].
Herein, the bandgap of CWSNs was extracted from the relation of the photon energy
and absorption coefficient based on the UV–vis-NIR absorption spectrum [14].
Compared with the reported CWS materials, the property characterizations of CWSNs
demonstrated that the bandgap and VB of CWSNs were 2.45 eV and 4.51 eV,
respectively, which enabled CWSNs to generate •OH for bacterial inactivation [28]; in
addition, the bandgap of CWSNs is larger than that of CWS materials,, which may be
due to their smaller size. Moreover, the generation of OH• was detected by using TA
through fluorescence characterization in water under visible light. The TA
fluorescence spectra showed that CWSNs can generate a large amount of OH• under
visible light. Notably, OH•, a highly toxic ROS, can kill bacteria by destroying
essential macromolecules and inducing oxidative lesions in the bacterial membrane [29].
S. aureus is a nosocomial bacterium that can cause different infections, from skin
and soft tissue infections to more serious and life-threatening infections, such as
sepsis[30]. In the present study, the CWSNs showed high antibacterial activity (~100%)
atlow CWSN concentration (1 μg/mL) for 2 h. The SEM images showed that CWSNs
can adhere to the cell membranes of S. aureus and result in damage and wrinkles in
the membrane structure. The MIC of CWSNs in LB broth is higher than their
antibacterial concentration in saline, and this can be attributed to the
biomacromolecules from the LB coating on the surface of CWSNs, which causes their
lower catalytic activity.
In addition, as antibiofilm nanomaterials, photocatalytic CWSNs can effectively
inhibit S. aureus biofilm formation. Thus, the biomass of adhesion biofilms decreased
with increasing concentrations of CWSNs. Property studies have demonstrated that
CWSNs can catalyze the generation of •OH under visible light. The biofilms were
reduced up to ~38% at 100 μg/mL or ~97% at 200 μg/mL CWSNs under visible-light.
However, biofilms were only reduced up to 72% at 100 μg/mL or ~46% at 200 μg/mL
CWSNs in the dark. Under visible light conditions, CLSM images show that the
biofilms are obviously destructed when they are exposed to a sufficient concentration
of CWSNs (200 μg/mL). Moreover, the CFU of S. aureus biofilms decreased with
increasing concentrations of CWSNs. Therefore, CWSNs have excellent antibacterial
properties and can inhibit bacterial surface adhesion to form biofilms in vitro.
It is difficult to incubate the traditional solution form of nanoagents on a wound
for a long time. Thus, there is a great need for new administration methods. The gel
form is a convenient method with many advantages, such as controlled release
performance, biodegradability and biocompatibility. The results showed that the
CWSN gel was successfully prepared and had a remarkable antibacterial effect in the
gel form on S. aureus bacterial coating plates. To evaluate the antibacterial activity of
CWSN gel in vivo, a wound infection model was established on the backs of BALB/c
mice according to a previous method[31]. In vivo data showed that the CWSN gel
achieved A 2 log bacterial inactivation efficiency in S. aureus infected mice and
efficiently promoted wound healing. The H&E and Masson staining results showed
that the length of the epithelial gap in the CWSN gel-treated groups was smaller than
that in the saline gel-treated groups. Furthermore, the saline gel groups maintained
obvious scabbing and abundant neutrophils compared with that of the CWSN gel-
treated groups. In addition, larger noninfected wounds were established and observed,
the wounds of the CWSN gel-treated groups were smaller than those of the saline
groups on the 4th and 8th days, and the wounds of the CWSN gel group were
completely healed on the 12th day. Therefore, the above results indicate that CWSN
gel not only inhibited biofilms but also promoted tissue regeneration and has excellent
wound healing abilities.
It is essential to evaluate the toxicity of nanoagents prior to their applications in
biomedicine. Cu and sulfur are essential elements in the human body, and ionic
tungsten species have low bioaccumulation and can be excreted from the body
through feces and urine[32-34]. Furthermore, after intravenous injection of CWSN saline
dispersions into mice, the photomicrographs of the major organs stained with H&E
show no abnormality at 14 days, which indicates that CWSNs have excellent
biocompatibility.
This work provides an antibiofilm agent for potentially inhibiting bacterial or
even drug-resistant bacterial biofilm formation on surfaces. CWSNs show good
biocompatibility for mammalian cells. This work provides a new photocatalytic
antibiofilm nanomaterial for the effective inhibition of bacterial biofilm formation.

Conclusion
Overall, CWSNs were prepared for efficient inhibition of S. aureus and biofilm
formation with a high efficiency by performing a photocatalytic generation of •OH
under ambient light. The CWSN gel can achieve an excellent efficiency in the in vivo
inactivation of S. aureus and promotes wound healing. This work provides a new
photocatalytic antibiofilm nanomaterial for the effective inhibition of bacterial biofilm
formation.

References
1. Iwase, T., et al., Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm
formation and nasal colonization. Nature., 2010. 465(7296): p. 346-9.
2. Foster, T.J., et al., Adhesion, invasion and evasion: the many functions of the surface proteins
of Staphylococcus aureus. Nat Rev Microbiol., 2014. 12(1): p. 49-62.
3. Li, Y., et al., A multifunctional Fenton nanoagent for microenvironment-selective anti-biofilm
and anti-inflammatory therapy. Mater Horiz, 2021. 8(4): p. 1264-1271.
4. Wang, Z., et al., Infection microenvironment-related antibacterial nanotherapeutic strategies.
Biomaterials, 2021: p. 121249.
5. Flemming, H.C. and J. Wingender, The biofilm matrix. Nat Rev Microbiol., 2010. 8(9): p.
623-633.
6. Chen, Z., et al., A Multinuclear Metal Complex Based DNase-Mimetic Artificial Enzyme:
Matrix Cleavage for Combating Bacterial Biofilms. Angew Chem Int Ed Engl., 2016. 55(36):
p. 10732-6.
7. Wani, F.A., et al., Resistance Patterns of Gram-Negative Bacteria Recovered from Clinical
Specimens of Intensive Care Patients. . Microorganisms, 2021. 9(11): p. 2246.
8. Xiu, W., et al., Recent development of nanomedicine for the treatment of bacterial biofilm
infections. View, 2020. 2(1).
9. Wang, L.S., A. Gupta, and V.M. Rotello, Nanomaterials for the Treatment of Bacterial
Biofilms. ACS Infect Dis., 2016. 2(1): p. 3-4.
10. Liu, C., et al., Rapid water disinfection using vertically aligned MoS(2) nanofilms and visible
light. Nat Nanotechnol., 2016. 11(12): p. 1098-1104.
11. Tao, J., M. Luttrell T Fau - Batzill, and M. Batzill, A two-dimensional phase of TiO₂ with a
reduced bandgap. Nat Chem. , 2011. 3(4): p. 296-300.
12. Ozel, F., et al., Hydrogen Evolution Catalyzed by Cu(2)WS(4) at Liquid-Liquid Interfaces.
ACS Appl Mater Interfaces., 2016. 8(39): p. 25881-25887.
13. Li, N., et al., Charge separation in facet-engineered chalcogenide photocatalyst: a selective
photocorrosion approach. Nanoscale, 2014. 6(16): p. 9695-702.
14. Shan, J., et al., Efficient Bacteria Killing by Cu(2)WS(4) Nanocrystals with Enzyme-like
Properties and Bacteria-Binding Ability. ACS Nano, 2019. 13(12): p. 13797-13808.
15. Rasool, K., et al., Antibacterial Activity of Ti₃C₂Tx MXene. ACS Nano, 2016. 10(3): p. 3674-
84.
16. Nguyen, T.H., et al., Rapid pathogen-specific recruitment of immune effector cells in the skin
by secreted toxins. . 2021(2058-5276 (Electronic)).
17. de Sousa, T.A.-O., et al., Genomic and Metabolic Characteristics of the Pathogenicity in
Pseudomonas aeruginosa. Int J Mol Sci., 2021. 22(23): p. 12892.
18. Dong, H., et al., Surface Modified Techniques and Emerging Functional Coating of Dental
Implants. Coatings, 2020. 10(11).
19. Cao, C., et al., Mesoporous Silica Supported Silver-Bismuth Nanoparticles as Photothermal
Agents for Skin Infection Synergistic Antibacterial Therapy. Small, 2020. 16(24): p. e2000436.
20. Blackman, L.D., et al., Approaches for the inhibition and elimination of microbial biofilms
using macromolecular agents. Chem Soc Rev, 2021. 50(3): p. 1587-1616.
21. Dong, H., et al., Polyethyleneimine modification of aluminum hydroxide nanoparticle
enhances antigen transportation and cross-presentation of dendritic cells. Int J Nanomedicine,
 2018. 13: p. 3353-3365.
22. Park, S.M., et al., Towards clinically translatable in vivo nanodiagnostics. Nat Rev Mater,
2017. 2(5): p. 17014.
23. Xiu, W., et al., Biofilm Microenvironment-Responsive Nanotheranostics for Dual-Mode
Imaging and Hypoxia-Relief-Enhanced Photodynamic Therapy of Bacterial Infections.
Research (Wash D C), 2020. 2020: p. 9426453.
24. Shi, L.A.-O., et al., Moldable Hyaluronan Hydrogel Enabled by Dynamic Metal-
Bisphosphonate Coordination Chemistry for Wound Healing. . Adv Healthc Mater., 2018.
7(5): p. 1700973.
25. Li, X., et al., Recent Advances in Synthesis and Biomedical Applications of Two-Dimensional
Transition Metal Dichalcogenide Nanosheets. . Small 2017. 13(5): p. 1602660.
26. Wainwright, M., et al., Photoantimicrobials-are we afraid of the light? Lancet Infect Dis.,
2017. 17(2): p. e49-e55.
27. Wu, S., et al., Biofilm‐Sensitive Photodynamic Nanoparticles for Enhanced Penetration and
Antibacterial Efficiency. Advanced Functional Materials, 2021. 31(33).
28. Jia, Q., et al., Hydrothermal synthesis of Cu2WS4 as a visible-light-activated photocatalyst in
the reduction of aqueous Cr(VI). Materials Letters, 2014. 117: p. 24-27.
29. Sun, H., et al., Graphene quantum dots-band-aids used for wound disinfection. ACS Nano,
2014. 8(6): p. 6202-6210.
30. Idrees, M., et al., Staphylococcus aureus Biofilm: Morphology, Genetics, Pathogenesis and
Treatment Strategies. . Int J Environ Res Public Health., 2021. 18(14): p. 7602.
31. Yuwen, L., et al., MoS(2)@polydopamine-Ag nanosheets with enhanced antibacterial activity
for effective treatment of Staphylococcus aureus biofilms and wound infection. Nanoscale,
2018. 10(35): p. 16711-16720.
32. Kim, B.E., D.J. Nevitt T Fau - Thiele, and D.J. Thiele, Mechanisms for copper acquisition,
distribution and regulation. Nat Chem Biol., 2008. 4(3): p. 176-185.
33. Komarnisky, L.A., T.K. Christopherson Rj Fau - Basu, and T.K. Basu, Sulfur: its clinical and
toxicologic aspects. Nutrition, 2003. 19(1): p. 54-61.
34. van der Voet, G.B., et al., Metals and health: a clinical toxicological perspective on tungsten
and review of the literature. Mil Med. 172(9): p. 1002-5.

Figure legand

Figure. 1. Characterization of the prepared CWSNs.

(A) TEM image and nanoparticle size distribution histogram of CWSNs. (B) HRTEM
images of CWSNs. (C) HAADF-STEM image of CWSNs and elemental mapping
images of Cu, W, and S. (D) EDX spectroscopy of the CWSNs. (E) XRD pattern of
CWSNs.
Abbreviations: CWSNs, Cu2WS4 nanocrystals; TEM, transmission electron
microscope; HRTEM, high-resolution TEM; EDX, energy dispersive X-ray; XRD, X-
ray diffraction.

Figure 2. The photocatalytic induction of ROS formation by CWSNs

(A) Schematic showing the CWSNs inactivating bacteria through visible light
photocatalytic ROS generation. (B) Bandgap of CWSNs, in which hʋ is the photon
energy and α is the absorption coefficient. (C) The VB edge of CWSNs measured by
using UPS. (D) The band position of CWSNs with respect to the ROS formation
(OH•) potential. (E) Fluorescence spectra of TA incubated with different experimental
groups.
Abbreviations: CWSNs, Cu2WS4 nanocrystals; ROS, reactive oxygen species; VB,
valence band. CB, conduction band; UPS, ultraviolet photoelectron spectroscopy.

Figure. 3. The antibacterial action of CWSNs in vitro

(A) Antibacterial activity of different concentrations of CWSNs in saline. (B) SEM
images of S. aureus treated with saline and 1 μg/mL CWSNs. (C) The regrowth
curves of S. aureus in LB broth under different concentrations of CWSNs. (D)
Concentration-dependent inhibition of bacterial growth in the presence of CWSNs in
LB.
Abbreviations: CWSNs, Cu2WS4 nanocrystals; LB broth, Luria-Bertani (LB) broth;
SEM, scanning electron microscope.

Figure. 4. Inhibition of S. aureus biofilms formation.

(A) The inactivation efficiency of S. aureus biofilms by CWSNs (1, 10, 100, and 200
μg/mL) under visible-light and in the dark. The biomass of crystal violet-stained S.
aureus biofilms was determined by measuring the absorbance at OD 590 nm. The
efficiency of CWSNs in inhibiting bacterial biofilm formation. (B) Photograph of S.
aureus biofilms after treatment with different concentrations of CWSNs followed by
crystal violet staining. (C) CLSM images of S. aureus biofilms after treatment with
different concentrations of CWSNs. (D) Photographs of bacterial broth plates and (E)
bacterial viability of S. aureus biofilms after treatment with different concentrations
of CWSNs.

Figure 5. Treatment of S. aureus-infected wounds.

(A) Photographs of the saline gel and CWSNs gel. (B) antibiofilm potential of the
saline gel and CWSN gel in vitro. (C) Schematic diagram of the in vivo infected
model construction and treatment. (D) Photographs and (E) area of the infected
wounds at different days (0, 1, 2, 3, 4) after the saline gel or CWSN gel treatment. (F)
Microphotographs of the H&E staining and Masson's trichrome staining slices of S.
aureus-infected tissues on the 4th day posttreatment. (G, H) Bacterial CFU of infected
tissues after treatments on the 4th day.

Figure 6. Biocompatibility of CWSNs

(A) Cell viability of CWSNs, which was determined by an LDH cytotoxicity assay.
(B) The body weight curves of the mice after the i.v. injection with saline (control)
and CWSNs. (C) H&E stained slices of major organs (heart, liver, spleen, lung, and
kidney) from the mice treated with saline and CWSNs at 14 days.