Preparation method and application of self-cascading reactive oxygen amplifier CHO@Cu / His-ZIF8

Abstract

This invention discloses a method for preparing and applying a self-cascaded reactive oxygen species amplifier (ROS) CHO@Cu / His-ZIF8. Belonging to the fields of nanomaterials and tumor therapy, the preparation steps are as follows: Histidine (His), 2-methylimidazole (2-MI), zinc nitrate, copper nitrate, and cholesterol oxidase are mixed uniformly in a specified ratio. After reacting for 24 hours, the mixture is washed, dialyzed, and dried to obtain the CHO@Cu / His-ZIF8 nanozyme. In this invention, the cholesterol oxidase activity, peroxidase activity (POD), and catalase activity of the CHO@Cu / His-ZIF8 nanozyme can form a cascade reaction, causing tumor cells to produce highly efficient reactive oxygen species and consume intratumoral cholesterol, thus inhibiting tumor cell growth and metastasis. Furthermore, the H2O2 produced by CHO-catalyzed cholesterol can be absorbed by Cu... 2+ The mediated Fenton-like reaction transforms into highly toxic hydroxyl radicals, amplifying oxidative damage to cancer cells. CHO@Cu / His-ZIF8 nanozymes exhibit tumor-specific killing effects on tumor cells, effectively accumulating at tumor sites while showing weak killing effects on normal sites, and possess good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and tumor therapy technology, and relates to a method for preparing and applying a self-cascaded reactive oxygen species amplifier (ROS) CHO@Cu / His-ZIF8 nanozyme; specifically, it relates to a method for preparing and applying a self-cascaded ROS amplifier CHO@Cu / His-ZIF8 nanozyme with multi-enzyme activity and tumor therapy effects; that is, a multifunctional self-cascaded ROS amplifier CHO@Cu / His-ZIF8 that can inhibit tumor growth and metastasis through a combination of self-enhanced chemodynamics (CDT) and cholesterol consumption, and its preparation method and application. Background Technology

[0002] Currently, tumor metastasis is the leading cause of cancer-related deaths. However, the efficacy of current treatments for tumor metastasis remains unsatisfactory. Although disrupting the tumor microenvironment can slow tumor progression, it cannot address the problem of tumor migration and invasion caused by the high motility of cancer cells. Tumor cell motility and migration are inextricably linked to the formation of lamellar pseudopodia and the integrity of lipid rafts. Lipid rafts are also a prerequisite for lamellar pseudopodia formation; when the integrity of lipid rafts is disrupted, lamellar pseudopodia formation is inhibited. Therefore, disrupting the lipid rafts of tumor cells may be an effective strategy to inhibit tumor cell metastasis. As a major component of lipid rafts, cholesterol is also overexpressed in the tumor microenvironment. It promotes cancer cell proliferation, enhances motility, induces epithelial-mesenchymal transition, and causes immunosuppression. Therefore, regulating cholesterol levels at the tumor site can not only affect lipid raft function but also effectively interfere with cancer cell proliferation and migration.

[0003] Cholesterol oxidase (CHO) is a natural enzyme with high specificity for cholesterol. It rapidly catalyzes the oxidation of cholesterol, converting it into products such as cholesterol-4-en-3-one, thereby lowering cholesterol levels in the body. However, as a natural enzyme, CHO is unstable under external environmental conditions, such as temperature and pH, and may also be affected by biodegradation or protein degradation, limiting its stability and durability in application. ZIF-8 is a metal-organic framework (MOF) composed of metal ions and organic ligands. The pore structure of ZIF-8 can be regulated according to the size and chemical properties of drugs, thereby achieving highly selective adsorption and release of drugs. It also has high thermal and chemical stability, maintaining its structural integrity in different biological environments, thus protecting the drug-loaded substance from external environmental influences. Therefore, encapsulating and mineralizing CHO with ZIF-8 helps improve its stability. However, current research still faces challenges such as low encapsulation efficiency and poor catalytic efficacy, leading to unsatisfactory therapeutic effects. Summary of the Invention

[0004] Purpose of the Invention: The purpose of this invention is to provide a method for preparing CHO@Cu / His-ZIF8, a self-cascaded reactive oxygen species amplifier, by further mineralizing cholesterol oxidase (CHO) through histidine (His)-modified Cu-ZIF-8. This method not only mimics the active site of natural peroxidase (POD) to achieve self-enhanced reactive oxygen species generation, but also converts cholesterol into H2O2. H2O2 is then converted into •OH through POD-like activity and simultaneously consumed through catalase-like activity to generate O2, promoting cholesterol consumption, degrading cholesterol to disrupt lipid rafts, and thus inhibiting migration. Another object of the present invention is to provide the application of the prepared self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 in antitumor and immunomodulatory therapy.

[0005] The technical solution of the present invention is as follows: The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 of the present invention includes the following steps: mixing the prepared ligand, metal salt and natural enzyme in a good solvent in a certain proportion and stirring, and then washing, dialysis and drying to finally obtain CHO@Cu / His-ZIF8 nanozyme.

[0006] Furthermore, the ligand is selected from one or two of 2-methylimidazole, histidine, 4-methylimidazole, imidazole, histamine, benzimidazole, and 2-ethylimidazole.

[0007] Furthermore, the metal salt is selected from one or two of zinc nitrate, copper nitrate, iron nitrate, manganese nitrate, molybdenum nitrate, and cobalt nitrate.

[0008] Furthermore, the natural enzyme is selected from one of glucose oxidase, lactate oxidase, catalase, cholesterol oxidase, or urate oxidase.

[0009] Furthermore, the molar ratio of the metal salt to the ligand is 0.2-2:1; The ratio of the natural enzyme to the sum of the masses of the metal salt and ligand is 1:5-30.

[0010] Furthermore, the stirring time is 24 hours.

[0011] Furthermore, the good solvent is one or two of N, N-dimethylformamide, methanol, and pure water.

[0012] Furthermore, the solvent used in the washing process is one or two of anhydrous ethanol, methanol, and pure water; The dialysis procedure is as follows: the washed mixed solution is placed in dialysis bags of 500 DA, 3000 DA, and 10000 DA for dialysis. The drying method is one of the following: oven drying, vacuum drying, or freeze drying.

[0013] Furthermore, during the preparation process, one of the following methods is selected for synthesis: solvothermal method, self-assembly, or biomineralization.

[0014] Furthermore, the application of a self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme prepared by the aforementioned method in anti-tumor and immunomodulatory therapy.

[0015] Furthermore, the CHO@Cu / His-ZIF8 nanozyme prepared in this invention possesses cholesterol oxidase activity, POD-like activity, and CAT-like activity. It is an artificially synthesized self-cascaded reactive oxygen species amplifier with multiple enzyme activities and exhibits extremely high affinity for the substrate H2O2. It can not only catalyze the generation of large amounts of ·OH from H2O2 in the tumor microenvironment to kill tumor cells, but also has CAT-like activity to alleviate intratumoral hypoxia and enhance the therapeutic effect of CDT. In addition, cholesterol oxidase can also consume intratumoral cholesterol, destroy the lipid rafts of tumor cells, and inhibit tumor cell metastasis. These effects can form a self-cascaded platform to induce tumor cell death and significantly enhance the effect on tumor migration.

[0016] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The CHO@Cu / His-ZIF8 nanozyme of the present invention simultaneously possesses cholesterol oxidase activity, POD-like activity, and CAT-like activity. These three enzyme activities can be cascaded into a self-reinforcing enzyme cascade reaction. First, the Cu²⁺-mediated Fenton-like reaction in the CHO@Cu / His-ZIF8 nanozyme can mimic peroxidase (POD) activity, exhibiting extremely high affinity for H₂O₂ and catalyzing the conversion of H₂O₂ into highly toxic ROS. Simultaneously, the CHO@Cu / His-ZIF8 nanozyme, by exerting CAT activity, can also generate oxygen, alleviate tumor hypoxia, and enhance the effect of CDT. In addition, the CHO@Cu / His-ZIF8 nanozyme further enhances POD activity by consuming cholesterol through cholesterol oxidase and generating H₂O₂. 2. The CHO@Cu / His-ZIF8 nanozyme of the present invention has a strong pro-tumor cell apoptosis effect on tumor cells at the cellular level through the combined action of cholesterol consumption and CDT, effectively promoting ROS accumulation and inhibiting tumor cell metastasis. 3. The CHO@Cu / His-ZIF8 nanozyme of the present invention... The CHO@Cu / His-ZIF8 nanozyme, when injected into mice via the tail vein, showed a more significant inhibitory effect on tumor growth than other control groups after a period of time, demonstrating that the CHO@Cu / His-ZIF8 nanozyme can exert a powerful anti-tumor effect through the combined action of cholesterol consumption and CDT; 4. The CHO@Cu / His-ZIF8 nanozyme of the present invention, when injected into mice via the tail vein, showed a more significant inhibitory effect on tumor metastasis than other control groups after a period of time, demonstrating that the CHO@Cu / His-ZIF8 nanozyme has a strong anti-tumor metastasis effect; 5. The CHO@Cu / His-ZIF8 nanozyme of the present invention, when injected into mice via the tail vein, showed a more significant immune recovery effect than other control groups after a period of time, demonstrating that the CHO@Cu / His-ZIF8 nanozyme has an immune recovery promoting effect; 6. The CHO@Cu / His-ZIF8 nanozyme of the present invention has good biocompatibility, and the HE staining results of the main organs in mouse experiments show that the material has no long-term toxicity to mice. Attached Figure Description

[0017] Figure 1 This is a TEM image of the CHO@Cu / His-ZIF8 nanozyme prepared in Example 1 of this invention; Figure 2 This is a particle size diagram of the CHO@Cu / His-ZIF8 nanozyme in the embodiments of the present invention; Figure 3 This is a structural diagram of the POD-like activity of CHO@Cu / His-ZIF8 nanozyme using TMB as a chromogenic substrate in an embodiment of the present invention; Figure 4This is a structural diagram of the CAT activity of CHO@Cu / His-ZIF8 nanozyme with H2O2 as substrate in an embodiment of the present invention; Figure 5 This is a schematic diagram of the ESR of the CHO@Cu / His-ZIF8 nanozyme with H2O2 as the substrate in this invention; Figure 6 This is a graph showing the relative cell viability of 4T1 cells after incubation with H2O2 and CHO@Cu / His-ZIF8 nanozyme at pH 6 in this invention. Figure 7 This is a graph showing the relative cell viability of 4T1 cells after incubation with cholesterol and CHO@Cu / His-ZIF8 nanozyme in this invention. Figure 8 This is a fluorescence image of ROS generation at the cellular level by the CHO@Cu / His-ZIF8 nanozyme, detected by the DCFH-DA probe in this invention. Figure 9 This is a schematic diagram illustrating the determination of intracellular cholesterol consumption in tumor cells by CHO@Cu / His-ZIF8 nanozyme under different conditions in this invention. Figure 10 These are fluorescence staining images of CHO@Cu / His-ZIF8 nanozymes on tumor cell actin under different conditions in this invention; Figure 11 This is a graph showing the ability of CHO@Cu / His-ZIF8 nanozyme to inhibit tumor cell migration and invasion under different conditions in this invention. Figure 12 This is a graph showing the changes in tumor volume in mice of different groups treated with CHO@Cu / His-ZIF8 nanozyme in vivo for anti-tumor therapy in this invention. Detailed Implementation

[0018] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0019] The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme of the present invention comprises the following steps: Histidine is dissolved in water and stirred for 5 minutes, then 2-methylimidazole solution dissolved in DMF is added dropwise and stirred for 10 minutes, then zinc nitrate and copper nitrate solutions dissolved in DMF are added dropwise and stirred for 15 minutes, and finally cholesterol oxidase is added and mixed in a good solvent and stirred at room temperature for 24 hours; after the reaction is completed, the mixture is washed 3 times with anhydrous ethanol and 2 times with pure water, and after dialysis and drying, CHO@Cu / His-ZIF8 nanozyme is obtained.

[0020] In the preparation process, the synthesis method is selected from one of solvothermal method, self-assembly or biomineralization. Preferably, the biomineralization method combines the compounds.

[0021] The metal salt is selected from one or two of zinc nitrate, copper nitrate, ferric nitrate, manganese nitrate, molybdenum nitrate, and cobalt nitrate; preferably, zinc nitrate and copper nitrate.

[0022] The ligand is selected from one or two of 2-methylimidazole, histidine, 4-methylimidazole, imidazole, histamine, benzimidazole, and 2-ethylimidazole; preferably, 2-methylimidazole and histidine.

[0023] Nanozymes are made to have natural enzyme-like activity by using bio-enzymes with natural enzyme activity. The natural enzyme is selected from one of glucose oxidase, lactate oxidase, catalase, cholesterol oxidase or urate oxidase, etc.; preferably, cholesterol oxidase.

[0024] The molar ratio of the metal salt to the ligand is 0.2-2:1; The ratio of the natural enzyme to the sum of the masses of the metal salt and ligand is 1:5-30; In the ligands used, the mass ratio of histidine to 2-methylimidazole is 1:1-8; preferably, it is 1:4. In addition, the mass ratio of 2-methylimidazole to a metal salt is 1:0.2-2; preferably, it is 1:0.45. The mass ratio of the two metal salts is 1:1-2, preferably 1:1.

[0025] The good solvent is one or two of N, N-dimethylformamide (DMF), methanol, and pure water, preferably DMF.

[0026] The washing solvent is one or two of anhydrous ethanol, methanol, and pure water, preferably anhydrous ethanol and pure water; The dialysis bags are available in sizes of 500 DA, 3000 DA, 10000 DA, etc., with 1000 DA being preferred. The method is as follows: the solution is placed in a dialysis bag of 500 DA, 3000 DA, 10000 DA, etc. for dialysis. The dialysis time is 8 hours to 36 hours, preferably 24 hours; The drying method is one of oven drying, vacuum drying, or freeze drying, with freeze drying being the preferred method.

[0027] The present invention also provides CHO@Cu / His-ZIF8 nanozyme prepared by the aforementioned preparation method.

[0028] The CHO@Cu / His-ZIF8 nanozyme prepared by this invention has a uniform particle size distribution and good stability.

[0029] The CHO@Cu / His-ZIF8 nanozyme prepared by this invention has peroxidase activity, catalase activity and cholesterol oxidase activity; it can generate hydroxyl radicals; and it can effectively generate ROS.

[0030] The CHO@Cu / His-ZIF8 nanozyme prepared in this invention can generate highly efficient ROS in tumor cells and consume intracellular cholesterol at the cellular level.

[0031] The application of a self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme prepared in this invention in anti-tumor therapy.

[0032] Example 1 illustrates the preparation method of CHO@Cu / His-ZIF8 nanozymes: Step (1) Dissolve histidine in water, add triethylamine, stir for 5 minutes to obtain solution A; Step (2) Add 2-methylimidazole solution dissolved in DMF dropwise to solution A, stir for 10 minutes to obtain solution B; Step (3) Add zinc nitrate and copper nitrate solutions dissolved in DMF dropwise to solution B, stir for 15 minutes, and obtain solution C; Step (4) Add cholesterol oxidase to solution C and stir at room temperature for 24 hours to obtain solution D; Step (5) Wash solution D three times with anhydrous ethanol and twice with pure water. Dialyze it with a 1000DA dialysis bag for 24 hours and freeze-dry it to obtain a loose product; namely, CHO@Cu / His-ZIF8 nanozyme.

[0033] The mass ratio of histidine to 2-methylimidazole used was 1:4, the mass ratio of 2-methylimidazole to zinc nitrate was 1:0.45, the mass ratio of zinc nitrate to copper nitrate was 1:1, and the concentration of TEA solution was 0.216 mol / L.

[0034] Example 2: Morphological characterization of CHO@Cu / His-ZIF8 nanozymes Figure 1 Transmission electron microscopy image of CHO@Cu / His-ZIF8 nanozyme; Figure 2 Particle size distribution of CHO@Cu / His-ZIF8 nanozyme; Depend on Figure 1 It can be seen that the CHO@Cu / His-ZIF8 nanozyme prepared by the above method is in granular form, with good dispersibility and uniform particle size distribution; Depend on Figure 2 The particle size of the CHO@Cu / His-ZIF8 nanozyme is approximately 100 nm.

[0035] In summary, morphology and particle size analysis using transmission electron microscopy and particle size analysis confirmed that the final product obtained was CHO@Cu / His-ZIF8 nanozyme, and that it had good dispersibility and uniform particle size distribution, indicating that the method provided by this invention can be used to prepare CHO@Cu / His-ZIF8 nanozyme.

[0036] Example 3 shows the detection of multiple enzyme activities of CHO@Cu / His-ZIF8 nanozymes. Figure 3 A schematic diagram of the POD-like activity of CHO@Cu / His-ZIF8 nanozyme with TMB as the chromogenic substrate; Figure 4 This is a schematic diagram of the CAT activity of CHO@Cu / His-ZIF8 nanozyme with H2O2 as substrate. Figure 5 A schematic diagram of the ESR of CHO@Cu / His-ZIF8 nanozyme with H2O2 as substrate; The method for detecting the POD-like activity of CHO@Cu / His-ZIF8 nanozyme is as follows: using TMB as a substrate, the POD-like activity of CHO@Cu / His-ZIF8 was measured in the presence of H2O2 and cholesterol; simply put, NaAc (pH = 4.55), TMB, CHO@Cu / His-ZIF8, H2O2 and cholesterol were mixed at room temperature, and a control group was set up. After the same time, the absorbance at 652 nm was measured simultaneously in a 96-well plate. Depend on Figure 3 It can be seen that the CHO@Cu / His-ZIF8 nanozyme can convert the colorless TMB substrate into a blue oxidized state, indicating that the CHO@Cu / His-ZIF8 nanozyme has effective POD-like activity and cholesterol oxidase activity, providing a strong basis for CDT therapy; The CAT-like activity of CHO@Cu / His-ZIF8 nanozyme was detected as follows: CAT activity of CHO@Cu / His-ZIF8 was determined at 37°C in H2O2 (0.4M) in HAc-NaAc buffer solution (pH 7.0); the oxygen content in the solution was measured using a portable dissolved oxygen meter. Depend on Figure 4 It can be seen that when the H2O2 concentration remains constant, the higher the concentration of CHO@Cu / His-ZIF8 nanozyme, the more oxygen is produced, indicating that CHO@Cu / His-ZIF8 nanozyme can react with H2O2 to produce oxygen, which may help alleviate tumor hypoxia. The method for detecting the ESR of CHO@Cu / His-ZIF8 nanozyme using H2O2 as a substrate is as follows: CHO@Cu / His-ZIF8, H2O2, and 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO) were mixed at room temperature and reacted, with DMPO (50 mmol / L) acting as a •OH scavenger. A quartz tube containing the sample was placed in the resonance chamber of an electron spin resonance (ESR) spectrometer, and the ESR spectrum of the spin adduct was subsequently acquired. Depend on Figure 5 It can be seen that the CHO@Cu / His-ZIF8 nanozyme has significant POD-like activity and can generate •OH.

[0037] In summary, the CHO@Cu / His-ZIF8 nanozyme exhibits POD-like activity, CAT-like activity, and cholesterol oxidase activity.

[0038] Example 4 demonstrates the effects of CHO@Cu / His-ZIF8 nanozyme on tumor cell viability and proliferation, and validates its cellular-level ROS production. Figure 6 The graph shows the relative cell viability of 4T1 cells after incubation with H2O2 and CHO@Cu / His-ZIF8 nanozyme at pH 6. Figure 7 The relative cell viability of 4T1 cells after incubation with cholesterol and CHO@Cu / His-ZIF8 nanozyme. Figure 8 The fluorescence image shows the generation of ROS at the cellular level by the CHO@Cu / His-ZIF8 nanozyme, as detected by the DCFH-DA probe. The cytotoxicity of CHO@Cu / His-ZIF8 nanozyme against tumor cells at the cellular level was assessed using the MTT assay. 4T1 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured until 80% confluence, and the culture medium was replaced with cholesterol, H2O2, and different concentrations of CHO@Cu / His-ZIF8 for 24 hours. Then, 10 μL of 5 mg / mL MTT solution was added to each well, and the plate was placed back into a CO2 incubator for another 4 hours. After incubation, the culture medium was gently aspirated, and 200 μL of DMSO was added to each well to dissolve the methylamine salt crystals produced by MTT reduction in the living cells. After incubation, the absorbance of each well was measured at 490 nm using a microplate reader. Depend on Figure 6 and Figure 7It was found that the cytotoxicity of different treatments on 4T1 cells was concentration-dependent; and when H2O2 and cholesterol coexisted with CHO@Cu / His-ZIF8, the cytotoxicity was significantly higher than that of CHO@Cu / His-ZIF8 alone, which proved that CHO@Cu / His-ZIF8 greatly enhanced the cytotoxicity of 4T1 cells under the combined action of cholesterol consumption and CDT. The method for detecting the ROS generation capacity of CHO@Cu / His-ZIF8 cells under different treatments at the cellular level is as follows: 4T1 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin; for fluorescence imaging, 4T1 cells (5 × 10⁶ cells per well) were... 4 Cells were seeded in 12-well plates for 24 h, and then cultured for 24 h in cholesterol, H2O2, CHO@Cu / His-ZIF8, CHO@Cu / His-ZIF8+cholesterol, and CHO@Cu / His-ZIF8+H2O2 solutions. The culture medium was then discarded, and the cells were washed with PBS. The cells were stained with DCFH-DA at 37°C in the dark for 30 min. The dye was then discarded, and the cells were washed with PBS and observed and photographed under a microscope. Depend on Figure 8 It was found that when cholesterol and H2O2 were used as substrates, the ROS generated by CHO@Cu / His-ZIF8 was more significant, indicating that CHO@Cu / His-ZIF8 can effectively inhibit tumor cells by consuming both H2O2 and cholesterol. This suggests that the CHO@Cu / His-ZIF8 nanozyme, under the combined action of cholesterol consumption and CDT, can promote the accumulation of ROS in tumor cells.

[0039] In summary, the CHO@Cu / His-ZIF8 nanozyme alleviates tumor hypoxia by consuming cholesterol and CDT in combination to generate oxygen, and generates a large amount of ROS to amplify the oxidative stress of tumors, ultimately promoting tumor cell apoptosis. Example 5 validates the effects of CHO@Cu / His-ZIF8 nanozyme on cholesterol depletion and anti-tumor cell metastasis. Figure 9 To determine the effect of CHO@Cu / His-ZIF8 nanozyme on cholesterol consumption in tumor cells under different conditions; Figure 10 Fluorescent staining of tumor cell actin by CHO@Cu / His-ZIF8 nanozymes under different conditions; Figure 11 Images showing the ability of CHO@Cu / His-ZIF8 nanozymes to induce tumor cell migration and invasion under different conditions; The method for detecting the consumption of intracellular cholesterol in tumor cells by CHO@Cu / His-ZIF8 nanozyme at the cellular level is as follows: 4T1 cells were cultured at 1×10⁶ cells per well. 6 Cells were seeded at a density of 100 cells per well in 6-well plates and cultured for 24 hours to promote cell adhesion. Subsequently, cells were treated with PBS, CHO, Cu / His-ZIF8, and CHO@Cu / His-ZIF8 for 24 hours. Cells were washed with PBS and intracellular cholesterol levels were measured using a cholesterol detection kit. Finally, the cholesterol content of each group was quantified by extrapolation to a pre-established standard curve. Depend on Figure 9 It can be seen that CHO@Cu / His-ZIF8 nanozyme can significantly reduce cholesterol levels in tumor cells, indicating that CHO@Cu / His-ZIF8 nanozyme can effectively inhibit cholesterol accumulation in tumor cells; The method for detecting the effect of CHO@Cu / His-ZIF8 on cellular actin levels under different treatments at the cellular level is as follows: 4T1 cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 12-well plates and cultured for 24 hours to promote cell adhesion. Cells were then treated with PBS, CHO, Cu / His-ZIF8, and CHO@Cu / His-ZIF8 for 24 hours. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then washed with PBS. After permeabilization with 0.5% Triton X-100 in PBS for 10 minutes, the cells were washed with PBS and incubated with phalloidin fluorescent staining solution for 30 minutes. Finally, the cells were observed and photographed using a fluorescence microscope.

[0040] Depend on Figure 10 It was found that in the control group, actin stress fibers in the cytoplasm were very prominent, and many invasive plate-like pseudopodia could be easily observed around the cells; in contrast, after treatment with CHO@Cu / His-ZIF8, the actin stress fibers in the cells became blurred and the membrane protrusions were sparse, indicating that the combined effect of cholesterol depletion and ROS production effectively inhibited the formation of invasive plate-like pseudopodia.

[0041] The method for detecting the ability of CHO@Cu / His-ZIF8 nanozyme to inhibit tumor cell migration and invasion at the cellular level is as follows: Transwell chambers containing 8μm pore size filter membranes are placed in 24-well plates. Serum-free culture medium is added to the lower chamber and pre-incubated at 37℃ for 40 min. 5 × 10⁶ cells are seeded per well in the upper chamber. 4Cells (suspended in 200 μL serum-free medium) were added to the lower chamber with 600 μL of medium containing 12% fetal bovine serum to construct a chemotactic gradient. The lower chamber medium was divided into four groups for treatment: PBS, CHO, Cu / His-ZIF8, and CHO@Cu / His-ZIF8. After the migration experiment, the migrated cells in the lower chamber were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with crystal violet. The upper chamber was rinsed with PBS, and the unmigrated cells were gently wiped with cotton swabs. The images of the migrated cells were observed under an optical microscope to assess the cell migration ability.

[0042] Depend on Figure 11 It can be seen that CHO@Cu / His-ZIF8 nanozyme can significantly inhibit the migration and invasion of tumor cells; In conclusion, the CHO@Cu / His-ZIF8 nanozyme inhibits tumor cell migration by consuming cholesterol, demonstrating its considerable potential in preventing cancer cell metastasis and progression. Example 6 is a study on the in vivo antitumor effect of CHO@Cu / His-ZIF8 nanozyme. Figure 12 Figure 1: Changes in tumor volume in mice of different groups treated with CHO@Cu / His-ZIF8 nanozyme in vivo for antitumor therapy. The specific implementation steps are as follows: To establish a subcutaneous tumor-bearing mouse model, each mouse was subcutaneously injected with 2×10⁻⁶ cells on its right back. 6 4T1 cells; when the tumor volume reached about 100 mm³, the tumor-bearing mice were randomly divided into 4 groups (n = 5): (1) control group (2) CHO group (3) Cu / His-ZIF8 group (4) CHO@Cu / His-ZIF8 group. PBS, CHO, Cu / His-ZIF8 and CHO@Cu / His-ZIF8 were injected intravenously every 1 day. Mouse weight and tumor size were measured and recorded every 1 day. After 14 days, the mouse tumor and major organs were removed and fixed with 4% paraformaldehyde solution. H&E staining, immunofluorescence staining and immunohistochemical staining were used to embed paraffin blocks. The section thickness was 8 μm for histological evaluation.

[0043] Depend on Figure 12 It can be concluded that: throughout the treatment process, the tumors in the PBS group showed significant rapid growth; in addition, the CHO group and Cu / His-ZIF8 group showed only mild anti-tumor activity, while CHO@Cu / His-ZIF8 significantly enhanced the tumor inhibition effect; the high tumor inhibition rate is due to the combined therapeutic effect of ROS production and cholesterol consumption. In summary, compared with CHO monotherapy and Cu / His-ZIF8 monotherapy, the cholesterol depletion generated by CHO@Cu / His-ZIF8 combined with CDT therapy can better inhibit tumor cell proliferation and mediate apoptosis. Furthermore, this method constructs a multifunctional CHO@Cu / His-ZIF8 nanozyme. In vitro experiments confirmed that this nanozyme possesses multiple enzymatic activities: the Cu²⁺-mediated Fenton-like reaction can mimic peroxidase (POD) activity, generating cytotoxic hydroxyl radicals (•OH); simultaneously, its catalase-like activity can alleviate the hypoxic tumor microenvironment through oxygen production, thereby enhancing the efficacy of chemodynamic therapy (CDT); concurrently, the oxygen generated by the nanozyme can further enhance the catalytic activity of CHO, promoting the generation of more H₂O₂, providing a substrate for peroxidase-like activity, forming a self-amplifying cycle of •OH generation; in addition, CHO can effectively deplete cholesterol in the tumor microenvironment. Steroids disrupt lipid raft integrity and pseudopodia formation, thereby significantly inhibiting tumor cell migration. This synergistic mechanism, combining oxidative stress amplification and cholesterol depletion, not only efficiently induces tumor cell apoptosis but also significantly inhibits tumor metastasis. In vivo studies have shown that the synthesized CHO@Cu / His-ZIF8 nanozyme can be effectively enriched at tumor sites and exhibits excellent biocompatibility, minimizing systemic toxicity. Animal experiments have also confirmed that the CHO@Cu / His-ZIF8 nanozyme demonstrates potent antitumor activity, especially in inhibiting tumor metastasis, showing broad application prospects.

Claims

1. A preparation method of a self-cascading reactive oxygen amplifier CHO@Cu / His-ZIF8, characterized in that, The process includes the following steps: mixing the prepared ligand, metal salt and natural enzyme in a good solvent in a certain proportion and stirring, followed by washing, dialysis and drying to finally obtain CHO@Cu / His-ZIF8 nanozyme.

2. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The ligand is selected from one or two of 2-methylimidazole, histidine, 4-methylimidazole, imidazole, histamine, benzimidazole, and 2-ethylimidazole.

3. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 according to claim 1, characterized in that, The metal salt is selected from one or two of zinc nitrate, copper nitrate, iron nitrate, manganese nitrate, molybdenum nitrate, and cobalt nitrate.

4. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The natural enzyme is selected from one of glucose oxidase, lactate oxidase, catalase, cholesterol oxidase, or urate oxidase.

5. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The molar ratio of the metal salt to the ligand is 0.2-2:1; The ratio of the natural enzyme to the sum of the masses of the metal salt and ligand is 1:5-30.

6. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The stirring time is 24 hours.

7. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The good solvent is one or two of N, N-dimethylformamide, methanol, and pure water.

8. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, The solvent used in the washing process is one or two of anhydrous ethanol, methanol, and pure water. The dialysis procedure is as follows: the washed mixed solution is placed in dialysis bags of 500 DA, 3000 DA, and 10000 DA for dialysis. The drying method is one of the following: oven drying, vacuum drying, or freeze drying.

9. The preparation method of the self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme according to claim 1, characterized in that, In the preparation process, one of the following methods is selected for synthesis: solvothermal method, self-assembly, or biomineralization.

10. The application of a self-cascaded reactive oxygen species amplifier CHO@Cu / His-ZIF8 nanozyme prepared by any one of the preparation methods described in claims 1-9 in antitumor and immunomodulatory therapy.

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