Bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies and its preparation

Abstract

This invention discloses a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation. The bilayer smart microneedle patch includes microneedle patch tips and a microneedle patch backing. The microneedle patch tips are formed from titanium carbide nanosheets loaded with cerium dioxide and grafted with 3-amino-1,2,4-triazole and glucose oxidase, and calcium carbonate nanoparticles loaded with aPD-1. The microneedle patch backing includes cross-linked, grafted human-like collagen. This bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies can effectively achieve precise targeted co-delivery of nanozymes and ICB antibodies to tumor sites, inhibiting melanoma growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies and its preparation. Background Technology

[0002] Immune checkpoint blockade (ICB) therapy for melanoma remains a key problem that researchers are committed to solving, mainly due to the following reasons: insufficient infiltration of cytotoxic T lymphocytes in immunosuppressive "cold tumors" and high expression of immunosuppressive factors, represented by M2 phenotype macrophages that promote tumor growth, resulting in low sensitivity of patients to ICB therapy, with only a small percentage of cancer patients benefiting from it.

[0003] Emerging ROS-based therapies can reprogram immunosuppressed "cold tumors" into immune-activated "hot tumors," thereby enhancing patient sensitivity to ICB therapy. Nanozyme-driven chemokinetic therapy (CDT), particularly based on the ROS-producing cascade reaction of glucose oxidase (GOx) and peroxidase (POD), has been widely reported. It not only depletes intratumoral glucose, cutting off the nutrient source of cancer cells and starving the tumor, but also provides abundant H2O2 to the tumor and lowers the pH of the tumor microenvironment, thereby enhancing POD ROS-producing activity. However, the H2O2 produced by glucose oxidation is easily decomposed into oxygen by endogenous catalase (CAT), thus reducing H2O2 concentration and ROS content. Furthermore, precise delivery of nanozymes and ICB antibodies to the tumor site to avoid off-target side effects, and how to protect the activity of ICB antibodies and nanozymes from being destroyed by ROS generated by CDT during co-delivery, and how to enable them to exert their efficient synergistic and programmed efficacy in treating melanoma, remain crucial challenges. Therefore, it is particularly important to construct a GOx and POD-based nanozyme that can self-supply H2O2 and inhibit its consumption, as well as a carrier for the precise co-delivery of nanozymes and ICB antibodies to tumor sites. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation. This bilayer smart microneedle patch includes a microneedle patch tip and a microneedle patch backing. The microneedle patch tip comprises titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase, and calcium carbonate nanoparticles loaded with aPD-1. The microneedle patch backing comprises cross-linked and grafted human-like collagen. This bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies can effectively achieve precise targeted co-delivery of nanozymes and ICB antibodies to tumor sites, inhibiting melanoma growth.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, characterized in that it includes a microneedle patch tip and a microneedle patch backing, wherein the microneedle patch tip is formed by titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase and calcium carbonate nanoparticles loaded with aPD-1, and the microneedle patch backing includes cross-linked grafted human-like collagen.

[0006] The above-mentioned bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody is characterized in that the preparation method of the microneedle patch tip includes:

[0007] Step 101: After etching the blocky Ti3AlC2 with hydrofluoric acid for 24-48 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. Peel the titanium carbon MXene multilayer nanosheets in tetrapropylammonium hydroxide solution for 48-96 hours, centrifuge and dry to obtain few-layer MXene nanosheets.

[0008] Step 102: The few-layer MXene nanosheets are ultrasonically and uniformly dispersed in water, preheated to obtain an MXene dispersion, cerium nitrate is dissolved in ethylene glycol to obtain a cerium nitrate solution, the cerium nitrate solution is added to the above MXene dispersion, the mixture is stirred and reacted, an ammonia solution is added, the reaction is continued for 1-6 hours, and the mixture is centrifuged and dried to obtain MXene nanosheets loaded with cerium dioxide.

[0009] Step 103: The MXene nanosheets loaded with cerium dioxide described in step 102 are ultrasonically dispersed in PBS solution, 3-amino-1,2,4-triazole is added, the mixture is stirred for 24-72 h, and then centrifuged and dried to obtain MXene@CeO2 nanosheets grafted with 3-AT.

[0010] Step 104: Dissolve glucose oxidase in PBS solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, activate for 60-120 min to obtain activated glucose oxidase system, add MXene@CeO2 nanosheets grafted with 3-AT as described in step 103 to the activated glucose oxidase system, react in an ice bath for 20-24 h, centrifuge and dry to obtain nanosheets grafted with 3-AT and GOx;

[0011] Step 105: Completely dissolve aPD-1 and CaCl2·2H2O in deionized water, then add...

[0012] NH4HCO3 was added, and the mixture was stirred for 6–21 h. After centrifugation, washing, and drying, calcium carbonate nanoparticles loaded with aPD-1 were obtained.

[0013] Step 106: The 3-AT and GOx grafted nanosheets described in Step 104 and the calcium carbonate nanoparticles loaded with aPD-1 described in Step 105 are ultrasonically dispersed in a polyvinyl alcohol solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip.

[0014] The above-mentioned bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody 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 tetrapropylammonium hydroxide solution is 2 to 4 times the mass of the titanium carbon MXene multilayer nanosheets.

[0015] The above-mentioned bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies is characterized in that, in step 102, the mass of the cerium nitrate solution is 1.2 to 3.2 times the mass of the few-layer MXene nanosheets, and the mass percentage of cerium nitrate in the cerium nitrate solution is 1% to 1.6%; in step 102, the mass of the water is 100 to 200 times the mass of the few-layer MXene nanosheets; in step 102, the volume of the ammonia solution is 0.01 to 0.07 times the mass of the few-layer MXene nanosheets. The volume unit of the ammonia solution is mL, and the unit of mass of the few-layer MXene nanosheets is mg; in step 103, the mass of the 3-amino-1,2,4-triazole is 1 to 4 times the mass of the MXene nanosheets loaded with cerium dioxide; in step 104, the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 46 to 48 times the mass of glucose oxidase, and the mass of the N-hydroxysuccinimide is 26 to 30 times the mass of glucose oxidase; in step 104, the grafted 3-AT...

[0016] The mass of MXene@CeO2 nanosheets is 0.5 to 2 times the mass of glucose oxidase.

[0017] The above-mentioned bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody is characterized in that, in step 105, the mass of aPD-1 is 0.001 to 0.003 times the mass of glucose oxidase in step 104; in step 105, the mass of CaCl2·2H2O is 1.5 to 5 times the mass of glucose oxidase in step 104; in step 105, the mass of NH4HCO3 is 15 to 150 times the mass of glucose oxidase in step 104; and in step 106, the mass of the polyvinyl alcohol solution is 50 to 160 times the mass of glucose oxidase, and the mass percentage of the polyvinyl alcohol solution is 6% to 10%.

[0018] The above-mentioned bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies is characterized in that the preparation method of the grafted human-like collagen includes: completely dissolving human-like collagen in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stirring and activating, then adding dopamine powder, stirring and reacting at room temperature, dialyzing with deionized water, and lyophilizing to obtain grafted human-like collagen.

[0019] The above-mentioned bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies is characterized in that the mass of water is 20 to 100 times the mass of human-like collagen protein; the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.15 times the mass of human-like collagen protein; the mass of N-hydroxysuccinimide is 0.69 times the mass of human-like collagen protein; the mass of dopamine is 1.5 to 3 times the mass of human-like collagen protein; and the mass of human-like collagen protein is 16 to 33 times the mass of glucose oxidase in the tip of the microneedle patch.

[0020] Furthermore, the present invention also provides a method for preparing the above-mentioned bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody, characterized in that it includes: dissolving the grafted human-like collagen to obtain a dissolved human-like collagen system; placing the dissolved human-like collagen system on the upper layer of the microneedle patch tip; adding hydrogen peroxide solution and oxyhemoglobin solution; stirring at room temperature to crosslink, thereby obtaining the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody.

[0021] The above method is characterized in that the dissolved human-like collagen system is a dissolved human-like collagen system obtained by dissolving grafted human-like collagen in water, and the mass percentage of grafted human-like collagen in the dissolved human-like collagen system is 2% to 5%.

[0022] The above method is characterized in that the volume of the hydrogen peroxide solution is 10 to 40 times the amount of human collagen protein in the grafted human collagen, the volume unit of the hydrogen peroxide solution is μL, the amount of human collagen protein is g, and the concentration of the hydrogen peroxide solution is 10 to 30 mM; the volume of the oxyhemoglobin solution is 20 to 50 times the amount of human collagen protein in the grafted human collagen, the volume unit of the oxyhemoglobin solution is μL, the amount of human collagen protein is g, and the concentration of the oxyhemoglobin solution is 20 to 60 mg / mL.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies of the present invention comprises a microneedle patch tip and a microneedle patch backing. The microneedle patch tip is formed by titanium carbide nanosheets loaded with cerium dioxide and grafted with 3-amino-1,2,4-triazole and glucose oxidase, and calcium carbonate nanoparticles loaded with aPD-1. The microneedle patch backing comprises cross-linked and grafted human-like collagen. This bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies can effectively achieve precise targeted co-delivery of nanozymes and ICB antibodies to tumor sites and inhibit melanoma growth.

[0025] 2. The preparation method of the bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies of the present invention cleverly uses titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase and calcium carbonate nanoparticles loaded with aPD-1 dispersed in polyvinyl alcohol solution to form needle tips with polyvinyl alcohol as the backbone, and grafted human collagen as the backing to obtain an improved bilayer microneedle patch.

[0026] 3. The bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies of the present invention comprises titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase, and calcium carbonate nanoparticles loaded with aPD-1 dispersed in a polyvinyl alcohol polymer material to form microneedle patch tips. It can effectively utilize its nanozymes to consume glucose in tumors, generate abundant ROS and reshape the immune microenvironment, and realize the slow release of ICB antibody aPD-1 in response to the acidic tumor microenvironment.

[0027] 4. The bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies of the present invention can comprehensively utilize the synergistic effects of starvation therapy, chemokinetic therapy and ICB to achieve highly efficient inhibition of melanoma growth.

[0028] 5. Furthermore, the bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies of the present invention also has the effect of promoting skin tissue regeneration after tumor ablation.

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0030] Figure 1 Electron microscopy and elemental distribution map of titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase, as shown in Example 1.

[0031] Figure 2 Schematic diagram of the structure of the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody in Example 1, and the results of needle tip morphology and solubility test;

[0032] Figure 3 This is a schematic diagram characterizing the intracellular and extracellular antioxidant, antibacterial, L929 cell proliferation and migration promotion capabilities of grafted human collagen in Example 1;

[0033] Figure 4 This is a schematic diagram showing the test results of the killing power, oxygen production, and ROS production capacity of titanium carbide nanosheets (MXene@CeO2@3-AT / GOx) grafted with 3-amino-1,2,4-triazole and glucose oxidase and loaded with cerium dioxide in Example 1 against melanoma cells.

[0034] Figure 5 A schematic diagram illustrating the mechanism by which a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies exerts its anti-tumor effect. Detailed Implementation

[0035] Example 1

[0036] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0037] Step 1: Provide the microneedle patch tips, specifically including:

[0038] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 24 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 72 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide and stirring at room temperature to exfoliate 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%, and the mass of the tetrapropylammonium hydroxide (TPAOH) solution is equal to the mass of the titanium carbon MXene nanosheets.

[0039] The mass of the MXene multilayer nanosheets is 3 times that of the tetrapropylammonium hydroxide (TPAOH) solution, which has a mass percentage concentration of 25%.

[0040] Step 102: 50 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60°C to obtain an MXene dispersion. 120 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the MXene dispersion, and the mixture was stirred for 10 minutes. Then, 2 ml of a 25% (w / w) ammonia solution was added, and the reaction was continued for 1 hour. The mixture was then centrifuged and dried to obtain MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2). The mass of water was 200 times the mass of the few-layer MXene nanosheets. The cerium nitrate solution contained 1.2% (w / w) cerium nitrate.

[0041] Step 103: Disperse 30 mg of the powdered MXene@CeO2 from Step 102 in 30 g of PBS solution with pH 7.4 using ultrasonication, add 90 mg of 3-amino-1,2,4-triazole (3-AT), stir for 24 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0042] Step 104: Dissolve 30 mg of glucose oxidase (GOx) in 30 g of PBS solution with pH 7.4, add 1.425 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.855 g of N-hydroxysuccinimide (NHS), and activate for 120 min to obtain an activated glucose oxidase system. Add 30 mg of the MXene@CeO2@3-AT nanosheets described in Step 103 to the activated glucose oxidase system, react in an ice bath for 24 h, centrifuge and dry to obtain titanium carbide nanosheets grafted with 3-amino-1,2,4-triazole and glucose oxidase and loaded with cerium dioxide, i.e., 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0043] Step 105: Dissolve 60 μg of aPD-1 and 100 mg of CaCl2·2H2O completely in 100 g of deionized water, add 750 mg of NH4HCO3, stir and react at 40 °C for 6 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1); the aPD-1 is an anti-mouse PD-1 antibody, catalog number 114114, purchased from Biolegend;

[0044] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 5g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain microneedle patch tips. The mass percentage of the polyvinyl alcohol solution is 6%. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205).

[0045] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 1g of human-like collagen (HLC) in water, adding 1.15g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.69g of N-hydroxysuccinimide (NHS), stirring and activating for 30min, then adding 2g of dopamine (DA) powder, stirring and reacting at room temperature for 12h, dialyzing with deionized water, and lyophilizing to obtain grafted human-like collagen; the mass of the water is 50 times the mass of the human-like collagen protein;

[0046] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 20 μL of 10 mM hydrogen peroxide solution and 50 μL of 20 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 3%.

[0047] Example 2

[0048] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0049] Step 1: Provide the microneedle patch tips, specifically including:

[0050] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 48 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 48 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide and stirring at room temperature to exfoliate 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 tetrapropylammonium hydroxide (TPAOH) solution is 2 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetrapropylammonium hydroxide (TPAOH) solution is 25%.

[0051] Step 102: 100 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60 °C to obtain an MXene dispersion. 126 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the above MXene dispersion, and the mixture was stirred and reacted for 10 minutes. Then, 1.5 ml of ammonia solution with a mass percentage of 25% was added, and the reaction was continued for 3 hours. After centrifugation and drying, MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2) were obtained. The mass of water was 100 times the mass of the few-layer MXene nanosheets. The mass percentage of cerium nitrate in the cerium nitrate solution was 1.26%.

[0052] Step 103: Disperse 30 mg of the powdered MXene@CeO2 from Step 102 in 30 g of PBS solution with pH 7.4 using ultrasonication, add 100 mg of 3-amino-1,2,4-triazole (3-AT), stir for 24 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0053] Step 104: Dissolve 60 mg of glucose oxidase (GOx) in 60 g of PBS solution at pH 7.4, add 2.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.6 g of N-hydroxysuccinimide (NHS), and activate for 90 min to obtain the activated glucose oxidase system. Add 30 mg of the solution described in step 103 to the activated glucose oxidase system.

[0054] MXene@CeO2@3-AT nanosheets were reacted in an ice bath for 24 h, then centrifuged and dried to obtain 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0055] Step 105: Dissolve 80 μg of aPD-1 and 100 mg of CaCl2·2H2O completely in 100 g of deionized water, add 1.2 g of NH4HCO3, stir and react at 40 °C for 15 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1).

[0056] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 3g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip. The mass percentage of the polyvinyl alcohol solution is 8%.

[0057] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 1g of human-like collagen (HLC) in water, adding 1.115g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.69g of N-hydroxysuccinimide (NHS), stirring and activating for 30min, then adding 1.5g of dopamine (DA) powder, stirring and reacting at room temperature for 18h, then dialyzing with deionized water and lyophilizing to obtain grafted human-like collagen; the mass of the water is 50 times the mass of the human-like collagen protein;

[0058] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 30 μL of 30 mM hydrogen peroxide solution and 20 μL of 50 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 5%.

[0059] Example 3

[0060] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0061] Step 1: Provide the microneedle patch tips, specifically including:

[0062] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 48 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 96 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a common method in this art. The commonly used method in this field is exfoliation, which can be as follows: the etched multilayer MXene nanosheets are dispersed in tetrapropylammonium hydroxide and stirred at room temperature to exfoliate the multilayer 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 tetrapropylammonium hydroxide (TPAOH) solution is 2.5 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetrapropylammonium hydroxide (TPAOH) solution is 25%.

[0063] Step 102: 60 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60 °C to obtain an MXene dispersion. 150 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the MXene dispersion, and the mixture was stirred for 10 minutes. Then, 4 ml of a 25% (w / w) ammonia solution was added, and the reaction was continued for 5 hours. The mixture was then centrifuged and dried to obtain MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2). The mass of the water was 167 times the mass of the few-layer MXene nanosheets. The cerium nitrate solution contained 1.5% (w / w) cerium nitrate.

[0064] Step 103: Disperse 30 mg of the powdered MXene@CeO2 from Step 102 in 30 g of PBS solution with pH 7.4 using ultrasonication, add 120 mg of 3-amino-1,2,4-triazole (3-AT), stir for 72 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0065] Step 104: Dissolve 50 mg of glucose oxidase (GOx) in 50 g of PBS solution with pH 7.4, add 2.375 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.425 g of N-hydroxysuccinimide (NHS), and activate for 60 min to obtain the activated glucose oxidase system. Add 50 mg of MXene@CeO2@3-AT nanosheets from step 103 to the activated glucose oxidase system, react in an ice bath for 24 h, centrifuge and dry to obtain 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0066] Step 105: Dissolve 100 μg of aPD-1 and 150 mg of CaCl2·2H2O completely in 100 g of deionized water, add 750 mg of NH4HCO3, stir and react at 40 °C for 20 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1).

[0067] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 4g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip. The mass percentage of the polyvinyl alcohol solution is 10%.

[0068] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 1g of human-like collagen (HLC) in water, adding 1.15g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.69g of N-hydroxysuccinimide (NHS), stirring and activating for 60min, then adding 2g of dopamine (DA) powder, stirring and reacting at room temperature for 12h, dialyzing with deionized water, and lyophilizing to obtain grafted human-like collagen; the mass of the water is 50 times the mass of the human-like collagen protein;

[0069] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 40 μL of 20 mM hydrogen peroxide solution and 20 μL of 60 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 5%.

[0070] Example 4

[0071] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0072] Step 1: Provide the microneedle patch tips, specifically including:

[0073] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 24 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 96 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a common method in this art. The commonly used method in this field is exfoliation, which can be as follows: the etched multilayer MXene nanosheets are dispersed in tetrapropylammonium hydroxide and stirred at room temperature to exfoliate the multilayer into few-layer MXene nanosheets; the mass of the hydrofluoric acid is 2.5 times the mass of the bulk Ti3AlC2, the mass percentage concentration of the hydrofluoric acid is 40%, the mass of the tetrapropylammonium hydroxide (TPAOH) solution is 4 times the mass of the titanium carbon MXene multilayer nanosheets, and the mass percentage concentration of the tetrapropylammonium hydroxide (TPAOH) solution is 25%.

[0074] Step 102: 80 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60°C to obtain an MXene dispersion. 150 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the MXene dispersion, and the mixture was stirred for 10 minutes. Then, 2.5 ml of a 25% (w / w) ammonia solution was added, and the reaction was continued for 4 hours. The mixture was then centrifuged and dried to obtain MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2). The mass of water was 125 times the mass of the few-layer MXene nanosheets. The cerium nitrate solution contained 1.5% (w / w) cerium nitrate.

[0075] Step 103: Disperse 50 mg of the powdered MXene@CeO2 from Step 102 in 50 g of PBS solution with pH 7.4 using ultrasonication, add 50 mg of 3-amino-1,2,4-triazole (3-AT), stir for 48 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0076] Step 104: Dissolve 50 mg of glucose oxidase (GOx) in 50 g of PBS solution with pH 7.4, add 2.375 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.425 g of N-hydroxysuccinimide (NHS), activate for 120 min to obtain the activated glucose oxidase system, add 30 mg of MXene@CeO2@3-AT nanosheets from step 103 to the activated glucose oxidase system, react in an ice bath for 20 h, centrifuge and dry to obtain 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0077] Step 105: Dissolve 80 μg of aPD-1 and 100 mg of CaCl2·2H2O completely in 100 g of deionized water, add 2 g of NH4HCO3, stir and react at 40 °C for 20 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1).

[0078] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 3g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip. The mass percentage of the polyvinyl alcohol solution is 8%.

[0079] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 1g of human-like collagen (HLC) in water, adding 1.15g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.69g of N-hydroxysuccinimide (NHS), stirring and activating for 30min, then adding 2g of dopamine (DA) powder, stirring and reacting at room temperature for 10h, then dialyzing with deionized water and lyophilizing to obtain grafted human-like collagen; the mass of the water is 50 times the mass of the human-like collagen protein;

[0080] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 20 μL of 30 mM hydrogen peroxide solution and 20 μL of 40 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 2%.

[0081] Example 5

[0082] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0083] Step 1: Provide the microneedle patch tips, specifically including:

[0084] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 24 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 72 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide and stirring at room temperature to exfoliate 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%, and the mass of the tetrapropylammonium hydroxide (TPAOH) solution is 3 times the mass of the titanium carbon MXene nanosheets.

[0085] The mass of the MXene multilayer nanosheets is 4 times that of the tetrapropylammonium hydroxide (TPAOH) solution, which has a mass percentage concentration of 25%.

[0086] Step 102: 60 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60 °C to obtain an MXene dispersion. 100 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the MXene dispersion, and the mixture was stirred for 10 minutes. Then, 4 ml of a 25% (w / w) ammonia solution was added, and the reaction was continued for 2 hours. After centrifugation and drying, MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2) were obtained. The mass of the water was 167 times the mass of the few-layer MXene nanosheets. The cerium nitrate solution contained 1% (w / w) of cerium nitrate.

[0087] Step 103: Disperse 30 mg of the powdered MXene@CeO2 from Step 102 in 30 g of PBS solution with pH 7.4 using ultrasonication, add 50 mg of 3-amino-1,2,4-triazole (3-AT), stir for 72 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0088] Step 104: Dissolve 30 mg of glucose oxidase (GOx) in 30 g of PBS solution with pH 7.4, add 1.425 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.855 g of N-hydroxysuccinimide (NHS), activate for 100 min to obtain the activated glucose oxidase system, add 50 mg of MXene@CeO2@3-AT nanosheets from step 103 to the activated glucose oxidase system, react in an ice bath for 20 h, centrifuge and dry to obtain 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0089] Step 105: Dissolve 70 μg of aPD-1 and 150 mg of CaCl2·2H2O completely in 100 g of deionized water, add 750 mg of NH4HCO3, stir and react at 40 °C for 18 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1).

[0090] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 3g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip. The mass percentage of the polyvinyl alcohol solution is 6%.

[0091] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 1g of human-like collagen (HLC) in water, adding 1.15g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.69g of N-hydroxysuccinimide (NHS), stirring and activating for 60min, then adding 3g of dopamine (DA) powder, stirring and reacting at room temperature for 12h, dialyzing with deionized water, and lyophilizing to obtain grafted human-like collagen; the mass of the water is 20 times the mass of the human-like collagen protein;

[0092] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 10 μL of 30 mM hydrogen peroxide solution and 20 μL of 20 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 3%.

[0093] Example 6

[0094] This embodiment describes a bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, and its preparation method, comprising the following steps:

[0095] Step 1: Provide the microneedle patch tips, specifically including:

[0096] Step 101: After etching the bulk Ti3AlC2 with hydrofluoric acid for 48 hours, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. The titanium carbon MXene multilayer nanosheets are then exfoliated in tetrapropylammonium hydroxide (TPAOH) solution for 96 hours, centrifuged, and dried to obtain few-layer MXene nanosheets. The etching is a commonly used etching method in the art, specifically: dispersing the bulk Ti3AlC2 powder in a hydrofluoric acid solution and stirring at room temperature to etch the intermediate Al layer; the exfoliation is a commonly used exfoliation method in the art, specifically: dispersing the etched multilayer MXene nanosheets in tetrapropylammonium hydroxide and stirring at room temperature to exfoliate 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%, and the mass of the tetrapropylammonium hydroxide (TPAOH) solution is 3 times the mass of the titanium carbon MXene nanosheets.

[0097] The mass of the MXene multilayer nanosheets is 3 times that of the tetrapropylammonium hydroxide (TPAOH) solution, which has a mass percentage concentration of 25%.

[0098] Step 102: 50 mg of few-layer MXene nanosheets were ultrasonically and uniformly dispersed in water and preheated to 60 °C to obtain an MXene dispersion. 160 mg of cerium nitrate (Ce(NO3)3) was dissolved in ethylene glycol to obtain a cerium nitrate solution. The cerium nitrate solution was added to the above MXene dispersion, and the mixture was stirred and reacted for 10 minutes. Then, 3.5 ml of ammonia solution with a mass percentage of 25% was added, and the reaction was continued for 6 hours. After centrifugation and drying, MXene nanosheets loaded with cerium dioxide (CeO2) (MXene@CeO2) were obtained. The mass of water was 200 times the mass of the few-layer MXene nanosheets. The mass percentage of cerium nitrate in the cerium nitrate solution was 1.6%.

[0099] Step 103: Disperse 20 mg of the powdered MXene@CeO2 from Step 102 in 20 g of PBS solution with pH 7.4 using ultrasonication, add 20 mg of 3-amino-1,2,4-triazole (3-AT), stir for 24 h, centrifuge and dry to obtain 3-AT grafted MXene@CeO2 nanosheets (MXene@CeO2@3-AT);

[0100] Step 104: Dissolve 20 mg of glucose oxidase (GOx) in 20 g of PBS solution with pH 7.4, add 0.95 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.57 g of N-hydroxysuccinimide (NHS), and activate for 120 min to obtain the activated glucose oxidase system. Add 20 mg of MXene@CeO2@3-AT nanosheets from step 103 to the activated glucose oxidase system, react in an ice bath for 24 h, centrifuge and dry to obtain 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx);

[0101] Step 105: Dissolve 50 μg of aPD-1 and 100 mg of CaCl2·2H2O completely in 100 g of deionized water, add 3 g of NH4HCO3, stir and react at 40 °C for 21 h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1 (CaCO3@aPD-1).

[0102] Step 106: The MXene@CeO2@3-AT / GOx nanosheets described in Step 104 and the CaCO3@aPD-1 nanoparticles described in Step 105 are ultrasonically dispersed in 2g of polyvinyl alcohol (PVA) solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip. The mass percentage of the polyvinyl alcohol solution is 7%.

[0103] Step 2: Providing grafted human-like collagen, specifically including: completely dissolving 500 mg of human-like collagen (HLC) in water, adding 0.575 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.345 g of N-hydroxysuccinimide (NHS), stirring and activating for 50 min, then adding 1.2 g of dopamine (DA) powder, stirring and reacting at room temperature for 18 h, then dialyzing with deionized water and lyophilizing to obtain grafted human-like collagen; the mass of the water is 100 times the mass of the human-like collagen protein;

[0104] Step 3: Dissolve the grafted human-like collagen to obtain a dissolved human-like collagen system. Place the dissolved human-like collagen system on the upper layer of the microneedle patch tip obtained in Step 1. Add 20 μL of 30 mM hydrogen peroxide solution and 20 μL of 20 mg / mL oxyhemoglobin solution to the upper layer of the dissolved human-like collagen system. Stir at room temperature to allow cross-linking, obtaining a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody. The dissolved human-like collagen system is obtained by dissolving the grafted human-like collagen in water, and the mass percentage of the grafted human-like collagen in the dissolved human-like collagen system is 3.5%.

[0105] Performance testing:

[0106] Electron microscopy image of titanium carbide nanosheets grafted with 3-amino-1,2,4-triazole and glucose oxidase and loaded with cerium dioxide (nanosheets grafted with 3-AT and GOX in step 104) in Example 1. Figure 1 As shown. Among them Figure 1 A and 1B are transmission electron microscopy images and crystal lattice images of CeO2 nanoparticles. Figure 1 C, D, E, and F are the elemental distribution diagrams for CeO2. Figure 1 G is a scanning electron microscope (SEM) image of the multilayer MXene nanosheets from step one, and 1H is a transmission electron microscope (TEM) image of the few-layer MXene nanosheets. Figure 1 I, J, K, and L show the elemental distribution of few-layer MXene, 1M and 1N represent MXene nanosheets (MXene@CeO2) loaded with CeO2 nanoparticles and their lattice, and 1O, P, Q, R, and S represent the corresponding elemental distributions. Figure 1 T represents MXene@CeO2 nanosheets loaded with 3-AT and GOx. IU, V, W, X, Y, and Z show the corresponding elemental distribution maps. The above images together comprehensively demonstrate the successful preparation of MXene@CeO2@3-AT / GOx.

[0107] The schematic diagram of the bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies in Example 1, along with the needle tip morphology and solubility test results, are shown below. Figure 2 As shown. According to Figure 2 As seen in A, B, and D, the dual-layer smart microneedle patch consists of two parts: a needle tip and a backing. It has a complete needle tip structure. Figure 2 As indicated by C, this bilayer smart microneedle patch exhibits rapid solubility, potentially enabling precise drug delivery to tumor sites and rapid dissolution to release the payload. The solubility test involved immersing the tip of the bilayer smart microneedle patch, co-loaded with nanozymes and ICB antibodies, in deionized water. After 2 minutes, the patch was removed, photographed, and its tip morphology recorded.

[0108] Figure 3This is a schematic diagram characterizing the intracellular and extracellular antioxidant, antibacterial, and L929 cell proliferation and migration-promoting abilities of the grafted human-like collagen in step two of Example 1. According to... Figure 3 As shown, the grafted human-like collagen exhibits excellent antioxidant activity in scavenging ROS. Compared to the control group, the intensity of the UV absorption peaks at 405nm (4A), 560nm (4B), and 510nm (4C) was reduced in the group treated with grafted human-like collagen (HD), demonstrating that the grafted human-like collagen of this invention has excellent scavenging activity against H2O2 and O2. ·- The ability of ·OH, Figure 3 D demonstrated that the grafted human-like collagen exhibited optimal resistance to both Gram-positive and Gram-negative bacteria under photothermal irradiation, and significantly promoted L929 cell proliferation and migration, potentially accelerating wound healing. The experimental methods used to characterize the process included:

[0109] Test methods for assessing the ability to remove H2O2 include:

[0110] Step 1: Mix 10 mL of 1 mM H2O2 with grafted human-like collagen (HD hydrogel) at 37°C for 2 h;

[0111] Step 2: Take 50 μL of supernatant and mix it with 100 μL of Ti(SO4)2 solution for 30 min; to obtain mixed system A; the Ti(SO4)2 solution is obtained by mixing 1.33 mL of Ti(SO4)2 with a mass percentage of 24% and 8.33 mL of H2SO4 in 50 mL of deionized water.

[0112] Step 3: Take 1 mL of the mixed system A and perform a full wavelength scan. The absorption peak intensity at 405 nm is used to characterize the ability to remove H2O2.

[0113] Remove O2 ·- The ability testing methods include:

[0114] Step 1: Mix riboflavin, methionine, nitrotetrazole blue and grafted human-like collagen (HD hydrogel) to obtain mixture system B; the concentration of riboflavin in mixture system B is 20 μM, the concentration of methionine is 12.5 mM, and the concentration of nitrotetrazole blue is 75 μM.

[0115] Step 2: Mix the mixture B under constant light intensity for 10 min, measure the full wavenumber scan curve of the solution, compare the absorbance of each group at 560 nm, and evaluate the gel's O2 scavenging ability. ·- The ability.

[0116] The method for testing the scavenging ability of ·OH includes: mixing FeSO4, H2O2 and salicylic acid (SA) solution into PBS, incubating at 37°C for 30 min, measuring the full wavenumber scan curve, recording the absorbance of the solution at 510 nm, and determining the scavenging ability of HD hydrogel for ·OH.

[0117] Methods for testing intracellular antioxidant activity include:

[0118] Step 1: Grafted human-like collagen (HD hydrogel) was incubated with Escherichia coli and Staphylococcus aureus, and then irradiated with 808nm near-infrared light for 10 min before being diluted and spread on solid culture medium. After 24 h, the plates were photographed and the bacterial count was recorded.

[0119] Step 2: Grafted human collagen and H2O2 were co-incubated in L929 cells. After 4 hours, DAPI and DCFH-DA probe were added, and the images were taken under a fluorescence microscope.

[0120] The method for testing the cell proliferation-promoting effect included: extracting grafted human collagen (HD hydrogel) in 1640 culture medium for 3 days, culturing L929 cells with the extract, adding AO / EB fluorescent dye after 24 hours, taking pictures under a fluorescence microscope, and detecting the cell proliferation-promoting effect of the gel.

[0121] The assay for promoting cell migration included: scratching L929 cells with a 100 μL pipette tip and recording the initial scratch area by photographing; then spreading grafted human collagen (HD hydrogel) on the cells; and recording the scratch size by microscopic photography after 48 h.

[0122] Figure 4 This is a schematic diagram illustrating the test results of the killing effect, oxygen production, and ROS production capacity of titanium carbide nanosheets (MXene@CeO2@3-AT / GOx) grafted with 3-amino-1,2,4-triazole and glucose oxidase on melanoma cells in Example 1. Figure 4 As shown in AM / PI, the nanosheets grafted with 3-AT and GOx of this invention (MXene@CeO2@3-AT / GOx) exhibit the best anti-tumor effect under the combined action of chemical kinetics and starvation therapy, resulting in the highest number of dead cells. Figure 4 As can be seen from JC-1, the 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx) of this invention can significantly reduce mitochondrial membrane potential and cause membrane damage; according to Figure 4 As can be seen from Ru(dpp)3Cl2, the 3-AT and GOx grafted nanosheets (MXene@CeO2@3-AT / GOx) of this invention can alleviate hypoxia; according to Figure 4As demonstrated by DCFH-DA, the 3-AT and GOx-grafted nanosheets (MXene@CeO2@3-AT / GOx) of this invention can generate ROS to the maximum extent. The testing process is as follows.

[0123] The assay method for testing the killing power against melanoma cells included: treating melanoma B16F10 cells with nanosheets grafted with 3-AT and GOx (MXene@CeO2@3-AT / GOx), and then adding...

[0124] Calcein-AM / PI live / dead cell dye was photographed under a fluorescence microscope to record its killing effect on melanoma cells.

[0125] The method for testing the effect on mitochondrial membrane potential included adding the JC-1 probe to nanosheets grafted with 3-AT and GOx (MXene@CeO2@3-AT / GOx), taking pictures under a fluorescence microscope, and recording the effect on mitochondrial membrane potential.

[0126] The test method for alleviating hypoxia included adding DAPI and Ru(dpp)3Cl2 oxygen indicator probe to nanosheets grafted with 3-AT and GOx (MXene@CeO2@3-AT / GOx), taking pictures under a fluorescence microscope, and recording the effect of alleviating hypoxia.

[0127] The method for testing the ability to generate ROS includes adding DAPI and DCFH-DA probes to nanosheets grafted with 3-AT and GOx (MXene@CeO2@3-AT / GOx), taking pictures under a fluorescence microscope, and recording the ability to generate ROS.

[0128] Figure 4In the table, Control represents the blank control group; MN represents the blank microneedles, which are prepared by adding a polyvinyl alcohol (PVA) solution dropwise into a PDMS mold, centrifuging, and then air-drying at room temperature to obtain blank microneedles; the concentration of the PVA solution is the same as in Example 1; MN / G represents GOx-loaded microneedles (MN / G), which are prepared by dissolving glucose oxidase GOx in a PVA solution, adding it dropwise into a PDMS mold, centrifuging, and then air-drying at room temperature to obtain GOx-loaded microneedles (MN / G); the amounts of glucose oxidase GOx and PVA are the same as in Example 1; MN / MCeG represents MCeG-loaded microneedles (MN / MCeG), which are prepared by ultrasonically dispersing the Mxene@CeO2 nanosheets obtained in step 102 of Example in water, and adding amino groups. Polyethylene glycol thiol (NH2-PEG-SH) was stirred at room temperature for 24 h, centrifuged and dried to obtain MXene@CeO2 nanosheets grafted with -NH2. Then, 30 mg of the MXene@CeO2 nanosheets grafted with -NH2 were added to the glucose oxidase system activated in step 104 of Example 1, reacted at room temperature for 24 h, centrifuged and dried to obtain MCeG. The MCeG was ultrasonically dispersed in 5 g of polyvinyl alcohol (PVA) solution to obtain the dispersed system. The dispersed system was placed in a PDMS mold, centrifuged and then naturally dried at room temperature to obtain MCeG-loaded microneedles (MN / MCeG). The concentration of the polyvinyl alcohol (PVA) solution was the same as in Example 1. MN / MCe3G is the nanosheet (MXene@CeO2@3-AT / GOx) grafted with 3-AT and GOx in step 104 of Example 1.

[0129] Figure 5The mechanism by which the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody exerts its anti-tumor effect is as follows: After the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody is inserted into skin tissue, it rapidly dissolves and releases MCE3G nanozyme and CP nanoparticles. CeO2 mimics catalase (CAT) to decompose endogenous hydrogen peroxide (H2O2) into oxygen (O2), alleviating tumor hypoxia. Glucose oxidase (GOx) catalyzes the oxidation and consumption of glucose, generating H2O2 and glucuronic acid. Simultaneously, 3-AT inhibits… H2O2 is decomposed by CAT within the tumor, creating a microenvironment with abundant H2O2 and a pH level around 4.5. Furthermore, CeO2 mimics peroxidase (POD), catalyzing the conversion of generated H2O2 into hydroxyl radicals (·OH), directly killing tumor cells through oxidative damage. This also transforms the immunosuppressed "cold" tumor into an immunostimulated "hot" tumor, reshaping the tumor's immune microenvironment. Subsequently, CaCO3 nanoparticles slowly release aPD-1 in response to the acidic tumor microenvironment, achieving immunosuppression of tumor growth. Thus, the bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies synergistically inhibits tumor growth through starvation therapy to block the tumor's energy source, chemokinetic therapy to generate abundant ROS, and combined with aPD-1 immunotherapy.

[0130] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A bilayer smart microneedle patch co-loaded with nanozymes and ICB antibodies, characterized in that, The device includes a microneedle patch tip and a microneedle patch backing. The microneedle patch tip comprises titanium carbide nanosheets loaded with cerium dioxide grafted with 3-amino-1,2,4-triazole and glucose oxidase, and calcium carbonate nanoparticles loaded with aPD-1. The microneedle patch backing comprises cross-linked grafted human-like collagen. The preparation method of the grafted human-like collagen includes: completely dissolving the human-like collagen in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stirring and activating, then... Dopamine powder was then added, and the mixture was stirred and reacted at room temperature. After dialysis with deionized water and lyophilization, grafted human-like collagen was obtained. The preparation method of the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody includes: dissolving the grafted human-like collagen to obtain a dissolved human-like collagen system; placing the dissolved human-like collagen system on the upper layer of the microneedle patch tip; adding hydrogen peroxide solution and oxyhemoglobin solution; stirring at room temperature to crosslink the mixture to obtain the bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody.

2. The bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody according to claim 1, characterized in that, The method for preparing the microneedle patch tip includes: Step 101: After etching the blocky Ti3AlC2 with hydrofluoric acid for 24-48 h, centrifuge until the supernatant pH is neutral, and dry to obtain titanium carbon MXene multilayer nanosheets. Peel the titanium carbon MXene multilayer nanosheets in tetrapropylammonium hydroxide solution for 48-96 h, centrifuge and dry to obtain few-layer MXene nanosheets. Step 102: The few-layer MXene nanosheets are ultrasonically and uniformly dispersed in water, preheated to obtain an MXene dispersion, cerium nitrate is dissolved in ethylene glycol to obtain a cerium nitrate solution, the cerium nitrate solution is added to the above MXene dispersion, the mixture is stirred and reacted, an ammonia solution is added, the reaction is continued for 1-6 h, and the mixture is centrifuged and dried to obtain MXene nanosheets loaded with cerium dioxide. Step 103: The MXene nanosheets loaded with cerium dioxide described in step 102 are ultrasonically dispersed in PBS solution, 3-amino-1,2,4-triazole is added, the mixture is stirred for 24-72 h, and then centrifuged and dried to obtain MXene@CeO2 nanosheets grafted with 3-AT. Step 104: Dissolve glucose oxidase in PBS solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, activate for 60-120 min to obtain activated glucose oxidase system, add MXene@CeO2 nanosheets grafted with 3-AT as described in step 103 to the activated glucose oxidase system, react in an ice bath for 20-24 h, centrifuge and dry to obtain nanosheets grafted with 3-AT and GOx; Step 105: Dissolve aPD-1 and CaCl2·2H2O completely in deionized water, add NH4HCO3, stir for 6~21h, centrifuge, wash, and dry to obtain calcium carbonate nanoparticles loaded with aPD-1. Step 106: The 3-AT and GOx grafted nanosheets described in Step 104 and the calcium carbonate nanoparticles loaded with aPD-1 described in Step 105 are ultrasonically dispersed in a polyvinyl alcohol solution to obtain a dispersed system. The dispersed system is placed in a PDMS mold, centrifuged, and then naturally dried at room temperature to obtain the microneedle patch tip.

3. The bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody according to claim 2, 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 tetrapropylammonium hydroxide solution is 2 to 4 times the mass of the titanium carbon MXene multilayer nanosheets.

4. The bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody according to claim 2, characterized in that, In step 102, the mass of the cerium nitrate solution is 1.2 to 3.2 times the mass of the few-layer MXene nanosheets, and the mass percentage of cerium nitrate in the cerium nitrate solution is 1% to 1.6%; in step 102, the mass of the water is 100 to 200 times the mass of the few-layer MXene nanosheets; in step 102, the volume of the ammonia solution is 0.01 to 0.07 times the mass of the few-layer MXene nanosheets, the volume of the ammonia solution is in mL, and the mass of the few-layer MXene nanosheets is in m³. g; In step 103, the mass of the 3-amino-1,2,4-triazole is 1 to 4 times the mass of the MXene nanosheets loaded with cerium dioxide; In step 104, the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 46 to 48 times the mass of glucose oxidase, and the mass of the N-hydroxysuccinimide is 26 to 30 times the mass of glucose oxidase; In step 104, the mass of the MXene@CeO2 nanosheets grafted with 3-AT is 0.5 to 2 times the mass of glucose oxidase.

5. The bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody according to claim 2, characterized in that, In step 105, the mass of aPD-1 is 0.001 to 0.003 times the mass of glucose oxidase in step 104; in step 105, the mass of CaCl2·2H2O is 1.5 to 5 times the mass of glucose oxidase in step 104; in step 105, the mass of NH4HCO3 is 15 to 150 times the mass of glucose oxidase in step 104; in step 106, the mass of the polyvinyl alcohol solution is 50 to 160 times the mass of glucose oxidase, and the mass percentage of the polyvinyl alcohol solution is 6% to 10%.

6. The bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody according to claim 1, characterized in that, The mass of the water is 20 to 100 times that of the human-like collagen protein; the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.15 times that of the human-like collagen protein; the mass of the N-hydroxysuccinimide is 0.69 times that of the human-like collagen protein; the mass of the dopamine is 1.5 to 3 times that of the human-like collagen protein; and the mass of the human-like collagen protein is 16 to 33 times that of the glucose oxidase in the tip of the microneedle patch.

7. A method for preparing a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody as described in claim 1, characterized in that, include: The grafted human-like collagen was dissolved to obtain a dissolved human-like collagen system. The dissolved human-like collagen system was placed on the upper layer of the microneedle patch tip, and hydrogen peroxide solution and oxyhemoglobin solution were added. The mixture was stirred at room temperature to crosslink, resulting in a bilayer smart microneedle patch co-loaded with nanozyme and ICB antibody.

8. The method according to claim 7, characterized in that, The dissolved human-like collagen system is a dissolved human-like collagen system obtained by dissolving grafted human-like collagen in water, and the mass percentage of grafted human-like collagen in the dissolved human-like collagen system is 2%~5%.

9. The method according to claim 7, characterized in that, The volume of the hydrogen peroxide solution is 10 to 40 times the mass of human-like collagen protein in the grafted human-like collagen, the volume unit of the hydrogen peroxide solution is μL, the mass unit of human-like collagen protein is g, and the concentration of the hydrogen peroxide solution is 10 to 30 mM; the volume of the oxyhemoglobin solution is 20 to 50 times the mass of human-like collagen protein in the grafted human-like collagen, the volume unit of the oxyhemoglobin solution is μL, the mass unit of human-like collagen protein is g, and the concentration of the oxyhemoglobin solution is 20 to 60 mg / mL.

Images