Mcm a nano-architecture, mcm a hydrogel and preparation method and application thereof

Abstract

The application discloses an MCMA nano architecture, an MCMA hydrogel and a preparation method and application thereof. In the application, chlorin e6 is grafted to the surface of mesoporous silica to obtain MSN@Ce6 (MC), so that the dispersibility of Ce6 is improved, then the surface is coated by using manganese dioxide nanosheets and nanosilver combined with chitosan, to obtain the MCMA nano architecture, and the MCMA hydrogel is further prepared by using the MCMA nano architecture, and the MCMA nano architecture and the MCMA hydrogel can be used for preparing synergistic PDT, PTT and CDT antibacterial drugs, so that bacteria can be better killed, and the MCMA nano architecture and the MCMA hydrogel can be retained in the body for a long time, and can play a long-term role.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug development technology, and in particular relates to an MCMA nanostructure with multi-mechanism synergistic antibacterial function, an MCMA hydrogel, its preparation method and application. Background Technology

[0002] Bacterial infections are one of the world's largest public health problems, and the various complications caused by wound infections can be life-threatening. With increasing bacterial resistance to antibiotics, many researchers have turned their attention to antibacterial nanomaterials. However, existing nanomaterials generally suffer from drawbacks such as poor stability, low water solubility, low biocompatibility, high preparation costs, and high toxicity. For example, the photosensitizer Ce6 has poor water solubility and is prone to aggregation; photothermal therapy (PTT) can damage normal tissue due to excessive heat; and high concentrations of nanosilver have tissue toxicity. Compared to traditional antibiotics, photodynamic therapy (PDT) rarely induces bacterial resistance. However, in complex abscess sites, the presence of a hypoxic microenvironment limits the bactericidal efficacy of oxygen-dependent PDT.

[0003] In recent years, phototherapy, which converts light energy into heat energy or generates reactive oxygen species (ROS) to kill bacteria, has received widespread attention due to its superior performance, strong tissue penetration, and few side effects. Summary of the Invention

[0004] The primary objective of this invention is to provide an MCMA nanostructure with multi-mechanism synergistic antibacterial function and its preparation method.

[0005] Another object of the present invention is to provide the above-mentioned MCMA hydrogel and its preparation method.

[0006] Another object of the present invention is to provide the application of the above-mentioned MCMA nanostructure or MCMA hydrogel in the preparation of antibacterial drugs.

[0007] This invention is achieved through a method for fabricating MCMA nanostructures, comprising the following steps: (1) Mix EDC, NHS, Ce6 and DMSO and stir for 30±2 min to obtain a carboxyl-activated Ce6 solution; (2) The Ce6 solution was mixed with the MSN-NH2 solution and stirred overnight. After centrifugation, washing and drying, MC was obtained. (3) 5 mg / mL potassium permanganate solution was added dropwise to 1.6±0.1 mg / mL MC aqueous solution. After the addition was complete, formamide was added and the mixture was sonicated at 200W power for 30±2 min. After centrifugation, washing and drying, MCM was obtained. (4) Disperse an equal volume of 2±0.1 mg / mL MCM in 4±0.5 mg / mL CS / Ag solution, stir slowly under vacuum for 12±1h, and obtain MCMA nanostructure by centrifugation, washing and drying.

[0008] Preferably, in step (1), the mass-to-volume ratio of EDC, NHS, Ce6, and DMSO is 15±1 mg mg: 10±1 mg mg: 5±1 mg mg: 5 mL.

[0009] Preferably, in step (2), the preparation process of MSN-NH2 includes the following steps: adding MSN powder to anhydrous toluene, ultrasonically dispersing until uniform, adding APTES and continuing ultrasonication until the solution is clear and transparent, heating and stirring at 110°C for 5-6 hours, and centrifuging, washing and drying the reaction product to obtain MSN-NH2; The concentration of the MSN-NH2 solution was 4 ± 0.5 mg / mL, and the volume ratio of Ce6 solution to MSN-NH2 solution was 1:1.

[0010] Preferably, in step (3), the volume ratio of the potassium permanganate solution, MC aqueous solution, and formamide is 12 mL ± 132 µL: 10 mL ± 120 µL: 1.2 mL ± 7.8 µL.

[0011] Preferably, in step (4), the preparation of CS / Ag includes the following specific steps: 60±2 mg CS is dissolved in 60±2 mL of 0.1% w / v acetic acid aqueous solution, and 4±1 mL of freshly prepared AgNO3 solution of 5±1 mg / mL is added dropwise while stirring. The mixture is heated to 95±1℃ while stirring, and 6±1 mg / mL of NaOH solution is added dropwise until the solution turns yellow. The reaction is stopped after 10±1 min, and the mixture is centrifuged, washed, and dried to obtain CS / Ag.

[0012] The present invention further discloses the MCMA nanostructure prepared by the above method.

[0013] This invention further discloses a method for preparing MCMA hydrogels using the aforementioned MCMA nanostructure, the method comprising the following steps: (1) The polymer was dissolved in 0.5±0.1 M acetic acid at 40±1℃ for 12±1 h to obtain a solution with a polymer concentration of 15±1% (w / v); wherein the polymer is CS and gelatin G in a mass ratio of 1:2. (2) The suspension of the above MCMA nanostructure was added to the solution under magnetic stirring. 2.5% of EDC relative to the total mass of CS-G was added to the solution as a crosslinking agent. The solution was treated at 50±1℃ for 30±2 min under stirring to gel and obtain MCMA hydrogel.

[0014] The present invention further discloses the MCMA hydrogel prepared by the above method.

[0015] This invention further discloses the application of the above-mentioned MCMA nanostructure or MCMA hydrogel in the preparation of antibacterial drugs.

[0016] Preferably, the bacteria include E. coli and S. aures.

[0017] Preferably, the MCMA nanostructure or MCMA hydrogel is used in the preparation of antibacterial drugs that synergistically induce PDT, PTT, and CDT antibacterial therapy.

[0018] This invention overcomes the shortcomings of existing technologies and provides an MCMA nanostructure, MCMA hydrogel, and their preparation method and applications with multi-mechanism synergistic antibacterial function. This invention grafts dihydroporphyrin e6 (Chlorine e6, Ce6) onto the surface of mesoporous silica (MSN) to obtain MSN@Ce6 (MC), thereby improving the dispersibility of Ce6 and enabling it to better exert photodynamic therapeutic effects. Then, it uses manganese dioxide (MnO2) nanosheets and chitosan (CS) combined with nanosilver (AgNPs) for surface coating to obtain the MSN@Ce6@MnO2-CS / Ag (MCMA) nanostructure.

[0019] This invention successfully synthesized an AgNP-doped synergistic PDT and PTT nanocomposite MCMA nanostructure, which serves as an effective strategy for synergistic antibacterial treatment of PDT, PTT, and CDT to promote the healing of infected wounds. Among these, MCMA's nanoplatform addresses the issues of Ce6's poor hydrolysis and easy aggregation leading to fluorescence quenching through MSN chemical grafting of Ce6. The MSN@Ce6 outer layer is coated with dual-responsive MnO2 to prevent premature exposure of the drug-loaded Ce6. PDT alone lacks sufficient depth of action, and hypoxic environments hinder its effectiveness. PTT's high temperature damages surrounding normal tissue. MnO2, acting as a photothermal agent and peroxidase, not only exerts photothermal effects and synergizes with PDT, but also generates oxygen, overcoming the anaerobic environment's hindrance to PDT. Since PDT and PTT have relatively short durations of action, constituting shock therapy, nanosilver is introduced to achieve thorough antibacterial action, serving as a chemical antibacterial agent for long-term antibacterial effects. Furthermore, the high toxicity associated with high concentrations of AgNPs is addressed after PDT and PTT, not only killing bacteria more effectively but also providing a long-term effect due to their prolonged retention time in the body.

[0020] Compared to the shortcomings and deficiencies of existing technologies, this invention has the following beneficial effects: Utilizing the photodynamic therapy (PDT), photothermal therapy (PTT), and AgNPs (AgNPs also refer to chemodynamic therapy (CDT)) of the MCMA nanostructure, it demonstrates the excellent antibacterial properties and good biocompatibility of the MCMA nanostructure under the synergistic effect of multiple mechanisms. Furthermore, combining it with hydrogels to form MCMA hydrogels can be used clinically to treat skin wound infections, providing new ideas and solutions for the treatment of chronic wound infections. The MCMA of this invention possesses multiple mechanisms of action, including ROS generation, GSH oxidation, POD-like properties, and photothermal effects, exhibiting good antibacterial effects against E. coli and S. aureus, demonstrating an excellent therapeutic index. Attached Figure Description

[0021] Figure 1 These are high-resolution TEM images; among them, (a) TEM image of MSN; (b) TEM image of MC; (c) TEM image of MCM; (d) corresponding elemental mapping of N, Mn, and O in MCM; (e) TEM image of MCMA; (f) high-resolution TEM image of MCMA; Figure 2 It is 808 nm (1 W / cm) 2Photothermal effect under laser irradiation (10 min); among which, (a) 808 nm laser power (0.5, 0.8, 1.0, 1.5 W / cm) 2 (a) Relationship between temperature and irradiation time of PBS solution containing MSN@Ce6@MnO2-CS / Ag (500 μg / mL); (b) MC, MCM, and MCMA (500 μg / mL) at 808 nm (1 W / cm²). 2 Photothermal effect under laser irradiation (10 min); Figure 3 It is 808 nm (1 W / cm) 2 Temperature difference curves under laser light; where (a) MCMA solutions of different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) at 808 nm (1 W / cm²) 2 (a) Heating curves under laser light; (b) Heating curves of MCMA solutions with different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) at 808 nm (1 W / cm²). 2 Temperature difference curve under laser; Figure 4 (a) Photothermal stability of MCMA (5 laser on / off cycles); (b) 808 nm (1 W / cm²) 2 Absorption spectrum of MCMA solution after laser irradiation; Figure 5 (a) shows the results of hemolytic toxicity assays of different concentrations of the compound. NaCl (0.9%) was used as a negative control and 1% Triton X-100 was used as a positive control. Figure 5 (b) is a picture of the hemolytic toxicity test. * indicates P < 0.05; ** indicates P < 0.01; Figure 6 Cell viability was measured by co-culturing L929 cells with different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) of MCMA with different concentrations of L929 (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) for 1 day using the CCK8 assay kit. * indicates P < 0.05, ** indicates P < 0.01; Figure 7 The images show bacterial colonies formed by S. aures and E. coli after co-incubation with PBS and 250 μg / mL MSN, MC, MCM, MCMA, S. aures, and E. coli for 24 h. Figure 8The images show bacterial colonies formed after mixing culture medium with or without 250 μg / mL MCMA and H2O2 solution with S. aures, treating the mixture under different light conditions, plating the bacteria, and incubating for 24 h. Figure 9 The images show E. coli bacterial colonies formed after mixing culture medium with or without 250 μg / mL MCMA and H2O2 solution, treating the mixture under different light conditions, plating the bacteria, and incubating for 24 h. Figure 10 Different concentrations of MCMA were co-incubated with *S. aureus* or *E. coli* and then treated with 808 nm (1 W / cm²). 2 10 min) and 660 nm (400 mW / cm 2 After laser treatment (5 min), bacteria were plated and incubated for 24 h. The images show the bacterial colonies formed by S. aures and E. coli. Figure 11 These are the results of a biofilm clearance experiment. MCMA and E. coli, S. aures After co-incubation, surface-dwelling bacteria were removed, and the cells were washed and fixed with PBS and methanol, then stained with crystal violet solution. After removing the surface stain, 95% ethanol was added to dissolve the crystal violet in the biofilm. Finally, the OD was measured. 570 The absorbance values ​​at the specified locations were recorded. (a) and (b) show the absorbance values ​​at different concentrations of MCMA and under different reaction conditions. E. coli, S. aures Biofilm clearance results; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; Figure 12 (a) and (c) Ellman's experimental results, using DTNB as the detection reagent, UV-vis absorption spectra of MCMA oxidizing GSH under different concentrations and different light conditions; (b) and (d) Ellman's analysis results, histograms of GSH loss from MCMA at different concentrations; histograms of GSH loss from 250 µg / mL MCMA under different light conditions. * indicates P < 0.05; *** indicates P < 0.001; Figure 13 (a) The POD-like catalytic activity of MCMA in solutions with different concentrations of H2O2 was determined using a TMB probe; (b) The POD-like catalytic activity of MCMA under different light conditions was determined. ** indicates P < 0.01; *** indicates P < 0.001; Figure 14 (a) MCMA was measured at 808 nm (1 W / cm²) using a DPBF probe. 2 ) and 660 nm (400 mW / cm2 (a) The UV-vis absorption spectrum of MCMA between 350 nm and 500 nm under laser irradiation was used to determine the ROS generation performance of MCMA. The detection time points were mins 0, 2, 4, 6, 8, and 10 under 808 nm irradiation, and mins 3 and 5 under 660 nm irradiation. (b) O2 generation by MCMA in solutions with different GSH concentrations was detected by oxidized cytochrome C. - The characteristics; Figure 15 This study used MB as the detection reagent to determine the characteristics of ·OH generated by MCMA. (a) UV-vis absorption spectra of ·OH generated by MCMA on MB degradation under different light conditions; (b) Absorption spectra of ·OH generated by MCMA on MB degradation at different concentrations; (c) Effect of different concentrations of H2O2 on the absorption spectra of ·OH generated by MCMA on MB degradation; (d) Effect of ·OH generated by MCMA treated with different concentrations of GSH on MB degradation. Figure 16 These are images of MCMA hydrogels adhering to different matrix materials, including: plastic, glass, metal, rubber, skin, and internal organs; Figure 17 It represents the percentage of hydrolysis of the MCMA hydrogel in PBS solution; Figure 18 These are the results of acute in vivo toxicity tests of MCMA; MCMA was administered intraperitoneally at doses of 4.5, 5.27, 6.95, 9.16, 12.08, and 15.92 g / kg (body weight), and the growth status of the mice was continuously observed and recorded. Figure 19 It is a record of infected wound photos taken every other day after medication, after the mouse wound infection model was successfully established; Figure 20 Images of healthy skin, infected skin, MCMA hydrogel, and mouse wound tissue treated under different light conditions with H&E and Masson staining; magnification: 100, scale bar: 50µm; Figure 21 These are H&E staining images of major organs of mice after treatment with MCMA hydrogels for infected wounds under different lighting conditions; magnification: 200, scale bar: 50µm. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Example 1: Fabrication of MCMA Nanostructures

[0024] 1. MSN Preparation (1) Stir 0.6 g cetyltrimethylammonium bromide (CTAB), 0.15 g triethanolamine (TEA) and 40 mL deionized water magnetically at 80 °C for 30 min. (2) Add 4.0 g of tetraethyl orthosilicate (TEOS) dropwise to the above solution. Stir the resulting mixture at 80°C (1000 rpm) for 4 h, centrifuge to collect the precipitate, and wash it three times with ethanol; (3) Reflux the above product in 26 mL of NH4NO3 (37% v / v) ethanol solution at 60°C for 12 h, centrifuge to collect the precipitate, and obtain mesoporous silica (MSN), which is then dried in an oven for later use.

[0025] 2. Preparation of amino-modified MSN (1) Add 50 mg of MSN powder to 10 mL of anhydrous toluene and ultrasonically disperse until uniform; (2) Add 1 mL of 3-aminopropyltriethoxysilane (APTES) and continue sonicating until the solution is clear and transparent. Heat and stir at 110°C for 6 h. (3) Centrifuge at 8000 rpm for 15 min, wash three times with anhydrous ethanol and collect the precipitate to obtain MSN-NH2, and then dry it under vacuum.

[0026] 3. Preparation of MSN@Ce6 (1) Disperse 20 mg MSN-NH2 in 5 mL of pure water using ultrasonication; (2) To activate the carboxyl group of Ce6, 15 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 10 mg of N-hydroxy-succinimide (NHS), 5 mg of Ce6 and 5 mL of dimethyl sulfoxide (DMSO) were mixed and stirred for 30 min. (3) Mix the two solutions and stir overnight on a magnetic stirrer. Then centrifuge at 8000 rpm for 15 min, wash three times with anhydrous ethanol, and vacuum dry to obtain MSN@Ce6 powder, denoted as MC.

[0027] 4. Preparation of MSN@Ce6@MnO2 (1) Disperse 16 mg MC in 10 mL of pure water by ultrasonication, and add 12 mL of potassium permanganate solution (5 mg / mL) dropwise while stirring. (2) Add 1.2 mL of formamide, sonicate at low power for 30 min, centrifuge at 8000 rpm for 15 min to collect the precipitate, wash three times with pure water, dry in an oven to obtain MSN@Ce6@MnO2, denoted as MCM.

[0028] 5. Preparation of CS / Ag (1) Dissolve 60 mg CS in 60 mL of acetic acid aqueous solution (0.1% w / v), add 4 mL of freshly prepared AgNO3 solution (5 mg / mL) dropwise while stirring, and heat to 95°C while stirring; (2) Add 6 mg / mL NaOH solution dropwise until the solution turns yellow, then stop the reaction after 10 min. Centrifuge to collect the precipitate, wash three times with pure water, dry in an oven, prepare a 4 mg / mL solution, and store at 4℃.

[0029] 6. Preparation of MSN@Ce6@MnO2-CS / Ag (1) Disperse an equal volume of 2 mg / mL MCM in a 4 mg / mL CS / Ag solution and stir slowly under vacuum for 12 h; (2) After stirring slowly under vacuum for 12 h, the precipitate was collected by centrifugation, washed three times with ultrapure water, and dried in an oven to obtain MSN@Ce6@MnO2-CS / Ag powder, which was denoted as MCMA.

[0030] Example 2: Preparation of MCMA hydrogel (1) CS and gelatin were dissolved in 0.5M acetic acid at 40℃ for 12 h. The polymer ratio was 15% (w / v) and the ratio of CS:G was 1:2. This was used to prepare CS-G hydrogel. (2) The MCMA suspension was added to the solution with magnetic stirring at 100 rpm. EDC (2.5% wt / wt relative to CS-G) was added to the solution as a crosslinking agent. The solution was treated with stirring at 50°C for 30 min. Gelation was performed to obtain MCMA hydrogel (MCMA content 250 μg / mL); (3) The MCMA hydrogel was formed into a film in a petri dish for later use. The same method was used to prepare a hydrogel without MCMA.

[0031] Effect Example

[0032] The following experiments were conducted using the product obtained in Example 1.

[0033] I. Characterization of Materials

[0034] 1. The morphology of the material was characterized using TEM (Tecnai F30, 300 kV).

[0035] MC, MCM, and MCMA were dispersed in pure water to prepare a 500 µg / mL solution. 1 mL of each solution was placed in a 1.5 mL EP tube, and then subjected to a NIR laser (808 nm, 1 W / cm²). 2 The sample dispersion was irradiated with a laser for 10 min, and the temperature change was monitored. Then, a certain volume of MCMA dispersion with a concentration of 500 μg / mL was prepared, and the dispersion was treated with lasers at concentrations of 0.5, 0.8, 1, and 1.5 W / cm². 2 The sample dispersion was irradiated with an 808 nm laser for 7 min, and the temperature change was monitored every 1 min. Different concentrations of MCMA dispersions were prepared, and then irradiated with an NIR laser for 10 min, with the temperature change monitored every 1 min. A certain volume of 500 μg / mL MCMA dispersion was prepared, irradiated with an 808 nm laser for 10 min, and then the laser was turned off, allowing the liquid to cool naturally for 10 min. This "irradiation-cooling" process was repeated 5 times, and the temperature change of the system was monitored. The change in absorbance of the dispersion before and after irradiation was measured using UV-Vis; absorbance can indirectly verify the stability of the sample.

[0036] The results are as follows Figure 1 As shown, Figure 1 The morphologies of MSN, MC, MCM and MCMA were characterized using TEM. Figure 1 In sample a, the MSNs exhibit uniform and regular spherical shapes with clearly visible mesoporous structures, and a particle size of approximately 39 nm. For example... Figure 1 As shown in b, the mesoporous image of MSN grafted with Ce6 did not change much and remained a regular and uniform sphere with no significant change in particle size, indicating that Ce6 was mostly loaded by chemical grafting or pore size loading. Figure 1 In c, MC is encapsulated by MnO2, and images of manganese dioxide nanosheets are visible on the outer layer of MC. Figure 1 In d, the mapping image of the key elements of MCM clearly shows the presence of N, Mn and O elements, indicating that MnO2 was successfully coated. Figure 1 e is a TEM image of MCMA, which has an irregular shape, with CS / Ag encapsulating the MCM and AgNPs particles visible on the surface. Figure 1 The f-measurement shows that the lattice spacing of AgNPs is approximately 0.243 nm, corresponding to the crystal plane of AgNPs. These results indicate that AgNPs were successfully loaded into the MCM composite.

[0037] 808 nm wavelength light has a high penetration depth in the skin, while tissues, blood, and water have low absorption intensity, making it one of the most important NIR wavelengths for PTT (photothermal radiation). Therefore, this invention uses an 808 nm laser to detect its photothermal properties. First, irradiation with 808 nm lasers of different powers was applied; after 7 minutes, it was shown that the temperature gradually increased with increasing power. Figure 2 a) The photothermal properties of MC, MCM and MCMA were measured, with the temperatures rising to 31.2°C, 58.8°C and 55.4°C, respectively. Figure 2 b proves that the heat generation originates from MnO2. The temperature of MCMA is slightly lower than that of MCM, which may be due to the CS / Ag coating on the surface of MnO2, which slightly hinders the light absorption of MnO2.

[0038] Using an 808 nm laser (1.0 W / cm²) 2 The temperature changes of different concentrations of MCMA were detected, and the results are as follows: Figure 3 As shown, the photothermal effect of MCMA is concentration-dependent, and it exhibits a stable temperature increase within 10 min ( Figure 3 a). At a concentration of 250 µg / mL, an 808 nm laser (1.0 W / cm²) was used. 2 After 10 minutes of irradiation, the temperature rise can reach 19.7 °C, while the temperature change of PBS is negligible. Figure 3 b).

[0039] Using an 808 nm laser (1.0 W / cm²) 2 The temperature change of the MCMA during laser on / off cycles was detected, and the results are as follows: Figure 4 As shown, the maximum temperature of the MCMA did not change significantly during the 5 laser on / off cycles, demonstrating that the MCMA has good photothermal conversion stability. Figure 4 a). Furthermore, the absorption curve of MCMA measured by UV-vis showed no significant change, indicating that MCMA possesses good material stability. Figure 4 b).

[0040] Therefore, MnO2 nanosheets can serve as an ideal PTA for PTT.

[0041] 2. Determination of hemolytic toxicity

[0042] Fresh healthy human blood was collected and diluted to 8% red blood cells for later use. Equal volumes of antibiotics at different concentration gradients (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) and 8% red blood cell suspension were incubated at 37 °C for 60 min. 0.9% NaCl and 8% red blood cells were used as negative controls, and 1% Triton X-100 and 8% red blood cells were used as positive controls. The mixture was centrifuged at 800 g for 10 min, and the OD490 of the supernatant was measured.

[0043] 3. Assay for cytotoxicity

[0044] The cytotoxicity of MCMA to mammalian cells was determined using the CCK8 assay. Mouse fibroblasts (L929 cells) were used as the cell line. Cells were seeded and treated with the drug: co-cultured for 24 h; then 200 μL of a solution containing 20 μL of CCK8 was added to each well, and the cells were incubated in the dark for 1–2 h. OD450 was measured at the end of the incubation.

[0045] The biosafety of MCMA was preliminarily evaluated through cytotoxicity studies. The results are as follows: Figure 5 , Figure 6 As shown, MCMA exhibits a hemolytic toxicity of less than 5% at a concentration of 1000 µg / mL, while its hemolytic toxicity is 1.2% at a concentration of 250 µg / mL, corresponding to the solution color. Figure 5 (a, 5b). L929 cells treated with 31.25–500 µg / mL MCMA showed a viability exceeding 79%. Treatment with 250 µg / mL MCMA resulted in a viability exceeding 82%. Furthermore, at a high concentration of 500 µg / mL, MCMA showed almost no toxicity to cells after 24 h of incubation, with a cell viability of approximately 79%. Figure 6 This demonstrates that MCMA has good biocompatibility.

[0046] 4. Determination of antibacterial properties of different materials

[0047] The logarithmic phase colonies were diluted with sterile liquid culture medium to a concentration of 1.0 × 10⁻⁶. 6 Bacterial suspensions of CFU / mL (OD600 = 0.1±0.02); 300 μL of different samples (MSN, MC, MCM, MCMA) and bacterial suspensions were mixed at 250 µg / mL, and the mixed bacterial suspensions were incubated at 37 ℃ for 1 h, followed by light treatment. The treated bacterial suspensions were then diluted by a certain factor and plated.

[0048] To study the antibacterial properties of each product, this invention co-incubated MSN, MC, MCM, and MCMA with *S. aureus* and *E. coli*, and then subjected them to light irradiation (808 nm, 1 W / cm²). 2660 nm, 400 mW / cm 2 ), 100 μL of bacterial suspension was used for plate counting to assess antibacterial activity. At the same concentration, the MCMA group had the fewest colonies, as shown in the results. Figure 7 As shown, MCMA exhibits the best antibacterial properties, and the components that make up MCMA have a synergistic antibacterial effect.

[0049] 5. Determination of antibacterial properties of MSN@Ce6@MnO2-CS / Ag under different conditions

[0050] Mix 300 μL of 250 µg / mL sample and 300 μL of bacterial culture. Incubate the mixed bacterial culture at 37 °C for 1 h. Then, divide it into 12 groups according to whether H2O2 (100 μM) was added and the light conditions: 1) Control; 2) Control + 808 nm + 660 nm; 3) MCMA; 4) MCMA + 808 nm; 5) MCMA + 660 nm; 6) MCMA + 808 nm + 660 nm; 7) H2O2; 8) H2O2 + 808 nm + 660 nm; 9) MCMA + H2O2; 10) MCMA + H2O2 + 808 nm; 11) MCMA + H2O2 + 660 nm; 12) MCMA + H2O2 + 808 nm + 660 nm; where 808 nm indicates the concentration of H2O2 (1 W / cm²) at 808 nm. 2 Laser irradiation for 10 minutes, 660 nm indicates the wavelength of 660 nm (400 mW / cm²). 2 Irradiate with laser for 5 minutes, dilute the treated bacterial solution by a certain factor, and then plate it.

[0051] The results are as follows Figure 8 , Figure 9 As shown, the H2O2 group exhibited weaker antibacterial activity compared to the blank control group. The MCMA + H2O2 group showed better antibacterial effect without laser irradiation than the group without H2O2, which may be related to the nanozyme activity of MCMA. (808 nm (1 W / cm²)) 2 After 10 minutes of laser irradiation, the antibacterial activity of the MCMA and MCMA + H2O2 groups was significantly enhanced compared to the group without 808 nm laser irradiation. Among them, the MCMA + H2O2 + 808 nm + 660 nm group showed the best antibacterial effect. The temperature rise of MCMA (250 µg / mL) after 10 minutes of light irradiation was approximately 45 °C. Therefore, MCMA does not cause any damage to normal human tissue when exerting its antibacterial effect.

[0052] 6. Determination of antibacterial properties of different concentrations of MSN@Ce6@MnO2-CS / Ag

[0053] Different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 µg / mL) of MCMA, H2O2 (100 μM), and bacterial culture were mixed in 200 μL each. The mixed bacterial culture was incubated at 37 °C for 1 h, followed by illumination (808 nm, 1 W / cm²). 2 And 660nm, 200 mW / cm 2 The treated bacterial solution was diluted a certain number of times and then coated onto a plate.

[0054] The results are as follows Figure 10 As shown, the antibacterial activity of MCMA is concentration-dependent. As the concentration of MCMA increases, its resistance to E. coli and S. aures increases, and the antibacterial rate against both is close to 100% at 250 μg / mL.

[0055] II. Biofilm clearance experiment

[0056] Biofilm removal assays are used to evaluate the ability of compounds to kill established S. aures and E. coli biofilms.

[0057] Take a sterile 96-well plate and add 200 μL of bacterial culture. Simultaneously, set up a control group (positive control: 100 μL of bacterial culture and culture medium added to each well; negative control: 200 μL of culture medium added to each well). Incubate at 37 ℃ for 96 h, changing the medium every 24 h. Dilute MCMA with culture medium to 0, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL. Remove the 96-well plate, discard the culture medium and airborne bacteria, wash the biofilm twice with PBS, and add 200 μL of the complex at different concentrations to each well, with 3 replicates per group. Add 200 μL of culture medium to the control group. Incubate at 37 ℃ for 1 h; then expose to light (808 nm, 1 W / cm²). 2 660 nm, 400 mW / cm 2 );

[0058] Remove surface-dwelling bacteria from the 96-well plate, then wash the biofilm twice with PBS. Afterward, add 100 μL of different concentrations of the complex and 100 μL of 100 μM H₂O₂ and incubate for 1 h. Then, treat with light and incubate for another 4 h. Fix with methanol 2 × 10⁻⁶ min, discard the methanol, wash and fix again, and stain with crystal violet solution for 15 min. Remove any floating crystal violet with PBS, then dissolve the crystal violet in the biofilm with 95% ethanol. Finally, measure the OD. 570 And record the results;

[0059] Remove the 96-well plate to remove surface airborne bacteria, then wash the biofilm twice with PBS. After illumination, the plates were divided into 12 groups based on the presence or absence of H2O2 and the illumination conditions: 1) Control (C); 2) Control + 808 nm + 660 nm (C86); 3) MCMA (M); 4) MCMA + 808 nm (M8); 5) MCMA + 660 nm (M6); 6) MCMA + 808 nm + 660 nm (M68); 7) H2O2 (H); 8) H2O2 + 808 nm + 660 nm (H86); 9) MCMA + H2O2 (MH); 10) MCMA + H2O2 + 808 nm (MH8); 11) MCMA + H2O2 + 660 nm (MH6); 12) MCMA + H2O2 + 808 nm + 660 nm (MH86). The control group consisted of samples treated with culture medium alone. The 808 nm value indicates the use of 808 nm (1 W / cm²). 2 Laser irradiation for 10 minutes, 660 nm indicates the wavelength of 660 nm (400 mW / cm²). 2 Laser irradiation for 5 min. After treatment under different conditions, incubation at 37℃ for 4 h. Fixation with methanol for 10 min, discarding the methanol, washing and fixing again, then staining with crystal violet solution for 15 min. Remove excess crystal violet with PBS, then dissolve the crystal violet in the biofilm with 95% ethanol. Finally, OD was measured. 570 The results were recorded; the positive control consisted of 100 μL of bacterial culture medium, and the negative control consisted of 00 μL of culture medium. Each group had three parallel groups.

[0060] Data processing, biofilm formation clearance rate (Disrubition rate) (%) = [1 - (Experimental group OD)] 570 / Negative control group OD 570 )]× 100%.

[0061] In clinical treatment, biofilms often form around chronic wounds and implants, leading to long-term, recurrent bacterial infections and chronic inflammation. To address this, this invention further evaluated the ability of the compound to clear existing biofilms at different concentrations and under different treatment conditions. The results are as follows: Figure 11 As shown, MCMA at 1000 µg / mL achieved clearance rates of 75% and 65.9% for the two biofilms, respectively. Figure 11 a) At a concentration of 250 µg / mL, clearance rates of 58% and 55.8% were achieved, respectively. Figure 11 b).

[0062] III. Determination of Antibacterial Mechanism

[0063] 1. Elman determination The oxidizing properties of MCMA for GSH were evaluated using the Elman assay. The Elman reagent (5,5′-dithiobis-(2-nitrobenzoic acid), DTNB) oxidizes the thiol group (-SH) to -SS-, producing a yellow product (2-nitro-5-thiobenzoic acid). 400 µL of MCMA bicarbonate buffer solution (0.1 M, pH = 7.4) at different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) was mixed with 400 µL of GSH (1 mM) bicarbonate buffer solution in 2 mL centrifuge tubes. The group without MCMA was considered the control group. The mixture was incubated at 37 °C with shaking at 150 rpm for 60 min. After adding 400 µL of DTNB (0.1 mM) solution and reacting for 2 min, the absorbance of the solution at 410 nm was recorded. To determine the effect of laser irradiation on the GSH oxidation ability of MCMA, MCMA (250 µg / mL) and GSH mixture were treated with no irradiation, irradiation at 808 nm and 660 nm alone, or irradiation at 808 nm and 660 nm synergistically, and the absorbance of the solution at 410 nm was recorded.

[0064] GSH is a thiol-containing tripeptide that plays an important role in bacterial antioxidant defense systems, indicating cellular oxidative stress. Under oxidative conditions, GSH is converted to GSSG. Furthermore, MnO2 nanosheets can undergo a redox reaction with GSH to generate MnO2. 2+ And GSSG. This is due to MnO2 in HCO3 − Oxidizing properties in the presence of a buffer. High temperature and pre-release of small amounts of Ag. + The synergistic effect causes bacterial membrane rupture, leading to leakage of internal bacterial substances. MnO2 nanosheets can also react with GSH at the rupture sites, accelerating the disruption of bacterial homeostasis and death.

[0065] GSH plays a crucial role in bacterial antioxidant defense systems because it protects cellular components from oxidative stress-induced damage, which can significantly weaken the bactericidal effect of POD-based nanomaterials in bacteria. GSH loss is necessary to maintain the antibacterial capacity of the materials. Here, an Ellman assay was performed to measure GSH loss. GSH oxidation was concentration-dependent; as the concentration of MCMA increased, GSH oxidation increased, indicating that MCMA can accelerate the oxidation of organothiols (R-SH) to disulfides (RSS−R). Figure 12 a, 12b). After different treatments, the loss of GSH under different illumination conditions was compared. The results showed that near-infrared irradiation can promote the loss of GSH ( Figure 12 c, 12d), to improve the antibacterial ability of the material. GSH oxidation may be one of the antibacterial mechanisms of MCMA.

[0066] 2. Determination of the activity of MSN@Ce6@MnO2-CS / Ag-type PODs

[0067] The POD-like activity of MCMA was assessed by oxidizing 3,3',5,5'-tetramethylbenzidine (TMB) to oxTMB in PBS solution (pH=7.4).

[0068] Equal volumes of MCMA (250 µg / mL) and H2O2 (100 μM) or PBS and TMB (3 mM) solutions were mixed, and the UV-Vis spectra of the color reaction were recorded over a certain time period (60 min) (the absorption peak of oxTMB was visible at 652 nm).

[0069] To determine the relationship between POD activity and concentration, the POD activity of MCMA at different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 µg / mL) was measured. Equal volumes of MCMA, H2O2 (100 μM), and TMB (3 mM) solutions of different concentrations were mixed, and the absorbance of the color reaction was recorded within a certain time (60 min).

[0070] To determine the relationship between the POD-like activity of MCMA and H2O2, the POD activity of H2O2 at different concentrations (0, 50, 100, 200, 400, 600, and 800 μM) was tested. Equal volumes of MCMA (250 µg / mL), H2O2 at different concentrations, and TMB (3 mM) solutions were mixed, and the absorbance of the color reaction was recorded over a certain time period (60 min).

[0071] The POD-like catalytic activity of MCMA can effectively kill bacteria, as determined by the TMB colorimetric detection method. In short, in the presence of H₂O₂, nanomaterials with POD-like properties can oxidize colorless TMB to a blue oxide (oxTMB), which has an absorption peak at 652 nm. Figure 13 In this process, MCMA can catalyze the oxidation of TMB. An absorption peak is visible at 652 nm. Figure 13 a); Furthermore, the catalytic activity was shown to be dependent on the laser treatment conditions and H2O2 concentration, with the highest catalytic activity observed after simultaneous irradiation at 808 nm and 660 nm, and increasing with increasing H2O2 concentration (0, 50, 100, 200, 400, 600, 800 μM). Figure 13 b). The result is consistent with the color change (blue) of the solution.

[0072] 3. ROS determination The absorbance value of 1,3-diphenylisobenzofuran (DPBF) increases with... 1 O2 oxidation reduces the concentration, making it suitable as a probe for detecting generated substances. 1 O2. Using a DPBF as a probe to detect optical triggering. 1 The generation of O2. 1 O2 can be captured by DPBF, resulting in a reduction in its absorption at 410 nm.

[144] ; Mix equal volumes of 250 μg / mL MCMA solution and 400 μg / mL DPBF acetonitrile solution for 3 min; use 808 nm (1 W / cm) in the dark. 2 10 min) and 660 nm (400 mW / cm 2 Centrifuge at 0, 2, 4, 6, 8, 10, 13, and 15 min respectively, collect the supernatant, and measure the absorbance of the supernatant at a wavelength of 410 nm to determine the ROS generation of the material.

[0073] 4. Determination of hydroxyl radicals

[0074] MB is a thiocyanate-based chromogenic reagent that reacts with ·OH to form hydroxymethylene blue (MB-OH), changing its color from blue to colorless. The fading of MB and the decrease in its characteristic absorbance value are positively correlated with the concentration of ·OH, making it suitable as a probe for detecting ·OH formation. The absorbance values ​​of different samples were recorded using a microplate reader.

[0075] Equal volumes of MCMA (250 μg / mL) were mixed with different concentrations of GSH (0, 8, 16, 24, 32, 40 mM), MB (20 µg / mL), and H2O2 (400 µM). The mixture was kept for 20 min to reach adsorption and desorption equilibrium. After centrifugation, the supernatant was collected and the absorbance at 665 nm was measured.

[0076] 5. Determination of superoxide anions

[0077] Oxidized cytochrome C can be oxidized by O2 - When oxidized cytochrome C is reduced to its reduced form, its absorbance at 550 nm increases, and the solution turns purple. The color change and decrease in characteristic absorbance of oxidized cytochrome C are related to O2. - The concentration of O2 is positively correlated, making it suitable as a method for detecting O2. - The generated probe. UV-Vis absorption spectra (500-600 nm) of different samples were recorded using UV-Vis.

[0078] Equal volumes of MCMA (250 μg / mL), different concentrations of GSH (0, 8, 16, 24, 32, 40 mM), cytochrome C (2 mg / mL) and H2O2 (400 µM) were mixed and kept for 20 min to reach adsorption and desorption equilibrium. After centrifugation, the supernatant was collected and the absorbance at 550 nm was measured.

[0079] Reactive oxygen species (ROS) are a weapon of neutrophils against bacterial pathogens and a central effector of oxidative stress in the treatment of skin and soft tissue infections. The ROS mechanism is considered one of the important toxic mechanisms associated with nanoparticle exposure, interfering with DNA and enzyme function, thereby disrupting normal metabolism and killing bacteria. This study assessed MCMA-induced oxidative stress by monitoring ROS production in the presence of MCMA using the DPBF probe, thus inferring whether MCMA can induce intracellular ROS production in bacteria. It has been reported that ROS levels are related to antibacterial efficiency due to their destructive effects on bacterial cell membranes. Increased levels of ROS (e.g., free radicals, O2) can lead to increased antibacterial activity. − Oxidative stress occurs when exposed to hydroxyl radicals (OH and H₂O₂). This invention uses DPBF as a probe to study the ability of MCMA to generate ROS under near-infrared irradiation; the photostability of DPBF ensures the reliability of this test. Figure 14 As shown, under irradiation with an 808 nm laser for 2, 4, 6, 8, and 10 min, and irradiation with a 660 nm laser for 3 and 5 min, the UV-vis measured ultraviolet absorption at 410 nm decreased sequentially, proving that MCMA can induce ROS generation under both 808 nm and 660 nm laser irradiation. Figure 14a). Therefore, this invention demonstrates that MCMA can induce ROS generation at 808 nm and 660 nm, attacking the bacterial membrane wall, thereby... 1 O2 penetrates the cell membrane, damages the interior of bacteria, and causes lipid peroxidation, thus producing an irreversible killing effect on bacteria.

[0080] In addition, this invention also detects O2 using oxidized cytochrome C. - The generation of O2 can be achieved through the generation of O2. - It reverts to a purple substance. The infected area becomes acidic due to bacterial fermentation in an anaerobic environment, producing acidic decomposition products and becoming rich in GSH. GSH reacts with MnO2 to simultaneously generate Mn. 2+ Mn 2+ Catalyzing the production of O2 from H2O2 - After GSH is reduced by MCMA, MnO2 is decomposed to generate Mn. 2+ And release CS / Ag, Mn 2+ It reacts with H2O2 to produce more O2 - This invention uses cytochrome C as O2. - The probe is used to detect O2 in the system. - The generation of O2 - The generation of O2 depends on the concentration of GSH, and an increase in GSH concentration leads to more O2. - This, in turn, increases the cytochrome C's response to O2. - The higher the GSH concentration, the more oxidized cytochrome C is consumed, resulting in the generation of more reduced cytochrome C, and a higher absorption peak at 550 nm. Figure 14 b).

[0081] Methylene blue (MB) is a dye that can be degraded by ·OH. MCMA and H2O2 solution can act as a POD-like agent. UV-Vis absorption values ​​at 500-800 nm were measured. After treatment under different light conditions, it was found that MCMA produced the most ·OH after irradiation at both 808 nm and 660 nm. Figure 15 c). This invention determined the formation of ·OH at different concentrations of MCMA and H2O2. The formation of ·OH was concentration-dependent on both MCMA and H2O2. GSH reacts with MnO2 to form Mn. 2 + Mn 2+ Catalyzing the formation of ·OH from H2O2 Figure 15 b, 15d). After the reaction of MCMA and GSH, they react with H2O2 to produce more ·OH ( Figure 15a) This invention uses MB as a probe for ·OH to detect the generation of ·OH in the system. The generation of ·OH depends on the concentration of GSH, and an increase in the concentration of GSH leads to the generation of more ·OH, which in turn increases the consumption of MB. Figure 15 a).

[0082] 6. Characterization of MSN@Ce6@MnO2-CS / Ag dressing

[0083] (1) Cut MCMA hydrogel into 1.5 cm diameter circles and adhere them to dressings on different substrate materials (plastic, glass, metal, rubber, skin, internal organs) to preliminarily assess their adhesion. Weigh 0.5 g of MCMA hydrogel and place it in a drying oven to remove water, then weigh the dehydrated MCMA hydrogel. The swelling degree (SD) of the hydrogel is calculated using the following formula: SD (%) = (m wet -m dried ) / m dried × 100%. Where, m wet It is the weight of the moist hydrogel, m dried This is the weight of the dried hydrogel.

[0084] (2) The in vitro hydrolysis properties of the hydrogel were investigated using PBS (pH = 7.4) at 37°C. The hydrogel was placed in the buffer solution and removed from the solution for further drying on each day of the test for a total of seven days. The percentage of weight loss was calculated using the following formula: Degradation (%) = (W t - W0) / W t × 100%. Where W0 is the initial weight of the hydrogel placed in the buffer medium, W t It is the weight of the dried hydrogel after hydrolysis and degradation.

[0085] MCMA hydrogels can adhere to the surfaces of various materials. For example... Figure 16 As shown, MCMA hydrogel can firmly adhere to the surfaces of plastics, alloys, iron sheets, glass, rubber, fingers, and organs without the need for other adhesives, demonstrating that MCMA has excellent bonding properties.

[0086] The swelling degree of MCMA hydrogel is 2.96 g / g, meaning that 1 g of dehydrated MCMA hydrogel can swell to 2.96 g. Figure 17 The percentage of hydrolysis of the MCMA hydrogel over time is shown. The weight loss due to hydrolysis of the resulting hydrogel increased over time, reaching 13.02% on day 7.

[0087] 7. Determination of in vivo toxicity (1) Six-week-old male Kunming rats, weighing 15±1 g, were purchased from the Experimental Animal Center of Lanzhou University School of Medicine. After being brought back, they were acclimatized for one week. Kunming rats weighing 20±1 g were randomly divided into groups of 10 each. (2) The mice were intraperitoneally injected with doses of 4.5, 5.27, 6.95, 9.16, 12.08, and 15.92 g / kg (mouse body weight), and their growth status was continuously observed and recorded. (3) Calculate the median lethal dose and 95% confidence interval concentration range based on the mortality rate using the Koch method.

[0088] 8. Construction of a mouse model of infectious full-thickness skin defect (1) Animal preparation: Male Kunming rats weighing 15±1 g were obtained from the Experimental Animal Center of Lanzhou University. After one week of acclimatization, the rats weighed 20±1 g. During the acclimatization period, the rats were fed and watered normally. They were fasted and water-free for 12 hours before the operation. (2) Bacterial preparation: Single colonies of *S. aureus* were picked from LB agar plates and added to centrifuge tubes containing 3 mL of LB liquid medium. The culture was incubated overnight at 37 °C in a shaker until the bacteria reached the logarithmic growth phase. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial concentration was adjusted to 1 × 10⁻⁶ using PBS buffer. 9 CFU / mL, stored in an ice box at 4 ℃ for later use; (3) Prepare a 4% chloral hydrate solution with physiological saline in advance, and administer anesthesia via intraperitoneal injection at a dose of 0.1 mL / g; (4) Use a shaver to shave the hair on the entire back, and then use hair removal cream to remove the hair completely. (5) On the back of the mouse, along the long axis of the body, prepare a full-thickness wound with a diameter of about 8 mm; (6) Bacterial inoculation: Use a pipette to add 100 μL of 1× 10⁻⁶ bacteria to each wound. 9 S. aureus bacterial solution at CFU / mL, and cover the wound with a sterile dressing; (7) Animal care: After surgery, provide normal feed and water, and closely observe the mice's vital signs and feeding status; (8) Wound infection: 48 h after S. aures inoculation, pale yellow pus was observed on the wound and wound edges, indicating successful wound infection. Normal skin and infected skin wound tissue were collected.

[0089] 9. Treatment grouping and wound treatment methods (1) Treatment grouping: Thirty mice were randomly divided into 5 groups of 6 mice each. The grouping was as follows: 1) MCMA hydrogel, denoted as MCMA; 2) MCMA hydrogel + 808 nm (1 W / cm) 2 5 min), recorded as MCMA + 808 nm; 3) MCMA hydrogel + 660 nm (400 mW / cm 2 5 min), recorded as MCMA + 660 nm; 4) MCMA hydrogel + 808 nm (1 W / cm 2 , 5min) + 660 nm (400 mW / cm 2 5 min), denoted as MCMA + 808 nm + 660 nm; 5) No drug treatment, denoted as Control; (2) The hydrogel dressing was circumcised into circular dressings with a diameter of 15 mm. For the group treated with 808 nm laser, the wound was exposed to 808 nm (1.0 W / cm²). 2 Laser irradiation for 5 min. For the group treated with 660 nm laser, the wound was exposed to 660 nm (400 mW / cm²) laser light. 2 Laser irradiation for 5 minutes. Dressings were changed every two days, and wounds were photographed on days 1, 3, 5, and 7 after the model was completed. (3) On day 7, wound tissue and internal organs of mice were collected and fixed in 4% paraformaldehyde for further analysis. H&E staining and Masson's trichrome staining were performed to evaluate the healing effect. In addition, major organs, including heart, liver, spleen, kidney and lung, were collected and stained with H&E.

[0090] This invention conducts further toxicological observations through in vivo toxicity testing. The invention includes an acute toxicity assay in mice, such as... Figure 18 As shown, the experimental results indicate that the median lethal dose (LD50) of compound MCMA is 10.52 g / kg (mouse body weight), and the 95% confidence interval concentration range is 8.774-12.612 g / kg (mouse body weight).

[0091] On the second day after bacterial inoculation of mouse wounds, the appearance of pus on the wound surface indicated successful establishment of the infection model. Drug administration and wound photography were performed every other day. By day 7 of drug administration, significant differences were observed among the five groups. Figure 19 As shown, minute damage was visible in the MCMA+ 808 nm group; the epithelium in the MCMA+ 808 nm+ 660 nm group was completely healed.

[0092] Epithelial formation marks the end of the wound healing process. For example... Figure 20 , 21As shown, intact epithelium and appendages are visible in the histological sections of healthy tissue, while inflammatory tissue is visible in infected skin. A natural and mature epidermal layer is present in the MCMA+ 808 nm+ 660 nm group, indicating complete wound healing. As mentioned above, the hydrogel exhibits shape adaptability and adhesion, ensuring a tight bond with the wound surface. Utilizing this property, the hydrogel can provide a biomimetic microenvironment for cell proliferation and migration, thereby facilitating the growth of new epidermis. Furthermore, due to the synergistic effects of PTT, PDT, and CDT, the hydrogel can promote the formation of new granulation tissue and blood vessels, contributing to tissue repair and angiogenesis. To further evaluate the wound healing effect of the hydrogel, tissues were stained using H&E and Masson trichrome staining. On day 7, the wound in the MCMA+ 808 nm+ 660 nm group showed more newly formed collagen fibers, demonstrating its better healing efficiency. To evaluate the in vivo toxicology of the hydrogel, H&E staining was performed on the major organs (including heart, liver, kidney, lung and spleen) of mice on day 7 after administration. The organ damage produced in all groups was negligible, which confirmed that the MCMA hydrogel is a safe wound dressing.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing MCMA nanostructures, characterized in that, The method includes the following steps: (1) Mix EDC, NHS, Ce6 and DMSO and stir for 30±2 min to obtain a carboxyl-activated Ce6 solution; (2) The Ce6 solution was mixed with the MSN-NH2 solution and stirred overnight. After centrifugation, washing and drying, MC was obtained. (3) Add 5 mg / mL potassium permanganate solution dropwise to 1.6±0.1 mg / mL MC aqueous solution. After the addition is complete, add formamide, sonicate for 30±2 min, centrifuge, wash and dry to obtain MCM; (4) Disperse an equal volume of 2±0.1 mg / mL MCM in 4±0.5 mg / mL CS / Ag solution, stir slowly under vacuum for 12±1h, and obtain MCMA nanostructure by centrifugation, washing and drying. MSN is mesoporous silica, and CS is chitosan. In step (4), the preparation of CS / Ag includes the following specific steps: 60±2 mg CS is dissolved in 60±2 mL of 0.1% w / v acetic acid aqueous solution, and 4±1 mL of freshly prepared AgNO3 solution of 5±1 mg / mL is added dropwise while stirring. The mixture is heated to 95±1℃ while stirring, and 6±1 mg / mL of NaOH solution is added dropwise until the solution turns yellow. The reaction is stopped after 10±1 min. After centrifugation, washing, and drying, CS / Ag is obtained.

2. The method as described in claim 1, characterized in that, In step (1), the mass-to-volume ratio of EDC, NHS, Ce6, and DMSO is 15±1 mg: 10±1 mg: 5±1 mg: 5 mL.

3. The method as described in claim 1, characterized in that, In step (2), the preparation process of MSN-NH2 includes the following steps: MSN powder is added to anhydrous toluene, ultrasonically dispersed until uniform, APTES is added and ultrasonically continued until the solution is clear and transparent, heated and stirred at 110°C for 5-6 h, and the reaction product is centrifuged, washed and dried to obtain MSN-NH2; The concentration of the MSN-NH2 solution was 4 ± 0.5 mg / mL, and the volume ratio of Ce6 solution to MSN-NH2 solution was 1:

1.

4. The method as described in claim 1, characterized in that, In step (3), the volume ratio of the potassium permanganate solution, MC aqueous solution and formamide is 12 mL ± 132 µL: 10 mL ± 120 µL: 1.2 mL ± 7.8 µL.

5. The MCMA nanostructure prepared by the method according to any one of claims 1 to 4.

6. A method for preparing MCMA hydrogel, characterized in that, The method includes the following steps: (1) The polymer was dissolved in 0.5±0.1 M acetic acid at 40±1℃ for 12±1 h to obtain a solution with a polymer concentration of 15±1% (w / v); wherein the polymer is CS and gelatin G in a mass ratio of 1:

2. (2) The suspension of the MCMA nanostructure described in claim 5 is added to the solution under magnetic stirring. 2.5% of EDC relative to the total mass of CS-G is added to the solution as a crosslinking agent. The solution is treated at 50±1℃ for 30±2 min under stirring to gel and obtain MCMA hydrogel.

7. The MCMA hydrogel prepared by the method of claim 6.

8. The application of the MCMA nanostructure of claim 5 or the MCMA hydrogel of claim 7 in the preparation of antibacterial drugs for synergistic PDT, PTT, and CDT antibacterial therapy; wherein PDT is photodynamic therapy, PTT is photothermal therapy, and CDT is chemikinetic therapy.

9. The application as described in claim 8, characterized in that, The bacteria include E. coli and S. aures .

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