Preparation method of core-shell microneedle patch loaded with dual nanoparticles

Abstract

This invention discloses a method for preparing a core-shell microneedle patch loaded with dual nanoparticles, comprising: preparing silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6; preparing calcium carbonate nanoparticles loaded with aPD-1 antibody; dispersing the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6 in a polyvinyl alcohol solution to prepare the shell structure of the microneedles; preparing a polymer hydrogel; dispersing the calcium carbonate nanoparticles loaded with aPD-1 antibody in the polymer hydrogel to prepare the core structure of the microneedles; and casting the polyvinyl alcohol solution onto the backing of the microneedles to finally obtain the core-shell microneedle patch loaded with dual nanoparticles. The core-shell microneedle patch loaded with dual nanoparticles prepared by this invention can deliver nanoparticles to the tumor site, where the nanoparticles respond to the tumor microenvironment and activate the body's own immune system, thereby achieving a therapeutic effect on tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a core-shell microneedle patch loaded with dual nanoparticles. Background Technology

[0002] Melanoma is a highly aggressive malignant tumor originating from melanocytes in pigmented areas of the skin, mucous membranes, eyes, and central nervous system. Although its incidence is relatively low, its high malignancy and tendency to metastasize result in a high mortality rate. Current main treatments include surgical resection, chemotherapy, and radiotherapy, but these methods cause significant trauma to normal tissues and often lead to poor treatment outcomes due to inherent drug resistance.

[0003] Immunotherapy for melanoma is a novel cancer treatment method that works by modulating the function of the patient's own immune system to treat melanoma. While immunotherapy is highly effective, only a small percentage of patients benefit from it due to issues such as low immunogenicity. 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 method for preparing a core-shell microneedle patch loaded with dual nanoparticles. This method involves dispersing silica nanoparticles loaded with oxyhemoglobin and Ce6 in a polyvinyl alcohol solution to obtain the shell structure of the microneedle patch. Calcium carbonate nanoparticles loaded with aPD-1 antibody are dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. This mixed solution is then cast into microneedles to prepare the core structure of the microneedle patch. Finally, a polyvinyl alcohol solution is cast onto the backing portion of the microneedle patch to obtain the core-shell microneedle patch loaded with dual nanoparticles. This core-shell microneedle patch delivers nanoparticles to the tumor site, where the nanoparticles respond to the tumor microenvironment and activate the body's own immune system, thereby achieving a therapeutic effect on tumors.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a core-shell microneedle patch loaded with dual nanoparticles, characterized by comprising: Preparation of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; Preparation of calcium carbonate nanoparticles loaded with aPD-1 antibody; The silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were dispersed in a polyvinyl alcohol solution to prepare the shell structure of the microneedle patch tip. A polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine was prepared by mixing N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol solution. The calcium carbonate nanoparticles loaded with aPD-1 antibody were dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to prepare a core-shell structure for the tip of the microneedle patch. Polyvinyl alcohol solution was cast onto a core-shell structure backing to obtain a core-shell microneedle patch loaded with dual nanoparticles.

[0006] The above-mentioned method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized by the preparation of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6, specifically including: Oxyhemoglobin solution, dihydroporphyrin E6 solution, tetraethoxysilane and bis-[3-(triethoxysilyl)propyl]-disulfide were added to a mixed solution of cyclohexane, n-hexanol and Triton X-100. Then ammonia was added dropwise, and the mixture was stirred overnight at room temperature. Acetone was then added to precipitate the nanoparticles. The nanoparticles were washed with ethanol and water and lyophilized to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6.

[0007] The method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that: the concentration of oxyhemoglobin in the oxyhemoglobin solution is 0.1 mg / mL to 20 mg / mL; the concentration of dihydroporphyrin E6 in the dihydroporphyrin E6 solution is 0.1 mg / mL to 2 mg / mL; the mass of dihydroporphyrin E6 is 0.1 to 0.5 times the mass of oxyhemoglobin; the volume of tetraethoxysilane is 0.1 to 0.3 times the mass of oxyhemoglobin, wherein the volume unit of tetraethoxysilane is mL, and the mass unit of oxyhemoglobin is mg; the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 0.25 to 0.5 times the mass of oxyhemoglobin, wherein the volume unit of bis-[3-(triethoxysilyl)propyl]-disulfide is mL, and the mass unit of oxyhemoglobin is mg. The mass unit is mg; the volume of cyclohexane is 30-40 times the mass of oxyhemoglobin, wherein the volume unit of cyclohexane is mL and the mass unit of oxyhemoglobin is mg; the volume of n-hexanol is 8-10 times the mass of oxyhemoglobin, wherein the volume unit of n-hexanol is mL and the mass unit of oxyhemoglobin is mg; the volume of Triton X-100 is 8-10 times the mass of oxyhemoglobin, wherein the volume unit of Triton X-100 is mL and the mass unit of oxyhemoglobin is mg; the volume of ammonia is 0.2-0.4 times the mass of oxyhemoglobin, wherein the volume unit of ammonia is mL and the mass unit of oxyhemoglobin is mg; the volume of acetone is 91-96 times the mass of oxyhemoglobin, wherein the volume unit of acetone is mL and the mass unit of oxyhemoglobin is mg.

[0008] The above-mentioned method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that the preparation of calcium carbonate nanoparticles loaded with aPD-1 antibody specifically includes: Calcium chloride was dissolved in ethanol to obtain a calcium chloride solution, and then aPD-1 antibody was added. The mixture was reacted in a vacuum environment at 35℃~45℃ for 24h~72h, centrifuged, and washed with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody.

[0009] The above-described method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that the concentration of calcium chloride in the calcium chloride solution is 1 mg / mL to 3 mg / mL; the volume of the aPD-1 antibody is 0.0005 to 0.0008 times the mass of calcium chloride, wherein the volume of the aPD-1 antibody is in mL and the mass of calcium chloride is in mg; and the mass of the ammonium bicarbonate is 30 to 50 times the mass of calcium chloride.

[0010] The above-mentioned method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized by dispersing silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 in a polyvinyl alcohol solution to prepare the shell structure of the microneedle patch tip, specifically including: Polyvinyl alcohol was dissolved in ultrapure water to obtain a polyvinyl alcohol solution; silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were dispersed in the polyvinyl alcohol solution to obtain system A; system A was placed in a PDMS mold, vacuumed and then centrifuged, and then allowed to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip.

[0011] The above-described method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 70 mg / mL to 210 mg / mL; the volume of the polyvinyl alcohol solution is 0.5 to 15 times the mass of the silica nanoparticles, wherein the volume of the polyvinyl alcohol solution is in mL and the mass of the silica nanoparticles is in mg.

[0012] The above-described method for preparing core-shell microneedle patches loaded with dual nanoparticles is characterized in that the concentration of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine in the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution is 11 mg / mL to 27 mg / mL; and the concentration of N1- The concentration of the polyvinyl alcohol solution mixed with (4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution is 120 mg / mL to 180 mg / mL, and the volume of the polyvinyl alcohol solution when mixed is 0.65 to 1.35 times the volume of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution.

[0013] The above-mentioned method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that calcium carbonate nanoparticles loaded with aPD-1 antibody are dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to prepare the core-shell structure of the microneedle patch tip, specifically including: The calcium carbonate nanoparticles loaded with the aPD-1 antibody were dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain system B. System B was then cast and filled into the cavity of a mold with a microneedle patch tip shell structure and allowed to dry naturally at room temperature to obtain the core-shell structure of the microneedle patch tip.

[0014] The above-mentioned method for preparing a core-shell microneedle patch loaded with dual nanoparticles is characterized in that the volume of the polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol is 0.5 to 15 times the mass of the calcium carbonate nanoparticles loaded with aPD-1 antibody, wherein the volume unit of the polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol is mL, and the mass unit of the calcium carbonate nanoparticles is mg.

[0015] Compared with the prior art, the present invention has the following advantages: 1. This invention constructs a core-shell structure for microneedles and adjusts the nanoparticle preparation process to first release silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6. Under the action of ultrasound, these nanoparticles generate reactive oxygen species, reshaping the immune microenvironment. Then, calcium carbonate nanoparticles loaded with aPD-1 antibody are released. This improves the efficiency of immune checkpoint inhibitors in treating tumors by reprogramming the immunosuppressive microenvironment.

[0016] 2. The core-shell microneedle patch loaded with dual nanoparticles prepared in this invention is used for ultrasound-assisted treatment. As a non-invasive tumor treatment method, sonodynamic therapy can effectively treat tumors that are large or located deep in the body. At the same time, compared with treatment methods such as surgery, chemotherapy, radiotherapy, and photothermal therapy, it causes less damage to normal tissues.

[0017] 3. The core-shell microneedle patch loaded with dual nanoparticles prepared by the method of the present invention can effectively alleviate the problem of poor treatment effect caused by high glutathione content and low pH in the tumor microenvironment. Silica nanoparticles can consume reduced glutathione in the tumor microenvironment, and calcium carbonate nanoparticles can alleviate the problem of low pH in the tumor microenvironment.

[0018] 4. The method of the present invention, wherein the polymer hydrogel and calcium carbonate particles crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol can effectively protect the activity of aPD-1 antibody and prevent the antibody activity from being destroyed by reactive oxygen species.

[0019] 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

[0020] Figure 1 This is a schematic diagram of the mechanism of the core-shell structured microneedle patch loaded with dual nanoparticles in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the core-shell structured microneedle patch loaded with dual nanoparticles in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the synthesis of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol in Example 1 of the present invention. Figure 4 This is an electron microscope image and particle size analysis diagram of the silica particles loaded with oxyhemoglobin and Ce6 in Example 1 of the present invention. Figure 5 This is an electron microscope image and particle size analysis diagram of calcium carbonate particles loaded with aPD-1 in Example 1 of the present invention; Figure 6 The hydrogen nuclear magnetic resonance spectrum of TSPBA in Example 1 of this invention; Figure 7 The diagram shows the core-shell microneedle fabrication process, confocal image, and mechanical strength test curve in Embodiment 1 of this invention. Figure 8 These are fluorescence images and quantitative fluorescence analysis diagrams illustrating the ability of SHC microspheres to alleviate tumor cell hypoxia in Example 1 of this invention. Figure 9 This is a graph showing the NDA fluorescence detection and quantitative fluorescence analysis of the glutathione consumption capacity of SHC microspheres in Example 1 of the present invention. Figure 10 Schematic diagram of the cell apoptosis-promoting ability of SHC microspheres in different groups of microneedle patches; Figure 11 This is a schematic diagram showing the CD4+ and CD8+ fluorescence staining of tumor cells in mice after treatment with different microneedle patches. Figure 12 A comparative graph showing the tumor treatment capabilities of different groups of microneedle patches in a melanoma animal model. Detailed Implementation

[0021] The present invention will be specifically described below through examples. These examples are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Experimental methods in the examples that do not specify specific conditions are generally performed under conventional conditions and conditions described in the manual, or according to the conditions recommended by the manufacturer; the equipment, materials, reagents, etc. used are commercially available unless otherwise specified.

[0022] This invention provides a method for preparing a core-shell structured microneedle patch loaded with dual nanoparticles, comprising: Step 1: Dissolve hemoglobin in phosphate buffer (pH 7.4), and bubble oxygen through to obtain an oxyhemoglobin solution; dissolve dihydroporphyrin E6 in N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add the oxyhemoglobin solution, dihydroporphyrin E6 solution, tetraethoxysilane, and bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of cyclohexane, n-hexanol, and Triton X-100, then add a small amount of ammonia, stir overnight at room temperature, add acetone to precipitate nanoparticles, wash three times each with ethanol and water, and lyophilize overnight. Silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were obtained; the concentration of oxyhemoglobin in the oxyhemoglobin solution was 0.1 mg / mL to 20 mg / mL; the concentration of dihydroporphyrin E6 in the dihydroporphyrin E6 solution was 0.1 mg / mL to 2 mg / mL; the mass of dihydroporphyrin E6 was 0.1 to 0.5 times the mass of oxyhemoglobin; the volume of tetraethoxysilane was 0.1 to 0.3 times the mass of oxyhemoglobin, wherein the volume of tetraethoxysilane is in mL, and the mass of oxyhemoglobin is... The mass unit is mg; the volume of the bis-[3-(triethoxysilyl)propyl]-disulfide is 0.25 to 0.5 times the mass of oxyhemoglobin, wherein the volume unit of the bis-[3-(triethoxysilyl)propyl]-disulfide is mL, and the mass unit of oxyhemoglobin is mg; the volume of the cyclohexane is 30 to 40 times the mass of oxyhemoglobin, wherein the volume unit of the cyclohexane is mL, and the mass unit of oxyhemoglobin is mg; the volume of the n-hexanol is 8 to 10 times the mass of oxyhemoglobin, wherein the volume unit of the n-hexanol is mg. The volume of the oxyhemoglobin is in mL, and the mass unit of oxyhemoglobin is in mg; the volume of the Triton X-100 is 8 to 10 times the mass of the oxyhemoglobin, wherein the volume unit of the Triton X-100 is mL, and the mass unit of the oxyhemoglobin is mg; the volume of the ammonia is 0.2 to 0.4 times the mass of the oxyhemoglobin, wherein the volume unit of the ammonia is mL, and the mass unit of the oxyhemoglobin is mg; the volume of the acetone is 91 to 96 times the mass of the oxyhemoglobin, wherein the volume unit of the acetone is mL, and the mass unit of the oxyhemoglobin is mg. Step 2: Dissolve calcium chloride in ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add aPD-1 antibody, and then place the beaker in a vacuum drying oven containing dry ammonium bicarbonate. React the reaction system in a vacuum environment at 35℃~45℃ for 24h~72h. Centrifuge the resulting suspension and wash it with ethanol 3~5 times to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles loaded with aPD-1 antibody in ethanol and store at room temperature for later use. The concentration of calcium chloride in the calcium chloride solution is 1mg / mL~3mg / mL; the volume of the aPD-1 antibody is 0.0005~0.0008 times the mass of calcium chloride, where the volume unit of aPD-1 antibody is mL and the mass unit of oxyhemoglobin is mg; the mass of the ammonium bicarbonate is 30~50 times the mass of calcium chloride. Step 3: Dissolve polyvinyl alcohol in ultrapure water to obtain a polyvinyl alcohol solution; disperse the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 in the polyvinyl alcohol solution to obtain System A; place System A in a PDMS mold, evacuate and then centrifuge, and allow it to air dry at room temperature to obtain the shell structure of the microneedle patch tip; the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 70 mg / mL to 210 mg / mL; the volume of the polyvinyl alcohol solution is 0.5 to 15 times the mass of the silica nanoparticles, where the volume of the polyvinyl alcohol solution is in mL and the mass of the silica nanoparticles is in mg; Step 4: Preparation of crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine: N,N,N',N'-tetramethyl-1,3-propanediamine and 4-(bromomethyl)phenylboronic acid are added to dimethylformamide. Then, the mixture is stirred in a water bath at 55℃~70℃ for 12~48 hours. After cooling to room temperature, the mixture is poured into tetrahydrofuran, producing a white insoluble substance. Centrifugation at room temperature, washing three times with tetrahydrofuran, and lyophilization overnight yielded the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; the mass of the 4-(bromomethyl)phenylboronic acid was 2.5 to 7 times the mass of N,N,N',N'-tetramethyl-1,3-propanediamine, and the mass unit of N,N,N',N'-tetramethyl-1,3-propanediamine was mg. The mass unit of phenylboronic acid is mg; the volume of dimethylformamide is 0.04 to 0.12 times the mass of N,N,N',N'-tetramethyl-1,3-propanediamine, and the volume of tetrahydrofuran is 0.5 to 1.5 times the mass of N,N,N',N'-tetramethyl-1,3-propanediamine, wherein the volume unit of dimethylformamide is mL, the volume unit of tetrahydrofuran is mL, and the mass unit of N,N,N',N'-tetramethyl-1,3-propanediamine is mg; Step 5: Dissolve the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 in deionized water to obtain an N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution; dissolve polyvinyl alcohol in deionized water to obtain a polyvinyl alcohol solution; mix the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution with the polyvinyl alcohol solution to obtain an N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution. A polymer hydrogel crosslinked with 3-diamine and polyvinyl alcohol; wherein the concentration of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine in the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution is 11 mg / mL to 27 mg / mL; the concentration of polyvinyl alcohol solution is 120 mg / mL to 180 mg / mL; the volume of polyvinyl alcohol solution is 0.65 to 1.35 times the volume of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution; Step 6: Disperse the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; The volume of the polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol is 0.5~15 times the mass of the calcium carbonate nanoparticles, wherein the volume unit of the polyvinyl alcohol solution is mL, and the mass unit of the calcium carbonate nanoparticles is mg; Step 7: Cast the polyvinyl alcohol solution onto the backing portion of the microneedle patch mold for which the core and shell have been cast in Step 6, and finally obtain the core-shell microneedle patch loaded with dual nanoparticles; the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 70 mg / mL to 210 mg / mL. Example 1

[0023] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 2 mg of hemoglobin in 5 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 0.25 mg of dihydroporphyrin E6 in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 5 mL of the oxyhemoglobin solution, 1 mL of the dihydroporphyrin E6 solution, 0.4 mL of tetraethoxysilane, and 0.5 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 62 mL of cyclohexane, 18.2 mL of n-hexanol, and 18.4 mL of Triton X-100, then add 0.4 mL of ammonia water dropwise, stir overnight at room temperature, add 190 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 200 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.15 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 10 g of dry ammonium bicarbonate. The reaction system is carried out in a vacuum environment at 45 °C for 24 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 80 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 0.5 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain System A; place System A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 200 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide; then, stir the mixture in a 55 °C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and freeze-dry overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 15 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain an N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution; dissolve 125 mg of polyvinyl alcohol in 1 mL of deionized water to obtain a polyvinyl alcohol solution; and dissolve 0.6 mL of... A solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine was mixed with 0.42 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.5 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 1 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 2

[0024] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 3 mg of hemoglobin in 3 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 0.6 mg of dihydroporphyrin E6 in 2 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 3 mL of oxyhemoglobin solution, 2 mL of dihydroporphyrin E6 solution, 0.4 mL of tetraethoxysilane, and 0.9 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 90 mL of cyclohexane, 25 mL of n-hexanol, and 26 mL of Triton X-100, then add 0.6 mL of ammonia water dropwise, stir overnight at room temperature, add 280 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 200 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.17 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 10 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 45 °C for 24 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 80 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 0.5 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain System A; place System A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 200 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide; then, stir the mixture in a 55 °C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and freeze-dry overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 15 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 125 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.4 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.28 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.5 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 0.68 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 3

[0025] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 0.3 mg of hemoglobin in 3 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 0.15 mg of dihydroporphyrin E6 in 1.5 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 3 mL of the oxyhemoglobin solution, 1.5 mL of the dihydroporphyrin E6 solution, 0.03 mL of tetraethoxysilane, and 0.15 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 12 mL of cyclohexane, 3 mL of n-hexanol, and 3 mL of Triton X-100, then add 0.1 mL of ammonia water dropwise, stir overnight at room temperature, add 28 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 100 mg of calcium chloride in 100 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.08 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 3 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 40 °C for 48 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 70 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 0.1 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain system A; place system A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 200 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide. Then, stir the mixture in a 55°C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and lyophilize overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 11 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 120 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.4 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.54 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 1 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 1 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 4

[0026] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 20 mg of hemoglobin in 1 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 2 mg of dihydroporphyrin E6 in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 1 mL of the oxyhemoglobin solution, 1 mL of the dihydroporphyrin E6 solution, 6 mL of tetraethoxysilane, and 8 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 600 mL of cyclohexane, 160 mL of n-hexanol, and 160 mL of Triton X-100, then add 8 mL of ammonia water, stir overnight at room temperature, add 1820 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 100 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.15 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 15 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 35 °C for 72 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 3.15g of polyvinyl alcohol in 15mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 1mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 15mL of polyvinyl alcohol solution to obtain system A; place system A in a PDMS mold, evacuate and then centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 200 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide; then, stir the mixture in a 55 °C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and freeze-dry overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 15 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 180 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.5 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.5 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.07 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 1 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 5

[0027] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 4 mg of hemoglobin in 2 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 0.8 mg of dihydroporphyrin E6 in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 2 mL of the oxyhemoglobin solution, 1 mL of the dihydroporphyrin E6 solution, 0.4 mL of tetraethoxysilane, and 1.2 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 135 mL of cyclohexane, 34 mL of n-hexanol, and 36 mL of Triton X-100, then add 0.9 mL of ammonia water dropwise, stir overnight at room temperature, add 370 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 200 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.15 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 10 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 45 °C for 24 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 80 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 0.5 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain System A; place System A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 300 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide; then, stir the mixture in a 55°C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained. Centrifuge at room temperature, wash three times with tetrahydrofuran, and freeze-dry overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 15 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 125 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.4 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.28 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.5 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 0.68 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 6

[0028] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 2.5 mg of hemoglobin in 1 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 1.2 mg of dihydroporphyrin E6 in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 1 mL of the oxyhemoglobin solution, 1 mL of the dihydroporphyrin E6 solution, 0.36 mL of tetraethoxysilane, and 0.625 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 78 mL of cyclohexane, 21 mL of n-hexanol, and 24 mL of Triton X-100, then add 0.6 mL of ammonia water dropwise, stir overnight at room temperature, add 240 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 200 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.15 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 10 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 45 °C for 24 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 80 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 2 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain system A; place system A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 350 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 900 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide; then, stir the mixture in a 55 °C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and freeze-dry overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 15 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 125 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.8 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.56 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.08 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 1.2 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles. Example 7

[0029] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps: Step 1: Dissolve 3.2 mg of hemoglobin in 1 mL of phosphate buffer (pH=7.4), and purge with oxygen for 20 min to obtain an oxyhemoglobin solution; dissolve 0.4 mg of dihydroporphyrin E6 in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution; add 1 mL of the oxyhemoglobin solution, 1 mL of the dihydroporphyrin E6 solution, 0.4 mL of tetraethoxysilane, and 0.9 mL of bis-[3-(triethoxysilyl)propyl]-disulfide to a mixed solution of 97 mL of cyclohexane, 26 mL of n-hexanol, and 27 mL of Triton X-100, then add 0.8 mL of ammonia water dropwise, stir overnight at room temperature, add 295 mL of acetone to precipitate the nanoparticles, wash three times each with ethanol and water, and freeze-dry overnight to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; the mass concentration of the ammonia water is 25%. Step 2: Dissolve 300 mg of calcium chloride in 200 mL of ethanol in a beaker covered with aluminum foil. Make some small holes in the aluminum foil, add 0.15 mL of aPD-1 antibody, and then place the beaker in a vacuum drying oven containing 10 g of dry ammonium bicarbonate. The reaction system is reacted in a vacuum environment at 45 °C for 24 hours. Centrifuge the obtained suspension and wash it three times with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody. Disperse the obtained calcium carbonate nanoparticles in ethanol and store them at room temperature for later use. Step 3: Dissolve 80 mg of polyvinyl alcohol in 1 mL of ultrapure water to obtain a polyvinyl alcohol solution; weigh 0.5 mg of the silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 described in Step 1 and disperse them in 1 mL of polyvinyl alcohol solution to obtain System A; place System A in a PDMS mold, evacuate and centrifuge, and allow it to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip; Step 4: Add 200 mg N,N,N',N'-tetramethyl-1,3-propanediamine and 600 mg 4-(bromomethyl)phenylboronic acid to 10 mL of dimethylformamide. Then, stir the mixture in a 55°C water bath for 24 hours; after cooling to room temperature, pour the mixture into 100 mL of tetrahydrofuran, and a white insoluble precipitate is obtained; centrifuge at room temperature, wash three times with tetrahydrofuran, and lyophilize overnight to obtain the crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; Step 5: Weigh 27 mg of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine synthesized in Step 4 and dissolve it in 1 mL of deionized water to obtain a solution of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; dissolve 125 mg of polyvinyl alcohol in 1 mL of deionized water. Water was used to obtain a polyvinyl alcohol solution; 0.8 mL of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution was mixed with 0.56 mL of polyvinyl alcohol solution to obtain a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol. Step 6: Weigh 0.5 mg of the calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in Step 2 and disperse them in 1 mL of a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain System B; Cast System B into the cavity of the mold with the microneedle patch tip shell structure in Step 3, evacuate and centrifuge, repeat the casting and filling several times, and then let it air dry at room temperature to obtain the core-shell structure of the microneedle patch tip; Step 7: Dissolve 280 mg of polyvinyl alcohol in 2 mL of deionized water, and cast the resulting polyvinyl alcohol solution into the backing part of the core-shell microneedle patch mold cast in Step 6, finally obtaining a core-shell microneedle patch loaded with dual nanoparticles.

[0030] Comparative Example 1 This comparative example is the same as Example 1, except that steps two, three, four, five, and seven are not included. In step one, 0.25 mg of dihydroporphyrin E6 is dissolved in 1 mL of N,N-dimethylformamide to obtain a dihydroporphyrin E6 solution. 1 mL of the dihydroporphyrin E6 solution, 0.4 mL of tetraethoxysilane, and 0.5 mL of bis-[3-(triethoxysilyl)propyl]-disulfide are added to a mixed solution of 62 mL of cyclohexane, 18.2 mL of n-hexanol, and 18.4 mL of Triton X-100. Then, 0.4 mL of ammonia is added dropwise, and the mixture is stirred overnight at room temperature. 190 mL of acetone is added to precipitate the nanoparticles. The nanoparticles are washed three times each with ethanol and water, and then lyophilized overnight to obtain silica nanoparticles loaded with dihydroporphyrin E6. The mass concentration of the ammonia is 25%. Step six involves dispersing 0.25 mg of silica nanoparticles loaded with dihydroporphyrin E6 obtained in step one in a polyvinyl alcohol solution to obtain system A. System A is then placed in a PDMS mold, evacuated, and centrifuged. The casting and filling process is repeated several times, and the mixture is then allowed to dry naturally at room temperature to obtain a microneedle patch containing only silica nanoparticles loaded with dihydroporphyrin E6.

[0031] Comparative Example 2 This comparative example is the same as Example 1, except that steps one, three, four, five, and seven are not included, and step six involves dispersing 2 mg of calcium carbonate nanoparticles loaded with aPD-1 antibody obtained in step two in 3 mL of polyvinyl alcohol solution with a concentration of 80 mg / mL to obtain system A. System A is placed in a PDMS mold, vacuumed, and then centrifuged. The casting and filling process is repeated several times, and then the mixture is allowed to dry naturally at room temperature to obtain a microneedle patch loaded only with calcium carbonate nanoparticles loaded with aPD-1 antibody.

[0032] Comparative Example 3 This comparative example is the same as Example 1, except that steps two, four, five, six, and seven are not included, and step three involves dispersing 2 mg of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6 from step one in 3 mL of a polyvinyl alcohol solution with a concentration of 80 mg / mL to obtain system A. System A is placed in a PDMS mold, vacuumed, and then centrifuged. The casting and filling process is repeated several times, and then the mixture is allowed to dry naturally at room temperature to obtain a microneedle patch loaded only with silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6.

[0033] Comparative Example 4 This comparative example is the same as Example 1, except that steps two, four, five, six, and seven are not included, and step three involves dispersing 0.15 mL of aPD-1 antibody and 2 mg of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin e6 as described in step one in 2.85 mL of a polyvinyl alcohol solution with a concentration of 80 mg / mL to obtain system A. System A is then placed in a PDMS mold to obtain a microneedle patch loaded with aPD-1 antibody and silica nanoparticles.

[0034] like Figure 10 As shown, the silica nanoparticles (CS group and CS+US group) loaded with only dihydroporphyrin E6 obtained by the method in Comparative Example 1 had weaker oxygen production, reactive oxygen species production, and tumor cell apoptosis ability. This is because the tumor hypoxic microenvironment, in Comparative Example 1, did not add oxygenated hemoglobin that can supply oxygen, thus failing to produce enough reactive oxygen species, resulting in a lower cell apoptosis rate.

[0035] like Figure 11 As shown, the microneedle patch of calcium carbonate nanoparticles loaded with aPD-1 antibody obtained by the method in Comparative Example 2 (S / SHC+US group) had a weaker effect on tumor treatment, and the expression of CD4+ T cells and CD8+ T cells in tumor tissue was low. This may be due to the severe hypoxia and strong immunosuppression in the tumor microenvironment, and the effect of using aPD-1 alone may be limited.

[0036] like Figure 11 As shown, the microneedle patch (S / CP) of silica nanoparticles loaded only with oxyhemoglobin and dihydroporphyrin e6 obtained by the method in Comparative Example 3 showed weaker therapeutic effects on tumors, with lower expression of CD4+ T cells and CD8+ T cells in tumor tissue. This may be because oxidative stress in the tumor microenvironment may promote the activation of immunosuppressive cells, which inhibit effector immune responses by expressing immune checkpoint molecules such as PD-L1.

[0037] like Figure 11 As shown, the microneedle patch loaded with aPD-1 and silica nanoparticles obtained by the method in Comparative Example 4 (S / SHCP+US group) showed weaker therapeutic effects on tumors, with lower expression of CD4+ T cells and CD8+ T cells in tumor tissue. This may be because oxidative stress in the tumor microenvironment may promote the activation of immunosuppressive cells, which suppress effector immune responses by expressing immune checkpoint molecules such as PD-L1, while aPD-1 antibody is destroyed by reactive oxygen species.

[0038] like Figure 11 and Figure 12 As shown, the core-shell microneedle patch loaded with nanoparticles obtained by the method in Example 1 (S / SHC+C / CP+US group) showed the best tumor treatment effect compared to the other groups in the comparative example.

[0039] Material characterization and performance testing: Figure 1 This diagram illustrates the mechanism of action of the core-shell structured microneedle patch loaded with dual nanoparticles in Example 1. Figure 1 As shown, the core-shell microneedle patch loaded with dual nanoparticles in Example 1 is a core-shell microneedle that enhances tumor immunogenicity and immune checkpoint blockade efficacy for the treatment of melanoma. It intelligently responds to the high levels of GSH and the acidic microenvironment of the tumor, programmatically releasing a sonosensitive agent and aPD-1: the microneedle shell responds to the high levels of GSH and the hypoxic microenvironment in the tumor by releasing O2, generating ROS under the action of ultrasound and the sonosensitive agent, thus reshaping the immune microenvironment. Subsequently, the core layer of the microneedle responds to ROS and the acidic microenvironment of the tumor by releasing aPD-1. This programmatic reshaping of the immune microenvironment enhances ICB.

[0040] Figure 2 This is a schematic diagram of the core-shell structured microneedle patch loaded with dual nanoparticles in Example 1. The diagram shows that the present invention prepares the shell structure of the microneedles by dispersing silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 in a polyvinyl alcohol solution, and prepares the core structure of the microneedles by dispersing calcium carbonate nanoparticles loaded with aPD-1 antibody in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol, and finally obtains the core-shell microneedle patch loaded with nanoparticles.

[0041] Figure 3 This is a schematic diagram illustrating the synthesis of the polymer hydrogel crosslinked with polyvinyl alcohol, N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine in Example 1. N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine was synthesized via a quaternization reaction of N1,N1,N3,N3-tetramethylpropane-1,3-diamine with 4-(bromomethyl)phenylboronic acid.

[0042] Figure 4 Figure 1 shows the electron microscope (EM) images and particle size analysis of the silica particles loaded with oxyhemoglobin and Ce6 in Example 1. Figures a and b are the EEM images and particle size analysis diagrams of the silica particles, respectively. As can be seen from the figures, the silica particles are spherical with a uniform particle size distribution, and the average particle size is 21–24 nm. Figure c shows the corresponding elemental spectrum, with uniform distribution of Si, O, C, N, and Fe elements, indicating that the SiO2 microspheres were successfully loaded with HbO2 and Ce6.

[0043] Figure 5Figure 1 shows the electron microscope (EM) images and particle size analysis diagrams of calcium carbonate particles loaded with aPD-1 in Example 1. Figures a and b are the EEM images and particle size analysis diagrams of silica particles, respectively. As can be seen from the figures, the silica particles are spherical with a uniform particle size distribution, and the average particle size is 800–1300 nm. Figure c shows the corresponding elemental spectrum, showing a uniform distribution of Ca, C, O, S, and N elements, indicating successful CP synthesis.

[0044] Figure 6 The 1H NMR spectrum of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine confirms the successful synthesis of TSPBA.

[0045] Figure 7 Figure 1 shows the fabrication process flow, confocal images, and mechanical strength test curves for the core-shell microneedles in Example 1. Figure a is a schematic diagram of the fabrication process flow for the core-shell microneedles; Figure b is a confocal image of the bottom view of the microneedle shell structure with a z-axis spacing of 200 μm; and Figure c is a 3D confocal image of the microneedle shell structure. Figures b and c are cross-sectional and stereoscopic images of the microneedle shell structure obtained using confocal microscopy, demonstrating the integrity of the microneedle shell structure. Figure d is a confocal image of the bottom view of the microneedle core-shell structure, showing the successful fabrication of the microneedle core-shell layer. Figure e evaluates the ability of the microneedles to penetrate skin tissue by assessing the mechanical strength of the microneedle shell and core-shell microneedles. The mechanical strength of the core-shell microneedles (2.35 N / needle) is significantly higher than that of the microneedle core (1.11 N / needle).

[0046] Figure 8 The images show fluorescence images and quantitative fluorescence analysis of the ability of SHC microspheres to alleviate tumor cell hypoxia in Example 1. When SHC microspheres were added to B16F10 cells, compared to 0 h, the red fluorescence decreased after 6 h of addition, and almost disappeared after 12 h, indicating effective relief of cellular hypoxia.

[0047] Figure 9 This is an NDA fluorescence detection and quantitative fluorescence analysis diagram of the ability of SHC microspheres to consume glutathione in cells in Example 1. After adding SHC microspheres to B16F10, the red fluorescence decreased to 54.3% at 12 h, and almost disappeared at 24 h, indicating that SHC can effectively reduce the intracellular glutathione content.

[0048] Figure 10Cell apoptosis was analyzed using AM / PI fluorescence staining, fluorescence imaging, quantitative fluorescence analysis, and flow cytometry. Compared with the control group, the SiO2@Ce6 group showed almost no red fluorescence and a cell survival rate of 99.8%, indicating that SiO2@Ce6 caused almost no cell damage. The apoptosis rate of the SiO2@Ce6+US group was 26.8%, confirming that Ce6-mediated SDT caused some damage to cells, but due to the hypoxic microenvironment, the anti-tumor effect was poor. In contrast, the SiO2@(HbO2+Ce6)+US group showed strong red fluorescence and only weak green fluorescence, with a 97.8% apoptosis rate, confirming that SiO2@(HbO2+Ce6)+US has excellent anti-tumor properties.

[0049] Figure 11 The tumor immune microenvironment remodeling effect was assessed by immunofluorescence staining of CD4+ T cells and CD8+ T cells in the tumor. In Example 1, the red fluorescence intensity of CD8+ and CD4+ T cells on the loaded nanoparticles and core-shell microneedle patches (S / SHC+C / CP+US group) was significantly increased, indicating that their infiltration into tumor tissue was increased and they had stronger anti-tumor immune activity.

[0050] Figure 12 This is a comparison of the tumor therapeutic capabilities of core-shell microneedles loaded with SHC microspheres and CP microspheres in an animal model of melanoma, as shown in Example 1. First, a subcutaneous mouse melanoma model was constructed, and the tumor volume was increased to approximately 100 mm². 3 Mice were randomly assigned to five groups (n=5): (1) Control group (2) Shell / CaCO3@aPD-1 group (S / CP) (3) Shell / SiO2@(HbO2+Ce6)+US group (S / SHC+US) (4) Shell / SiO2@(HbO2+Ce6)+aPD-1+US group (S / SHCP+US) (5) Shell / SiO2@(HbO2+Ce6)+Core / CaCO3@aPD-1+US group (S / SHC+C / CP+US). In the ultrasound treatment group, each mouse underwent ultrasound treatment 12 h after microneedle treatment. The Control group used empty microneedles made of 20% PVA solution.

[0051] The control group showed almost no inhibitory effect on melanoma growth, and tumor development remained uncontrolled. The Shell / CaCO3@aPD-1 group (S / CP) showed limited inhibitory effect on melanoma growth. The Shell / SiO2@(HbO2+Ce6)+US group (S / SHC+US) and the Shell / SiO2@(HbO2+Ce6)+aPD-1+US group (S / SHCP+US) showed significant inhibitory effects on melanoma growth. In contrast, the Shell / SiO2@(HbO2+Ce6)+Core / CaCO3@aPD-1+US group (S / SHC+C / CP+US) exhibited superior anti-tumor effects, significantly inhibiting tumor growth, reducing tumor volume to near disappearance, and showing no signs of tumor recurrence during this treatment cycle.

[0052] 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 method for preparing a core-shell microneedle patch loaded with dual nanoparticles, characterized in that, include: Preparation of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6; Preparation of calcium carbonate nanoparticles loaded with aPD-1 antibody; The silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were dispersed in a polyvinyl alcohol solution to prepare the shell structure of the microneedle patch tip. A polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine was prepared by mixing N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol solution. The calcium carbonate nanoparticles loaded with aPD-1 antibody were dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to prepare a core-shell structure for the tip of the microneedle patch. Polyvinyl alcohol solution was cast onto a core-shell structure backing to obtain a core-shell microneedle patch loaded with dual nanoparticles.

2. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 1, characterized in that, The preparation of silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 specifically includes: Oxyhemoglobin solution, dihydroporphyrin E6 solution, tetraethoxysilane and bis-[3-(triethoxysilyl)propyl]-disulfide were added to a mixed solution of cyclohexane, n-hexanol and Triton X-100. Then ammonia was added dropwise, and the mixture was stirred overnight at room temperature. Acetone was then added to precipitate the nanoparticles. The nanoparticles were washed with ethanol and water and lyophilized to obtain silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6.

3. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 2, characterized in that, The concentration of oxyhemoglobin in the oxyhemoglobin solution is 0.1 mg / mL to 20 mg / mL; the concentration of dihydroporphyrin E6 in the dihydroporphyrin E6 solution is 0.1 mg / mL to 2 mg / mL; the mass of dihydroporphyrin E6 is 0.1 to 0.5 times the mass of oxyhemoglobin; the volume of tetraethoxysilane is 0.1 to 0.3 times the mass of oxyhemoglobin, wherein the volume of tetraethoxysilane is in mL and the mass of oxyhemoglobin is in mg; the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 0.25 to 0.5 times the mass of oxyhemoglobin, wherein the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is in mL and the mass of oxyhemoglobin is in mg; the volume of cyclohexane... The volume of the cyclohexane is 30-40 times that of oxyhemoglobin, wherein the volume unit of cyclohexane is mL and the mass unit of oxyhemoglobin is mg; the volume of the n-hexanol is 8-10 times that of oxyhemoglobin, wherein the volume unit of n-hexanol is mL and the mass unit of oxyhemoglobin is mg; the volume of the Triton X-100 is 8-10 times that of oxyhemoglobin, wherein the volume unit of Triton X-100 is mL and the mass unit of oxyhemoglobin is mg; the volume of the ammonia is 0.2-0.4 times that of oxyhemoglobin, wherein the volume unit of ammonia is mL and the mass unit of oxyhemoglobin is mg; the volume of the acetone is 91-96 times that of oxyhemoglobin, wherein the volume unit of acetone is mL and the mass unit of oxyhemoglobin is mg.

4. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 1, characterized in that, The preparation of calcium carbonate nanoparticles loaded with aPD-1 antibody specifically includes: Calcium chloride was dissolved in ethanol to obtain a calcium chloride solution, and then aPD-1 antibody was added. The mixture was reacted in a vacuum environment at 35℃~45℃ for 24h~72h, centrifuged, and washed with ethanol to obtain calcium carbonate nanoparticles loaded with aPD-1 antibody.

5. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 4, characterized in that, The concentration of calcium chloride in the calcium chloride solution is 1 mg / mL to 3 mg / mL; the volume of the aPD-1 antibody is 0.0005 to 0.0008 times the mass of calcium chloride, wherein the volume of the aPD-1 antibody is in mL and the mass of calcium chloride is in mg; the mass of the ammonium bicarbonate is 30 to 50 times the mass of calcium chloride.

6. The method for preparing the core-shell microneedle patch loaded with dual nanoparticles according to claim 1, characterized in that, Silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were dispersed in a polyvinyl alcohol solution to prepare the shell structure of the microneedle patch tip, specifically including: Polyvinyl alcohol was dissolved in ultrapure water to obtain a polyvinyl alcohol solution; silica nanoparticles loaded with oxyhemoglobin and dihydroporphyrin E6 were dispersed in the polyvinyl alcohol solution to obtain system A; system A was placed in a PDMS mold, vacuumed and then centrifuged, and then allowed to dry naturally at room temperature to obtain the shell structure of the microneedle patch tip.

7. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 6, characterized in that, The concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 70 mg / mL to 210 mg / mL; the volume of the polyvinyl alcohol solution is 0.5 to 15 times the mass of the silica nanoparticles, wherein the volume of the polyvinyl alcohol solution is in mL and the mass of the silica nanoparticles is in mg.

8. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 1, characterized in that, The concentration of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine in the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution is 11 mg / mL to 27 mg / mL; the concentration of the polyvinyl alcohol solution mixed with the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution is 120 mg / mL to 180 mg / mL, and the volume of the polyvinyl alcohol solution during mixing is 0.65 to 1.35 times the volume of the N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine solution.

9. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 1, characterized in that, Calcium carbonate nanoparticles loaded with aPD-1 antibody were dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to prepare the core-shell structure of the microneedle patch tip, specifically including: The calcium carbonate nanoparticles loaded with the aPD-1 antibody were dispersed in a polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol to obtain system B. System B was then cast and filled into the cavity of a mold with a microneedle patch tip shell structure and allowed to dry naturally at room temperature to obtain the core-shell structure of the microneedle patch tip.

10. The method for preparing a core-shell microneedle patch loaded with dual nanoparticles according to claim 9, characterized in that, The volume of the polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol is 0.5 to 15 times the mass of the calcium carbonate nanoparticles loaded with aPD-1 antibody. The volume of the polymer hydrogel crosslinked with N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine and polyvinyl alcohol is in mL, and the mass of the calcium carbonate nanoparticles is in mg.

Images