*3.1. Characterization of MoS2-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, MoS2 converted light energy into heat energy, thereby killing the bacteria by actively trapping the cells through Van. MoS2-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 MoS2 nanosheets, the morphology and thickness of MoS2 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 MoS2 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 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<sup>−</sup>1, and a distinct peak at 1000 cm<sup>−</sup><sup>1</sup> 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.

**Figure 2.** Synthesis and characterization of MoS2-Van-FITC@CS. ( **A**) Synthesis illustration of MoS2- Van-FITC@CS. (**B**) SEM images of MoS2 NPs. ( **C**) Zeta potential of MoS2, MoS2-Van, MoS2-Van-FITC, and MoS2-Van-FITC@CS. ( **D**) UV-vis absorption spectra of MoS2, Van, MoS2-Van, MoS2-Van-FITC, and MoS2-Van-FITC@CS. (**E**) FT-IR spectrometry of MoS2, Van, MoS2-Van, MoS2-Van-FITC, and MoS2-Van-FITC@CS. (**F**) Digital images of I (Milli-water), II (FITC), III (MoS2), IV (MoS2-Van), V (MoS2-Van-FITC), and VI (MoS2-Van-FITC@CS) under bright and UV light. ( **G**) Fluorescence emission spectra of MoS2-Van-FITC@CS.

#### *3.2. In Vitro Photothermal Efficiency*

MoS2 is a photothermal agen<sup>t</sup> 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 *τs* 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.

**Figure 3.** In vitro photothermal efficiency. (**A**) The photothermal responses of MoS2-Van-FITC@CS. Different concentrations (50, 100, 200 and 400 μg/mL) were exposed to NIR irradiation (808 nm; 2 W/cm2). PBS served as a control. (**B**) The photothermal responses of MoS2-Van-FITC@CS (400 μg/mL), which was exposed to different power density of NIR irradiation (808 nm; 0.5, 1, 1.5 and 2 W/cm2). (**C**) The heating and cooling curves of MoS2-Van-FITC@CS (400 μg/mL) and PBS (laser irradiation at 808 nm). (**D**) The linear regression between the cooling period and −ln(*θ*) of the driving force temperature. Results shown are mean ± SD, *n* = 3.

#### *3.3. 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-positive bacteria. MoS2-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

density and light time using power densities of 0.5, 1, and 1.5 W/cm2, and light times of 0, 150, and 300 s. We observed that a power density of 1.5 W/cm<sup>2</sup> and a light time of 300 s inhibited bacterial growth. As shown in Figure 4, the inhibition rate of bacteria in the absence of MoS2 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 MoS2 (100 μg/mL, 1.5 W/cm2, 300 s). After treatment with MoS2-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/cm2. 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.

**Table 1.** Antibacterial activities with MICs values test (μg/mL).


Data are average values of at least three replicates.

**Figure 4.** In vitro antibacterial activity. (**A**) The results of the CFU assay for the blank group. PBS as a blank group. (**B**) The results of the CFU assay for MoS2 (100 μg/mL) for different times (0, 150 and 300 s) at different power densities (0.5, 1 and 1.5 W/cm2). (**C**) The results of the CFU assay for MoS2-Van-FITC (100 μg/mL) for different times (0, 150 and 300 s) at different power densities (0.5, 1 and 1.5 W/cm2). (**D**) Quantitative statistical results of (**A**–**C**) (PD = Power Density). graphed using the Origin software. Results shown are mean ± SD, *n* = 3.

## *3.4. 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 [46–48]. 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 μg/mL. However, as the concentration increased, the number of bacteria decreased, showing the excellent antibacterial ability of MoS2-Van-FITC (Figure 5). The results of cellular uptake assays indicated that MoS2-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.

**Figure 5.** Fluorescence microscopy images of *S. aureus* cultures treated with MoS2-Van-FITC at various concentrations (15, 30, 45 μg/mL) for 12 h. PBS served as a control for the blank group. The cells stained with DAPI for 30 min were fluorescent blue, and those stained with MoS2-Van-FITC were fluorescent green. Scale bar = 15 μm. (DAPI, Ex = 358 nm and Em = 461 nm; FITC, Ex = 490 nm and Em = 525 nm).

#### *3.5. 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.

**Figure 6.** Confocal fluorescence microscopy assay (**A**). *S. aureus* cultures after treatment with MoS2- Van NPs + NIR (100 μg/mL). Van solution (1 μg/mL), MoS2 NPs + NIR (100 μg/mL) and MoS2-Van (100 μg/mL) served as control groups. PBS served as a blank group. The cells were stained with SYTO 9 (green fluorescence) and PI (red fluorescence) for 30 min. The cells underwent NIR irradiation at 808 nm (1.5 W/cm2, 6 min). The results of the apoptotic assay by flow cytometry analysis were statistically analyzed by CytExpert software (version 2.4.0.28) (**B**). Scale bar = 15 μm. The data are expressed as mean ± SD (*n* = 3).

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 played its own role after NIR irradiation, and the apoptotic rate was 94.51%. The increased antibacterial activity of MoS2-Van NPs was further confirmed by the quantitative analysis of viable and non-viable cells via flow cytometry.

## *3.6. 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 MoS2- 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 MoS2. Taken collectively, these findings indicate that MoS2-Van-FITC NPs have very effective antibacterial activity compared to MoS2 NPs and Van alone.

**Figure 7.** SEM images of *S. aureus* cells. The bacterial cultures were treated with MoS2-Van-FITC NPs at various concentrations (25, 50 and 100 μg/mL). The bacterial cultures treated with Van (1 μg/mL) and MoS2 NPs + NIR (100 μg/mL) served as control groups. PBS + NIR served as a blank group. The red squares indicate the enlarged regions. The blue area is the elemental analysis chart. The cells underwent NIR irradiation at 808 nm (1.5 W/cm2, 6 min).

#### *3.7. 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<sup>2</sup> for 6 min, achieved a good therapeutic effect. The MoS2 group had the largest relative wound area, which did not heal. However, MoS2 decreased the relative wound area after NIR irradiation. MoS2-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. MoS2 was least effective, and the healing of mice treated with MoS2 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 MoS2-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 MoS2-Van-FITC@CS hydrogel combined with photothermolysis significantly promoted wound healing in mice.
