Preparation method and application of manganese tetraoxide / GSDME complex

Abstract

The present invention relates to a preparation method of a manganese tetraoxide / GSDME complex and its application, which can effectively solve the problem of tumor cell re-proliferation and normal tissue damage after radiotherapy. The technical solution is to firstly prepare a manganese tetraoxide / GSDME complex in the presence of Mn 3 O 4 The surface of the nanoparticles is modified with GSDME protein, and a manganese tetraoxide / GSDME complex is prepared by cross-linking tannic acid with the GSDME protein. The preparation method of the invention is stable and reliable, and is easy to operate. 3 O 4 The / GSDME complex has the advantages of good biocompatibility and low toxicity and side effects. It can achieve efficient intracellular delivery of GSDME protein, activate the Caspase-3 / GSDME signaling pathway, convert radiotherapy-induced tumor cell apoptosis into pyroptosis to inhibit tumor regeneration; and utilize the difference in intracellular GSH content between tumor cells and normal cells to inhibit the expression of Mn in tumor cells. 3 O 4 It can enhance radiosensitization by downregulating GSH, while Mn 3 O 4 It is an innovation in tumor radiotherapy drugs to exert its nanozyme activity to eliminate ROS produced by radiotherapy and achieve radiation protection for normal tissues.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, in particular to a preparation method of a manganese tetraoxide / GSDME complex and application thereof. Background Art

[0002] Cancer is the leading cause of death. Since 2000, the incidence and mortality of cancer in China have increased year by year, seriously threatening human health and causing a huge economic burden on society. Radiotherapy (RT) is a major way to treat malignant tumors. More than 50% of cancer patients receive radiotherapy clinically. From a molecular perspective, radiotherapy causes direct or reactive oxygen species (ROS)-dependent damage to DNA, ultimately leading to cell death. In order to reduce damage to normal tissues during radiotherapy, low-dose fractionation therapy is often used to allow normal tissues to recover from sublethal radiation damage and allow surviving cells to repopulate in normal tissues. However, surviving tumor cells will also proliferate during the treatment interval, and tumor cell reproliferation is considered to be the main cause of tumor recurrence and treatment failure after radiotherapy. Selectively inhibiting tumor cell reproliferation can help improve the therapeutic effect of radiotherapy.

[0003] It has been reported that dying tumor cells can generate powerful growth-stimulating signals to promote the proliferation of nearby surviving tumor cells. As the executioner in the process of cell death by apoptosis, cysteine-specific protease-3 (Caspase-3) is closely related to tumor re-growth. Caspase-3 releases growth-promoting signaling factors PGE2 and VEGF-A through the Caspase-3 / iPLA2 / AA / PGE2 and Caspase-3 / PKCd / Akt / VEGF-A signaling pathways, strongly stimulating the proliferation of tumor cells. Inhibition of Caspase 3 can improve the efficacy of tumor radiotherapy. Both apoptosis and pyroptosis are programmed cell death pathways that are dependent on Caspase-3. When the tumor suppressor gene Gasdermin E (GSDME) is highly expressed, active Caspase-3 cleaves it, releasing the N-terminal domain to perforate the cell membrane, leading to cell pyroptosis. When the expression level of GSDME is low, it leads to the classic death pathway of tumor cells, namely apoptosis. Therefore, increasing the GSDME content in tumor cells is expected to activate the Caspase-3 / GSDME signaling pathway, convert radiotherapy-mediated cell apoptosis into pyroptosis, and inhibit tumor re-growth.

[0004] Under the premise of ensuring that radiotherapy is effective in treating tumors, it is urgent to prevent radiation-induced normal tissue damage. Manganese tetraoxide nanoparticles (Mn3O4) have the activity of multiple biological enzymes, such as superoxide dismutase, catalase, etc., which can effectively remove multiple reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide and hydroxyl free radicals. Moreover, Mn3O4 can react with glutathione (GSH) highly expressed in tumor cells, reduce GSH levels and increase ROS content. Therefore, Mn3O4 can be used as a radiotherapy protective agent to remove ROS induced by high-dose X-ray radiation in normal tissues to protect normal tissues, while in tumor cells, it can increase ROS content by downregulating GSH levels to improve the efficacy of radiotherapy.

[0005] In order to achieve efficient cytoplasmic delivery of GSDME, a Mn3O4 / GSDME complex was prepared by self-assembly of GSDME and polyphenols (tannic acid) on Mn3O4. The Mn3O4 / GSDME complex can respond to the acidic environment of the lysosome and degrade and escape the lysosome to release GSDME and Mn3O4 into the cytoplasm. In normal cells with low GSH content, Mn3O4 exerts its nanozyme activity to remove ROS generated by radiotherapy in normal cells, reduce DNA damage, and protect normal tissues; in tumor cells with high GSH content, Mn3O4 is degraded by high levels of GSH, its nanozyme activity is lost, GSH content is downregulated, intracellular ROS content is increased, and the effect of tumor radiotherapy is enhanced. More importantly, intracellular GSDME protein can induce radiotherapy-mediated cell apoptosis to pyroptosis and inhibit Caspase-3-mediated tumor cell repopulation. There are no related reports so far. Summary of the invention

[0006] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a method for preparing a manganese tetraoxide / GSDME complex and its application, which can effectively solve the problems of tumor cell reproliferation and normal tissue damage after radiotherapy.

[0007] The technical solution provided by the present invention is that the preparation method of the complex is to first modify the GSDME protein on the surface of Mn3O4 nanoparticles, and prepare the manganese tetraoxide / GSDME complex through the cross-linking effect of tannic acid and GSDME protein, which specifically includes the following steps:

[0008] 1) Preparation of Mn3O4 nanoparticles: 1.25-5 g of manganese acetate tetrahydrate was dissolved in 35-140 mL of N, N-dimethylformamide, and then transferred to a reactor, heated at 160 °C for 6-10 h, cooled naturally to room temperature, and then centrifuged at 5000-12000 rpm for 5-10 min to collect the product, washed three times with anhydrous ethanol, and dried at 40-60 °C for 10-15 h to obtain Mn3O4 nanoparticles;

[0009] 2) Preparation of GSDME-modified Mn3O4: Dissolve 2.5-7.5 mg Mn3O4 in 1-3 mL ultrapure water, then dissolve 15-45 mg GSDME in 250-750 μL ultrapure water, mix Mn3O4 and GSDME by ultrasonic for 2 min, transfer to a 10-20 mL round-bottom flask, and react in an oil bath at 37 °C for 8-12 h. After the reaction is completed, centrifuge at 8000-12000 rpm for 10-20 min to collect the product, wash it three times with ultrapure water to remove the unreacted GSDME protein, and obtain GSDME-modified Mn3O4;

[0010] 3) Preparation of Mn3O4 / GSDME complex: resuspend the GSDME-modified Mn3O4 prepared in step 2) in 1-3 mL ultrapure water, and dissolve 4-12 mg tannic acid in 25-75 μL ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 10-20 mL round-bottom flask and react in an oil bath at 37 °C for 3-6 h. After the reaction is completed, collect the product by centrifugation at 8000-12000 rpm for 10-20 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain a Mn3O4 / GSDME complex.

[0011] Furthermore, in the step 1), the particle size of the Mn3O4 nanoparticles is 5 to 15 nm, and in the step 3), the particle size of the Mn3O4 / GSDME complex is 100 to 200 nm.

[0012] Application of the Mn3O4 / GSDME complex prepared by the method of the present invention in the preparation of anti-tumor drug injections.

[0013] Application of the Mn3O4 / GSDME complex prepared by the method of the present invention in the preparation of radiotherapy protective agents.

[0014] The Mn3O4 / GSDME complex prepared by the method of the present invention is used in the preparation of tumor radiotherapy drugs that enhance tumor radiosensitivity by down-regulating the intracellular GSH content of tumor cells.

[0015] The invention discloses an application of the Mn3O4 / GSDME complex prepared by the method of the invention in preparing a drug for inhibiting tumor cell re-proliferation.

[0016] The Mn3O4 / GSDME complex prepared by the method of the present invention is used in the preparation of a combined immunotherapy drug for inducing tumor cell pyroptosis.

[0017] The preparation method of the invention is stable and reliable, and is easy to operate. The prepared Mn3O4 / GSDME complex has the advantages of good biocompatibility, small toxic side effects, etc., can realize efficient intracellular delivery of GSDME protein, activate Caspase-3 / GSDME signaling pathway, and convert radiotherapy-induced tumor cell apoptosis into pyroptosis to inhibit tumor re-growth; and by utilizing the difference in intracellular GSH content between tumor cells and normal cells, Mn3O4 in tumor cells can enhance radiotherapy sensitization by down-regulating GSH, while Mn3O4 in normal cells can exert its nanozyme activity to clear ROS generated by radiotherapy, thereby realizing radiation protection of normal tissues. The invention is an innovation in tumor radiotherapy drugs, and has huge social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a transmission electron microscope image of Mn3O4 and Mn3O4 / GSDME composite of the present invention.

[0019] Figure 2 It is the potential diagram of Mn3O4 and Mn3O4 / GSDME complex of the present invention.

[0020] Figure 3 These are transmission electron micrographs of Mn3O4 treated with different concentrations of GSH according to the present invention.

[0021] Figure 4 This is a diagram of the conversion of GSH to GSSG after treatment with different concentrations of Mn3O4 in the present invention.

[0022] Figure 5 Fluorescence imaging of intracellular ROS in 4T1 cells after different treatments of the present invention, scale bar: 50 μm.

[0023] Figure 6 Fluorescence imaging of intracellular ROS in Hs578Bst cells after different treatments of the present invention, scale bar: 50 μm.

[0024] Figure 7 These are representative photos of 4T1 cells of the present invention after different treatments. Arrows indicate pyroptotic cells. Scale bar: 10 μm.

[0025] Figure 8 This is a graph showing the levels of PGE2 and VEGF-A secreted in the supernatant of 4T1 cells of the present invention after different treatments. DETAILED DESCRIPTION

[0026] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0027] Example 1

[0028] The present invention, in its specific implementation, specifically comprises the following steps:

[0029] 1) Preparation of Mn3O4 nanoparticles: 1.25 g of manganese acetate tetrahydrate was dissolved in 35 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, centrifuged at 5000 rpm for 10 min to collect the product, washed three times with anhydrous ethanol, and dried at 40 °C for 15 h to obtain Mn3O4 nanoparticles;

[0030] 2) Preparation of GSDME-modified Mn3O4: 2.5 mg Mn3O4 was dissolved in 1 mL ultrapure water, and then 15 mg GSDME was dissolved in 250 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 10 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 8 h. After the reaction was completed, the product was collected by centrifugation at 8000 rpm for 20 min, and washed three times with ultrapure water to remove the unreacted GSDME protein to obtain GSDME-modified Mn3O4.

[0031] 3) Preparation of Mn3O4 / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 1 mL of ultrapure water, and dissolve 4 mg of tannic acid in 25 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 10 mL round-bottom flask and react in an oil bath at 37 °C for 4 h. After the reaction is completed, collect the product by centrifugation at 8000 rpm for 20 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain Mn3O4 / GSDME complex.

[0032] Example 2

[0033] The present invention, in its specific implementation, specifically comprises the following steps:

[0034] 1) Preparation of Mn3O4 nanoparticles: 2.5 g of manganese acetate tetrahydrate was dissolved in 70 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, and centrifuged at 12000 rpm for 5 min to collect the product, washed three times with anhydrous ethanol, and dried at 50 °C for 12 h to obtain Mn3O4 nanoparticles;

[0035] 2) Preparation of GSDME-modified Mn3O4: 5 mg Mn3O4 was dissolved in 2 mL ultrapure water, and then 30 mg GSDME was dissolved in 500 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 10 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 10 h. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 min, and washed three times with ultrapure water to remove unreacted GSDME protein to obtain GSDME-modified Mn3O4.

[0036] 3) Preparation of Mn3O4 / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 2 mL of ultrapure water, and dissolve 8 mg of tannic acid in 50 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 10 mL round-bottom flask and react in an oil bath at 37 °C for 3 h. After the reaction is completed, collect the product by centrifugation at 10,000 rpm for 15 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain a Mn3O4 / GSDME complex.

[0037] Example 3

[0038] The present invention, in its specific implementation, specifically comprises the following steps:

[0039] 1) Preparation of Mn3O4 nanoparticles: 5 g of manganese acetate tetrahydrate was dissolved in 140 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, and centrifuged at 12000 rpm for 10 min to collect the product, washed three times with anhydrous ethanol, and dried at 60 °C for 10 h to obtain Mn3O4 nanoparticles;

[0040] 2) Preparation of GSDME-modified Mn3O4: 7.5 mg Mn3O4 was dissolved in 3 mL ultrapure water, and then 45 mg GSDME was dissolved in 750 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 20 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 12 h. After the reaction was completed, the product was collected by centrifugation at 12000 rpm for 10 min, and washed three times with ultrapure water to remove unreacted GSDME protein to obtain GSDME-modified Mn3O4.

[0041] 3) Preparation of Mn3O4 / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 3 mL of ultrapure water, and dissolve 12 mg of tannic acid in 75 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 20 mL round-bottom flask and react in an oil bath at 37 °C for 6 h. After the reaction is completed, collect the product by centrifugation at 12,000 rpm for 10 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain a Mn3O4 / GSDME complex.

[0042] The preparation method of the present invention is simple, and the nanozyme activity of Mn3O4 is used to remove ROS in normal cells during radiotherapy to protect normal cells, and at the same time, the GSH of tumor cells is reduced to increase the ROS content to enhance the killing efficiency of tumor cells; and the transformation of tumor cells from apoptosis to pyroptosis can be achieved by increasing the intracellular GSDME content, thereby inhibiting the re-proliferation of tumor cells after radiotherapy. After repeated tests, consistent results were obtained. Taking Example 1 as an example, the relevant test data are as follows:

[0043] 1. Characterization of Mn3O4 and Mn3O4 / GSDME composites by transmission electron microscopy:

[0044] Mn3O4 and Mn3O4 / GSDME complex were dissolved in ultrapure water to prepare 0.1 mg / mL solutions, and then 10 μL was dropped on the copper grid. After the liquid evaporated, this operation was repeated 3 times and the morphology was observed by transmission electron microscopy. The results are as follows: Figure 1 shown.

[0045] From the transmission electron microscope image, it can be observed that the size of Mn3O4 nanoparticles is relatively uniform, with a spherical structure and a particle size of about 10 nm; the particle size of the Mn3O4 / GSDME complex is significantly increased, and its particle size is about 180 nm, proving the successful preparation of the Mn3O4 / GSDME complex.

[0046] 2. Determination of the potential of Mn3O4 and Mn3O4 / GSDME complexes:

[0047] Take appropriate amount of Mn3O4 and Mn3O4 / GSDME complex and disperse them in water. Use laser nanoparticle size analyzer to measure the potential. Figure 2 shown.

[0048] The potential of Mn3O4 nanoparticles is about -5 mV. After modification with GSDME, the potential of the Mn3O4 / GSDME complex is reduced to -14 mV, which proves the successful preparation of the Mn3O4 / GSDME complex.

[0049] 3. GSH concentration-dependent in vitro degradation of Mn3O4:

[0050] 1 mg of Mn3O4 was weighed and dissolved in 1 mL of ultrapure water. Then different amounts of GSH were added to the solution. The final concentrations of GSH were 0 mM, 1 mM, and 10 mM, respectively. After 6 h of reaction, the morphology of Mn3O4 particles was observed using a transmission electron microscope. The results are shown in Figure 2. Figure 3 shown.

[0051] It was observed from the transmission electron microscopy images that under the action of 1 mM GSH, the Mn3O4 particles were partially decomposed and the particle size was reduced; under the action of 10 mM GSH, there were no visible particles in the field of view and the Mn3O4 particles were completely degraded. The above results show that Mn3O4 can be degraded by GSH.

[0052] IV. Mn3O4 concentration-dependent GSH content reduction experiment:

[0053] Different amounts of Mn3O4 particles were added to the 10 mM GSH solution. The final concentrations of Mn3O4 were 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL and 80 μg / mL, respectively. After 6 h of reaction, the solution was centrifuged at 12000 rpm for 10 min. The supernatant was collected and the GSSG and GSH contents in the sample were determined using the GSH and GSSG detection kit. The results are shown in Figure 4 shown.

[0054] It was found that with the increase of Mn3O4 concentration, the GSSG content gradually increased, indicating that Mn3O4 can oxidize GSH to GSSG and reduce the GSH level.

[0055] 5. Investigate the effect of Mn3O4 on the intracellular ROS content of normal cells and tumor cells undergoing radiotherapy:

[0056] 4T1 cells and Hs578Bst cells were passaged into six-well plates, and the cells were cultured overnight and incubated with Mn3O4-containing medium for 6 h, where the Mn3O4 concentration was 10 μg / mL. After the drug incubation, the cells were washed 3 times with PBS, and fresh medium was added before X-ray irradiation (6 Gy). After the cells were cultured for 24 h, 1 mL of DCFH-DA (10 μM) probe was added and incubated with the cells for 20 min. After washing three times with medium, 1 mL of fresh medium was added and the cells were observed under a laser confocal microscope.

[0057] like Figure 5 As shown in Figure 2, compared with the 4T1 cells in the radiotherapy group alone, the intracellular fluorescence signal of the 4T1 cells receiving radiotherapy was significantly enhanced after 6 h of Mn3O4 pretreatment, indicating that the ROS content increased. Figure 6 As shown, after radiotherapy, the intracellular ROS content of Hs578Bst cells increased significantly; the intracellular fluorescence signal of cells in the Mn3O4+ radiotherapy group was significantly weakened, indicating that Mn3O4 can effectively remove ROS generated by radiotherapy, thereby protecting normal cells.

[0058] 6. Laser confocal microscopy to observe cell morphology:

[0059] 4T1 cells were passaged into culture dishes, and various preparations were added to the cells for 6 h after overnight culture, with a Mn3O4 concentration of 10 μg / mL. After the drug incubation, the cells were washed three times with PBS, and fresh culture medium was added before X-ray irradiation (6 Gy). After the radiotherapy, the cells were placed in an incubator for another 48 h and then observed under a laser confocal microscope.

[0060] like Figure 7 As shown in the figure, after 4T1 cells were treated with Mn3O4 / GSDME and radiotherapy, the cells showed pyroptosis characteristics with cell membrane expansion under the microscope; while 4T1 cells treated with Mn3O4 and radiotherapy only showed apoptosis characteristics with cell membrane shrinkage and the production of apoptotic bodies, indicating that GSDME can convert radiotherapy-induced cell apoptosis into pyroptosis.

[0061] 7. Experiment on the determination of the content of growth-promoting signal factors PGE2 and VEGF-A by ELISA:

[0062] 4T1 cells were passaged into six-well plates, and various preparations were added to the cells for 6 h after overnight culture, with a Mn3O4 concentration of 10 μg / mL. After the drug incubation, the cells were washed three times with PBS, and fresh culture medium was added before X-ray irradiation (6 Gy). After the radiotherapy, the cells were placed in an incubator and cultured for 48 h, and then the cell supernatant was collected and centrifuged at 2000 rpm for 20 min to carefully collect the supernatant. The PGE2 and VEGF-A contents of each sample were detected by mouse prostaglandin E2 (PGE2) enzyme-linked immunosorbent assay (ELISA) kit and mouse vascular endothelial growth factor A (VEGF-A) enzyme-linked immunosorbent assay (ELISA) kit.

[0063] like Figure 8 As shown in the figure, compared with untreated cells, the levels of growth-promoting signal factors PGE2 and VEGF-A in 4T1 cells increased significantly after radiotherapy, indicating that radiotherapy can induce tumor cells to release growth-promoting signal factors; after GSDME treatment, the secretion of PGE2 and VEGF-A by 4T1 cells decreased significantly, which is beneficial to inhibit the re-proliferation of tumor cells after radiotherapy.

[0064] It can be seen from the above experiments that compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The Mn3O4 / GSDME complex provided by the present invention has good biocompatibility and stability, can efficiently load GSDME protein, and can achieve localized release in the cytoplasm;

[0066] (2) The Mn3O4 / GSDME complex provided by the present invention can exert multiple nanozyme activities in normal cells with low GSH content to remove ROS generated by radiotherapy; at the same time, it increases ROS production by consuming intracellular GSH in tumor cells, thereby protecting normal cells while enhancing the tumor cell killing effect and reducing the toxic side effects of radiotherapy;

[0067] (3) The Mn3O4 / GSDME complex provided by the present invention can efficiently deliver GSDME into cells, convert Caspase3-mediated cell apoptosis into cell pyroptosis, inhibit the re-proliferation of tumor cells after radiotherapy, and effectively prevent tumor recurrence after radiotherapy.

[0068] The invention is easy to operate, the method is stable and reliable, and the prepared Mn3O4 / GSDME complex can be targeted to tumor tissues through the EPR effect. In normal cells, Mn3O4 nanoparticles exert nanozyme activity, remove ROS generated by normal cells after radiotherapy, and protect normal cells; in tumor cells, Mn3O4 increases ROS content by downregulating intracellular GSH levels, thereby enhancing the radiotherapy effect. More importantly, intracellular delivery of GSDME protein inhibits Caspase-3-mediated tumor re-growth by reversing the cell apoptosis pathway, thereby improving the treatment efficiency of tumors. It is an innovation in radiotherapy drugs with huge economic and social benefits.

[0069] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for preparing a manganese tetraoxide / GSDME complex, characterized in that: The preparation method of the complex is to first modify the GSDME protein on the surface of Mn3O4 nanoparticles, and prepare the manganese tetraoxide / GSDME complex through the cross-linking effect of tannic acid and GSDME protein, which specifically includes the following steps: 1) Preparation of Mn3O4 nanoparticles: 1.25-5 g of manganese acetate tetrahydrate was dissolved in 35-140 mL of N, N-dimethylformamide, and then transferred to a reactor, heated at 160 °C for 6-10 h, cooled naturally to room temperature, and then centrifuged at 5000-12000 rpm for 5-10 min to collect the product, washed three times with anhydrous ethanol, and dried at 40-60 °C for 10-15 h to obtain Mn3O4 nanoparticles; 2) Preparation of GSDME-modified Mn3O4: Dissolve 2.5-7.5 mg Mn3O4 in 1-3 mL ultrapure water, then dissolve 15-45 mg GSDME in 250-750 μL ultrapure water, mix Mn3O4 and GSDME by ultrasonic for 2 min, transfer to a 10-20 mL round-bottom flask, react in an oil bath at 37 °C for 8-12 h, after the reaction is completed, collect the product by centrifugation at 8000-12000 rpm for 10-20 min, wash three times with ultrapure water to remove unreacted GSDME protein, and obtain GSDME-modified Mn3O4; 3) Preparation of manganese tetraoxide / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 1-3 mL ultrapure water, and dissolve 4-12 mg tannic acid in 25-75 μL ultrapure water. Ultrasonicate and mix the two for 2 min, then transfer them to a 10-20 mL round-bottom flask and react in an oil bath at 37 °C for 3-6 h. After the reaction is completed, collect the product by centrifugation at 8000-12000 rpm for 10-20 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain manganese tetraoxide / GSDME complex.

2. The method for preparing the manganese tetraoxide / GSDME complex according to claim 1, characterized in that: The following steps are involved: 1) Preparation of Mn3O4 nanoparticles: 1.25 g of manganese acetate tetrahydrate was dissolved in 35 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, centrifuged at 5000 rpm for 10 min to collect the product, washed three times with anhydrous ethanol, and dried at 40 °C for 15 h to obtain Mn3O4 nanoparticles; 2) Preparation of GSDME-modified Mn3O4: 2.5 mg Mn3O4 was dissolved in 1 mL ultrapure water, and then 15 mg GSDME was dissolved in 250 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 10 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 8 h. After the reaction was completed, the product was collected by centrifugation at 8000 rpm for 20 min, and washed three times with ultrapure water to remove the unreacted GSDME protein to obtain GSDME-modified Mn3O4. 3) Preparation of manganese tetraoxide / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 1 mL of ultrapure water, and dissolve 4 mg of tannic acid in 25 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 10 mL round-bottom flask and react in an oil bath at 37 °C for 4 h. After the reaction is completed, collect the product by centrifugation at 8000 rpm for 20 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain manganese tetraoxide / GSDME complex.

3. The method for preparing the manganese tetraoxide / GSDME complex according to claim 1, characterized in that: The following steps are involved: 1) Preparation of Mn3O4 nanoparticles: 2.5 g of manganese acetate tetrahydrate was dissolved in 70 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, and centrifuged at 12000 rpm for 5 min to collect the product, washed three times with anhydrous ethanol, and dried at 50 °C for 12 h to obtain Mn3O4 nanoparticles; 2) Preparation of GSDME-modified Mn3O4: 5 mg Mn3O4 was dissolved in 2 mL ultrapure water, and then 30 mg GSDME was dissolved in 500 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 10 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 10 h. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 min, and washed three times with ultrapure water to remove unreacted GSDME protein to obtain GSDME-modified Mn3O4. 3) Preparation of manganese tetraoxide / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 2 mL of ultrapure water, and dissolve 8 mg of tannic acid in 50 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 10 mL round-bottom flask and react in an oil bath at 37 °C for 3 h. After the reaction is completed, collect the product by centrifugation at 10,000 rpm for 15 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain manganese tetraoxide / GSDME complex.

4. The method for preparing the manganese tetraoxide / GSDME complex according to claim 1, characterized in that: The following steps are involved: 1) Preparation of Mn3O4 nanoparticles: 5 g of manganese acetate tetrahydrate was dissolved in 140 mL of N, N-dimethylformamide, then transferred to a reactor, heated at 160 °C for 8 h, cooled naturally to room temperature, and centrifuged at 12000 rpm for 10 min to collect the product, washed three times with anhydrous ethanol, and dried at 60 °C for 10 h to obtain Mn3O4 nanoparticles; 2) Preparation of GSDME-modified Mn3O4: 7.5 mg Mn3O4 was dissolved in 3 mL ultrapure water, and then 45 mg GSDME was dissolved in 750 μL ultrapure water. Mn3O4 and GSDME were ultrasonically mixed for 2 min and then transferred to a 20 mL round-bottom flask. The mixture was reacted in an oil bath at 37 °C for 12 h. After the reaction was completed, the product was collected by centrifugation at 12000 rpm for 10 min, and washed three times with ultrapure water to remove the unreacted GSDME protein to obtain GSDME-modified Mn3O4. 3) Preparation of manganese tetraoxide / GSDME complex: Resuspend the GSDME-modified Mn3O4 prepared in step 2) in 3 mL of ultrapure water, and dissolve 12 mg of tannic acid in 75 μL of ultrapure water. After ultrasonic mixing for 2 min, transfer the two to a 20 mL round-bottom flask and react in an oil bath at 37 °C for 6 h. After the reaction is completed, collect the product by centrifugation at 12,000 rpm for 10 min, and wash it three times with ultrapure water to remove unreacted tannic acid to obtain manganese tetraoxide / GSDME complex.

5. The method for preparing the manganese tetraoxide / GSDME complex according to any one of claims 1 to 4, characterized in that: The particle size of the Mn3O4 nanoparticles in step 1) is 5 to 15 nm, and the particle size of the manganese tetraoxide / GSDME complex in step 3) is 100 to 200 nm.

6. Use of the manganese tetraoxide / GSDME complex prepared by the method according to any one of claims 1 to 4 in the preparation of anti-tumor drug injections.

7. Use of the manganese tetraoxide / GSDME complex prepared by the method according to any one of claims 1 to 4 in the preparation of a radiotherapy protective agent.

8. Use of the manganese tetraoxide / GSDME complex prepared by the method according to any one of claims 1 to 4 in the preparation of a tumor radiotherapy drug that enhances tumor radiosensitization by downregulating the intracellular GSH content of tumor cells.

9. Use of the manganese tetraoxide / GSDME complex prepared by the method according to any one of claims 1 to 4 in the preparation of a drug for inhibiting tumor cell re-proliferation.

10. Use of the manganese tetraoxide / GSDME complex prepared by the method according to any one of claims 1 to 4 in the preparation of a drug for inducing tumor cell pyroptosis combined with immunotherapy.

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