Programmed co-delivery oxygen vacancy engineered self-oxygen-supply nano platform and ICB antibody core-shell structure microneedle and preparation method thereof

Abstract

The invention discloses a core-shell structure microneedle of a programmed co-delivery oxygen vacancy engineered self-oxygen-supply nano platform and an ICB antibody and a preparation method of the core-shell structure microneedle, a needle tip of the core-shell structure microneedle comprises a shell layer and a core layer located in the shell layer, and the shell layer is a PVA shell layer capable of being rapidly dissolved; mXene (at) RuO2 nanosheets grafted with GOx and HbO2 are dispersed in a PVA solution and then poured in a PDMS mold to be dried, and the composite material is prepared. The core layer is a core layer with ROS responsiveness, SiO2 nanoparticles loaded with aPD-1 are dispersed in a PVA solution, and the SiO2 nanoparticles loaded with aPD-1 and a TSPBA solution are jointly poured in a PVA shell layer capable of being rapidly dissolved. The core-shell structure microneedle can programmatically release an enzymatic cascade reaction nano-platform ROS generator MRuGH and an ICB antibody, the antibody activity is protected, programmed immunotherapy of malignant tumors is controlled in a time-space mode, and ICB treatment sensitivity is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a method for preparing recombinant collagen microspheres through photo-crosslinking. Background Art

[0002] Immune checkpoint blockade (ICB) therapy has made great progress in the treatment of various types of tumors. However, clinical data show that less than 20% of patients benefit from it due to the immunosuppressive "cold tumor" microenvironment. Therefore, converting immunosuppressive "cold tumors" into immune-activated "hot tumors" is a key challenge in the treatment of malignant tumors with ICB.

[0003] As a new therapeutic modality, reactive oxygen species (ROS) can not only directly kill tumors, but also induce immunogenic cell death, activate dendritic cell maturation, and promote cytotoxic T lymphocyte infiltration, thereby reshaping the immune microenvironment and improving the sensitivity of ICB therapy. Chemodynamic therapy (CDT) mediated by an enzymatic cascade reaction nanoplatform based on glucose oxidase (GOx) can self-supply hydrogen peroxide and reduce tumor pH, further generating toxic hydroxyl radicals. In addition, sonodynamic therapy (SDT) can activate sonosensitizers to produce singlet oxygen, and has been widely studied for its superior spatiotemporal control, deep tissue penetration, and minimal side effects. However, the GOx catalytic process and SDT reaction are highly dependent on oxygen, and the hypoxic microenvironment of the tumor greatly restricts the reaction. In addition, defect engineering, especially oxygen vacancy engineering, has been considered an effective strategy to improve catalytic performance. Therefore, it is crucial to construct an oxygen-deficient self-supplying nanoplatform to generate abundant ROS through CDT and SDT mediated by it, thereby reshaping the immune microenvironment.

[0004] However, when ROS nanogenerators and ICB antibodies are co-delivered, ROS will inevitably damage the antibody activity, and the ROS remodeling of the immune microenvironment may not be completed when the ICB antibody is effective, and the ICB efficacy is not effectively improved. Therefore, it is necessary to construct an intelligent system that co-delivers and programmatically releases oxygen vacancies engineered self-oxygen nanoplatforms and ICB antibodies to protect antibody activity and improve the sensitivity of ICB treatment of malignant tumors. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a core-shell structure microneedle for programmed co-delivery of oxygen vacancy engineered self-oxygen supply nano-platform and ICB antibody and its preparation method in view of the above-mentioned deficiencies of the prior art. The core-shell structure microneedle can programmatically release the enzymatic cascade reaction nano-platform ROS generator MRuGH and ICB antibody, protect the antibody activity, and control its programmed immunotherapy of malignant tumors in time and space, significantly improving the sensitivity of ICB treatment.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a core-shell structure microneedle for programmed co-delivery of oxygen vacancy engineered self-oxygen supply nanoplatform and ICB antibody, characterized in that the needle tip of the core-shell structure microneedle includes a shell layer and a core layer located in the shell layer, the shell layer is a rapidly dissolvable PVA shell layer, which is made by dispersing MXene@RuO2 nanosheets grafted with GOx and HbO2 in a PVA solution and then casting it in a PDMS mold and drying it; the core layer is a ROS-responsive core layer, which is made by dispersing SiO2 nanoparticles loaded with aPD-1 in a PVA solution and casting it together with a TSPBA solution in the rapidly dissolvable PVA shell layer.

[0007] The above-mentioned core-shell structure microneedle for programmed co-delivery of oxygen vacancy engineered self-oxygen supply nanoplatform and ICB antibody is characterized in that the core-shell structure microneedle regulates the degradation rate of the nuclear layer in response to ROS and SiO2 in response to the tumor microenvironment with high expression of glutathione, so that the core-shell structure microneedle can programmed co-deliver oxygen vacancy engineered self-oxygen supply nanoplatform and ICB antibody, and can produce abundant ROS through SDT and CDT under the stimulation of ultrasound and the activation of glucose in the tumor microenvironment, thereby reshaping the immune microenvironment.

[0008] Furthermore, the present invention provides a method for preparing the core-shell structure microneedle, characterized in that it comprises the following steps:

[0009] Step 1: etching the blocky titanium aluminum carbide compound with hydrofluoric acid, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0010] Step 2, ultrasonically disperse the few-layer MXene nanosheets described in step 1 in water, then add RuCl3 aqueous solution, then dropwise add NaOH solution, stir to react, centrifuge and dry to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0011] Step 3, ultrasonically dispersing the MXene@RuO2 nanosheets obtained in step 2 in water, adding amino polyethylene glycol thiol, stirring, centrifugation and drying to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0012] Step 4: Ultrasonic dispersion of the MXene@RuO2 nanosheet grafted with -NH2 obtained in step 3 in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adding GOx and HbO2 after activation, reacting in an ice bath and centrifuging and drying to obtain MXene@RuO2 nanosheet grafted with GOx and HbO2;

[0013] Step 5, the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 are uniformly dispersed in a PVA aqueous solution, cast in a PDMS mold, and dried to obtain a rapidly soluble PVA shell layer;

[0014] Step 6, polyethylene glycol octylphenyl ether, n-hexanol and cyclohexane are mixed to obtain an organic solution; aPD-1, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide are mixed and shaken well, and then added to the organic solution, and ammonia water is added. After stirring at room temperature overnight, pure acetone is added, precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0015] Step 7: Ultrasonically disperse the aPD-1 loaded SiO2 nanoparticles obtained in step 6 in the PVA aqueous solution, then cast them together with the TSPBA solution in the rapidly soluble PVA shell layer obtained in step 5, and dry them naturally to obtain core-shell structure microneedles.

[0016] The above method is characterized in that the etching time in step 1 is 12h to 96h, and the stripping time is 12h to 96h.

[0017] The above method is characterized in that the mass concentration of the RuCl3 aqueous solution in step 2 is 5 mg / mL to 20 mg / mL, and the volume of the RuCl3 aqueous solution is 20 to 40 times the mass of the few-layer MXene nanosheets, wherein the unit of volume is mL and the unit of mass is g; the concentration of the NaOH solution is 0.5M to 3M, and the volume of the NaOH solution is 3 to 6 times the mass of the few-layer MXene nanosheets, wherein the unit of volume is mL and the unit of mass is g; the stirring reaction time is 1h to 10h.

[0018] The above method is characterized in that the mass of the amino polyethylene glycol thiol group in step three is 0.3 to 3 times the mass of the MXene@RuO2 nanosheets, and the stirring time is 1 h to 48 h.

[0019] The above method is characterized in that the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in step 4 is (0.5-2):1, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 30-60 times the mass of the MXene@RuO2 nanosheet grafted with -NH2, and the activation time is 20min-300min; the mass ratio of GOx and HbO2 is 1:1, the mass of GOx is 0.3-3 times the mass of the MXene@RuO2 nanosheet grafted with -NH2, and the ice bath reaction time is 1h-72h.

[0020] The above method is characterized in that the mass concentration of the PVA aqueous solution in step five is 5% to 20%, and the ratio of the volume of the PVA aqueous solution to the mass of the MXene@RuO2 nanosheets grafted with GOx and HbO2 is (0.5 to 5):1, where the unit of volume is mL and the unit of mass is mg.

[0021] The above method is characterized in that the volume of n-hexanol in step six is ​​1 to 1.5 times the volume of polyethylene glycol octyl phenyl ether, and the volume of cyclohexane is 2 to 6 times the volume of polyethylene glycol octyl phenyl ether; the volume of tetraethyl orthosilicate is 0.5 to 1.5 times the mass of aPD-1, the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 1 to 3 times the mass of aPD-1, and the volume of polyethylene glycol octyl phenyl ether is 30 to 50 times the mass of aPD-1. The concentration of the ammonia water is 30%, the volume of the ammonia water is 1 to 2 times the mass of aPD-1, and the volume of the pure acetone is 500 to 1000 times the mass of aPD-1, wherein the unit of volume is μL and the unit of mass is μg.

[0022] The above method is characterized in that the mass concentration of the PVA aqueous solution in step seven is 5% to 20%, and the volume of the PVA aqueous solution is 2 to 3 of the mass of the SiO2 nanoparticles loaded with aPD-1, wherein the unit of volume is μL and the unit of mass is μg; the mass concentration of the TSPBA solution is 1% to 5%, and the volume ratio of the PVA aqueous solution dispersed with nanoparticles and the TSPBA solution during pouring is (1 to 3): (1 to 4).

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

[0024] 1. The oxygen-deficient engineered self-oxygenating enzymatic cascade reaction nanoplatform MRuGH of the present invention can generate abundant ROS through SDT and CDT mediated by it under ultrasound irradiation and tumor glucose activation.

[0025] 2. The core-shell structure microneedle of the present invention can programmatically release the enzymatic cascade reaction nanoplatform ROS generator MRuGH and ICB antibody, protect the antibody activity, and control its programmed immunotherapy for malignant tumors in time and space, significantly improving the sensitivity of ICB treatment.

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The scanning electron microscope images of the MXene multilayer nanosheets prepared in Example 1 of the present invention, the transmission electron microscope images and element distribution diagrams of the few-layer MXene nanosheets, RuO2, MXene@RuO2 nanosheets and MXene@RuO2 nanosheets grafted with GOx and HbO2.

[0028] Figure 2 This is the XPS spectrum of the MXene@RuO2 nanosheets prepared in Example 1 of the present invention.

[0029] Figure 3 Schematic diagram of the structure of the core-shell structure microneedle prepared in Example 1 of the present invention, needle tip morphology and solubility test; HM gel backing structure characterization, intracellular antioxidant, L929 cell proliferation and migration promotion ability.

[0030] Figure 4 The killing ability of nanozymes on melanoma cells, changes in mitochondrial membrane potential, production of ROS and ability to alleviate hypoxia under different treatments. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0032] The needle tip of a core-shell structure microneedle for programmed co-delivery of an oxygen vacancy engineered self-oxygen supply nanoplatform and an ICB antibody of the present invention comprises a shell layer and a core layer located in the shell layer, wherein the shell layer is a rapidly soluble PVA shell layer, which is made by dispersing MXene@RuO2 nanosheets grafted with GOx and HbO2 in a PVA solution and then casting in a PDMS mold and drying; the core layer is a ROS-responsive core layer, which is made by dispersing SiO2 nanoparticles loaded with aPD-1 in a PVA solution and casting together with a TSPBA solution in the rapidly soluble PVA shell layer; the core-shell structure microneedle regulates the degradation rate of the core layer in response to ROS and the SiO2 in response to the tumor microenvironment with high expression of glutathione, so that the core-shell structure microneedle can programmed co-deliver an oxygen vacancy engineered self-oxygen supply nanoplatform and an ICB antibody, and can generate abundant ROS through SDT and CDT under the stimulation of ultrasound and the activation of glucose in the tumor microenvironment, thereby reshaping the immune microenvironment.

[0033] Example 1

[0034] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0035] Step 1: etching the blocky titanium aluminum carbide compound (Ti3AlC2) with hydrofluoric acid for 72 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide (TMAOH) solution for 48 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0036] Step 2: Ultrasonically disperse 0.1 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 3 mL of a 10 mg / mL RuCl3 aqueous solution, and then drop 0.6 mL of a 1 M NaOH solution, stir the reaction for 6 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0037] Step 3: Ultrasonic disperse 20 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 20 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 36 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0038] Step 4: Ultrasonic disperse 20 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.86 g of N-hydroxysuccinimide (NHS), activate for 20 min, add 30 mg of GOx and 30 mg of HbO2, react in an ice bath for 24 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0039] Step 5: Evenly disperse 20 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 in 20 mL of a 15% PVA aqueous solution, then cast it in a PDMS mold and dry it to obtain a rapidly soluble PVA shell layer;

[0040] Step 6, 3 mL of polyethylene glycol octylphenyl ether (Triton X-100), 3 mL of n-hexanol and 6 mL of cyclohexane were mixed to obtain an organic solution; 60 μg of aPD-1, 60 μL of tetraethyl orthosilicate (TEOS) and 100 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 70 μL of 30% ammonia water was added, and 30 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0041] Step 7. Ultrasonically disperse 60 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step 6 in 150 μL of a 15% PVA aqueous solution. Take 150 μL of the PVA aqueous solution with dispersed nanoparticles and 50 μL of a 2% TSPBA solution and pour them into the rapidly soluble PVA shell layer obtained in step 5. Dry naturally to obtain a core-shell structure microneedle.

[0042] Example 2

[0043] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0044] Step 1: etching the blocky titanium aluminum carbide compound with hydrofluoric acid for 48 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 96 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0045] Step 2: Ultrasonically disperse 0.05 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 1 mL of a 20 mg / mL RuCl3 aqueous solution, and then drop 0.3 mL of a 1 M NaOH solution, stir the reaction for 4 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0046] Step 3: Ultrasonic disperse 20 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 15 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 16 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0047] Step 4: Ultrasonic disperse 40 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 1.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.0 g of N-hydroxysuccinimide (NHS), activate for 25 min, add 12 mg of GOx and 12 mg of HbO2, react in an ice bath for 48 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0048] Step 5: 40 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 were uniformly dispersed in 20 mL of a 15% PVA aqueous solution, then cast in a PDMS mold and dried to obtain a rapidly soluble PVA shell layer;

[0049] Step 6, 2 mL of polyethylene glycol octylphenyl ether (Triton X-100), 3 mL of n-hexanol and 10 mL of cyclohexane were mixed to obtain an organic solution; 50 μg of aPD-1, 25 μL of tetraethyl orthosilicate (TEOS) and 150 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 50 μL of 30% ammonia water was added, and 40 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0050] Step 7. Ultrasonically disperse 50 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step 6 in 150 μL of a 20% PVA aqueous solution. Take 40 μL of the PVA aqueous solution with dispersed nanoparticles and 160 μL of a 4% TSPBA solution and pour them into the rapidly soluble PVA shell layer obtained in step 5. Dry naturally to obtain a core-shell structure microneedle.

[0051] Example 3

[0052] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0053] Step 1: etching the blocky titanium aluminum carbide compound with hydrofluoric acid for 96 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 96 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0054] Step 2: Ultrasonically disperse 0.1 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 3 mL of a 5 mg / mL RuCl3 aqueous solution, and then drop 0.6 mL of a 0.5 M NaOH solution, stir the reaction for 5 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0055] Step 3: Ultrasonic disperse 30 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 60 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 24 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0056] Step 4: Ultrasonic disperse 30 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 1.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.46 g of N-hydroxysuccinimide (NHS), activate for 30 min, add 30 mg of GOx and 30 mg of HbO2, react in an ice bath for 24 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0057] Step 5: 30 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 were uniformly dispersed in 15 mL of a 12% PVA aqueous solution, then cast in a PDMS mold and dried to obtain a rapidly soluble PVA shell layer;

[0058] Step 6, 0.9 mL of polyethylene glycol octylphenyl ether (Triton X-100), 1 mL of n-hexanol and 5 mL of cyclohexane were mixed to obtain an organic solution; 30 μg of aPD-1, 45 μL of tetraethyl orthosilicate (TEOS) and 30 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 30 μL of 30% ammonia water was added, and 30 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0059] Step seven, ultrasonically uniformly disperse 30 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step six in 60 μL of a 15% PVA aqueous solution, take 60 μL of the PVA aqueous solution dispersed with the nanoparticles and 60 μL of a 2% TSPBA solution and pour them together in the rapidly soluble PVA shell layer obtained in step five, dry naturally, and obtain a core-shell structure microneedle.

[0060] Example 4

[0061] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0062] Step 1: etching the blocky titanium aluminum carbide compound with hydrofluoric acid for 12 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 12 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0063] Step 2: Ultrasonically disperse 0.1 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 3 mL of 15 mg / mL RuCl3 aqueous solution, and then drop 0.3 mL of 3 M NaOH solution, stir the reaction for 3 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0064] Step 3: Ultrasonic disperse 25 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 20 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 1 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0065] Step 4: Ultrasonic disperse 50 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 1.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.7 g of N-hydroxysuccinimide (NHS), activate for 50 min, add 40 mg of GOx and 40 mg of HbO2, react in an ice bath for 72 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0066] Step 5: Evenly disperse 50 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 in 200 mL of a 5% PVA aqueous solution, then cast it in a PDMS mold and dry it to obtain a rapidly soluble PVA shell layer;

[0067] Step 6, 3 mL of polyethylene glycol octylphenyl ether (Triton X-100), 3 mL of n-hexanol and 18 mL of cyclohexane were mixed to obtain an organic solution; 60 μg of aPD-1, 60 μL of tetraethyl orthosilicate (TEOS) and 100 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 120 μL of 30% ammonia water was added, and 30 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0068] Step 7. Ultrasonically disperse 60 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step 6 in 120 μL of a 13% PVA aqueous solution. Take 80 μL of the PVA aqueous solution with dispersed nanoparticles and 120 μL of a 5% TSPBA solution and pour them into the rapidly soluble PVA shell layer obtained in step 5. Dry naturally to obtain a core-shell structure microneedle.

[0069] Example 5

[0070] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0071] Step 1: etching a block of titanium aluminum carbide compound (Ti3AlC2) with hydrofluoric acid for 72 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 72 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0072] Step 2: Ultrasonically disperse 0.1 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 3 mL of a 5 mg / mL RuCl3 aqueous solution, and then drop 0.5 mL of a 1 M NaOH solution, stir the reaction for 1 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0073] Step 3: Ultrasonic disperse 50 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 15 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 48 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0074] Step 4: Ultrasonic disperse 50 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 2.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 2.12 g of N-hydroxysuccinimide (NHS), activate for 20 min, add 20 mg of GOx and 20 mg of HbO2, react in an ice bath for 24 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0075] Step 5: 40 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 are uniformly dispersed in 200 mL of a 20% PVA aqueous solution, then cast in a PDMS mold and dried to obtain a rapidly soluble PVA shell layer;

[0076] Step 6, 3 mL of polyethylene glycol octylphenyl ether (Triton X-100), 3 mL of n-hexanol and 10 mL of cyclohexane were mixed to obtain an organic solution; 70 μg of aPD-1, 60 μL of tetraethyl orthosilicate (TEOS) and 100 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 100 μL of 30% ammonia water was added, and 40 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0077] Step seven, ultrasonically disperse 70 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step six in 200 μL of 10% PVA aqueous solution, take 100 μL of PVA aqueous solution dispersed with nanosheets and 100 μL of TSPBA solution with a mass concentration of 1%, and pour them together in the rapidly soluble PVA shell layer obtained in step five, dry naturally, and obtain core-shell structure microneedles.

[0078] Example 6

[0079] The method for preparing the core-shell structure microneedle of this embodiment specifically comprises the following steps:

[0080] Step 1: etching a block of titanium aluminum carbide compound (Ti3AlC2) with hydrofluoric acid for 72 hours, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution for 48 hours, centrifuging and drying to obtain a few-layer MXene nanosheets;

[0081] Step 2: Ultrasonically disperse 0.1 g of the few-layer MXene nanosheets described in step 1 in 15 mL of water, then add 4 mL of a 20 mg / mL RuCl3 aqueous solution, and then drop 0.6 mL of a 1 M NaOH solution, stir the reaction for 10 h, and centrifuge to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets;

[0082] Step 3: Ultrasonic disperse 20 mg of the MXene@RuO2 nanosheets obtained in step 2 in water, add 60 mg of amino polyethylene glycol thiol (NH2-PEG-SH), stir for 24 h, and centrifuge to obtain MXene@RuO2 nanosheets grafted with -NH2;

[0083] Step 4: Ultrasonic disperse 30 mg of the MXene@RuO2 nanosheets grafted with -NH2 obtained in step 3 in water, add 0.75 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.5 g of N-hydroxysuccinimide (NHS), activate for 300 min, add 90 mg of GOx and 90 mg of HbO2, react in an ice bath for 1 h, and then centrifuge and dry to obtain MXene@RuO2 nanosheets grafted with GOx and HbO2 (MXene@RuO2@GOx-HbO2, MRuGH);

[0084] Step 5: 30 mg of the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 are uniformly dispersed in 100 mL of a 12% PVA aqueous solution, then cast in a PDMS mold and dried to obtain a rapidly soluble PVA shell layer;

[0085] Step 6, 3 mL of polyethylene glycol octylphenyl ether (Triton X-100), 4 mL of n-hexanol and 10 mL of cyclohexane were mixed to obtain an organic solution; 90 μg of aPD-1, 60 μL of tetraethyl orthosilicate (TEOS) and 100 μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTEPDS) were mixed and shaken well, and then added to the organic solution, 150 μL of 30% ammonia water was added, and 60 mL of pure acetone was added after stirring at room temperature overnight, and the mixture was precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1;

[0086] Step 7. Ultrasonically disperse 90 μg of the aPD-1-loaded SiO2 nanoparticles obtained in step 6 in 200 μL of a 5% PVA aqueous solution. Take 50 μL of the PVA aqueous solution dispersed with nanosheets and 150 μL of a 5% TSPBA solution and pour them into the rapidly soluble PVA shell layer obtained in step 5. Dry naturally to obtain a core-shell structure microneedle.

[0087] Performance Test:

[0088] Taking the enzymatic cascade reaction nanoplatform MRuGH prepared in Example 1 as an example, according to Figure 1 As shown, Figure 1 a is a transmission electron microscope image of a few-layer MXene nanosheet. Figure 1 b, c, d, and e show the element distribution diagrams of few-layer MXenes. Figure 1 f, g are transmission electron microscopy images and lattices of RuO2 nanoparticles. Figure 1 h, i, j are the corresponding element distribution diagrams of RuO2. Figure 1 k is the MXene nanosheet loaded with RuO2 nanoparticles, Figure 1l is the interface-induced heterojunction diagram of RuO2 nanoparticles and MXene nanosheets. Figure 1 m, n, o, p are the corresponding element distributions. Figure 1 q is MXene@RuO2 nanosheets loaded with GOx and HbO2, Figure 1 r, s, t, u, v, and w show the corresponding element distribution diagrams, all of which prove the successful preparation of MXene@RuO2@GOx / HbO2.

[0089] according to Figure 2 As shown, RuO2 nanoparticles have no EPR signal, proving that no oxygen vacancies are generated. However, MXene nanosheets have weaker peaks, proving that a small amount of oxygen vacancies exist. When RuO2 nanoparticles are loaded on MXene nanosheets, the EPR signal intensity is significantly enhanced, proving that the MXene@RuO2 of the present invention has abundant oxygen vacancies (2a). Figure 2 The g value of 1.998 in b further proves the existence of oxygen vacancies. Figure 2 In the XPS spectra of MXene@RuO2 in c, the Ru3d peak moves to a lower peak position than that of RuO2, indicating that the presence of oxygen vacancies makes more Ru 4+ is reduced to a lower valence state.

[0090] according to Figure 3 As shown, the SiO2 nanoparticles of the present invention have a complete morphology (3a), 3b, c, d, and e show the element distribution diagram of SiO2. The core-shell microneedle of the present invention has a complete needle tip structure (3f), and 3g proves the core-shell structure of the microneedle. Figure 3 h is the controlled release of ICB antibody in SiO2 nanoparticles and core-shell microneedles. The core-shell microneedles can control the ICB antibody to achieve large-scale release starting at 24h. Figure 3 i is the SDS-page image of bovine serum albumin mimic protein in core-shell microneedles and SiO2 nanoparticles, indicating that core-shell microneedles can significantly protect antibody activity.

[0091] according to Figure 4 As shown, the MRuGH prepared by the present invention can cause CRT exposure and HMGB1 release, indicating that it can successfully induce immunogenic cell death (4a). Figure 4 b, c, and d are diagrams showing that MRuGH promotes the maturation of dendritic cells. MRuGH can significantly upregulate the expression of CD80 and CD86, indicating that it can promote the maturation of dendritic cells. Figure 4 e, f, and g are the secretion levels of inflammatory factors TNF-α, INF-γ, and IL-6 in the supernatant after co-culture of MRuGH and macrophages, indicating that MRuGH can successfully reprogram the phenotype of macrophages and reshape the immune microenvironment.

[0092] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A core-shell microneedle for programmed co-delivery of oxygen vacancy engineered self-oxygen supply nanoplatform and ICB antibody, characterized in that: The needle tip of the core-shell structure microneedle includes a shell layer and a core layer located in the shell layer. The shell layer is a rapidly dissolvable PVA shell layer, which is made by dispersing MXene@RuO2 nanosheets grafted with GOx and HbO2 in a PVA solution and then casting it in a PDMS mold and drying it; the core layer is a ROS-responsive core layer, which is made by dispersing SiO2 nanoparticles loaded with aPD-1 in a PVA solution and casting it together with a TSPBA solution in the rapidly dissolvable PVA shell layer.

2. The core-shell structure microneedle of a programmed co-delivery oxygen vacancy engineered self-oxygen supply nanoplatform and ICB antibody according to claim 1, characterized in that: The core-shell structure microneedle can programmatically co-deliver the self-oxygen supply nanoplatform engineered with oxygen vacancies and ICB antibodies by regulating the degradation rate of the nuclear layer in response to ROS and SiO2 in response to the tumor microenvironment with high glutathione expression. It can produce abundant ROS through SDT and CDT under the stimulation of ultrasound and the activation of glucose in the tumor microenvironment, thereby reshaping the immune microenvironment.

3. A method for preparing the core-shell structure microneedle as claimed in claim 1 or 2, characterized in that: The following steps are involved: Step 1: etching the blocky titanium aluminum carbide compound with hydrofluoric acid, then centrifuging until the pH of the supernatant is neutral, and drying to obtain titanium carbon MXene multilayer nanosheets; exfoliating the titanium carbon MXene multilayer nanosheets in a tetramethylammonium hydroxide solution, centrifuging and drying to obtain a few-layer MXene nanosheets; Step 2, ultrasonically disperse the few-layer MXene nanosheets described in step 1 in water, then add RuCl3 aqueous solution, then dropwise add NaOH solution, stir to react, centrifuge and dry to obtain RuO2-loaded MXene nanosheets, i.e., MXene@RuO2 nanosheets; Step 3, ultrasonically dispersing the MXene@RuO2 nanosheets obtained in step 2 in water, adding amino polyethylene glycol thiol, stirring, centrifugation and drying to obtain MXene@RuO2 nanosheets grafted with -NH2; Step 4: Ultrasonic dispersion of the MXene@RuO2 nanosheet grafted with -NH2 obtained in step 3 in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adding GOx and HbO2 after activation, reacting in an ice bath and centrifuging and drying to obtain MXene@RuO2 nanosheet grafted with GOx and HbO2; Step 5, the MXene@RuO2 nanosheets grafted with GOx and HbO2 obtained in step 4 are uniformly dispersed in a PVA aqueous solution, cast in a PDMS mold, and dried to obtain a rapidly soluble PVA shell layer; Step 6, polyethylene glycol octylphenyl ether, n-hexanol and cyclohexane are mixed to obtain an organic solution; aPD-1, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide are mixed and shaken well, and then added to the organic solution, and ammonia water is added. After stirring at room temperature overnight, pure acetone is added, precipitated and centrifuged to obtain SiO2 nanoparticles loaded with aPD-1; Step 7: Ultrasonically disperse the aPD-1 loaded SiO2 nanoparticles obtained in step 6 in the PVA aqueous solution, then cast them together with the TSPBA solution in the rapidly soluble PVA shell layer obtained in step 5, and dry them naturally to obtain core-shell structure microneedles.

4. The method according to claim 3, characterized in that The etching time in step 1 is 12 hours to 96 hours, and the stripping time is 12 hours to 96 hours.

5. The method according to claim 3, characterized in that: The mass concentration of the RuCl3 aqueous solution in step 2 is 5 mg / mL to 20 mg / mL, and the volume of the RuCl3 aqueous solution is 20 to 40 times the mass of the few-layer MXene nanosheets, where the unit of volume is mL and the unit of mass is g; The concentration of the NaOH solution is 0.5M to 3M, and the volume of the NaOH solution is 3 to 6 times the mass of the few-layer MXene nanosheets, wherein the unit of volume is mL and the unit of mass is g; the stirring reaction time is 1h to 10h.

6. The method according to claim 3, characterized in that The mass of the aminopolyethylene glycol thiol group in step 3 is 0.3 to 3 times the mass of the MXene@RuO2 nanosheets, and the stirring time is 1 h to 48 h.

7. The method according to claim 3, characterized in that The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in step 4 is (0.5-2):1, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 30-60 times the mass of the MXene@RuO2 nanosheet grafted with -NH2, and the activation time is 20min-300min; the mass ratio of GOx and HbO2 is 1:1, the mass of GOx is 0.3-3 times the mass of the MXene@RuO2 nanosheet grafted with -NH2, and the ice bath reaction time is 1h-72h.

8. The method according to claim 3, characterized in that The mass concentration of the PVA aqueous solution in step five is 5% to 20%, and the ratio of the volume of the PVA aqueous solution to the mass of the MXene@RuO2 nanosheets grafted with GOx and HbO2 is (0.5 to 5):1, where the unit of volume is mL and the unit of mass is mg.

9. The method according to claim 3, characterized in that: In step six, the volume of n-hexanol is 1 to 1.5 times the volume of polyethylene glycol octyl phenyl ether, and the volume of cyclohexane is 2 to 6 times the volume of polyethylene glycol octyl phenyl ether; the volume of tetraethyl orthosilicate is 0.5 to 1.5 times the mass of aPD-1, the volume of bis-[3-(triethoxysilyl)propyl]-disulfide is 1 to 3 times the mass of aPD-1, the volume of polyethylene glycol octyl phenyl ether is 30 to 50 times the mass of aPD-1, the concentration of the ammonia water is 30%, the volume of the ammonia water is 1 to 2 times the mass of aPD-1, and the volume of the pure acetone is 500 to 1000 times the mass of aPD-1, wherein the unit of volume is μL, and the unit of mass is μg.

10. The method according to claim 3, characterized in that: The mass concentration of the PVA aqueous solution in step seven is 5% to 20%, and the volume of the PVA aqueous solution is 2 to 3 of the mass of the SiO2 nanoparticles loaded with aPD-1, where the unit of volume is μL and the unit of mass is μg; the mass concentration of the TSPBA solution is 1% to 5%, and the volume ratio of the PVA aqueous solution dispersed with nanoparticles and the TSPBA solution during pouring is (1 to 3): (1 to 4).

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