Synthesis of a Two-Dimensional Molybdenum Disulfide Nanosheet and Ultrasensitive Trapping of Staphylococcus Aureus for Enhanced Photothermal and Antibacterial Wound-Healing Therapy

Photothermal therapy has been widely used in the treatment of bacterial infections. However, the short photothermal effective radius of conventional nano-photothermal agents makes it difficult to achieve effective photothermal antibacterial activity. Therefore, improving composite targeting can significantly inhibit bacterial growth. We inhibited the growth of Staphylococcus aureus (S. aureus) by using an extremely low concentration of vancomycin (Van) and applied photothermal therapy with molybdenum disulfide (MoS2). This simple method used chitosan (CS) to synthesize fluorescein 5(6)-isothiocyanate (FITC)-labeled and Van-loaded MoS2-nanosheet hydrogels (MoS2-Van-FITC@CS). After modifying the surface, an extremely low concentration of Van could inhibit bacterial growth by trapping bacteria synergistically with the photothermal effects of MoS2, while FITC labeled bacteria and chitosan hydrogels promoted wound healing. The results showed that MoS2-Van-FITC@CS nanosheets had a thickness of approximately 30 nm, indicating the successful synthesis of the nanosheets. The vitro antibacterial results showed that MoS2-Van-FITC with near-infrared irradiation significantly inhibited S. aureus growth, reaching an inhibition rate of 94.5% at nanoparticle concentrations of up to 100 µg/mL. Furthermore, MoS2-Van-FITC@CS could exert a healing effect on wounds in mice. Our results demonstrate that MoS2-Van-FITC@CS is biocompatible and can be used as a wound-healing agent.


Introduction
Bacterial infections are consistently ranked as one of the leading causes of human mortality, with infection rates, mortality rates, and hospitalization costs increasing annually [1][2][3]. The high prevalence of bacterial infections has led to the misuse of antibiotics, resulting in the emergence of superbugs as bacteria become resistant to treatment. Unfortunately, superbugs are arising at a rate much faster than that of new antibiotic discovery, thereby leading to a growing threat of untreatable bacterial infections [4][5][6][7][8]. Therefore, new treatments are urgently needed.
With the rapid development of technologies in the fields of modern nanotechnology and biomedicine, numerous antibacterial inorganic nanoparticles (NPs), such as silver, gold, copper nanoparticles, alumina, zinc oxide, magnesium oxide, silica titanium dioxide, and graphene oxide NPs, as well as their composites, have been used in antibacterial therapy [3,[9][10][11]. For example, two-dimensional (2D) graphene-based nanocomposites and

Synthesis of MoS2 Nanosheets
MoS2 nanosheets were synthesized by liquid ultrasonic stripping [29][30][31][32]. In brief, 0.5 g of MoS2 was combined with 50 mL of 45% ethanol to form the dispersion system of MoS2 (concentration, 10 µg/mL), which was sonicated in a water bath for 12 h. The solution was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and processed by a rotary evaporator to yield a thin film. The film was weighed, and MoS2 was resuspended in deionized water to form the MoS2 nanosheet aqueous solution.

Synthesis of MoS2-Van-FITC
Van is a heptapeptide-containing glycopeptide antibiotic with a primary amine moiety that binds covalently to FITC [33]. In brief, 4 mL of the MoS2 nanosheet aqueous solution (concentration, 0.002 g/mL) was aliquoted, 0.2 mg of Van was added to the solution, and the volume was adjusted to 20 mL. The solution was mixed on a magnetic stirrer at 250 rpm for 6 h and centrifuged at 12,000 rpm for 15 min. The precipitate was resuspended in 20 mL of pure water, and 1 mL of FITC was added. The solution was further mixed for 12 h. MoS2-Van-FITC was obtained by centrifugation.

Synthesis of the MoS2-Van-FITC@CS Hydrogel
To prepare the hydrogel, 300 mg of CS was added to 18 mL of 0.1 mol HCl solution and mixed on a magnetic stirrer until the CS solution was clarified. Thereafter, 2 mg of sodium β-glycerophosphate was added to 2 mL of 0.001 g/mL MoS2-Van-FITC solution, dissolved completely, and mixed for 4 h [27,34,35]. The MoS2-Van-FITC@CS hydrogel was obtained by heating in a water bath at 37 °C for 1 h.

Synthesis of MoS 2 Nanosheets
MoS 2 nanosheets were synthesized by liquid ultrasonic stripping [29][30][31][32]. In brief, 0.5 g of MoS 2 was combined with 50 mL of 45% ethanol to form the dispersion system of MoS 2 (concentration, 10 µg/mL), which was sonicated in a water bath for 12 h. The solution was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and processed by a rotary evaporator to yield a thin film. The film was weighed, and MoS 2 was resuspended in deionized water to form the MoS 2 nanosheet aqueous solution.

Synthesis of MoS 2 -Van-FITC
Van is a heptapeptide-containing glycopeptide antibiotic with a primary amine moiety that binds covalently to FITC [33]. In brief, 4 mL of the MoS 2 nanosheet aqueous solution (concentration, 0.002 g/mL) was aliquoted, 0.2 mg of Van was added to the solution, and the volume was adjusted to 20 mL. The solution was mixed on a magnetic stirrer at 250 rpm for 6 h and centrifuged at 12,000 rpm for 15 min. The precipitate was resuspended in 20 mL of pure water, and 1 mL of FITC was added. The solution was further mixed for 12 h. MoS 2 -Van-FITC was obtained by centrifugation.

Synthesis of the MoS 2 -Van-FITC@CS Hydrogel
To prepare the hydrogel, 300 mg of CS was added to 18 mL of 0.1 mol HCl solution and mixed on a magnetic stirrer until the CS solution was clarified. Thereafter, 2 mg of sodium β-glycerophosphate was added to 2 mL of 0.001 g/mL MoS 2 -Van-FITC solution, dissolved completely, and mixed for 4 h [27,34,35]. The MoS 2 -Van-FITC@CS hydrogel was obtained by heating in a water bath at 37 • C for 1 h.

Characterization
Ultraviolet-visible (UV-vis) absorption spectra were recorded by UV-vis spectroscopy (model S-3100, Scinco Co., Daejeon, Korea). Fluorescence spectra of MoS 2 -Van-FITC NPs were recorded by fluorescence spectrophotometry (model RF-5301PC, Shimadzu Corp., Kyoto, Japan). Fourier transform infrared (FT-IR) spectroscopy was performed with an FT-IR spectrometer (model Nicolette is50, Thermo Fisher Scientific, Waltham, MA, USA). The ultrastructural characteristics of synthesized MoS 2 were observed via scanning electron microscopy (SEM; model S-4800, Hitachi, Tokyo, Japan). The Brookhaven Zeta Pals instrument was used to obtain zeta potential measurements and to characterize the optical properties of MoS 2 -Van-FITC (Brookhaven Instruments Corp., Holtsville, NY, USA). NPs of different concentrations were illuminated by using NIR irradiation at 808 nm at different power densities for 15 min, and the temperature was detected with an infrared camera with an accuracy of 0.1 • C [36,37].

In Vitro Antibacterial Assays
The minimum inhibitory concentration (MIC) of MoS 2 -Van-FITC NPs was determined using the 96-well microtitration plate dilution method. In brief, 100 µL of LB medium was added to each well of a sterile 96-well plate, and 100 µL of the drug solution was added to the first well, mixed, and diluted in multiples until the last well was mixed. Thereafter, 100 µL of the mixture was discarded, followed by the addition of 100 µL of the bacterial diluent to each well (final density of bacteria, 1 × 10 6 CFU/mL). The positive controls were kanamycin and ampicillin. The cells of the NIR group were NIR irradiated (1.5 W/cm 2 ) for 6 min and cultured at 37 • C for 12 h. The OD value was measured with a microplate reader [38]. The three independent measurements were averaged, and each treatment group had three wells.
Log-phase S. aureus cultures were inoculated (1:40) into medium containing MoS 2 -Van-FITC (100 µg/mL) or MoS 2 (100 µg/mL) and incubated in a shaking incubator at 37 • C for 12 h. The irradiation power densities were 0.5 W/cm 2 , 1 W/cm 2 , and 1.5 W/cm 2 , and the irradiation times were 0 s, 150 s, and 300 s. Thereafter, the cells were coated on solid medium. The number of live bacteria was calculated using the CFU counting method.

Cellular Uptake Assays
To confirm the effect of Van in capturing S. aureus cells, we performed cellular uptake assays. Log-phase S. aureus cultures were incubated with different concentrations (15,30,45 µg/mL) of MoS 2 -Van-FITC NPs for 12 h. The cells treated with PBS served as the control. S. aureus cells were harvested and stained with 4 -6-diamidino-2-phenylindole (DAPI; concentration, 5 µg/mL) for 15 min. Thereafter, the cells were washed twice with PBS, and the red and blue channels were examined under a fluorescence microscope.

LIVE-DEAD Assays
To further investigate the antibacterial ability of the drug, we conducted LIVE-DEAD assays. Log-phase S. aureus cultures were inoculated (1:40) in medium containing MoS 2 -Van-FITC (100 µg/mL), MoS 2 (100 µg/mL), or Van (1 µg/mL) and incubated in a shaking incubator at 37 • C for 12 h. The irradiation power density was 1.5 W/cm 2 , and the irradiation time was 300 s. Thereafter, the cells were stained with SYTO9 and propidium iodide for 30 min in the dark, washed twice with PBS, and red and green channels were examined under a fluorescence microscope. The number of non-viable bacterial cells was determined by the CytoFLEX system (Beckman Coulter, Brea, CA, USA) [2,39].

Cell Integrity Assays
Log-phase S. aureus cultures were treated with different concentrations (25,50, 100 µg/mL) of MoS 2 -Van-FITC NPs. Van (1 µg/mL) and MoS 2 (100 µg/mL) served as the controls. The PBS-treated group served as the blank. The irradiation power density was 1.5 W/cm 2 , and the irradiation time was 6 min. The cells were collected by centrifugation at 4000 rpm for Nanomaterials 2022, 12, 1865 5 of 17 10 min, washed twice with PBS, and fixed with 2.5% glutaraldehyde at 4 • C for 12 h. The cells were subjected to gradient dehydration with different concentrations of ethanol (35%, 50%, 70%, 80%, 95%, 100%) for 20 min each time and then placed into acetone [40,41]. The specimens were observed under a scanning electron microscope.

Establishment of the Wound Mice Model
Kunming female mice (~25 g body weight; 4-5 weeks old) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) and housed under standard environmental conditions (temperature, 22 ± 3 • C; humidity, 55 ± 5%; 12-h dark/12-h light cycles). All animals were housed according to the guidelines in the "Guide for the Care and Use of Laboratory Animals". All animal studies were approved by Suzhou University (Suzhou, China) (approval number: 202010A415). After 7 days of acclimatization, an oval wound of approximately 1.5 cm in length was made by shaving the back of each mouse under anesthesia and adding 100 µL of activated S. aureus cells (1 × 10 6 CFU/mL) dropwise to each wound for two consecutive days. The wound was treated after inflammation [14,42].

Wound Healing Assays
The mice whose wounds were infected with S. aureus were randomly divided into five groups (n = 5-7 mice) as follows: blank group, MoS 2 group, MoS 2 -Van-FITC@CS group, NIR MoS 2 group, and NIR MoS 2 -Van-FITC@CS group. The wound site of the blank group was treated with 100 µL of PBS, that of the MoS 2 group was treated with 100 µL of MoS 2 each day, that of the MoS2-Van-FITC@CS group was treated with 100 µL of MoS 2 -Van-FITC@CS hydrogel each day, that of the NIR MoS 2 group was treated with MoS 2 , followed by irradiation at 1.5 W/cm 2 for 5 min, and that of the NIR MoS 2 -Van-FITC@CS group was treated with MoS 2 -Van-FITC@CS, followed by irradiation at 1.5 W/cm 2 for 5 min. The mice were photographed daily for eight consecutive days. The mice were euthanized, and their epidermises were harvested for hematoxylin-eosin staining [43].

Safety Evaluation of MoS 2 -Van-FITC@CS
To examine the toxic effects of NIR and MoS 2 -Van-FITC@CS on the heart, liver, spleen, lungs, and kidneys of mice, the wounded mice were divided into four groups as follows: control group, NIR irradiation group, MoS 2 -Van-FITC@CS group, and MoS 2 -Van-FITC@CS + NIR group. The irradiation power density was 1.5 W/cm 2 , the wavelength was 808 nm, and the irradiation time was 6 min. The mice were treated until their wounds healed, and they were euthanized 30 days after the end of treatment. Their organs were collected, and the cross-sections were stained with hematoxylin-eosin [44].

Characterization of MoS 2 -Van-FITC@CS
The nanocarriers were exfoliated by liquid phase ultrasound. The composite nanomaterials conjugated to FITC-labeled Van showed strong antibacterial effects. After irradiation, MoS 2 converted light energy into heat energy, thereby killing the bacteria by actively trapping the cells through Van. MoS 2 -Van-FITC had good antibacterial and wound-promoting abilities through the temperature-sensitive hydrogel formed with chitosan ( Figure 2A). To determine the synthesis of two-dimensional MoS 2 nanosheets, the morphology and thickness of MoS 2 NPs were observed via SEM ( Figure 2B). The results of SEM showed that the flake NPs, which had a thickness of approximately 40 nm, were uniformly distributed. Zeta potentiometry can be used to determine the solid-liquid interfacial electrical properties of dispersed systems of particulate matter, so we can determine the successful synthesis of materials by using the potential changes of nanomaterials. The zeta potential of MoS 2 was −21.4 ± 1.2 mV, whereas the loading of Van resulted in a potential of −8.3 ± 1.9 mV and a zeta potential of −33 ± 0.8 mV after labeling with FITC ( Figure 2C). The synthesis of MoS 2 -Van-FITC@CS causes changes in the structure of a single component as a result of electron leaps between electronic energy levels in the valence and molecular orbitals, which can be observed by UV-vis spectrum ( Figure 2D). In the UV-vis spectrum, MoS 2 NPs were observed to have an absorption peak near 808 nm, showing a longitudinal surface plasmon resonance band, which indicated photothermal effects, whereas Van did not show an absorption peak near 808 nm. Van was adsorbed on MoS 2 , after which this characteristic peak significantly shifted. After FITC was decorated on the Van surface, the characteristic peak of MoS 2 -Van-FITC was significantly shifted, and a new characteristic peak was observed near 450 nm. MoS 2 -Van-FITC@CS also showed a shift in the characteristic peak compared to MoS 2 . FTIR spectroscopy allows the observation of the functional groups and chemical bonds contained in the material, in order to be able to determine the successful synthesis of MoS 2 -Van-FITC nanocomposites. FT-IR spectral analysis showed that the presence of Van resulted in an amino peak near 3000 cm −1 , and a distinct peak at 1000 cm −1 in the fingerprint region after the loading of FITC ( Figure 2E). A significant change was also found in the fingerprint region after the wrapping of chitosan. The fluorescence properties of MoS 2 -Van-FITC@CS are shown in Figure 2F,G. MoS 2 -Van-FITC@CS emitted fluorescence under UV light irradiation at 465 nm, and the fluorescence properties of the nanomaterials were further confirmed by the fluorescence spectra. Taken collectively, these findings indicate that MoS 2 , which has photothermal properties, was successfully synthesized, Van was successfully loaded, and the surface was modified by FITC. The morphological features of the MoS 2 -Van-FITC@CS hydrogel were indicative of its successful synthesis.
Nanomaterials 2022, 12, x FOR PEER REVIEW 6 of 18 a potential of −8.3 ± 1.9 mV and a zeta potential of −33 ± 0.8 mV after labeling with FITC ( Figure 2C). The synthesis of MoS2-Van-FITC@CS causes changes in the structure of a single component as a result of electron leaps between electronic energy levels in the valence and molecular orbitals, which can be observed by UV-vis spectrum ( Figure 2D). In the UV-vis spectrum, MoS2 NPs were observed to have an absorption peak near 808 nm, showing a longitudinal surface plasmon resonance band, which indicated photothermal effects, whereas Van did not show an absorption peak near 808 nm. Van was adsorbed on MoS2, after which this characteristic peak significantly shifted. After FITC was decorated on the Van surface, the characteristic peak of MoS2-Van-FITC was significantly shifted, and a new characteristic peak was observed near 450 nm. MoS2-Van-FITC@CS also showed a shift in the characteristic peak compared to MoS2. FTIR spectroscopy allows the observation of the functional groups and chemical bonds contained in the material, in order to be able to determine the successful synthesis of MoS2-Van-FITC nanocomposites.
FT-IR spectral analysis showed that the presence of Van resulted in an amino peak near 3000 cm −1 , and a distinct peak at 1000 cm −1 in the fingerprint region after the loading of FITC ( Figure 2E). A significant change was also found in the fingerprint region after the wrapping of chitosan. The fluorescence properties of MoS2-Van-FITC@CS are shown in Figure 2F,G. MoS2-Van-FITC@CS emitted fluorescence under UV light irradiation at 465 nm, and the fluorescence properties of the nanomaterials were further confirmed by the fluorescence spectra. Taken collectively, these findings indicate that MoS2, which has photothermal properties, was successfully synthesized, Van was successfully loaded, and the surface was modified by FITC. The morphological features of the MoS2-Van-FITC@CS hydrogel were indicative of its successful synthesis.

In Vitro Photothermal Efficiency
MoS 2 is a photothermal agent that produces a large amount of heat to kill bacteria in the NIR region of 808 nm [45,46]. Therefore, we measured its photothermal conversion efficiency to understand the photothermal properties of MoS 2 . As expected, MoS 2 -Van-FITC acted as a good photothermal nanomaterial under NIR irradiation in that it converted light energy into heat energy, resulting in rapid warming ( Figure 3A,B). Furthermore, the MoS 2 -Van-FITC concentration was 400 µg/mL, m PBS (m D ) was 1.0 g, C H 2 O (C D ) was 4.2 J/g/ • C, ∆T max was 47.3 • C ( Figure 3C), I was 2 W, and τ s was 296 s ( Figure 3D). Thus, the photothermal conversion efficiency (η) of MoS 2 -Van-FITC was 52%. Figure S1 shows the thermal images of MoS 2 -Van-FITC at different concentrations under an NIR irradiation at 808 nm. In summary, MoS 2 -Van-FITC has good photothermal conversion efficiency and can be used as a photothermal nanomaterial for killing bacteria.

In vitro Photothermal Efficiency
MoS2 is a photothermal agent that produces a large amount of heat to kill bacteria in the NIR region of 808 nm [45,46]. Therefore, we measured its photothermal conversion efficiency to understand the photothermal properties of MoS2. As expected, MoS2-Van-FITC acted as a good photothermal nanomaterial under NIR irradiation in that it converted light energy into heat energy, resulting in rapid warming ( Figure 3A,B). Furthermore, the MoS2-Van-FITC concentration was 400 µg/mL, mPBS (mD) was 1.0 g, CH2O (CD) was 4.2 J/g/°C, ΔTmax was 47.3 °C (Figure 3C), I was 2 W, and was 296 s ( Figure 3D). Thus, the photothermal conversion efficiency (η) of MoS2-Van-FITC was 52%. Figure S1 shows the thermal images of MoS2-Van-FITC at different concentrations under an NIR irradiation at 808 nm. In summary, MoS2-Van-FITC has good photothermal conversion efficiency and can be used as a photothermal nanomaterial for killing bacteria.

In Vitro Antibacterial Activity
We used S. aureus and E. coli as Gram-positive and Gram-negative bacteria, respectively, in subsequent experiments (Table 1). In the MIC test, we found that MoS2-Van-FITC + NIR had the highest killing effect against S. aureus, which may have been related to the fact that Van is a narrow-spectrum antibiotic that is only effective against Gram-

In Vitro Antibacterial Activity
We used S. aureus and E. coli as Gram-positive and Gram-negative bacteria, respectively, in subsequent experiments (Table 1). In the MIC test, we found that MoS 2 -Van-FITC + NIR had the highest killing effect against S. aureus, which may have been related to the fact that Van is a narrow-spectrum antibiotic that is only effective against Gram-positive bacteria. MoS 2 -Van-FITC + NIR showed the effects of common antibiotics at low doses, and the inhibition of growth made it difficult for bacteria to develop resistance (Table 1). Thermal images of the test in MIC were showed in Figure S2. Figure 4 shows the thermogram of the solution temperature increase after NIR irradiation. We found that our nanomaterials had a stronger growth inhibition ability against S. aureus. In the CFU test, we screened the power Nanomaterials 2022, 12, 1865 8 of 17 density and light time using power densities of 0.5, 1, and 1.5 W/cm 2 , and light times of 0, 150, and 300 s. We observed that a power density of 1.5 W/cm 2 and a light time of 300 s inhibited bacterial growth. As shown in Figure 4, the inhibition rate of bacteria in the absence of MoS 2 was low, and the survival rate of bacteria was 89%. However, the survival rate of bacteria gradually decreased after NIR irradiation, and the survival rate of bacteria was only 4.2% after treatment with MoS 2 (100 µg/mL, 1.5 W/cm 2 , 300 s). After treatment with MoS 2 -Van-FITC + NIR (100 µg/mL), the survival rate of bacteria was 0.9% after an irradiation time of 300 s at a power density of 1.5 W/cm 2 . In the in vitro antibacterial test, the nanomaterials inhibited the growth of S. aureus cells, which played a role in the elimination of bacteria from the wound, thereby speeding up wound healing.

Cellular Uptake Assays
Van targets Gram-positive bacteria by binding to the hydrogen bond of the terminal D-Ala-D-Ala sequence of the cytosolic peptide of bacteria. It is also a heptapeptide-containing glycopeptide antibiotic with a primary amine moiety that binds covalently to FITC [46][47][48]. Van can label FITC on the bacterial surface; therefore, we verified the targeting of Van by DAPI staining. When MoS2-Van-FITC was used at a concentration of 15 µg/mL, most of the bacteria were labeled, similar to higher concentrations of 30 µg/mL and 45

Cellular Uptake Assays
Van targets Gram-positive bacteria by binding to the hydrogen bond of the terminal D-Ala-D-Ala sequence of the cytosolic peptide of bacteria. It is also a heptapeptide-containing glycopeptide antibiotic with a primary amine moiety that binds covalently to FITC [46][47][48]. Van can label FITC on the bacterial surface; therefore, we verified the targeting of Van by DAPI staining. When MoS 2 -Van-FITC was used at a concentration of 15 µg/mL, most of the bacteria were labeled, similar to higher concentrations of 30 µg/mL and 45 µg/mL. However, as the concentration increased, the number of bacteria decreased, showing the excellent antibacterial ability of MoS 2 -Van-FITC ( Figure 5). The results of cellular uptake assays indicated that MoS 2 -Van-FITC successfully targeted bacteria and showed excellent antibacterial ability, thereby achieving our goal of using combined chemotherapy and photothermal therapy to inhibit bacterial growth.

Fluorescent Staining Analysis of Antibacterial Activity
The CFU assay can detect only viable bacteria to determine the antibacterial activity of nanomaterials. To further examine the antibacterial activity of nanomaterials, we determined the number of viable and non-viable cells using the LIVE-DEAD assay to examine the antibacterial activity of MoS2-Van NPs. Viable bacterial cells were stained green, whereas non-viable bacterial cells were stained red ( Figure 6A). No cell death was observed in the blank group. However, cell death was observed in the Van group, indicating that Van can inhibit bacterial growth. Similar results were obtained for the cells treated with MoS2 + NIR and MoS2-Van, with the MoS2-Van + NIR group exhibiting stronger inhibition of bacterial growth after NIR irradiation.
To further confirm that MoS2-Van+NIR reduced the survival of bacteria, the number of viable and non-viable bacterial cells was quantified via flow cytometry. As shown in Figure 6B, the apoptotic rate of the blank + NIR group was 0.78%. The apoptotic rate of MoS2 + NIR (100 µg/mL) after NIR irradiation was 53.95%. When the concentration of Van was 1 µg/mL, the apoptotic rate was 49.68%. The apoptotic rate of MoS2-Van (100 µg/mL) was 67.24% without irradiation, which was mainly due to the effects of Van, but MoS2 also

Fluorescent Staining Analysis of Antibacterial Activity
The CFU assay can detect only viable bacteria to determine the antibacterial activity of nanomaterials. To further examine the antibacterial activity of nanomaterials, we determined the number of viable and non-viable cells using the LIVE-DEAD assay to examine the antibacterial activity of MoS 2 -Van NPs. Viable bacterial cells were stained green, whereas non-viable bacterial cells were stained red ( Figure 6A). No cell death was observed in the blank group. However, cell death was observed in the Van

Cell Integrity Study
Based on our findings, MoS2-Van NPs + NIR showed efficient antibacterial activity against S. aureus. Because photothermal action mainly targets the bacterial cell surface, and the cell surface is also the site of action of Van, we speculate that changes in cell integrity may be the main mechanism behind the induction of apoptosis in bacteria. As such, we investigated the effects of MoS2-Van NPs + NIR on the cellular integrity of S. aureus by SEM.
The integrity of bacterial cells was examined via SEM, as shown in Figure 7. S. aureus cells in the blank group had normal cell morphology, including intact cell membranes. The results showed that NIR irradiation alone did not affect the structure of cells. The rupture and shrinkage of cells could be clearly seen after treatment with Van and MoS2 in the control group, and the enlarged area showed that the cells did not have intact cell membranes. In addition, there was cell leakage. In the MoS2-Van-FITC group, we observed more severe cell damage, even at a concentration of 25 µg/mL, compared to the blank group. As the concentration increased, the cell damage increased, indicating that To further confirm that MoS 2 -Van+NIR reduced the survival of bacteria, the number of viable and non-viable bacterial cells was quantified via flow cytometry. As shown in Figure 6B, the apoptotic rate of the blank + NIR group was 0.78%. The apoptotic rate of MoS 2 + NIR (100 µg/mL) after NIR irradiation was 53.95%. When the concentration of Van was 1 µg/mL, the apoptotic rate was 49.68%. The apoptotic rate of MoS 2 -Van (100 µg/mL) was 67.24% without irradiation, which was mainly due to the effects of Van, but MoS 2 also played its own role after NIR irradiation, and the apoptotic rate was 94.51%. The increased antibacterial activity of MoS 2 -Van NPs was further confirmed by the quantitative analysis of viable and non-viable cells via flow cytometry.

Cell Integrity Study
Based on our findings, MoS 2 -Van NPs + NIR showed efficient antibacterial activity against S. aureus. Because photothermal action mainly targets the bacterial cell surface, and the cell surface is also the site of action of Van, we speculate that changes in cell integrity may be the main mechanism behind the induction of apoptosis in bacteria. As such, we investigated the effects of MoS 2 -Van NPs + NIR on the cellular integrity of S. aureus by SEM.
The integrity of bacterial cells was examined via SEM, as shown in Figure 7. S. aureus cells in the blank group had normal cell morphology, including intact cell membranes. The results showed that NIR irradiation alone did not affect the structure of cells. The rupture and shrinkage of cells could be clearly seen after treatment with Van and MoS 2 in the control group, and the enlarged area showed that the cells did not have intact cell membranes. In addition, there was cell leakage. In the MoS 2 -Van-FITC group, we observed more severe cell damage, even at a concentration of 25 µg/mL, compared to the blank group. As the concentration increased, the cell damage increased, indicating that MoS 2 -Van-FITC had a stronger antibacterial effect. The results from the elemental analysis chart showed that the bacterial surface did contain elemental sulfur and molybdenum, indicating that the bacterial surface contained MoS 2 . Taken collectively, these findings indicate that MoS 2 -Van-FITC NPs have very effective antibacterial activity compared to MoS 2 NPs and Van alone.

In Vivo Wound Healing Evaluation
We established a wound-healing mouse model and examined the pro-wound healing effects by directly applying NPs combined with irradiation. As shown in Figure 8, the MoS2-Van-FITC@CS hydrogel alone was slightly therapeutic, and the rate of wound healing increased with irradiation ( Figure 8A). Heating, which increased the temperature of the nanomaterial to 50 °C by irradiation at 1.5 W/cm 2 for 6 min, achieved a good

In Vivo Wound Healing Evaluation
We established a wound-healing mouse model and examined the pro-wound healing effects by directly applying NPs combined with irradiation. As shown in Figure 8, the MoS 2 -Van-FITC@CS hydrogel alone was slightly therapeutic, and the rate of wound healing increased with irradiation ( Figure 8A). Heating, which increased the temperature of the nanomaterial to 50 • C by irradiation at 1.5 W/cm 2 for 6 min, achieved a good therapeutic effect. The MoS 2 group had the largest relative wound area, which did not heal. However, MoS 2 decreased the relative wound area after NIR irradiation. MoS 2 -Van-FITC@CS hydrogel + NIR had the best therapeutic effect after NIR irradiation, where the relative wound area was reduced to 20.8%. The weight of mice in all groups decreased and then increased. The reason for the decrease in weight was likely due to the appearance of wounds, but as treatment progressed, the mice recovered and regained the weight. MoS 2 was least effective, and the healing of mice treated with MoS 2 was similar to that of the controls ( Figure 8B). Immunohistochemical analysis was performed to examine the epidermis from the different groups of mice, and the abnormal histological features could be clearly seen in the stained sections ( Figure 8D). No changes in morphology were observed in the epidermises of mice in the control group and the other four groups. The epidermises showed no significant damage compared to those of normal mice, indicating that the elevated temperature of the nanosheets during the photothermal treatment did not cause significant damage to the wounds. Therefore, the therapeutic effect of the MoS 2 -Van-FITC@CS hydrogel combined with NIR irradiation showed that there was no harm to the mice and their wound healing was accelerated. The preliminary photothermal imaging of mice also demonstrated the thermal response of NPs. In summary, the MoS 2 -Van-FITC@CS hydrogel combined with photothermolysis significantly promoted wound healing in mice.

Biosafety Evaluation
The evaluation of toxicity is important for each new drug. As such, we evaluated the safety of our hydrogels in vivo. We applied the hydrogels to the wounds of healthy mice, followed by NIR irradiation, and performed a routine histological analysis of the major organs ( Figure 9). Compared with the controls, no significant organ damage, tissue edema, cell death, or inflammatory cell infiltration was observed in the examined organs after treatment with NIR irradiation, MoS 2 -Van-FITC@CS, and MoS 2 -Van-FITC@CS + NIR, indicating that MoS 2 -Van-FITC@CS + NIR was non-toxic in mice.  The evaluation of toxicity is important for each new drug. As such, we evaluated the safety of our hydrogels in vivo. We applied the hydrogels to the wounds of healthy mice, followed by NIR irradiation, and performed a routine histological analysis of the major organs ( Figure 9). Compared with the controls, no significant organ damage, tissue edema, cell death, or inflammatory cell infiltration was observed in the examined organs after treatment with NIR irradiation, MoS2-Van-FITC@CS, and MoS2-Van-FITC@CS + NIR, indicating that MoS2-Van-FITC@CS + NIR was non-toxic in mice. Figure 9. In vivo toxicity evaluation. The hematoxylin-eosin-stained images of major organs following different treatments of normal mice. Group 1 was the no treatment group, group 2 was the NIR irradiation (1.5 W/cm 2 , 6 min) group, group 3 was the MoS2-Van-FITC@CS group and group 4 was the MoS2-Van-FITC@CS + NIR (1.5 W/cm 2 , 6 min) group.

Conclusions
In this study, the Van-modified MoS2-loaded nanosystem, which was encapsulated in a chitosan hydrogel, was established to examine its antibacterial activity and wound healing ability. The results showed that the thickness of MoS2 NPs was <100 nm, whereas other experiments revealed that the surface of MoS2 was successfully modified by Van. The antimicrobial activity was enhanced when Van was labeled. The photothermal characterization experiments confirmed that MoS2 had good photothermal conversion efficiency, and cellular uptake assays verified the active capture of S. aureus by Van, which significantly improved the photothermal inhibition of bacterial growth. The results of in vitro experiments indicated that NIR could significantly increase the antibacterial activity of MoS2 NPs, whereas those of flow cytometry showed that NPs could increase the apoptotic rate of bacterial cells. The morphological features of bacterial cells treated with MoS2-Van-FITC NPs and NIR irradiation were examined, and NPs were observed to disrupt the integrity of the bacterial cell wall. Furthermore, MoS2-Van-FITC combined with NIR Figure 9. In vivo toxicity evaluation. The hematoxylin-eosin-stained images of major organs following different treatments of normal mice. Group 1 was the no treatment group, group 2 was the NIR irradiation (1.5 W/cm 2 , 6 min) group, group 3 was the MoS 2 -Van-FITC@CS group and group 4 was the MoS 2 -Van-FITC@CS + NIR (1.5 W/cm 2 , 6 min) group.

Conclusions
In this study, the Van-modified MoS 2 -loaded nanosystem, which was encapsulated in a chitosan hydrogel, was established to examine its antibacterial activity and wound healing ability. The results showed that the thickness of MoS 2 NPs was <100 nm, whereas other experiments revealed that the surface of MoS 2 was successfully modified by Van. The antimicrobial activity was enhanced when Van was labeled. The photothermal characterization experiments confirmed that MoS 2 had good photothermal conversion efficiency, and cellular uptake assays verified the active capture of S. aureus by Van, which significantly improved the photothermal inhibition of bacterial growth. The results of in vitro experiments indicated that NIR could significantly increase the antibacterial activity of MoS 2 NPs, whereas those of flow cytometry showed that NPs could increase the apoptotic rate of bacterial cells. The morphological features of bacterial cells treated with MoS 2 -Van-FITC NPs and NIR irradiation were examined, and NPs were observed to disrupt the integrity of the bacterial cell wall. Furthermore, MoS 2 -Van-FITC combined with NIR irradiation could disrupt the cell morphology, induce apoptosis, and affect cell proliferation in vitro. In a wound healing assay, the MoS 2 -Van-FITC@CS hydrogel could accelerate wound healing. In summary, MoS 2 in combination with NIR irradiation shows good applicability in the inhibition of bacterial growth, and the CS hydrogel in combination with a photothermal agent that actively traps S. aureus can disinfect the wound and maintain a moist environment to accelerate wound healing.