Dual-function microneedles that intelligently respond to tumor microenvironment and preparation method thereof

Abstract

The present invention discloses a dual-function microneedle that intelligently responds to the tumor microenvironment and a preparation method. The raw materials of the dual-function microneedle that intelligently responds to the tumor microenvironment include a needle tip loaded with nanosheets and nanoparticles and methacrylic anhydride-grafted hyaluronic acid; the needle tip loaded with nanosheets and nanoparticles is a secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles dispersed in a mixed solution of polyvinyl alcohol and polyvinylpyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles. The dual-function microneedle that intelligently responds to the tumor microenvironment has intelligent responsiveness to the tumor microenvironment and can achieve precise delivery of nanoenzymes and ICB antibodies to the tumor site, achieving efficient inhibition of tumor growth and promoting skin tissue regeneration after tumor ablation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a dual-function microneedle that intelligently responds to the tumor microenvironment and a preparation method thereof. Background Art

[0002] Melanoma kills an increasing number of people worldwide each year, placing significant financial and emotional strain on patients. Immune checkpoint blockade (ICB) therapy has significantly improved survival rates for patients with advanced melanoma, yet less than 40% of patients benefit. This is primarily due to insufficient infiltration of cytotoxic T lymphocytes within the immunosuppressive "cold tumor" microenvironment, high expression of tumor-promoting M2 phenotype macrophages, and low sensitivity to ICB therapy.

[0003] Studies have reported that ROS can reprogram immunosuppressive "cold tumors" into immune-activated "hot tumors," thereby increasing patients' sensitivity to ICB treatment. Nanozyme-driven chemodynamic therapy (CDT), especially the cascade reaction based on glucose oxidase (GOx) and peroxidase (POD) to produce ROS, has been widely reported. It not only consumes intratumoral glucose to cut off the nutrient source of cancer cells to starve the tumor, but also provides abundant H2O2 to the tumor and reduces the pH of the tumor microenvironment, thereby enhancing the activity of POD to produce ROS. However, the glucose oxidation process is highly dependent on oxygen, and the hypoxic microenvironment of the tumor limits its activity. In addition, glutathione (GSH), which is highly expressed in the tumor microenvironment, can consume the generated ROS and weaken oxidative stress. Therefore, it is necessary to construct a GOx- and POD-based nanozyme that can alleviate hypoxia and deplete GSH. The research and development of biomedical materials that can achieve precise delivery of nanozymes and ICB antibodies to tumor sites to avoid immune-related adverse events caused by systemic administration, promote tissue regeneration after tumor ablation, and protect the activity of ICB antibodies and nanozymes from ROS destruction when they are co-delivered, so that they can efficiently and synergistically exert their efficacy in treating melanoma has received widespread attention. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and provide a dual-functional microneedle that intelligently responds to the tumor microenvironment and a preparation method thereof. The dual-functional microneedle that intelligently responds to the tumor microenvironment of the present invention comprises raw materials including a needle tip loaded with nanosheets and nanoparticles and methacrylic anhydride grafted hyaluronic acid; the needle tip loaded with nanosheets and nanoparticles is a secondary grafted MXene nanosheet loaded with RuO2 and SiO2 nanoparticles loaded with aPD-1 dispersed in a mixed solution of polyvinyl alcohol and polyvinylpyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles. The dual-functional microneedle that intelligently responds to the tumor microenvironment has intelligent responsiveness to the tumor microenvironment, can achieve precise delivery of nanozymes and ICB antibodies to the tumor site, achieve efficient inhibition of tumor growth, and promote skin tissue regeneration after tumor ablation.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a dual-functional microneedle that intelligently responds to the tumor microenvironment, characterized in that the raw materials include a needle tip loaded with nanosheets and nanoparticles and methacrylic anhydride grafted hyaluronic acid; the needle tip loaded with nanosheets and nanoparticles is a needle tip loaded with nanosheets and nanoparticles obtained by secondary grafting MXene nanosheets loaded with RuO2 and SiO2 nanoparticles loaded with aPD-1 dispersed in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone.

[0006] In addition, the present invention also provides a method for preparing a dual-functional microneedle that intelligently responds to the tumor microenvironment, which is characterized by comprising:

[0007] Providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0008] Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0009] A system containing the methacrylic anhydride grafted hyaluronic acid powder is placed on the upper layer of the needle tip loaded with nanosheets and nanoparticles, and ammonium persulfate, tannic acid and FeCl3 are added to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0010] The above method is characterized in that it specifically includes:

[0011] Step 1: providing the secondary grafted RuO2-loaded MXene nanosheets, comprising:

[0012] Step 101: etching a block of titanium aluminum carbide compound with a hydrofluoric acid solution for 48 to 96 hours, centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium aluminum carbide MXene multilayer nanosheets; exfoliating the MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 48 to 96 hours, and centrifuging and drying to obtain a few-layer MXene nanosheets;

[0013] Step 102: ultrasonically disperse the few-layer MXene nanosheets in water, add RuCl3 aqueous solution, dropwise add NaOH solution, stir and react for 3-5 hours at room temperature, and centrifuge and dry to obtain RuO2-loaded MXene nanosheets;

[0014] Step 103: ultrasonically dispersing the RuO2-loaded MXene nanosheets in water, adding amino polyethylene glycol thiol, stirring for 18 to 24 hours, and centrifugally drying to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0015] Step 104: Glucose oxidase and HbO2 are dissolved in water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, and after activation for 30 to 120 minutes, MXene@RuO2 nanosheets grafted with -NH2 are added, the mixture is reacted in an ice bath for 20 to 24 hours, and the mixture is centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets;

[0016] Step 2: providing the aPD-1 loaded SiO2 nanoparticles, comprising:

[0017] Step 201: mixing polyethylene glycol octylphenyl ether, n-hexanol, and cyclohexane to obtain an organic mixed system;

[0018] Step 202: aPD-1 is mixed with ethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide, shaken and added to the organic mixed system to obtain a mixed system B;

[0019] Step 203: Add ammonia water to the mixed system B, stir at room temperature overnight, add pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1;

[0020] Step 3: providing the needle tip loaded with nanosheets and nanoparticles, comprising:

[0021] Step 301: ultrasonically disperse the secondary grafted RuO2-loaded MXene nanosheets and the aPD-1-loaded SiO2 nanoparticles in a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system;

[0022] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles;

[0023] Step 4: providing the methacrylic anhydride grafted hyaluronic acid powder, comprising:

[0024] Step 401: Dissolve hyaluronic acid in water, add methacrylic anhydride solution, adjust the pH to 8.5-10 with NaOH, and stir for 24-48 hours. Then, dialyze with deionized water and freeze-dry to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0025] Step 5: Obtain dual-function microneedles that intelligently respond to the tumor microenvironment, including:

[0026] Step 501: Place the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate solution, TA solution and FeCl3 solution, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0027] The above method is characterized in that, in step 101, the mass of the hydrofluoric acid is 2 to 4 times the mass of the bulk Ti3AlC2, and the mass of the tetramethylammonium hydroxide solution is 2 to 4 times the mass of the titanium dioxide carbon MXene multilayer nanosheets.

[0028] The above method is characterized in that, in step 102, the volume of the water is 150 to 300 times the mass of the few-layer MXene nanosheets, the unit of the water volume is mL, and the unit of the mass of the few-layer MXene nanosheets is g; in step 102, the volume of the RuCl3 aqueous solution is 20 to 80 times the mass of the few-layer MXene nanosheets, so the volume unit of the RuCl3 aqueous solution is mL, and the unit of the mass of the few-layer MXene nanosheets is g; in step 102, the volume of the NaOH solution is 10 to 40 times the mass of the few-layer MXene nanosheets, the unit of the NaOH solution volume is mL, and the unit of the mass of the few-layer MXene nanosheets is g; in step 103, the mass of the aminopolyethylene glycol thiol group is 0.3 to 2 times the mass of the RuO2-loaded MXene nanosheets;

[0029] The above method is characterized in that, in step 104, the mass ratio of the glucose oxidase and the -NH2-grafted MXene@RuO2 nanosheets is 1:(0.5~4); in step 104, the mass of the HbO2 is 1 times the mass of the glucose oxidase; in step 104, the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 10~90 times the mass of the glucose oxidase; in step 104, the mass of the N-hydroxysuccinimide is 7~60 times the mass of the glucose oxidase; in step 104, the mass of the water is 500~4000 times the mass of the glucose oxidase.

[0030] The above method is characterized in that, in step 201, the volume of n-hexanol is 1 to 1.5 times the volume of polyethylene glycol octylphenyl ether, and the volume of cyclohexane is 3 to 6.5 times the volume of polyethylene glycol octylphenyl ether; in step 202, the mass of aPD-1 is 0.001 to 0.004 times the mass of the MXene@RuO2 nanosheet grafted with -NH2; in step 202, the volume of tetraethyl orthosilicate is 0.2 to 0.9 times the mass of aPD-1, the unit of the volume of tetraethyl orthosilicate is μL, and the unit of the mass of aPD-1 is μg; in step 202, the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 0.4 to 1.6 times the mass of aPD-1, The unit of the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is μL, and the unit of the mass of aPD-1 is μg; in step 202, the volume of the polyethylene glycol octylphenyl ether is 0.008 to 0.04 times the mass of aPD-1, the unit of the volume of the polyethylene glycol octylphenyl ether is mL, and the unit of the mass of aPD-1 is μg; in step 203, the volume of the ammonia water is 0.2 to 2 times the mass of aPD-1, the unit of the volume of the ammonia water is μL, and the unit of the mass of aPD-1 is μg; in step 203, the volume of the pure acetone is 0.1 to 0.4 times the mass of aPD-1, the unit of the volume of the pure acetone is mL, and the unit of the mass of aPD-1 is μg.

[0031] The above method is characterized in that, in step 301, the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 7% to 10%, and the mass percentage of PVP is 13% to 20%; in step 301, the mass of the PVA / PVP mixed aqueous solution is 66 to 250 times the mass of the MXene@RuO2 nanosheets grafted with -NH2 in step 104.

[0032] The above method is characterized in that, in step 401, the volume of the methacrylic anhydride solution is 0.8 to 4 times the mass of the hyaluronic acid, the volume unit of the methacrylic anhydride solution is mL, and the mass unit of the hyaluronic acid is g.

[0033] The above method is characterized in that, in step 501, the volume of the ammonium persulfate solution is 0.14 to 0.22 times the volume of the grafted hyaluronic acid system, the volume of the TA solution is 0.06 to 0.24 times the volume of the grafted hyaluronic acid system, and the volume of the FeCl3 solution is 0.2 to 1.5 times the volume of the grafted hyaluronic acid system; in step 501, the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 0.14 to 0.22 times the volume of the grafted hyaluronic acid system. The mass percentage concentration is 3%; in step 501, the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.001-0.008 g / mL, and the concentration of the FeCl3 solution is 0.02-0.05 g / mL; in step 501, the volume of the grafted hyaluronic acid system is 15-30 times the mass of the MXene@RuO2 nanosheets grafted with -NH2 in step 104, the unit of the volume of the grafted hyaluronic acid system is μL, and the unit of the mass of the MXene@RuO2 nanosheets grafted with -NH2 is mg.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. The dual-functional microneedle that intelligently responds to the tumor microenvironment of the present invention comprises raw materials including a needle tip loaded with nanosheets and nanoparticles and methacrylic anhydride grafted hyaluronic acid; the needle tip loaded with nanosheets and nanoparticles is a needle tip loaded with nanosheets and nanoparticles obtained by dispersing secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinylpyrrolidone. The dual-functional microneedle that intelligently responds to the tumor microenvironment has intelligent responsiveness to the tumor microenvironment, can achieve precise delivery of nanozymes and ICB antibodies to the tumor site, achieve efficient inhibition of tumor growth and promote skin tissue regeneration after tumor ablation.

[0036] 2. The dual-functional microneedle of the present invention that intelligently responds to the tumor microenvironment comprises dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles. The ability of the nanoenzymes in the needle tip loaded with nanosheets and nanoparticles to produce ROS in the hypoxic microenvironment of the tumor can be fully utilized. At the same time, the SiO2 nanoparticles can respond to the tumor microenvironment with high GSH expression, thereby achieving GSH depletion and increasing ROS levels.

[0037] 3. The preparation method of the dual-functional microneedle that intelligently responds to the tumor microenvironment of the present invention comprises placing a system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, and then adding ammonium persulfate, tannic acid and FeCl3, which can effectively realize the construction of a microneedle with intelligent response containing a backing and a needle tip structure.

[0038] 4. The dual-functional microneedle of the present invention that intelligently responds to the tumor microenvironment can comprehensively utilize the synergistic effects of starvation therapy, chemodynamic therapy and ICB.

[0039] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0040] Figures in the specification

[0041] Figure 1 The electron microscopy image and element distribution of RuO2-loaded MXene nanosheets grafted with GOx and HbO2 in Example 1;

[0042] Figure 2 This is a morphological image of the dual-functional microneedle that intelligently responds to the tumor microenvironment in Example 1;

[0043] Figure 3 Schematic diagram of the tumor cell killing effect of RuO2-loaded MXene nanosheets grafted with GOx and HbO2;

[0044] Figure 4 Schematic diagram of the mechanism of dual-functional microneedles that intelligently respond to the tumor microenvironment. DETAILED DESCRIPTION

[0045] Example 1

[0046] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0047] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0048] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0049] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0050] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0051] Step 101: After etching the bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 72 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 96 hours, centrifuged and dried to obtain few-layer MXene nanosheets. The etching comprises: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is 3 times the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is 3 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0052] Step 102: ultrasonically disperse 0.05 g of the few-layer MXene nanosheets in 15 mL of water, add 3 mL of RuCl3 aqueous solution, dropwise add 0.5 mL of 1 M NaOH solution, stir and react for 4 h at room temperature, and centrifuge to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%; the room temperature is 20-25°C;

[0053] Step 103: ultrasonically disperse 20 mg of MXene@RuO2 powder in 20 g of water, add 20 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 24 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0054] Step 104: 10 mg of glucose oxidase (GOx) and 10 mg of HbO2 were dissolved in 20 g of water, 0.465 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.285 g of N-hydroxysuccinimide (NHS) were added, and after activation for 30 min, 20 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 24 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0055] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0056] Step 201: 1.17 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.8 mL of n-hexanol, and 7.5 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0057] Step 202: 80 μg of aPD-1 is mixed with 40 μL of tetraethyl orthosilicate (TEOS) and 60 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken, and added to the organic mixed system to obtain a mixed system B;

[0058] Step 203: Add 20 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 20 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0059] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0060] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles (SiO2@aPD-1 nanoparticles) in 5 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 8%, and the mass percentage of PVP is 15%;

[0061] Step 302: Add the hydrogel premix system to a silicone rubber microneedle mold, centrifuge, and naturally dry at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the aPD-1 is an anti-mouse PD-1 antibody, item number 114114, purchased from Biolegend; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0062] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0063] Step 401: Dissolve 1 g of hyaluronic acid (HA) in 100 mL of water, add 2 mL of methacrylic anhydride (MA) solution, adjust the pH to 8.5 with NaOH, and react with stirring at 4° C. for 24 hours. The mixture is dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0064] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0065] Step 501: Place 600 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 100 μL of ammonium persulfate solution, 125 μL of TA solution and 125 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.001 g / mL, and the concentration of the FeCl3 solution is 0.02 g / mL.

[0066] Example 2

[0067] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0068] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0069] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0070] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0071] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0072] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0073] Step 101: After etching the bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 48 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 72 hours, centrifuged and dried to obtain few-layer MXene nanosheets. The etching comprises: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is twice the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is twice the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0074] Step 102: ultrasonically disperse 0.1 g of the few-layer MXene nanosheets in 15 mL of water, add 4 mL of RuCl3 aqueous solution, dropwise add 3 mL of 1 M NaOH solution, stir and react at room temperature for 4 h, and centrifuge to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%;

[0075] Step 103: Ultrasonic dispersion of 40 mg of MXene@RuO2 powder in 40 g of water, addition of 15 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stirring for 24 h, and centrifugation to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0076] Step 104: 10 mg of glucose oxidase (GOx) and 10 mg of HbO2 were dissolved in 40 g of water, 0.92 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.57 g of N-hydroxysuccinimide (NHS) were added, and after activation for 60 min, 40 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 24 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0077] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0078] Step 201: 1.77 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.8 mL of n-hexanol, and 8 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0079] Step 202: 60 μg of aPD-1 is mixed with 35 μL of tetraethyl orthosilicate (TEOS) and 65 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken well, and added to the organic mixed system to obtain a mixed system B;

[0080] Step 203: Add 40 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 15 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0081] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0082] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles SiO2@aPD-1 nanoparticles in 3 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 7%, and the mass percentage of PVP is 13%;

[0083] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0084] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0085] Step 401: dissolving 1.5 g of hyaluronic acid (HA) in 100 mL of water, adding 3 mL of methacrylic anhydride (MA) solution, adjusting the pH to 9 with NaOH, and stirring the mixture at 4° C. for 24 h. The mixture was dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0086] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0087] Step 501: Place 650 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 100 μL of ammonium persulfate solution, 150 μL of TA solution and 100 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.001 g / mL, and the concentration of the FeCl3 solution is 0.03 g / mL.

[0088] Example 3

[0089] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0090] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0091] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0092] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0093] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0094] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0095] Step 101: After etching a bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 96 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 96 hours, centrifuged and dried to obtain few-layer MXene nanosheets. The etching comprises: dispersing bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is 4 times the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is 4 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0096] Step 102: ultrasonically disperse 0.08 g of the few-layer MXene nanosheets in 15 mL of water, add 3 mL of a RuCl3 aqueous solution, dropwise add 3 mL of a 1 M NaOH solution, stir and react at room temperature for 5 h, and centrifuge to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%;

[0097] Step 103: ultrasonically disperse 30 mg of MXene@RuO2 powder in 30 g of water, add 60 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 24 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0098] Step 104: 30 mg of glucose oxidase (GOx) and 30 mg of HbO2 were dissolved in 30 g of water, 0.698 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.428 g of N-hydroxysuccinimide (NHS) were added, and after activation for 30 min, 30 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 24 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0099] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0100] Step 201: 1.06 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.08 mL of n-hexanol, and 4.5 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0101] Step 202: 100 μg of aPD-1 is mixed with 24 μL of tetraethyl orthosilicate (TEOS) and 45 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken well, and added to the organic mixed system to obtain a mixed system B;

[0102] Step 203: Add 50 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 12 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0103] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0104] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles SiO2@aPD-1 nanoparticles in 2 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 9%, and the mass percentage of PVP is 15%;

[0105] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0106] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0107] Step 401: dissolving 1.0 g of hyaluronic acid (HA) in 100 mL of water, adding 4 mL of methacrylic anhydride (MA) solution, adjusting the pH to 8.5 with NaOH, and stirring the mixture at 4° C. for 48 h. The mixture was dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0108] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0109] Step 501: Place 690 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 100 μL of ammonium persulfate solution, 50 μL of TA solution and 150 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.005 g / mL, and the concentration of the FeCl3 solution is 0.05 g / mL.

[0110] Example 4

[0111] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0112] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0113] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0114] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0115] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0116] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0117] Step 101: After etching the bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 48 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 96 hours, centrifuged and dried to obtain few-layer MXene nanosheets. The etching comprises: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is 2 times the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is 3 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0118] Step 102: ultrasonically disperse 0.1 g of the few-layer MXene nanosheets in 15 mL of water, add 2 mL of a RuCl3 aqueous solution, dropwise add 2 mL of a 1 M NaOH solution, stir and react at room temperature for 3 h, and centrifuge to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%;

[0119] Step 103: Ultrasonic dispersion of 30 mg of MXene@RuO2 powder in 30 g of water, addition of 50 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stirring for 20 h, and centrifugation to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0120] Step 104: 60 mg of glucose oxidase (GOx) and 60 mg of HbO2 were dissolved in 30 g of water, 0.698 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.428 g of N-hydroxysuccinimide (NHS) were added, and after activation for 90 min, 30 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 24 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0121] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0122] Step 201: 1.06 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.08 mL of n-hexanol, and 4.5 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0123] Step 202: 120 μg of aPD-1 is mixed with 30 μL of tetraethyl orthosilicate (TEOS) and 50 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken well, and added to the organic mixed system to obtain a mixed system B;

[0124] Step 203: Add 45 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 15 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0125] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0126] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles (SiO2@aPD-1 nanoparticles) in 5 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 9%, and the mass percentage of PVP is 15%;

[0127] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0128] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0129] Step 401: dissolving 1.0 g of hyaluronic acid (HA) in 100 mL of water, adding 4 mL of methacrylic anhydride (MA) solution, adjusting the pH to 8.5 with NaOH, and stirring the mixture at 4° C. for 48 h. The mixture was dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0130] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0131] Step 501: Place 900 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 150 μL of ammonium persulfate solution, 130 μL of TA solution and 120 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.0008 g / mL, and the concentration of the FeCl3 solution is 0.04 g / mL.

[0132] Example 5

[0133] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0134] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0135] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0136] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0137] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0138] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0139] Step 101: After etching a bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 96 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 96 hours, centrifuged and dried to obtain few-layer MXene nanosheets. The etching comprises: dispersing bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is 4 times the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is 3 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0140] Step 102: ultrasonically disperse 0.06 g of the few-layer MXene nanosheets in 15 mL of water, add 5 mL of RuCl3 aqueous solution, dropwise add 1.5 mL of 1 M NaOH solution, stir and react for 3 h at room temperature, and centrifuge to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%;

[0141] Step 103: ultrasonically disperse 20 mg of MXene@RuO2 powder in 20 g of water, add 40 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 24 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0142] Step 104: 20 mg of glucose oxidase (GOx) and 20 mg of HbO2 were dissolved in 30 g of water, 0.698 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.428 g of N-hydroxysuccinimide (NHS) were added, and after activation for 120 min, 30 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 24 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0143] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0144] Step 201: 1.30 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.60 mL of n-hexanol, and 5 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0145] Step 202: 40 μg of aPD-1 is mixed with 24 μL of tetraethyl orthosilicate (TEOS) and 45 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken, and added to the organic mixed system to obtain a mixed system B;

[0146] Step 203: Add 80 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 13 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0147] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0148] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles SiO2@aPD-1 nanoparticles in 3 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 10%, and the mass percentage of PVP is 20%;

[0149] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0150] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0151] Step 401: dissolving 0.8 g of hyaluronic acid (HA) in 100 mL of water, adding 3 mL of methacrylic anhydride (MA) solution, adjusting the pH to 10 with NaOH, and stirring the mixture at 4° C. for 24 h. The mixture was dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0152] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0153] Step 501: Place 690 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 150 μL of ammonium persulfate solution, 125 μL of TA solution and 125 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.002 g / mL, and the concentration of the FeCl3 solution is 0.04 g / mL.

[0154] Example 6

[0155] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0156] This embodiment provides a method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, comprising the following steps:

[0157] Step 1: providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder;

[0158] Step 2: Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles;

[0159] Step 3: Place the system containing the methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate, tannic acid and FeCl3, and obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

[0160] The preparation method of the secondary grafted RuO2-loaded MXene nanosheets includes:

[0161] Step 101: After etching a bulk titanium aluminum carbide compound (Ti3AlC2) with a hydrofluoric acid solution for 72 hours, centrifuge until the pH of the supernatant is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The MXene multilayer nanosheets are exfoliated in a tetramethylammonium hydroxide (TMAOH) solution for 48 hours, and centrifuge and dry to obtain few-layer MXene nanosheets. The etching comprises: dispersing bulk Ti3AlC2 powder in a hydrofluoric acid solution, stirring the reaction at room temperature to etch the middle Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide, stirring the reaction at room temperature, and exfoliating the multilayers into few-layer MXene nanosheets; the mass of the hydrofluoric acid is twice the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetramethylammonium hydroxide (TMAOH) solution is twice the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetramethylammonium hydroxide (TMAOH) solution is 25%;

[0162] Step 102: ultrasonically disperse 0.07 g of the few-layer MXene nanosheets in 15 mL of water, add 2 mL of RuCl3 aqueous solution, dropwise add 1 mL of 1 M NaOH solution, stir and react at room temperature for 4 h, and centrifuge and dry to obtain RuO2-loaded MXene nanosheets (MXene@RuO2, MRu); the mass percentage concentration of RuCl3 in the RuCl3 aqueous solution is 1%;

[0163] Step 103: ultrasonically disperse 30 mg of MXene@RuO2 powder in 30 g of water, add 30 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 18 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0164] Step 104: 25 mg of glucose oxidase (GOx) and 25 mg of HbO2 were dissolved in 30 g of water, 0.698 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.428 g of N-hydroxysuccinimide (NHS) were added, and after activation for 50 min, 30 mg of MXene@RuO2 nanosheets grafted with -NH2 were added. The mixture was reacted in an ice bath for 20 h, and centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets, i.e., RuO2-loaded MXene nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0165] The preparation method of the aPD-1-loaded SiO2 nanoparticles comprises:

[0166] Step 201: 1.6 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1.8 mL of n-hexanol, and 7 mL of cyclohexane were added to a round-bottom flask to obtain an organic mixture system;

[0167] Step 202: 50 μg of aPD-1 is mixed with 45 μL of tetraethyl orthosilicate (TEOS) and 80 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS), shaken well, and added to the organic mixed system to obtain a mixed system B;

[0168] Step 203: Add 25 μL of 30% ammonia water to the mixed system B, stir at room temperature overnight, add 18 mL of pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1 (SiO2@aPD-1, SP);

[0169] The method for obtaining the needle tip loaded with nanosheets and nanoparticles specifically includes:

[0170] Step 301: ultrasonically disperse the MXene@RuO2@GOx-HbO2 nanosheets and the aPD-1-loaded SiO2 nanoparticles (SiO2@aPD-1 nanoparticles) in 3 g of a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 8%, and the mass percentage of PVP is 15%;

[0171] Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; the polyvinyl alcohol model is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the molecular weight of polyvinyl pyrrolidone is 44,000 to 54,000;

[0172] The preparation method of the methacrylic anhydride grafted hyaluronic acid powder specifically comprises:

[0173] Step 401: Dissolve 1.2 g of hyaluronic acid (HA) in 100 mL of water, add 1 mL of methacrylic anhydride (MA) solution, adjust the pH to 8.5 with NaOH, and react with stirring at 4° C. for 24 hours. The mixture is dialyzed against deionized water and freeze-dried to obtain methacrylic anhydride-grafted hyaluronic acid powder.

[0174] The method for obtaining a dual-functional microneedle that intelligently responds to the tumor microenvironment specifically includes:

[0175] Step 501: Place 800 μL of the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add 150 μL of ammonium persulfate solution, 50 μL of TA solution and 150 μL of FeCl3 solution to the upper layer of the grafted hyaluronic acid system, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is 3%; the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.005 g / mL, and the concentration of the FeCl3 solution is 0.05 g / mL.

[0176] Performance testing:

[0177] Figure 1 This is the electron microscope image and element distribution of RuO2-loaded MXene nanosheets grafted with GOx and HbO2 in Example 1. Figure 1 A is a scanning electron microscopy image of a multilayer MXene nanosheet, and 1B is a transmission electron microscopy image of a few-layer MXene nanosheet. Figure 1 C, D, E and F are the element distribution diagrams of few-layer MXene. Figure 1 G and 1H are transmission electron microscopy images and lattices of RuO2 nanoparticles. Figure 1 I, J and K are the corresponding element distribution diagrams of RuO2. Figure 1 L is the transmission electron microscopy image of MXene nanosheets loaded with RuO2 nanoparticles, 1M, N, O and P are the corresponding element distributions, Figure 1 Q is the transmission electron microscopy image of MXene@RuO2 nanosheets loaded with GOx and HbO2, Figure 1 R, S, T, U, V and W show the corresponding element distribution diagrams. Figure 1 It can be seen that RuO2-loaded MXene nanosheets grafted with GOx and HbO2, namely MXene@RuO2@GOx / HbO2, were successfully prepared.

[0178] Figure 2 This is a morphological diagram of the dual-functional microneedle that intelligently responds to the tumor microenvironment in Example 1. Figure 2 A is the overall shape, according to Figure 2 A shows that the dual-functional microneedle of the present invention that intelligently responds to the tumor microenvironment is composed of a needle tip and a backing. Figure 2 B shows that the microneedle of the present invention has a complete needle tip structure. Figure 2 C is a schematic diagram of solubility performance, according to Figure 2 As shown in Figure 3, the microneedles of the present invention exhibit rapid solubility, promising the potential for precise drug delivery to tumor sites and rapid dissolution to release their payload. The solubility test involved placing the tip of a dual-function microneedle that intelligently responds to the tumor microenvironment in deionized water for 2 minutes, removing it and photographing its morphology. Figure 2 D and Figure 2 E are the FTIR and NMR images of HA grafted with MA in Example 1, respectively. Figure 2 D and Figure 2 E shows that MA was successfully grafted onto HA. Figure 2 F is the photothermal conversion diagram of the microneedles after TA / Fe catalytic cross-linking. TA / Fe can convert light into heat under 808nm irradiation, and is expected to be highly effective in antibacterial treatment. Figure 2 G is a schematic diagram of the performance of microneedles in cell proliferation and migration. Figure 2 G shows that the microneedle can clear intracellular ROS, significantly promote L929 cell proliferation and migration, and achieve the function of promoting wound healing.

[0179] The method for testing the cell proliferation promoting effect includes: extracting methacrylic anhydride-grafted hyaluronic acid in 1640 culture medium for 3 days, culturing L929 cells with the extract, and after 24 hours, adding AO / EB fluorescent dye, taking pictures under a fluorescence microscope, and detecting its cell proliferation promoting effect.

[0180] The test method for promoting cell migration includes: scratching L929 cells with a 100μL pipette tip and taking photos to record the initial scratch area, spreading the dual-function microneedles on the cells, and taking photos under a microscope 48 hours later to record the scratch size.

[0181] Figure 3 Schematic diagram of the tumor cell killing effect of RuO2-loaded MXene nanosheets (MN / MRuGH) grafted with GOx and HbO2. Figure 3It can be seen that the RuO2-loaded MXene nanosheets grafted with GOx and HbO2 can produce ROS by consuming glucose, peroxidase and oxidase, killing melanoma cells, and causing a decrease in mitochondrial membrane potential and membrane damage. The DCFH-DA probe further proves that the RuO2-loaded MXene nanosheets grafted with GOx and HbO2 can produce abundant ROS, and catalase-like enzymes and oxygenated hemoglobin can alleviate hypoxia.

[0182] The testing process includes:

[0183] The method for testing the killing ability of melanoma cells includes: treating melanoma B16F10 cells with RuO2-loaded MXene nanosheets grafted with GOx and HbO2, adding Calcein-AM / PI live cell / dead cell dyes, taking pictures under a fluorescence microscope, and recording the killing ability of melanoma cells.

[0184] The test method for the effect on mitochondrial membrane potential includes: adding JC-1 probe to RuO2-loaded MXene nanosheets grafted with GOx and HbO2, taking pictures under a fluorescence microscope, and recording the effect on mitochondrial membrane potential.

[0185] The test method for the effect of alleviating hypoxia includes: adding DAPI and Ru(dpp)3Cl2 oxygen indicator probes to the RuO2-loaded MXene nanosheets grafted with GOx and HbO2, taking pictures under a fluorescence microscope, and recording the effect of alleviating hypoxia.

[0186] The ROS-generating ability test method includes: adding DAPI and DCFH-DA probes to RuO2-loaded MXene nanosheets grafted with GOx and HbO2, taking pictures under a fluorescence microscope, and recording the ability to generate ROS.

[0187] Figure 3 Control refers to the blank control group, MN refers to blank microneedles, MN / G refers to microneedles loaded with GOx only, MN / MRuG refers to microneedles loaded with MRuG; MN / MRuGH refers to microneedles loaded with MRuGH.

[0188] The MN preparation method comprises: adding the PVA / PVP mixed aqueous solution described in Example 1 to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain blank microneedles;

[0189] The MN / G preparation method includes: dissolving GOx in a PVA / PVP mixed aqueous solution, adding the solution to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain GOx-loaded microneedles; the amounts of GOx and PVA / PVP mixed aqueous solution used are the same as those in Example 1.

[0190] The preparation method of MN / MRuG includes: dissolving GOx in water, adding EDC and NHS for activation, adding the -NH2-grafted MXene@RuO2 nanosheets in Example 1, reacting in an ice bath for 24 hours, and centrifuging and drying to obtain GOx-grafted RuO2-loaded MXene nanosheets (MXene@RuO2@GOx, MRuG).

[0191] Figure 4 Schematic diagram of the mechanism of dual-function microneedles that intelligently respond to the tumor microenvironment, Figure 4 A is a schematic diagram of the dual-function microneedle structure and needle tip dissolution. Figure 4 B. Schematic diagram of the preparation of SiO2 nanoparticles loaded with aPD-1 and their release of aPD-1 in response to GSH. Figure 4 C is a schematic diagram of the preparation process of RuO2-loaded titanium carbide nanosheets grafted with GOx and HbO2. Figure 4 D is a schematic diagram of the efficient inhibition mechanism of tumor growth by dual-function microneedles that intelligently respond to the tumor microenvironment. Figure 4 As can be seen, the polyvinyl alcohol and polyvinyl pyrrolidone (PVA / PVP) in the dual-functional microneedles, which intelligently respond to the tumor microenvironment, rapidly dissolve after penetrating the skin tissue, releasing the nanozyme and ICB antibody to the tumor site. Oxyhemoglobin (HbO2) molecules respond to the tumor's hypoxic microenvironment with controlled oxygen release. Glucose oxidase (GOx) catalyzes glucose oxidation, consuming it and starving the tumor while simultaneously lowering the tumor pH and producing abundant hydrogen peroxide (H2O2). Subsequently, RuO2-mimicking peroxidase (POD) catalyzes H2O2 to hydroxyl radicals (·OH) under chemical kinetics (CDT), directly killing cancer cells and reshaping the immune microenvironment, transforming immunosuppressive "cold tumors" into immune-activated "hot tumors." Furthermore, the ICB antibody loaded into glutathione (GSH)-sensitive SiO2 nanoparticles responds to intratumoral GSH depletion, triggering biodegradation and controlled, slow release of the antibody. This protects the antibody's activity from ROS destruction and controls the nanozyme and ICB antibody's synergistic effect in treating melanoma through starvation, CDT, and immunotherapy.

[0192] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A dual-function microneedle that intelligently responds to the tumor microenvironment, characterized in that: The raw materials include a needle tip loaded with nanosheets and nanoparticles and methacrylic anhydride grafted hyaluronic acid. The preparation method of the dual-functional microneedle includes: placing a system containing methacrylic anhydride grafted hyaluronic acid powder on the upper layer of the needle tip loaded with nanosheets and nanoparticles, adding ammonium persulfate, tannic acid and FeCl3, to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment; the needle tip loaded with nanosheets and nanoparticles is a needle tip loaded with nanosheets and nanoparticles obtained by dispersing secondary grafted MXene nanosheets loaded with RuO2 and SiO2 nanoparticles loaded with aPD-1 in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone.

2. A method for preparing a dual-function microneedle that intelligently responds to the tumor microenvironment, characterized in that: include: Providing secondary grafted RuO2-loaded MXene nanosheets, aPD-1-loaded SiO2 nanoparticles, and methacrylic anhydride-grafted hyaluronic acid powder; Dispersing the secondary grafted RuO2-loaded MXene nanosheets and aPD-1-loaded SiO2 nanoparticles in a mixed solution of polyvinyl alcohol and polyvinyl pyrrolidone to obtain a needle tip loaded with nanosheets and nanoparticles; A system containing the methacrylic anhydride grafted hyaluronic acid powder is placed on the upper layer of the needle tip loaded with nanosheets and nanoparticles, and ammonium persulfate, tannic acid and FeCl3 are added to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

3. The method according to claim 2, characterized in that Specifically include: Step 1: providing the secondary grafted RuO2-loaded MXene nanosheets, comprising: Step 101: etching a block of titanium aluminum carbide compound with a hydrofluoric acid solution for 48 to 96 hours, centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium aluminum carbide MXene multilayer nanosheets; exfoliating the MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 48 to 96 hours, and centrifuging and drying to obtain a few-layer MXene nanosheets; Step 102: ultrasonically disperse the few-layer MXene nanosheets in water, add RuCl3 aqueous solution, dropwise add NaOH solution, stir and react for 3-5 hours at room temperature, and centrifuge and dry to obtain RuO2-loaded MXene nanosheets; Step 103: ultrasonically dispersing the RuO2-loaded MXene nanosheets in water, adding amino polyethylene glycol thiol, stirring for 18 to 24 hours, and centrifugally drying to obtain MXene@RuO2 nanosheets grafted with -NH2; Step 104: Glucose oxidase and HbO2 are dissolved in water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, and after activation for 30 to 120 minutes, MXene@RuO2 nanosheets grafted with -NH2 are added, the mixture is reacted in an ice bath for 20 to 24 hours, and the mixture is centrifuged and dried to obtain secondary grafted RuO2-loaded MXene nanosheets; Step 2: providing the aPD-1 loaded SiO2 nanoparticles, comprising: Step 201: mixing polyethylene glycol octylphenyl ether, n-hexanol, and cyclohexane to obtain an organic mixed system; Step 202: aPD-1 is mixed with ethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide, shaken and added to the organic mixed system to obtain a mixed system B; Step 203: Add ammonia water to the mixed system B, stir at room temperature overnight, add pure acetone, precipitate, and centrifuge and wash to obtain SiO2 nanoparticles loaded with aPD-1; Step 3: providing the needle tip loaded with nanosheets and nanoparticles, comprising: Step 301: ultrasonically disperse the secondary grafted RuO2-loaded MXene nanosheets and the aPD-1-loaded SiO2 nanoparticles in a PVA / PVP mixed aqueous solution to obtain a hydrogel premix system; Step 302: adding the hydrogel premix system to a silicone rubber microneedle mold, centrifuging, and naturally drying at room temperature to obtain a needle tip loaded with nanosheets and nanoparticles; Step 4: providing the methacrylic anhydride grafted hyaluronic acid powder, comprising: Step 401: Dissolve hyaluronic acid in water, add methacrylic anhydride solution, adjust the pH to 8.5-10 with NaOH, and stir for 24-48 hours. Then, dialyze with deionized water and freeze-dry to obtain methacrylic anhydride-grafted hyaluronic acid powder. Step 5: Obtain dual-function microneedles that intelligently respond to the tumor microenvironment, including: Step 501: Place the grafted hyaluronic acid system on the upper layer of the needle tip loaded with nanosheets and nanoparticles, add ammonium persulfate solution, TA solution and FeCl3 solution, and stir to form a gel to obtain a dual-functional microneedle that intelligently responds to the tumor microenvironment.

4. The method according to claim 3, characterized in that In step 101, the mass of the hydrofluoric acid is 2 to 4 times the mass of the bulk Ti3AlC2, and the mass of the tetramethylammonium hydroxide solution is 2 to 4 times the mass of the titanium dioxide carbon MXene multilayer nanosheets.

5. The method according to claim 3, characterized in that In step 102, the volume of the water is 150 to 300 times the mass of the few-layer MXene nanosheets, the unit of the water volume is mL, and the unit of the mass of the few-layer MXene nanosheets is g; in step 102, the volume of the RuCl3 aqueous solution is 20 to 80 times the mass of the few-layer MXene nanosheets, so the volume of the RuCl3 aqueous solution is mL, and the unit of the mass of the few-layer MXene nanosheets is g; in step 102, the volume of the NaOH solution is 10 to 40 times the mass of the few-layer MXene nanosheets, the unit of the NaOH solution volume is mL, and the unit of the mass of the few-layer MXene nanosheets is g; In step 103, the mass of the amino polyethylene glycol thiol group is 0.3 to 2 times the mass of the RuO2-loaded MXene nanosheets.

6. The method according to claim 3, characterized in that In step 104, the mass ratio of the glucose oxidase and the -NH2-grafted MXene@RuO2 nanosheets is 1:(0.5-4); in step 104, the mass of the HbO2 is 1 times the mass of the glucose oxidase; in step 104, the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 10-90 times the mass of the glucose oxidase; in step 104, the mass of the N-hydroxysuccinimide is 7-60 times the mass of the glucose oxidase; in step 104, the mass of the water is 500-4000 times the mass of the glucose oxidase.

7. The method according to claim 3, characterized in that In step 201, the volume of n-hexanol is 1 to 1.5 times the volume of polyethylene glycol octylphenyl ether, and the volume of cyclohexane is 3 to 6.5 times the volume of polyethylene glycol octylphenyl ether; in step 202, the mass of aPD-1 is 0.001 to 0.004 times the mass of the MXene@RuO2 nanosheets grafted with -NH2; in step 202, the volume of tetraethyl orthosilicate is 0.2 to 0.9 times the mass of aPD-1, the unit of the volume of tetraethyl orthosilicate is μL, and the unit of the mass of aPD-1 is μg; in step 202, the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 0.4 to 1.6 times the mass of aPD-1, and the bis-[3-(triethoxysilyl)propyl]-disulfide is 0. The unit of the volume of [triethoxysilyl]propyl-disulfide is μL, and the unit of the mass of aPD-1 is μg; in step 202, the volume of the polyethylene glycol octylphenyl ether is 0.008 to 0.04 times the mass of aPD-1, the unit of the volume of the polyethylene glycol octylphenyl ether is mL, and the unit of the mass of aPD-1 is μg; in step 203, the volume of the ammonia water is 0.2 to 2 times the mass of aPD-1, the unit of the volume of the ammonia water is μL, and the unit of the mass of aPD-1 is μg; in step 203, the volume of the pure acetone is 0.1 to 0.4 times the mass of aPD-1, the unit of the volume of the pure acetone is mL, and the unit of the mass of aPD-1 is μg.

8. The method according to claim 3, characterized in that In step 301, the PVA / PVP mixed aqueous solution is a PVA / PVP mixed aqueous solution obtained by dissolving polyvinyl alcohol and polyvinyl pyrrolidone in water, and the mass percentage of PVA in the mixed aqueous solution is 7% to 10%, and the mass percentage of PVP is 13% to 20%; in step 301, the mass of the PVA / PVP mixed aqueous solution is 66 to 250 times the mass of the MXene@RuO2 nanosheets grafted with -NH2 in step 104.

9. The method according to claim 3, characterized in that In step 401, the volume of the methacrylic anhydride solution is 0.8 to 4 times the mass of the hyaluronic acid. The volume unit of the methacrylic anhydride solution is mL, and the mass unit of the hyaluronic acid is g.

10. The method according to claim 3, characterized in that In step 501, the volume of the ammonium persulfate solution is 0.14 to 0.22 times the volume of the grafted hyaluronic acid system, the volume of the TA solution is 0.06 to 0.24 times the volume of the grafted hyaluronic acid system, and the volume of the FeCl3 solution is 0.2 to 1.5 times the volume of the grafted hyaluronic acid system; in step 501, the grafted hyaluronic acid system is a grafted hyaluronic acid system obtained by dissolving methacrylic anhydride grafted hyaluronic acid powder in water, and the mass percentage concentration of methacrylic anhydride grafted hyaluronic acid powder in the grafted hyaluronic acid system is is 3%; in step 501, the concentration of the ammonium persulfate solution is 0.02 g / mL, the concentration of the TA solution is 0.001-0.008 g / mL, and the concentration of the FeCl3 solution is 0.02-0.05 g / mL; in step 501, the volume of the grafted hyaluronic acid system is 15-30 times the mass of the MXene@RuO2 nanosheets grafted with -NH2 in step 104, the unit of the volume of the grafted hyaluronic acid system is μL, and the unit of the mass of the MXene@RuO2 nanosheets grafted with -NH2 is mg.

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