Materials

MoS2 was acquired from Nanjing XFNANO Materials Tech Co. Ltd. CS (Deacetylation degree 90%) was obtained from Shanghai yuanye Bio-Technology Co. Ltd. Tetrabutylammonium hydroxide (~ 25% in H2O), N-(3-Dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDCI), p-toluenesulfonic acid, 3-mercaptopropionic acid, methanol, N-hydroxy succinimide (NHS), N-methylpyrrolidone (NMP), N, N-dimethylformamide (DMF), and tert-butyl nitrite were purchased from Aladdin Biochemical Technology Co., Ltd. (China).

Mouse fibroblasts (L929), Human umbilical vein endothelial cells (HUVECs), Escherichia coli (E. coli, ATCC 25,923), and Staphylococcus aureus (S. aureus, ATCC 25,922) were obtained from the American Type Culture Collection (ATCC). Methyl thiazolyl diphenyl-tetrazolium bromide (MTT), Griess reagent kit, ATP assay kit, BCA Protein assay kit, 3-Amino,4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM DA), and Calcein/PI cell viability/cytotoxicity assay kit were purchased from beyotime biotechnology (China). Live/dead bacteria double staining kit was obtained from Shanghai Beibo Biotechnology Co. Ltd. Mouse anti-CD31 and anti-VEGFα were bought from proteintech (China).

Preparation and characterization of MoS2 nanosheets

MoS2 nanosheets were prepared by liquid-phase exfoliation. First, bulk MoS2 was dispersed in a tetrabutylammonium hydroxide (TBAOH) solution at a concentration of 5 mg mL-1 and stirred overnight at room temperature to reduce the interlayer forces between the MoS2 sheets. Afterwards, the bulk MoS2 was rinsed twice with ethanol to remove residual TBAOH. The resulting precipitate was collected in a 50 mL centrifuge tube and dispersed in an 80% aqueous solution of NMP. The precipitate was then completely dispersed through bath sonication, and subsequently exfoliated into MoS2 nanosheets using probe sonication (JY92-IIDN, Scientz) for 10 h with a 3 s on/off cycle. The solution was centrifuged for 45 min at 5000 rpm to remove any unexfoliated MoS2 particles. The resultant supernatant, containing the exfoliated nanosheets, was further centrifuged at 15,000 rpm for 30 min to remove excess organic solvent. Finally, the obtained precipitate was re-dispersed in deionized water, resulting in a transparent and homogeneous dark-green solution of MoS2 nanosheets. The structure of MoS2 nanosheets was characterized using a UV-visible photometer (Ultrospec 7000, Biochrom) and Raman spectrometer (Horiba Evolution, HORIBA Scientific). The surface morphology of MoS2 nanosheets was observed using scanning electron microscopy (SEM, SU8010, HITACHI) and transmission electron microscopy (TEM, FEI Tecnai G2 F20, Thermo Fisher). The particle size and zeta potential of MoS2 were analyzed using a malvern particle size analyzer (Zetasizer Nano ZS-90, Malvern Panalytical).

Preparation and characterization of SNO-CS@MoS2

As previously reported, 3-(nitroso) propionic acid (SNO) was initially produced prior to the synthesis of SNO-CS [39]. Briefly, an excess of tert-butyl nitrite (4 mmol) and 212 mg 3-mercaptopropionic acid were mixed in a 2 mL solution of DMF, and the reaction mixture was kept in an ice bath under a nitrogen atmosphere for 6 h. Afterward, any unreacted tert-butyl nitrite was removed by vacuuming for 1 h to obtain the SNO solution. The distinctive peak of SNO was characterized using a UV-visible photometer.

To prepare organic solvent-soluble CS, p-toluene sulfonic acid and CS were dissolved in 5 mL of deionized water at a CS concentration of 10 mg mL-1. The resulting solution was stirred overnight at room temperature. The undissolved CS was then removed by centrifugation at 2000 rpm for 15 min, and the supernatant was lyophilized to obtain p-toluene sulfonic acid-modified CS. The preparation of SNO-CS mainly involves the amide condensation reaction between the amino group on CS and the carboxyl group on SNO. First, 1 mL SNO (1 mol mL-1), 170.5 mg EDC, and 126.5 mg NHS were mixed in 10 mL DMF, then stirred in an ice bath for 30 min before adding 50 mg p-toluene sulfonic acid-modified CS and stirring for another 20 h in 0 ℃. The resulting mixture was then rinsed three times with methanol, freeze-dried, and stored at -20 °C. The SNO-CS structure was characterized using an FT-IR spectrometer (ALPHAII, Bruker) and a UV-visible photometer. Different amounts of SNO (concentrations ranging from 25, 50, 75, 100, 125.0, and 166.7 μmol L-1) and a condensation agent were added to quantify the grafting amount of SNO in DMF and stirred for 30 min. Equal amounts of p-toluene sulfonic acid-modified CS were dissolved in DMF and reacted in an ice bath for 20 h to conduct the amidation reaction. The concentration and standard fitting curves of SNO were obtained using the characteristic peak of the SNO group in the UV-Vis spectrum at 358 nm. Based on the fitting curve, the grafting efficiency of SNO on CS can be calculated, and the grafting ratio can be determined.

To prepare SNO-CS@MoS2, SNO-CS was first re-dispersed in deionized water at a concentration of 3 mg mL-1, and then gradually added into the MoS2 nanosheet dispersion (1.0 mg mL-1). The reaction mixture was kept in an ice bath for 4 h. Afterwards, excess SNO-CS in the supernatant was removed. The precipitate was collected and dried in a vacuum drying oven to obtain a black powdery product. CS@MoS2 was prepared using a parallel method. The structures of the SNO-CS@MoS2 and CS@MoS2 nanosheets were characterized using a UV-visible photometer and FT-IR spectrometer. The surface morphology of the SNO-CS@MoS2 nanosheets was observed using SEM and TEM. The particle size of SNO-CS@MoS2 and CS@MoS2, as well as the zeta potential of SNO-CS@MoS2, CS@MoS2, SNO-CS, and CS was analyzed using a Malvern particle size analyzer.

In vitro photothermal effect of SNO-CS@MoS2

To evaluate the in vitro photothermal performance, SNO-CS@MoS2 (200 μg mL-1) was placed in a 2 mL plastic centrifuge tube, and then irradiated using an 808 NIR laser at 0.50, 0.75, or 1.00 W cm-2 for 10 min. The temperature values and thermal images were recorded at 15 s intervals using an infrared thermal imager camera (FLIR ONE Pro, TELEDYNE FLIR). Similarly, the dependence of photothermal performance on concentration was also examined. To calculate the photothermal conversion efficiency (η) [40], ultra-pure water and 200 μg mL-1 of SNO-CS@MoS2 were first irradiated using an 808 NIR laser at 1.0 W cm-2 for 15 min, then cooled down naturally for another 20 min. Finally, η was calculated using the following equation:

$$\eta =\frac_-_\right)-_}^_}\right)}$$

(1)

$$\theta =\frac_}_-_}$$

(4)

$$_=hs\left(_-_\right)$$

(5)

where hs can be calculated using Eq. (2), where m is the mass of the SNO-CS@MoS2 solution (g), Cwater is the heat capacity of water (J/g·°C), and τs is the sample system time constant (s). τs can be calculated according to formula (3–4); in formula (3), θ is the dimensionless driving force, and t is the time. Moreover, in formula (4), Tmax represents the maximum steady-state temperature of the mixed solution, Twater represents the maximum steady-state temperature of the water, and Tamb represents the room temperature. Q0 represents the background energy input without a photothermal agent and is calculated using Eq. (5).

To evaluate the photothermal stability of SNO-CS@MoS2, freshly synthesized MoS2, CS@MoS2, or SNO-CS@MoS2 dispersed in PBS were kept at 4 °C for five days. Then, they underwent continuous irradiation and cooling for three cycles using an 808 nm NIR laser to assess the photothermal stability of these materials.

In vitro NO release profiles of SNO-CS@MoS2

The NO release profiles of SNO-CS@MoS2 were detected using a Griess reagent kit. Briefly, different concentrations of SNO-CS@MoS2 (100.0, 150.0, and 200.0 μg mL-1 in PBS) were placed in a 2 mL of the plastic centrifuge tube and then irradiated using an 808 NIR laser at an intensity of 1.0 W cm-2 for 10 min. At predetermined intervals, 100 μL of the solution was removed and mixed with 50 μL Griess reagent. After an additional half-hour of incubation at 37 °C, the amount of NO produced was determined by measuring the absorbance of the mixture at 540 nm using a microplate reader (SpectroMAX M5, Molecular Devices). To further investigate the self-release behavior of SNO-CS@MoS2 in the application scenarios, where SNO-CS@MoS2 releases a large amount of NO in response to photothermal action and exerts efficient antibacterial effects, then residual SNO-CS@MoS2 slowly produces trace amounts of NO under physiological conditions to promote wound healing, SNO-CS@MoS2 exposed to 10 min of NIR light irradiation was collected and then placed in an incubator at 37 °C. At regular intervals, 50 μL of the mixture was extracted to determine the amount of NO produced.

In vitro antibacterial property

The bacterial strains used in this study included S. aureus, methicillin-resistant S. aureus (MRSA), E. coli and extended-spectrum beta-lactamase (ESBL)-producing E. coli. Bacterial single colonies were transferred to tryptone soy broth (TSB) liquid culture medium and incubated overnight at 37 °C for 12 h. The bacteria were grown to the logarithmic stage, collected, washed, and then diluted with sterile PBS to OD600 = 0.1 for subsequent antibacterial experiments. To evaluate the antibacterial effect of SNO-CS@MoS2, 500 μL of bacteria was first incubated with different volumes of SNO-CS@MoS2 (1 mg mL-1) at 37 °C for 30 min, and then illuminated with an 808 nm laser at 1 W cm-2 density for 10 min. The bacterial suspension was then gradually diluted stepwise from 106 to 101 and uniformly inoculated onto TSB plates. After incubation at 37 °C for 18 h, the number of bacterial colonies was counted to evaluate the antibacterial effect. The bacterial survival rate is expressed as log10 CFU mL-1. Bacteria treated with PBS, MoS2 (100, 200, or 400 μg mL-1), or CS@MoS2 (100, 200, or 400 μg mL-1) served as controls. SNO-CS@MoS2 (100, 200, or 400 μg mL-1) was used as a control without NIR light irradiation.

Live/dead staining was performed to further evaluate the antibacterial activity of SNO-CS@MoS2. SYTO-9 and PI were used to distinguish viable and nonviable bacterial cells. Briefly, 200 μL SNO-CS@MoS2 (1 mg mL-1) was mixed with 800 μL of bacterial suspension (108 CFU mL-1) in an eppendorf tube and at 37 °C for 30 min. Then, the bacteria suspension was irradiated as described above. The bacteria were then collected by centrifugation and stained with 100 μL of live/dead backlight bacterial viability kit for 30 min in the dark. Finally, the stained bacteria were washed twice with PBS and observed under a confocal fluorescence microscope (A1, Nikon) at 60× magnification to capture fluorescent staining images. SEM was used to observe changes in bacterial morphology. The bacteria were treated as described above, then fixed with glutaraldehyde at room temperature for 4 h. After fixation, the bacteria were washed thrice with 0.85% NaCl solution and dehydrated in an increasing gradient of ethanol concentrations (20%, 40%, 60%, 80%, 90%, and 100%) in sequence for 15 min. The bacteria were then sputtered with gold before SEM examination. To quantify protein leakage from bacteria following SNO-CS@MoS2 treatment, the bacteria were first treated as described above. The supernatant was collected to measure protein concentration using an enhanced BCA protein assay kit and a microplate reader at 560 nm. Variations in intracellular ATP levels in response to the SNO-CS@MoS2 therapy were evaluated using an improved ATP assay kit. The bacteria were initially treated as described previously, collected, and lysed with 200 μL lysis buffer. Finally, the supernatant was collected and quantified using a luminance-mode microplate reader.

Cell cultivation and in vitro cytotoxicity assay

L929 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin-streptomycin (P&S, Gibco) at 37 ℃, 5% CO2. HUVECs were cultured in endothelial cell medium (ECM, Sciencell) containing 5% (v/v) FBS, 1% (v/v) growth factor (ECGS/ECGF), and 1% (v/v) P&S at 37 ℃, 5% CO2.

The cytotoxicity of SNO-CS@MoS2 against L929 cells was assessed using the MTT assay. L929 cells were seeded at a density of 5 × 103 cells per well in 96-well plates overnight and then treated with SNO-CS@MoS2 at 10, 20, 50, 100, 200, and 500 μg mL-1 doses for another 24 h. Next, 10 μL of MTT reagent was added to each well and incubated for another 4 h. After carefully removing the supernatant from each well, 150 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance of each well at 570 nm was measured using a microplate reader. The live/dead cell-staining assay was performed as follows. Briefly, after 1, 3, or 5 days of incubation with SNO-CS@MoS2 (200 μg mL-1), L929 cells were first stained with calcein-AM and PI double fluorescent stain for 30 min in the dark and then imaged using an inverted fluorescence microscope (Axio Observer 3 materials, ZEISS). Cells treated with PBS, SNO-CS (200 μg mL-1), and MoS2 (200 μg mL-1) served as controls.

Hemolysis assay

Whole blood was extracted from healthy rat hearts by using an anticoagulant. To prepare red blood cell suspensions (RBCs), whole blood was first centrifuged, and the obtained RBCs were then redispersed in PBS. To evaluate the hemolysis rate of SNO-CS@MoS2, 1 mL of SNO-CS@MoS2 at concentrations ranging from 10 to 500 μg mL-1 was first mixed with 20 μL of RBCs and incubated at 37 °C for 4 h. Following centrifugation at 3000 rpm for 15 min, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance of each well was measured at 542 nm using a spectrophotometric microplate reader. RBCs treated with SNO-CS and MoS2 (50–400 μg mL-1) served as the control, PBS was used as a negative control, and double-distilled water was used as a positive control.

Intracellular NO release

The intracellular NO generation behavior of SNO-CS@MoS2 in the absence of NIR irradiation was assessed as follows. Briefly, HUVECs were inoculated onto 96-well plates at a density of 1 × 103 cells per well overnight. Then, 20 μL of SNO-CS-@MoS2 (1 mg mL-1) was added, and the cells were cultured for another 6 h. The cells were then exposed to 100 μL of DAF-FM DA (1 × 10− 3 M) for 30 min in the dark. Finally, cells were washed twice with PBS and observed under an inverted fluorescence microscope. Cells treated with PBS, SNO-CS (30 μg mL-1), and MoS2 (200 μg mL-1) served as the control, SNO-CS, and MoS2 groups, respectively.

Cell scratching healing

Cell scratch experiments were performed to assess the effect of SNO-CS@MoS2 on promoting wound healing. Briefly, L929 cells were seeded at 1.5 × 105 cells per well into 12-well plates and cultured to 80% confluence. The cell monolayer was then scraped with a sterile pipette tip (200 μL) and washed three times with PBS. Subsequently, the cells were incubated with fresh serum-free medium containing SNO-CS@MoS2 (200 μg mL-1) for 6, 18, or 24 h. After that, the cells were stained with crystal violet and observed under an inverted microscope. Cells treated with PBS, SNO-CS (30 μg mL-1), and MoS2 (200 μg mL-1) served as control, SNO-CS and MoS2 groups.

Angiogenesis assay

To assess the angiogenic potential of SNO-CS@MoS2, a tube formation assay was performed. Briefly, HUVECs (3.5 × 103) were seeded overnight in 48-well plates, then exposed to SNO-CS@MoS2 (200 μg mL-1)-containing molecular cellular and developmental biology 131 (MCDB131, pricella) medium (containing 2% (v/v) FBS and 1% (v/v) P&S) for 12 h. Afterward, the cells were digested and reseeded in 48-well plates pre-spread with matrix gel for 3 h. The cells were then stained with calcein fluorescent dye and observed using an inverted fluorescence microscope. The total length and the number of junctions were assessed by using the Image J software with an angiogenesis analyser plugin. Cells treated with PBS, SNO-CS (30 μg mL-1), and MoS2 (200 μg mL-1) served as the control, SNO-CS, and MoS2 groups, respectively. Besides, HUVECs were treated as described above. The cells were then collected to measure the mRNA and protein expression levels of CD31 and VEGF by qRT-PCR and western blotting. The primer sequences for angiogenesis-related genes (CD31 and VEGFα) are listed in Table S1.

In vivo treatment of S. aureus-infected full-thickness cutaneous wound model

All studies involving animals were reported following the ARRIVE guidelines and conducted according to National Research Council guidelines for the care and use of laboratory animals. Sprague-Dawley rats (eight weeks, Male) were obtained from Biotech Co. Ltd. (Beijing, China) and acclimatized for one week prior to the study. To establish the S. aureus-infected full-thickness cutaneous wound model, the rats anesthetized with an intramuscular injection of ketamine (100 mg mL-1) and xylazine (20 mg mL-1) at 1 mL kg-1 of body weight. The back of rats were shaved, and a round full-thickness cutaneous wound (10 × 10 mm) area was created using a punch, followed by inoculation with 10 μL of S. aureus suspension (108 CFU mL-1) into the wound. Subsequently, all rats were randomly divided into five groups (n = 8): (1) PBS + NIR, (2) SNO-CS + NIR, (3) MoS2 + NIR, (4) SNO-CS@MoS2, and (5) SNO-CS@MoS2 + NIR. The wound area was treated by direct injection of 200 μL of PBS or the materials three times during the entire treatment period. Rats in NIR light irradiation group were irradiated with an 808 nm NIR laser at 1 W cm-2 for 5 min. Thermal images were acquired by an infrared thermal imager every 50 s. On day 1, the antibacterial activity in vivo was measured by swabbing the infected wound for 10 s and counting the colonies in PBS. The wound area was photographed using a digital camera on days 0, 3, 7, 9, and 11 to determine the wound closure rate (WCR). The WCR was calculated using the following equation, where S0 represents the wound area on day 0, and S represents the wound area on the photographed day.

$$WCR \left(\%\right)=\frac_-S\right)}_} \times 100\%$$

At the end of treatment, the rats were sacrificed, and blood was collected using a standard vein blood collection technique for hematology analysis. The safety of the treatment process was assessed by comparing the number of red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST). The wound tissues and viral organs were fixed, embedded, sectioned, and stained with hematoxylin and eosin (H&E), Masson’s trichrome, and anti-CD31 antibodies. All sections were observed under an inverted fluorescence microscope.

Statistical analysis

The normal distribution of the data was verified using the Kolmogorov-Smirnov test. Significant differences in these variables were detected using a one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.

Results and discussionPreparation and characterization of SNO-CS@MoS2

The preparation of SNO-CS@MoS2 mainly includes three steps (Scheme 1 A): 1) exfoliation of bulk MoS2 into mono- and few-layer 2D nanosheets of MoS2, (2) preparation of SNO-modified CS (SNO-CS), and (3) electrostatic adsorption of SNO-CS on the surface of MoS2 nanosheets. To prepare mono- and few-layer MoS2, TBAOH was employed as the intercalation agent to reduce the van der Waals interactions between the bulk MoS2 layers [41]. Subsequently, high-frequency ultrasound was employed to strip multiple layers of bulk MoS2 into mono- and few-layer MoS2 through acoustic cavitation [42]. The resulting mono- and few-layer MoS2 were then redispersed in deionized water, resulting in a homogenous and transparent dark green suspension. The successful preparation of mono- and few-layer MoS2 was confirmed by UV-vis absorption spectroscopy, morphological characterization, and Raman spectroscopy.

Figure 1 A displays the UV-vis absorption spectrum of the MoS2 nanosheet, depicting characteristic absorption bands at 663 and 618 nm, which arise from the direct excitonic transitions at the K point of the Brillouin zone [43]. The presence of bands at 666 and 608 nm confirms the existence of mono- and few-layer MoS2, indicating the successful exfoliation of bulk MoS2 into nanosheets [44]. The microstructure and morphology of MoS2 nanosheets were characterized by SEM and TEM as shown in Fig. 1G-H, revealing a single or few-layered sheet-like structure with a smooth surface. Figure 1B displays Raman spectra of bulk MoS2 and MoS2 nanosheets, which reveal the characteristic peaks of MoS2: E12g and A1g, where the E12g peak is the in-plane bending mode, and the A1g peak is the out-of-plane phonon mode related mostly to the stretching of the sulfur atoms [45]. It has been reported that the E12g and A1g peaks in mono- and few-layer MoS2 typically exhibit a redshift and blueshift, respectively, along with a decrease in intensity compared to that of bulk MoS2 [44]. Notably, our sample exhibited a similar trend, as evidenced by the E12g and A1g peaks of bulk MoS2 at 381 and 406 cm-1, respectively. In contrast, the E12g and A1g peaks of the resulting MoS2 nanosheets shifted to 380 and 403 cm-1, respectively, and the intensities of the two peaks were reduced, confirming the successful preparation of mono- and few-layer MoS2.

The preparation of SNO-CS involves amide condensation reactions between the carboxyl groups on SNO and the amino groups on CS (Fig. S1). The UV-vis absorption and FT-IR spectra of SNO-CS confirmed the successful synthesis of SNO-CS. It has been reported that the S-NO group has a characteristic absorption band at 335–338 cm-1 in UV-vis spectrum, which is attributed to the allowed n0→ π* transition [46]. The characteristic absorption band of the S-NO group can be observed at 330–350 cm-1 in the UV-vis spectrum of SNO-CS (Fig. 1D). The synthesis of SNO-CS was further verified by FT-IR spectra in Fig. 1E. SNO-CS exhibits absorption spectra similar to pure CS [47]. Furthermore, in the spectra of SNO-CS, the characteristic bands found at 1516 and 1634 cm-1 correspond to the I and II bonds of C = O, respectively. The peak representing the N-H bond is also observed in the range of 3500–3700 cm-1, which together prove the amide bond existence [48]. Furthermore, SNO-CS also demonstrated a new peak at 686 cm-1 corresponding to the -S-N = bond [30]. The above analysis proves the successful combination of SNO-CS. Furthermore, the grafting rates of 3-(nitroso) propionic acid on CS were calculated using the UV-vis spectra and a standard curve of 3-(nitroso) propionic acid (Fig. S2A–B). The grafting rates were concentration-dependent, and at a concentration of 125 μmol L-1, the grafting efficiency of 3-(nitroso) propionic acid was 10.31%, resulting in a maximum grafting ratio of 15.46% (Fig. S2C). The above SNO-CS were used for the subsequent synthesis of SNO-CS@MoS2.

Through electrostatic interactions, we synthesized SNO-CS-coated MoS2 (SNO-CS@MoS2) following the successful creation of mono- and few-layer MoS2 and SNO-CS. The successful preparation of SNO-CS@MoS2 was confirmed by the following results:1) zeta potential and particle size distribution, 2) morphological characterization, 3) elemental mapping, 4) UV-vis absorption spectroscopy, and 5) FT-IR spectra. In detail, the zeta potential value charged from − 21.07 to 20.03 mV (Fig. 1C), and the average size increased from 127.5 to 341.1 nm (Fig. S3A-B) after coating of SNO-CS on MoS2. Additionally, the microstructures of MoS2 and SNO-CS@MoS2 were characterized by SEM and TEM in Fig. 1G-H. Compared to pure MoS2, SNO-CS@MoS2 exhibited thicker and more uniformly dispersed nanostructures with a rough and vague surface. The above experiments proved that the combination of SNO-CS and MoS2 resulted in significant changes in the surface morphology and roughness of the original MoS2. Through measurements of TEM images, the average size of pure MoS2 is approximately 120 nm, which is consistent with the results obtained from DLS measurements. However, the average size of SNO-CS@MoS2 is approximately 260 nm, which is smaller than the results obtained from DLS measurements. The size difference of the SNO-CS@MoS2 measured by TEM and DLS was mainly attributed to DLS presenting wet samples. High-resolution TEM was further used to analyze the structure. The TEM images showed a clear lattice structure of MoS2 (Fig. 1I).The clarity of the lattice structure in SNO-CS@MoS2 is reduced due to the adsorption of SNO-CS (Fig. 1J). Elemental mapping was conducted to determine the elemental composition of SNO-CS@MoS2 in Fig. 1K and Table S2. The results confirmed the presence of Mo, S, C, N, and O, further confirming SNO-CS loading. The above results were further verified by the UV-vis absorption spectra in Fig. 1D and FT-IR spectra in Fig. 1E. The UV-vis absorption spectra of SNO-CS@MoS2 showed a shift in the absorption band of pure MoS2 from 450 to 437 cm-1 after SNO-CS modification. The characteristic absorption band of the S-NO group at 330–350 cm-1 was also observed. Moreover, UV-vis-NIR absorbance spectra of MoS2 showed strong UV to NIR absorbance, which was not affected by the surface SNO-CS modification (Fig. S4). The mass extinction coefficient of MoS2 nanosheets at 800 nm was calculated to be 7.45 L g-1 cm-1, similar to values measured by other researchers [49]. In addition, FT-IR spectra clearly demonstrated that SNO-CS@MoS2 had absorption spectra similar to those of SNO-CS and pure MoS2. All the above results prove the successful synthesis of SNO-CS@MoS2. Furthermore, the loading amount of SNO-CS on MoS2 was estimated using thermogravimetric analysis (TGA), as demonstrated in Fig. 1F. The TGA curve of MoS2 shows significant weight loss between 400 and 500 °C due to the oxidation of MoS2 to MoO3. The thermal decomposition profile of SNO-CS@MoS2 is like SNO-CS, it gradually decreases between 250 and 600 ºC, and the weight loss below 150 °C can be attributed to moisture vaporization. According to TGA data, it was estimated that ~ 14% weight of SNO-CS was loaded onto the MoS2 nanosheets.

Furthermore, to demonstrate that the synergism of SNO-CS on the PTT antibacterial activity of MoS2 mainly originates from NO, we synthesized CS@MoS2 using similar methods. In addition, the successful preparation of CS@MoS2 was confirmed by particle size and zeta potential analysis, as displayed in Fig. S3C-D. CS@MoS2 presents a positively charged nanostructure with an average size of 212 nm. Long-term stability tests were conducted on MoS2, CS@MoS2, and SNO-CS@MoS2. Fig. S5A-B depict photographs of the solution color and particle size change at different time points (day 1, 3, and 5) for freshly synthesized MoS2, CS@MoS2, and SNO-CS@MoS2 in the PBS solution. MoS2, CS@MoS2, and SNO-CS@MoS2 exhibited good dispersibility on day 1. However, MoS2 aggregated on day 3, and the average particle size increased to approximately 1 μm. In contrast, CS@MoS2 and SNO-CS@MoS2 exhibited high dispersion and stability on day 5 (Fig. S5C). This experiment demonstrated that the interlayer re-stacking of MoS2 can be inhibited after CS or SNO-CS modification, and SNO-CS@MoS2 exhibits superior stability and shows good prospects for application in vivo.

Fig. 1

Spectroscopic characterization of SNO-CS@MoS2 nanosheets. (A) UV-vis absorbance spectra of MoS2 nanosheets. (B) Raman spectra of bulk MoS2 and MoS2 nanosheets. (C) Zeta potential of SNO-CS, MoS2 nanosheets and SNO-CS@MoS2 nanosheets. D-E) FT-IR spectra and UV-vis spectra of CS, SNO-CS, MoS2 nanosheets and SNO-CS@MoS2 nanosheets. F) TGA curves of MoS2 nanosheets, SNO-CS and SNO-CS@MoS2 nanosheets. G-H) SEM and TEM of MoS2 nanosheets and SNO-CS@MoS2 nanosheets. I-J) High-resolution TEM images of MoS2 nanosheets and SNO-CS@MoS2 nanosheets. K) Elemental mappings of Mo, S, C, N and O of SNO-CS@MoS2 nanosheets

Photothermal performance and NO generation behavior

After successfully preparing SNO-CS@MoS2, we systematically evaluated the photothermal properties of SNO-CS@MoS2. Figure 2 A-B display the temperature increase of SNO-CS@MoS2, CS@MoS2, MoS2, and SNO-CS under 808 nm NIR light irradiation. The results demonstrate that MoS2 has excellent photothermal conversion performance, and the coatings of CS and SNO-CS did not affect the photothermal performance of MoS2. Studies have demonstrated that temperatures over 50 °C kill bacteria by inactivating enzymes related to bacterial life activities [3]. The temperature of SNO-CS@MoS2 solution increased by 25 °C after 6 min of irradiation, indicating that it could achieve effective antibacterial performance in vivo. In addition, the results in Fig. 1C-D show that the photothermal properties of SNO-CS@MoS2 nanosheets depend on the concentration and laser intensity. Under a fixed laser power (1.0 W cm-2), the solution is heated with increasing concentrations. Moreover, the solution temperature increased with the laser intensity at a fixed concentration (200 μg mL-1). The photothermal conversion efficiency (PCE) of the freshly prepared nanomaterials is calculated in Fig. 2E and S6C. The PCE of SNO-CS@MoS2 was 23.75%, barely similar to that of CS@MoS2 (24.37%) and MoS2 (22.87%), and is related to the high photothermal stability of freshly prepared MoS2. The long-term photothermal stability of SNO-CS@MoS2 was also investigated. After five days of setting, three heating/cooling cycles under 808 nm radiation were applied to MoS2, CS@MoS2, and SNO-CS@MoS2 (Fig. 2F and S6A-B). The temperature of pure MoS2 reached only 34 °C after 10 min of irradiation, which was much lower than that on the first day (49.57 °C). In contrast, CS@MoS2 and SNO-CS@MoS2 exhibit high photothermal stability across all three switching cycles. This result confirmed that the long-term photothermal stability of MoS2 nanosheets was greatly improved after chitosan or SNO-CS modification. In conclusion, the above investigations depicted that SNO-CS@MoS2 possesses excellent photothermal characteristics and is a promising photothermal agent for both in vivo and in vitro applications.

After demonstrating the excellent photothermal performance of SNO-CS@MoS2, we further evaluated the NO-releasing behavior of SNO-CS@MoS2 using the Griess assay. Quantitative NO investigation was conducted using a standard curve of NO (Fig. S7). Figure 2G depicts that under 808 nm NIR irradiation (1 W cm− 2), the amount of NO generated by SNO-CS@MoS2 was much higher than that stored at 4 °C, indicating effective control of NO release by NIR laser irradiation. Furthermore, NO release from the SNO-CS@MoS2 nanosheets was concentration-dependent (Fig. 2H). The amount of released NO increased with higher concentrations. Among them, 200 μg mL− 1 of SNO-CS@MoS2 can release nearly 7 μmol L− 1 of NO after 8 min of NIR irradiation, fully meeting the effective antibacterial concentration requirements.

In addition to the efficient release of NO by laser irradiation, we further demonstrated that SNO-CS@MoS2 can produce low concentrations of NO in a physiological environment. To detect the NO release under the physiological environment, we placed RSNO, SNO-CS and SNO-CS@MoS2 in PBS under natural light at 37 ℃. The results showed that the NO release from the three groups was basically the same, indicating that the slow release of RSNO was mainly related to natural light and room temperature [50, 51] (Fig. S8). Furthermore, we collected the SNO-CS@MoS2 solution after 10 min of irradiation and monitored its long-term NO-release behavior at 37 °C. As illustrated in Fig. 2I, SNO-CS@MoS2 can release low concentrations of NO after the removal of NIR irradiation, and the increased amount of NO could be cumulative to 6.88 μmol L− 1 from 10 min to 18 h. In conclusion, the above experiments have demonstrated that SNO-CS@MoS2 nanosheets possess the ability to generate NO under photothermal control and exhibit sustained NO generation in a physiological environment.

Fig. 2

The photothermal effect and NO generation behavior of SNO-CS@MoS2 nanosheets. (A) Infrared thermal images of SNO-CS, MoS2, CS@MoS2 and SNO-CS@MoS2 (concentration: [MoS2] = 200 μg mL-1, [SNO-CS] = 30 μg mL-1) under NIR irradiation (808 nm, 1 W cm-2). and (B) corresponding temperature profiles. (C) Temperature profiles of SNO-CS@MoS2 in different concentration (12, 25, 50, 100 and 200 μg mL-1) under NIR irradiation (808 nm, 1 W cm-2). (D) Temperature profiles of SNO-CS@MoS2 at different light intensity under an 808 nm laser (0.5, 0.75 and 1 W cm-2). (E) The cooling time plot versus − ln(?) of MoS2 and SNO-CS@MoS2 nanosheets (200 μg mL-1). (F) Temperature elevations of SNO-CS@MoS2 nanosheets for three NIR light irradiations cycles under an 808 nm laser (200 μg mL-1, 1 W cm-2). (G) NO release curves of SNO-CS@MoS2 nanosheets under NIR irradiation (808 nm, 1 W cm-2) or stored at 4 °C. (H) NO release curves of SNO-CS@MoS2 nanosheets in different concentration (100, 150 and 200 μg mL-1) under NIR irradiation (808 nm, 1 W cm-2). (I) Accumulation NO release within 18 h on SNO-CS@MoS2 nanosheets after 10 min of irradiation (808 nm, 1 W cm-2)

In vitro antibacterial effects of SNO-CS@MoS2

The above studies have demonstrated the photothermal property and NO-producing capability of SNO-CS@MoS2, which prompted further investigation into its antibacterial properties. The antibacterial experiment against Gram-positive S. aureus and Gram-negative E. coli was conducted using the plate counting method. As displayed in Fig. 3A-D, in the absence of a NIR laser, the PBS and MoS2 treatment groups exhibited no antibacterial effect, whereas the CS@MoS2 and SNO-CS@MoS2 groups demonstrated some antibacterial effect, which may be attributed to the inherent antibacterial property of CS [52]. After 10 min of exposure to NIR irradiation, the survival rate of bacteria in the MoS2 and CS@MoS2 groups displayed a significant downward trend compared with the corresponding groups without irradiation treatment. The CS@MoS2 + NIR group exhibited log reductions of 3.29 for S. aureus and 1.92 for E. coli, indicating that the local hyperthermia produced by PTT of MoS2 exhibited high antibacterial effects. The SNO-CS@MoS2 + NIR exerted the greatest antibacterial effect. Furthermore, the bacterial colonies of S. aureus and E. coli significantly decreased by 4.07 and 4.71 log, respectively, demonstrating that a high concentration of NO significantly enhances the antibacterial effect of PTT. Moreover, the synergistic effect of NO on MoS2 PTT antibacterial therapy was concentration-dependent. As displayed in Fig. S9-10, the synergistic effect of SNO-CS@MoS2 was more obvious at low concentrations (100 μg mL− 1) while inconspicuous at high concentrations (400 μg mL− 1). Moreover, an increase in the concentration of SNO-CS@MoS2 led to higher PTT temperatures, posing a risk of tissue damage. Therefore, we selected 200 μg mL− 1 as the final concentration. The live/dead staining technique confirmed the synergistic antibacterial action. The proportion of living and dead bacteria can be directly observed in the fluorescence images (Fig. 3E-H). Only red fluorescent spots were observed in S. aureus and E. coli of the SNO-CS@MoS2 + NIR group, implying that SNO-CS@MoS2 treatment with NIR killed almost all bacteria. Intense green fluorescence was still observed in MoS2 + NIR and CS@MoS2 + NIR groups. This research further demonstrates the feasibility and efficacy of the synergistic antibacterial activity of NO and PTT. Furthermore, the main issue with antibacterial agents is bacterial multidrug resistance. We further conducted in vitro antibacterial experiments against resistant bacteria using plate counting method. The results are generally align with the findings mentioned above (Fig. S11).

Fig. 3

Antibacterial effect of SNO-CS@MoS2. (A) Photographs of bacterial colonies formed by S. aureus after various treatment; (B) The corresponding bacterial viabilities of S. aureus; (C) Photographs of bacterial colonies formed by E. coli after various treatment; (D) The corresponding bacterial viabilities of E. coli; E-F) Fluorescent images of S. aureus and corresponding statistical data; G-H) Fluorescent images of E. coli and corresponding statistical data. (Laser: 808 nm, 1.0 W cm-2, 10 min; Concentration: 200 μg mL-1)

After confirming the excellent synergistic antibacterial performance of SNO-CS@MoS2, experiments were conducted to further investigate the antibacterial mechanisms. Firstly, changes in the morphology and membrane integrity of S. aureus and E. coli after SNO-CS@MoS2 treatment were observed using SEM. As demonstrated in Fig. 4A-B, in the presence or absence of NIR laser, both S. aureus and E. coli in the PBS treatment groups exhibited intact spherical morphology, characteristic rod-like structures, smooth surfaces, and intact cell membrane structures. Comparatively, MoS2 + NIR and CS@MoS2 + NIR caused varying degrees of membrane contraction and deformation. In contrast, treatment with SNO-CS@MoS2 + NIR resulted the most pronounced deformation and loss of cell integrity in S. aureus and E. coli (shown by red arrows), leading to bacteria death owi