Preparation of 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster comparable with vancomycin and application of 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster in infection

Abstract

The invention discloses a preparation method of a 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster comparable with vancomycin and an application of the 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster in infection, and the 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster is used for treating methicillin-resistant staphylococcus aureus. The result shows that the 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster can be adsorbed on the surface of bacteria, down-regulate a Na < + > / H < + > transporter on a cell membrane, influence the potential of the cell membrane, reduce the activity of bacterial ATPase synthetase and the generation of ATP, and finally show the effect of killing the bacteria. The 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster is good in antibacterial stability, low in cytotoxicity and good in blood compatibility, and in a rat wound infection model experiment, the gold nanocluster has a relatively remarkable curative effect of promoting wound healing, is mainly characterized by reduction of local inflammation, increase of generation of blood vessels and collagenous fibers, down-regulation of inflammatory factors and the like, and can be used for preparing a drug for treating wound healing. And no obvious side effect is observed during the treatment period. The 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster can be used as a material comparable to vancomycin and used for treating infection caused by methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] The invention relates to the preparation of a 2-mercapto-1,3,4-thiadiazole-gold nanocluster and is used for treating methicillin-resistant Staphylococcus aureus infection, belonging to the field of nanomedicine. Background Art

[0002] Conventional methods of screening microbial metabolites or compound libraries have not produced new drugs in more than 30 years. Therefore, there is an urgent need to find new ways to deal with the increasingly serious microbial resistance. Advances in nanotechnology have shown great potential in combating drug-resistant strains. Nanomaterials are comparable in size to biomolecules and bacterial intracellular structures and can be designed into new treatments. Currently, nanomaterials used for antibacterial applications include silver nanoparticles, magnetic nanoparticles, metal oxide (such as CuO, ZnO or TiO2) nanoparticles, and carbon-based nanomaterials. However, some materials only show good antibacterial activity with the assistance of substrates, ultrasound or light stimulation. In addition, most of the above nanomaterials have very limited applications in the medical field due to their low antibacterial activity.

[0003] When the size of nanoparticles is reduced to nearly one nanometer or sub-nanometer, ultrasmall nanoclusters begin to have interesting physical and chemical properties, such as antibacterial activity. It is worth mentioning that gold nanoclusters have been widely studied due to their excellent chemical stability and high biocompatibility. However, the nature of the surface ligands can greatly affect the antibacterial effect of gold nanoclusters. There are methods to modify gold nanoclusters with cationic ligands to enhance their interaction with bacteria with negative potentials. However, cationic antibacterial agents have a more serious hemolytic phenomenon in vivo. There are also methods to design gold nanoclusters through oxygen vacancies in the surface structure, which can capture a large number of electrons to interact with the absorbed O2 and promote the generation of reactive oxygen species, thereby killing bacteria. However, reactive oxygen species lack intrinsic selectivity in distinguishing normal flora from pathogenic bacteria, which also makes them lose the necessary selectivity as ideal antibacterial agents. In general, different surface functionalizations give gold nanoclusters different antibacterial properties. In the present invention, we prepared 2-mercapto-1,3,4-thiadiazole-gold nanoclusters by a one-step method. The gold clusters have strong bactericidal properties against methicillin-resistant Staphylococcus aureus. They initially adhere to the surface of the bacterial membrane and then downregulate the Na + / H + Antiporters cause membrane potential imbalance and respiratory chain inhibition, ultimately leading to bacterial cell death. Compared with vancomycin, this unique effect gives gold nanoclusters extraordinary antibacterial efficacy in the MRSA-infected rat epidermal model. In summary, the gold nanoclusters prepared by the present invention elucidate new insights into the antibacterial mechanism and provide an effective strategy for the development of new antibacterial drugs.

[0004] The invention constructs a new method for preparing 2-mercapto-1,3,4-thiadiazole-gold nanoclusters and using the nanoclusters for treating methicillin-resistant Staphylococcus aureus infection. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing 2-mercapto-1,3,4-thiadiazole-gold nanoclusters by using 2-mercapto-1,3,4-thiadiazole as a modified ligand, and to establish a new method for treating methicillin-resistant Staphylococcus aureus infection.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The object of the present invention is achieved in this way. A 2-mercapto-1,3,4-thiadiazole-gold nanocluster is characterized in that it is prepared by the following method: a 10 mg / mL chloroauric acid solution is added to a 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L), mixed and placed in a dark place for incubation for 60 minutes. After the reaction is completed, the reaction solution is dialyzed and purified with a dialysis bag with a molecular weight of 3500 to obtain a 2-mercapto-1,3,4-thiadiazole-gold nanocluster with a diameter of 1.8 nm.

[0008] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters obtained above can be adsorbed on the surface of methicillin-resistant Staphylococcus aureus and downregulate the Na + / H + transporter, thereby affecting the cell membrane potential, reducing the activity of bacterial ATPase synthase and the production of ATP, and ultimately producing a bactericidal effect.

[0009] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters obtained above have good and stable killing effect on methicillin-resistant Staphylococcus aureus, weak toxicity to human epidermal cells, and good blood compatibility.

[0010] The aqueous solution of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters obtained above is light yellow, and the characteristic absorption peak of 2-mercapto-1,3,4-thiadiazole appears at 280nm in the UV-visible spectrum. Yellow fluorescence is generated under UV light, and the maximum excitation wavelength and emission wavelength are 330nm and 537nm respectively. The quantum yield is 2.88% and the fluorescence lifetime is 8.3ns.

[0011] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters obtained above have good anti-methicillin-resistant Staphylococcus aureus activity comparable to vancomycin, low toxicity to mammalian cells, long-lasting antibacterial activity and good blood compatibility.

[0012] The invention discloses an application of the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters in the preparation of a drug for treating skin wound infection.

[0013] The invention discloses a new method for treating methicillin-resistant Staphylococcus aureus infection by using 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0014] The application is characterized in that: in an in vivo rat skin wound infection model, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters show significant wound healing efficacy, characterized by reduced local inflammation, angiogenesis, collagen fiber deposition, down-regulation of inflammatory factors, and no significant side effects observed during treatment. In an in vivo rat wound infection model, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters have significant wound healing efficacy comparable to vancomycin, characterized by shortened wound healing time, reduced inflammatory cells and related inflammatory factors, inhibited increase in inflammatory cells and anti-inflammatory factors, increased blood vessel growth and angiogenic factors, increased collagen fiber synthesis, and no significant side effects were observed during treatment.

[0015] Specifically, the present invention adopts the following technical solutions:

[0016] (I) Preparation of 2-mercapto-1,3,4-thiadiazole-gold nanocluster materials

[0017] All glassware used in the following process was soaked in aqua regia, thoroughly washed with double distilled water, and dried. The preparation process of 2-mercapto-1,3,4-thiadiazole-gold nanocluster material is as follows: add 10 mg / mL chloroauric acid solution to 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L), mix well and incubate in the dark for 60 minutes. After the reaction is completed, the reaction solution is dialyzed and purified with a dialysis bag with a molecular weight of 3500 to obtain 2-mercapto-1,3,4-thiadiazole-gold nanocluster with a diameter of 1.8 nm.

[0018] (II) In vitro anti-methicillin-resistant Staphylococcus aureus activity of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters

[0019] 2.1 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the growth of methicillin-resistant Staphylococcus aureus

[0020] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (0, 0.5, 1, 2 μg / mL) were introduced into the system and mixed with 0.5 McLaren-degree MRSA turbid solution (1.5×10 8CFU / mL), co-cultured at 35°C for 0-20 h, and the OD600 absorbance value was measured using an enzyme-labeled instrument.

[0021] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (0, 8, 10, 12 μg) and vancomycin sensitive paper (30 μg) were introduced into the system and then attached to a turbid solution of methicillin-treated Staphylococcus aureus (1.5×10 8 The plates were plated with 100 μg / mL (400 μg / mL) and incubated at 35°C for 20 h. The inhibition zones were measured with a vernier caliper.

[0022] 2.2 Effects of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the cell wall and membrane of methicillin-resistant Staphylococcus aureus

[0023] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) were introduced into the system and mixed with 0.5 McFarland's MRSA turbid solution (1.5×10 8 CFU / mL), and PBS blank group (20mmol / L, pH=7.4), were co-cultured at 35℃ for 20h, and the methicillin-resistant Staphylococcus aureus was determined by transmission electron microscopy and scanning electron microscopy, and the morphological changes of methicillin-resistant Staphylococcus aureus were observed; at the same time, the culture medium was centrifuged at 10000rpm for 2min, the supernatant was discarded, and 1ml of double distilled water was used to reconstitute it, and Hoechst33342 (10μg / mL) and propidium iodide (0.3mg / mL) dyes were added to observe the changes in the cell membrane of methicillin-resistant Staphylococcus aureus.

[0024] 2.3 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on methicillin-resistant Staphylococcus aureus Na + / H + The impact of transporters

[0025] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) were introduced into the system and mixed with 0.5 McFarland's MRSA turbid solution (1.5×10 8 CFU / mL), and PBS blank group (20mmol / L, pH=7.4), were co-cultured at 35℃ for 20h. A protein sample of bacteria was extracted using a protein extraction kit, and the Na of MRSA was detected by mass spectrometry. + / H + Changes in transporters; one sample of bacterial RNA was extracted using an RNA extraction kit and RT-PCR was used to detect methicillin-resistant Staphylococcus aureus Na + / H + Changes in transporter genes.

[0026] 2.4 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the zeta potential of methicillin-resistant Staphylococcus aureus

[0027] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) were introduced into the system and mixed with 0.5 McFarland's MRSA turbid solution (1.5×10 8 CFU / mL), and PBS blank group (20mmol / L, pH=7.4), were co-cultured at 35℃ for 20h, and the zeta potential change of methicillin-resistant Staphylococcus aureus was detected by zeta potential meter.

[0028] 2.5 Effects of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on ATP and ATP synthase of methicillin-resistant Staphylococcus aureus

[0029] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) were introduced into the system and mixed with 0.5 McFarland's MRSA turbid solution (1.5×10 8 CFU / mL), and PBS blank group (20mmol / L, pH=7.4), were co-cultured at 35℃ for 20h, one sample of bacterial ATP was extracted with ATP extraction kit, and the change of ATP of methicillin-resistant Staphylococcus aureus was detected by microplate reader; one sample of bacterial ATP synthase was extracted with ATP synthase extraction kit, and the change of ATP synthase of methicillin-resistant Staphylococcus aureus was detected by microplate reader.

[0030] 2.6 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on staphyloxanthin in methicillin-resistant Staphylococcus aureus

[0031] 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) were introduced into the system and mixed with 0.5 McFarland's MRSA turbid solution (1.5×10 8 CFU / mL), and PBS blank group (20mmol / L, pH=7.4), were co-cultured at 35℃ for 20h, and the absorbance change of staphyloxanthin in methicillin-resistant Staphylococcus aureus was detected by microplate reader.

[0032] (III) Toxicity of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters

[0033] 3.1 Toxicity detection of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on mammalian cells

[0034] (1) Collect human epidermal cells HaCaT in the logarithmic growth phase, digest the cells and prepare a suspension, dilute to 5000 cells / well, add 100 μL of cell suspension to each well of a 96-well plate, set a zero well, and record the inoculation time. Add 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (1, 2, 4, 8, 10, 20 μg / mL) and PBS blank group (20 mmol / L, pH = 7.4), place in a 37°C, 5% CO2 incubator for 24 h, then add 20 μL CCK8 reagent, continue to incubate in a 37°C, 5% CO2 incubator for 1 h, measure the absorbance at a wavelength of 450 nm with an enzyme reader, record the OD of each well, and calculate the survival rate.

[0035] (2) Collect human epidermal cells HaCaT in the logarithmic growth phase, digest the cells and prepare a suspension, dilute to 5000 cells / well, add 100 μL of cell suspension to each well of a 96-well plate, set a zero well, and record the inoculation time. Add 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2,8 μg / mL) and PBS blank group (20 mmol / L, pH = 7.4), place in a 37°C, 5% CO2 incubator for 24 h, then add 200 μL of phalloidin (Alexa Fluor 568), continue incubation in a 5% CO2 incubator for 2 h, finally add 10 μL of DAPI working solution, and observe the cell morphology using a laser confocal microscope.

[0036] (3) Collect 1 mL of mouse blood in an EDTA-stable anticoagulant tube, centrifuge at 1000 rpm for 10 minutes, and wash three times with PBS to remove the released hemoglobin. After removing the supernatant, resuspend the red blood cells (2% w / w), place them in a 1.5 mL centrifuge tube, and add 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16, 20 μg / mL). PBS, double distilled water and 1% Triton were used as controls. After incubation at 37°C for 2 hours and centrifugation at 1000 rpm for 10 minutes, 100 μL of supernatant was added to a 96-well plate, and the absorbance value (OD) at 540 nm was measured on a multifunctional microplate reader.

[0037] (IV) 2-Mercapto-1,3,4-thiadiazole-gold nanoclusters for treating infections caused by methicillin-resistant Staphylococcus aureus

[0038] 4.1 Rat wound model of methicillin-resistant Staphylococcus aureus infection

[0039] 4.4.1 Rat wound modeling

[0040] (1) Experimental animal selection: Healthy male rats, weighing 200-250 g, were housed in an animal barrier center and randomly divided into three groups: blank group, 2-mercapto-1,3,4-thiadiazole-gold nanocluster low-dose group, 2-mercapto-1,3,4-thiadiazole-gold nanocluster low- and high-dose groups, and vancomycin group.

[0041] (2) Preparation of anesthetic drugs: 2wt% sodium pentobarbital;

[0042] (3) Preoperative preparation: after anesthesia, the back is shaved and hair removed, disinfected, and draped;

[0043] (4) Surgical method: Use surgical scissors to make a 1.5 cm long incision on the back, separate the skin tissue, and form a circular wound;

[0044] (5) Establishment of skin infection model: 200 μL of methicillin-resistant Staphylococcus aureus (1.5×10 5 CFU / mL);

[0045] (6) Drug treatment: Low-dose and high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters were dripped onto the wound, and vancomycin (16 μg / mL) and PBS were used as positive and negative control groups.

[0046] 4.4.2 Infected wound healing area

[0047] ① Specimen collection: After 12 days, the experimental rats were killed with sodium pentobarbital, the back hair was shaved, the healed skin and the surrounding 0.5 cm were cut, washed with saline, fixed in 4% formalin for 12 h, embedded in wax blocks, and sliced ​​at 5 μm thickness for hematoxylin & eosin (HE) staining, Masson staining, and immunohistochemistry.

[0048] ② Comparison of wound healing: On the day of surgery and at different observation points of drug administration, the weight, wound size and healing area of ​​the rats before and after treatment were measured and recorded. The wound size was determined by measuring the ruler. The calculation method of wound size is as follows: [D12 wound area) / D0 wound area]×100%.

[0049] ③ Observation of toxicity in important organ tissues: After 12 days, the experimental rats were euthanized using sodium pentobarbital to observe the safety of low-dose and high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on important organ tissues such as the heart, liver, spleen, lungs, and kidneys.

[0050] Advantages of the present invention:

[0051] (1) The present invention uses 2-mercapto-1,3,4-thiadiazole as a template to in-situ synthesize gold nanocluster materials. The preparation method is green and environmentally friendly, simple and fast to operate, and has good reproducibility.

[0052] (2) The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters prepared by the present invention exhibit yellow fluorescence (maximum emission wavelength of 537 nm), a quantum yield of 2.88%, a fluorescence lifetime of 8.3 ns, and a large Stokes shift (207 nm).

[0053] (3) In vitro experiments showed that it exhibited excellent anti-methicillin-resistant Staphylococcus aureus activity comparable to that of vancomycin, low toxicity to mammalian cells, long-lasting antibacterial activity, and good blood compatibility.

[0054] (4) In vivo experiments further verified that 2-mercapto-1,3,4-thiadiazole-gold nanoclusters can effectively promote wound healing and repair in animal models, showing significant therapeutic effects in promoting wound proliferation, anti-inflammation, and promoting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The following are the appearance comparison pictures of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters under visible light (A) and ultraviolet light (B).

[0056] Figure 2 This is the UV-visible absorption spectrum of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0057] Figure 3 This is the fluorescence spectrum of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0058] Figure 4 Transmission electron microscopy image (A) and particle size distribution diagram (B) of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0059] Figure 5 This is the fluorescence lifetime diagram of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0060] Figure 6 This is the X-ray photoelectron spectrum of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. In the figure: A is the 4f map of gold, and B is the 2p map of sulfur.

[0061] Figure 7 The antibacterial effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on methicillin-resistant Staphylococcus aureus. In the figure: A is the antibacterial curve of different concentrations of gold clusters (0, 0.5, 1, 1.5, 2 μg / mL), and B is the drug sensitivity picture of different contents of gold clusters (ad: 0, 8, 10, 12 μg) and vancomycin (e: 30 μg).

[0062] Figure 8The scanning electron micrograph of the effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on methicillin-resistant Staphylococcus aureus. In the figure: A is methicillin-resistant Staphylococcus aureus treated with injection water, B is methicillin-resistant Staphylococcus aureus treated with low-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, and C is methicillin-resistant Staphylococcus aureus treated with high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0063] Fig. 9 Transmission electron microscopy images of the effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on methicillin-resistant Staphylococcus aureus. In the figure: A is methicillin-resistant Staphylococcus aureus treated with injection water, B is methicillin-resistant Staphylococcus aureus treated with low-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, and C is methicillin-resistant Staphylococcus aureus treated with high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0064] Fig.10 This is a fluorescence staining diagram of the effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on methicillin-resistant Staphylococcus aureus. In the figure: A is Hoechst33342 / PI stained with injection water for methicillin-resistant Staphylococcus aureus, B is Hoechst33342 / PI methicillin-resistant Staphylococcus aureus treated with low-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, and C is Hoechst33342 / PI methicillin-resistant Staphylococcus aureus treated with high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0065] Fig.11 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the Na+ binding of methicillin-resistant Staphylococcus aureus + / H + In the figure: A is the Na of methicillin-resistant Staphylococcus aureus treated with injection water + / H + Mass spectrometry results of transporters. B is the Na of MRSA treated with low doses of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. + / H + Mass spectrometry results of transporters. C is the Na of MRSA treated with high doses of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. + / H + Mass spectrometry results of the transporter.

[0066] Fig.12 Effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the Na+ binding of methicillin-resistant Staphylococcus aureus + / H+ Influence of transporter genes. In the figure: A is the Na of methicillin-resistant Staphylococcus aureus treated with injection water + / H + Gene expression of transporters. B is the Na of MRSA treated with low dose of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. + / H + Gene expression of transporters, C is the Na of MRSA treated with high doses of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters + / H + Gene expression of transporters.

[0067] Fig.13 The effect of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the zeta potential of methicillin-resistant Staphylococcus aureus. In the figure: A is the zeta potential of methicillin-resistant Staphylococcus aureus treated with injection water, B is the zeta potential of methicillin-resistant Staphylococcus aureus treated with low-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, and C is the zeta potential of methicillin-resistant Staphylococcus aureus treated with high-dose 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0068] Fig.14 Effects of different concentrations of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on ATP of methicillin-resistant Staphylococcus aureus (af: 0, 2, 4, 8, 16, 32 μg / mL).

[0069] Fig.15 The effects of different concentrations of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on the ATP synthase of methicillin-resistant Staphylococcus aureus (af: 0, 2, 4, 8, 16, 32 μg / mL).

[0070] Fig.16 This is the antibacterial stability diagram of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0071] Fig.17 This is a graph showing the effects of different concentrations of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on HaCaT cells (ag: 0, 1, 2, 4, 8, 10, 20 μg / mL).

[0072] Fig.18 The graph shows the effect of different concentrations of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters on mouse red blood cells (ah: 0, 2, 4, 8, 16, 20 μg / mL, water and 0.1% Triton X 100).

[0073] Fig.19The rat skin wound model and the healing diagram are shown in the figure. In the figure: A is the wound healing diagram, and B is the wound healing curve diagram.

[0074] Fig. 20 The figure is a diagram of the bacterial infection model and healing of rat skin wounds. In the figure: A is a blood plate diagram of wound bacteria, and B is a quantitative bar graph of wound bacterial colony counts.

[0075] Fig.21 The skin tissue staining of the skin wound on days 3, 7, and 12 after treatment. In the figure: A is the H&E staining image; B is the epidermal thickness analysis bar graph (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0076] Fig. 22 The skin tissue staining of the skin wound on days 3 and 12 after treatment. In the figure: A is the Masson staining image; B is the collagen deposition analysis bar graph (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0077] Fig.23 The immune status of skin wound skin tissue on day 12 after treatment. A: CD86 and CD206 immunohistofluorescence images; B: Quantitative analysis of CD86 in immunohistochemical results; C: Quantitative analysis of CD206 in immunohistochemical results (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0078] Fig.24 The immune status of skin wound skin tissue on the 7th day after treatment. A: IL-6 and IL-10 immunohistofluorescence images; B: Quantitative analysis of IL-6 in immunohistochemical results; C: Quantitative analysis of IL-10 in immunohistochemical results (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0079] Fig.25 The immune status of skin wound skin tissue on days 3, 7 and 12 after treatment. In the figure, A: immunohistofluorescence images of iNOS on days 3 and 7; B: quantitative analysis of iNOS in immunohistochemical results on days 3 and 7; C: immunohistofluorescence images of CD31 on day 12; D: quantitative analysis of CD31 in immunohistochemical results on day 12 (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose group and vancomycin group).

[0080] Fig.26The immune status of skin wound skin tissue on days 3 and 12 after treatment. In the figure, A: immunohistochemical images of TNF-α on days 3 and 12; B: immunohistochemical images of IL-1β on days 3 and 12; C: immunohistochemical images of TGF-β on days 3 and 12; D: quantitative analysis of TNF-α in immunohistochemical results on days 3 and 12; E: quantitative analysis of IL-1β in immunohistochemical results on days 3 and 12; F: quantitative analysis of TGF-β in immunohistochemical results on days 3 and 12 (blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose group and vancomycin group).

[0081] Fig. 27 The immune status of the skin tissue of the skin wound on the 12th day after treatment. In the figure, A: immunohistochemical image of HIF-1α on the 12th day; B: immunohistochemical image of HIF-1α on the 12th day; C: immunohistochemical image of VEGF on the 12th day; D: quantitative analysis of VEGF in the immunohistochemical results on the 12th day; E: quantitative analysis of α-SAM in the immunohistochemical results on the 12th day; F: quantitative analysis of α-SAM in the immunohistochemical results on the 12th day (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0082] Fig.28 The immune status of the skin tissue of the skin wound on the 12th day after treatment. In the figure, A: immunohistochemical image of MMP on the 12th day; B: immunohistochemical image of MMP on the 12th day; C: immunohistochemical image of MPO on the 12th day; D: quantitative analysis of MPO in the immunohistochemical results on the 12th day (ad: blank group, MTD-AuNCs low-dose group, MTD-AuNCs high-dose and vancomycin group).

[0083] Fig.29 This is the HE staining image of important organ tissues on the 14th day after treatment. DETAILED DESCRIPTION

[0084] Example 1:

[0085] Add 10 mg / mL chloroauric acid solution (4 mL) to 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L, 4 mL), mix well and incubate in the dark for 60 minutes. After the reaction is completed, the reaction solution is dialyzed and purified using a dialysis bag with a molecular weight of 3500 to obtain 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, which are light yellow under visible light (see Figure 1 A in the figure), produces yellow fluorescence under ultraviolet light (see Figure 1B), the UV-visible spectrum shows a characteristic absorption peak at 280nm (see Figure 2 ), the maximum excitation wavelength and emission wavelength are 330nm and 537nm respectively (see Figure 3 ). Example 2:

[0086] Add 10 mg / mL chloroauric acid solution (4 mL) to 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L, 4 mL), mix well and incubate in the dark for 60 minutes. After the reaction is completed, dialyze the reaction solution with a dialysis bag with a molecular weight of 3500 to purify it, and obtain 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. Figure 4 As shown, the gold cluster is an elliptical nanoparticle with a diameter of 1.8 nm (in the figure: A is a transmission electron microscope image, and B is a particle size distribution diagram).

[0087] Example 3:

[0088] Add 10 mg / mL chloroauric acid solution (4 mL) to 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L, 4 mL), mix well and incubate in the dark for 60 minutes. After the reaction is completed, the reaction solution is dialyzed and purified using a dialysis bag with a molecular weight of 3500 to obtain 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. The resulting solution was subjected to fluorescence lifetime measurement, and the fluorescence lifetime of the gold cluster was measured to be 8.32 ns (see Figure 5 ).

[0089] Example 4:

[0090] Add 10 mg / mL chloroauric acid solution (4 mL) to 65 mmoL / L 2-mercapto-1,3,4-thiadiazole NaOH solution (0.3 mmoL / L, 4 mL), mix well and incubate in the dark for 60 minutes. After the reaction is completed, the reaction solution is dialyzed and purified with a dialysis bag with a molecular weight of 3500 to obtain 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. The obtained solution is freeze-dried to obtain a powder, and the obtained powder is subjected to X-ray photoelectron spectroscopy. The 4f peaks of gold appear at 84.0 eV and 87.8 eV, and the 2p peak of sulfur appears at 162.6 eV (see Figure 6 A in the figure is the 4f diagram of gold, and B is the 2p diagram of sulfur).

[0091] Example 5:

[0092] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (0, 0.5, 1, 2 μg / mL) prepared in Example 1 were added with a suspension of methicillin-resistant Staphylococcus aureus (1.5×108 CFU / mL), co-cultured at 35°C for 0-20h, and the OD 600 absorbance value was measured using an ELISA reader. Figure 7 As shown, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 μg / mL) significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

[0093] Example 6:

[0094] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (0, 8, 10, 12 μg) prepared in Example 1 and vancomycin sensitive paper (30 μg) were attached to a plate coated with a turbid solution of methicillin-resistant Staphylococcus aureus (1.5×10 8 CFU / mL) plates were incubated at 35°C for 20 h, and the inhibition zone was measured using a vernier caliper. Figure 8 As shown, the inhibition zone of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (12 μg) was similar to that of vancomycin (30 μg).

[0095] Example 7:

[0096] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2.8 μg / mL) prepared in Example 1 were added to the PBS blank group (20 mmol / L, pH = 7.4) and the methicillin-resistant Staphylococcus aureus suspension (1.5×10 8 CFU / mL), incubated at 35°C for 20 h, centrifuged the culture solution at 10,000 rpm for 2 min, discarded the supernatant, and fixed the bacteria with 2.5 wt% glutaraldehyde, and then measured by scanning electron microscopy and transmission electron microscopy. Fig. 9 and Fig.10 As shown, the methicillin-resistant Staphylococcus aureus cells were severely damaged after treatment with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0097] Example 8:

[0098] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2.8 μg / mL) prepared in Example 1 were added to the PBS blank group (20 mmol / L, pH = 7.4) and the methicillin-resistant Staphylococcus aureus suspension (1.5×10 8 CFU / mL), incubate at 35℃ for 20h, centrifuge the culture solution at 10000rpm for 2min, discard the supernatant, re-dissolve the bacteria with double distilled water, add Hoechst33342 (10μg / mL) and propidium iodide (0.3mg / mL), incubate for 15min, and observe with a fluorescence microscope. Fig.11 As shown, the color of propidium iodide staining of methicillin-resistant Staphylococcus aureus bacteria treated with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters is dark.

[0099] Example 9:

[0100] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) prepared in Example 1 were added to the PBS blank group (20 mmol / L, pH = 7.4) and the methicillin-resistant Staphylococcus aureus suspension (1.5×10 8 CFU / mL), incubate at 35°C for 20 h, centrifuge the culture solution at 10,000 rpm for 2 min, discard the supernatant, extract the bacterial protein sample using a protein extraction kit, and then perform mass spectrometry detection, such as Fig.12 As shown, the Na of MRSA in the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) treatment groups + / H + The transporter was significantly downregulated; another sample of bacterial RNA was extracted using an RNA extraction kit and tested using RT-PCR, such as Fig.13 As shown, the Na of MRSA in the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) treatment groups + / H + Significant downregulation of transporter genes.

[0101] Example 10:

[0102] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) prepared in Example 1 were added to the PBS blank group (20 mmol / L, pH = 7.4) and the methicillin-resistant Staphylococcus aureus suspension (1.5×10 8 CFU / mL), incubated at 35°C for 20 h, and then the zeta potential was measured. Fig.13 As shown, the zeta potential of methicillin-resistant Staphylococcus aureus in the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 and 8 μg / mL) treatment group increased significantly.

[0103] Example 11:

[0104] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16 and 32 μg / mL) prepared in Example 1 were added to the PBS blank group (20 mmol / L, pH = 7.4) and the methicillin-resistant Staphylococcus aureus suspension (1.5×10 8 CFU / mL), incubate at 35°C for 20 h, centrifuge the culture solution at 10,000 rpm for 2 min, discard the supernatant, extract the bacterial sample using an ATP extraction kit, and then test the ATP content, such as Fig.14As shown in Figure 2, the ATP content of methicillin-resistant Staphylococcus aureus in the groups treated with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16 and 32 μg / mL) gradually decreased; another bacterial sample was extracted using an ATPase synthase extraction kit, and then ATPase synthase detection was performed, as shown in Figure 2. Fig.15 As shown, the ATPase synthase content of methicillin-resistant Staphylococcus aureus in the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16 and 32 μg / mL) treatment groups gradually decreased.

[0105] Example 12:

[0106] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 μg / mL) prepared in Example 1 were then mixed with a PBS blank group (20 mmol / L, pH = 7.4) and methicillin-resistant Staphylococcus aureus suspension (1.5 × 10 8 CFU / mL), and co-cultured at 35°C for 20 h. The OD 600 absorbance value was measured using an ELISA reader. Fig.16 As shown, the activity of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2 μg / mL) in inhibiting methicillin-resistant Staphylococcus aureus remained stable.

[0107] Example 13:

[0108] Take the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (1, 2, 4, 8, 10 and 20 μg / mL) prepared in Example 1, and then add human epidermal cell suspension (HaCaT, 5000 / well) and PBS blank group (20 mmol / L, pH = 7.4), respectively, and place in a 37°C, 5% CO2 incubator for 24 h, then add 20 μL CCK8 reagent, and continue to incubate in a 37°C, 5% CO2 incubator for 1 h. Use a microplate reader to measure the absorbance at a wavelength of 450 nm and record the OD of each well to calculate the survival rate, as shown in FIG. Fig.17 As shown, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (1, 2, 4, 8, 10 and 20 μg / mL) had no obvious toxic side effects.

[0109] Example 14:

[0110] The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16, 20 μg / mL) prepared in Example 1 were taken, and PBS (20 mmol / L, pH = 7.4), double distilled water and 1% Triton were used as controls, and then red blood cells (2% w / w) were added and resuspended. After incubation at 37°C for 3 h, centrifugation was performed at 1000 rpm for 10 min, and 100 μL of the supernatant was added to a 96-well plate, and the absorbance value (OD) at 540 nm was measured on a multifunctional microplate reader. Fig.18 As shown, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (2, 4, 8, 16, 20 μg / mL) have good blood cell compatibility.

[0111] Example 15:

[0112] After clarifying the in vitro efficacy of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, we selected SD rats for skin wound infection modeling and randomly divided them into 4 groups: PBS blank group, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters low-dose group, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters high-dose group and vancomycin group. The specific modeling method is as follows: the rats were anesthetized with 2% amobarbital, and a 1.5 cm long dorsal skin incision was made with surgical scissors, and then 200 μL of methicillin-resistant Staphylococcus aureus suspension was added to the wound for 24 hours. Then, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (10 and 20 μg / mL), vancomycin (32 μg / mL) and PBS (20 mmol / L, pH=7.4) prepared in Example 1 were added, and the medication was continued for 12 days. The wound size was measured on days 0, 1, 3, 5, 7, 10, and 12, respectively. Fig.19 As shown, the wound healing of the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (10 and 20 μg / mL) group and the vancomycin group was significantly better than that of the blank group. Example 16:

[0113] Rats were anesthetized with 2wt% amobarbital, and a 1.5cm long dorsal skin incision was made with surgical scissors. Then 200μL of methicillin-resistant Staphylococcus aureus suspension was added to the wound and infected for 24h. Then 100μL of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (10 and 20μg / mL), vancomycin (32μg / mL) and PBS (20mmol / L, pH=7.4) prepared in Example 1 were added. The wound was moistened with 100μL of sterile saline on days 0, 7 and 12, and inoculated on a blood culture plate and incubated at 35°C for 20h. Fig. 20 As shown, the number of methicillin-resistant Staphylococcus aureus clones in the blood culture plate treated with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (10 and 20 μg / mL) gradually decreased and was less than that in the blank group.

[0114] Example 17:

[0115] After 3, 7, and 12 days of medication, samples were collected, mainly Fig.19 The skin wound in A and the surrounding 0.5 cm were washed with normal saline, fixed in 4wt% formalin for 12 h, embedded in wax, and sliced ​​at 5 μm thickness for hematoxylin & eosin (HE) staining and Masson staining. The tissue was then stained with immunofluorescence and immunohistochemistry, and CD86, CD206, IL-6, IL-10, iNOS, CD31, TNF-α, IL-1β, TGF-β, HIF-1α, VEGF, α-SAM, MMP and MPO in the tissue samples were analyzed.

[0116] Example 18:

[0117] Skin wound epithelialization and granulation tissue formation are the main indicators for evaluating the wound healing process. On days 3, 7, and 12, we observed the following through tissue HE staining and Masson staining: Fig.21 The results are shown in Fig.21 As shown in A, on the 3rd day, the PBS blank group, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, and vancomycin groups all showed more neutrophil infiltration in the wound, red blood cells were visible, and the skin tissue only regenerated immature granulation tissue without epithelium or epithelial destruction. On the 7th day, the neutrophil infiltration decreased, the number of new blood vessels increased, and the proportion of collagen fibers was higher in the 2-mercapto-1,3,4-thiadiazole-gold nanocluster group and the vancomycin group. On the 12th day, continuous epithelium and skin appendages were observed in the 2-mercapto-1,3,4-thiadiazole-gold nanocluster group and the vancomycin group. These analysis results were also analyzed by Fig.21 In addition, tissue sections were stained with MT to examine the deposition of collagen during the healing process. Fig. 22 As shown in A, on the 3rd day, the 2-mercapto-1,3,4-thiadiazole-gold nanocluster group and the vancomycin group showed more collagen fibers compared with the blank group. On the 12th day, the collagen fibers of the 2-mercapto-1,3,4-thiadiazole-gold nanocluster group and the vancomycin group were darker in color and more densely arranged. These analysis results were also Fig. 22The analysis of B in the experiment confirmed this. We also used macrophage M1-M2 polarization regulators CD86 and CD206, non-specific mucosal inflammatory factor inducible nitric oxide synthase (iNOS), platelet endothelial cell adhesion molecule-1 (CD31), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), transforming growth factor-β (TGF-β), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), α-smooth muscle actin (α-SMA), matrix metalloproteinase-9 (MMP-9), and myeloperoxidase (MPO) as indicators for evaluating wound healing mediated by 2-thiol-1,3,4-thiadiazole-gold nanoclusters. Fig.23 As shown in A, on the 7th day of treatment, compared with the blank group, the expression of CD86 in the wound treated with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin was significantly reduced, and the expression of CD206 was significantly increased. Therefore, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters can promote the transformation of macrophages M1 to M2. The analysis results are shown in Fig.23 B and C. Interleukin-6 (IL-6) and interleukin-10 (IL-10) are pro-inflammatory and anti-inflammatory factors, respectively, reflecting the prognosis of infection. Fig.24 As shown in A, compared with the blank group, the expression of pro-inflammatory factor IL-6 in the wound treated with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin was significantly reduced, and the expression of anti-inflammatory factor IL-10 was significantly increased. The analysis results are shown in Fig.24 B and C in Figure 2. Changes in the nonspecific mucosal inflammatory factor inducible nitric oxide synthase (iNOS) are shown in Figure 2. Fig.25 .like Fig.25 As shown in Figure 25A, from the 3rd to the 7th day of treatment, the iNOS level after treatment with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin gradually decreased compared with the blank group. The analysis results are shown in Figure 25C. Fig.25 B in the figure shows the formation of new blood vessels in wound tissue after immunohistochemical CD31 staining. Compared with the blank group, an increase in new blood vessels was observed using 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin. The analysis results are shown in Fig.25 D in. Cytokines such as TNF-α, IL-1β and TGF-β are often used as indicators for regulating inflammatory responses and immune cell activity. Fig.26 As shown in A, B, and C in Figure 3, from the 3rd to the 12th day of treatment, compared with the blank group, the levels of TNF-α and IL-1β after treatment with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin gradually decreased, and the level of TGF-β gradually increased. Fig.26D, E and F in Figure 1. HIF-1α subunit is regulated by hypoxia and regulates the activity of HIF-1, activating downstream VEGF, thereby promoting angiogenesis. In addition, α-SMA is also involved in regulating fibroblast proliferation and the synthesis of extracellular matrix proteins. Fig. 27 As shown in A, B, and C in Figure 1, on the 12th day of treatment, the levels of HIF-1α, VEGF, and α-SMA after treatment with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin gradually increased compared with the blank group. Fig. 27 D, E, and F. MMP-9 and MPO are often used as prognostic indicators of inflammation, such as Fig.28 As shown in A and B in Figure 1, on the 12th day of treatment, the levels of MMP-9 and MPO after treatment with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or vancomycin gradually decreased compared with the blank group. Fig.28 C and D. In general, the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters prepared in Example 1 have more significant anti-inflammatory, pro-proliferation and pro-angiogenesis effects than the blank group, thereby showing a more significant effect of accelerating skin wound healing.

[0118] Example 19:

[0119] SD rats were killed with sodium pentobarbital 12 days after administration, and the safety of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters (10 and 20 μg / mL) prepared in Example 1 on important organs and tissues such as heart, liver, spleen, lung, and kidney was observed pathologically. Fig.29 As shown, no obvious toxic reaction was found in HE staining of various organ tissues.

Claims

1. A 2-mercapto-1,3,4-thiadiazole-gold nanocluster comparable to vancomycin, characterized by: It was prepared by the following method: a 10 mg / mL chloroauric acid solution was added to a 65 mmol / L 0.3 mmol / L NaOH solution of 2-mercapto-1,3,4-thiadiazole, the mixture was mixed and incubated in the dark for 60 minutes. After the reaction was completed, the reaction solution was dialyzed and purified using a dialysis bag with a molecular weight of 3500. The average particle size of the obtained 2-mercapto-1,3,4-thiadiazole-gold nanoclusters was 1.8 nm.

2. The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters comparable to vancomycin according to claim 1, characterized in that: The obtained 2-mercapto-1,3,4-thiadiazole-gold nanoclusters can be adsorbed on the surface of methicillin-resistant Staphylococcus aureus and downregulate the Na + / H + transporter, thereby affecting the cell membrane potential, reducing the activity of bacterial ATPase synthase and the production of ATP, and ultimately producing a bactericidal effect.

3. The 2-mercapto-1,3,4-thiadiazole-gold nanoclusters comparable to vancomycin according to claim 1 or 2, characterized in that: The obtained 2-mercapto-1,3,4-thiadiazole-gold nanoclusters have good and stable killing effect on methicillin-resistant Staphylococcus aureus, weak toxicity to human epidermal cells, and good blood compatibility.

4. Use of the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters according to any one of claims 1 to 3 in the preparation of drugs for treating skin wound infections.

5. The use according to claim 4, characterized in that: In the in vivo rat skin wound infection model, 2-mercapto-1,3,4-thiadiazole-gold nanoclusters showed significant wound healing effects, characterized by reduced local inflammation, angiogenesis, collagen fiber deposition, downregulation of inflammatory factors, and no significant side effects were observed during the treatment period.

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