Arsenic in-situ growth-metal organic framework material as well as preparation method and application thereof

Abstract

The invention discloses an arsenic in-situ growth-metal organic framework material which is arsenic oxide nanoparticles formed in situ on the surface of a metal organic framework material by performing ligand exchange on an arsenic compound and the metal organic framework material, the arsenic compound is selected from sodium arsenite, disodium arsenate heptahydrate, arsenic trioxide and the like; the metal organic framework material is selected from ZIF-8 and the like. The invention also discloses bionic nano-particles for in-situ growth of arsenic. The bionic nano-particles are formed by coating an arsenic in-situ growth-metal organic framework material with a cell membrane. The invention further discloses application of the arsenic in-situ growth-metal organic framework material and the bionic nanoparticles in preparation of drugs for treating cancers. The arsenic in-situ growth-metal organic framework material disclosed by the invention is bionic nano-enzyme with enzyme-like activity and dual epigenetic regulation and control effects, and is uniform in morphology and relatively small in particle size; and due to cell membrane coating, the bone marrow targeting property is realized, the problem of insufficient targeting property of an arsenic compound is solved, and the tumor immunotherapy effect is enhanced.

Description

Technical Field

[0001] This invention relates to an arsenic in-situ grown metal-organic framework material, its preparation method and application, belonging to the field of nano-preparation technology. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant tumor characterized by impaired differentiation of hematopoietic stem cells, leading to the uncontrolled clonal proliferation of immature myeloid progenitor cells and primitive cells in the bone marrow, peripheral blood, and other tissues. Unlike solid tumors, AML is a diffuse malignant tumor in which leukemia cells or leukemia stem cells (LSCs) circulate in the peripheral blood or bone marrow microenvironment. Therefore, local treatments, including surgery, radiotherapy, and phototherapy, are not suitable for leukemia. Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment option, but it is limited by high morbidity and mortality. In recent years, with advances in the genomic and epigenomic characterization of AML, several novel targeted therapies have been developed and approved. However, despite achieving remission after treatment, nearly 50% of AML patients relapse, and the 5-year overall survival rate is less than 30%. Addressing the treatment challenges of leukemia, such as poor drug targeting and difficulty in penetrating the bone marrow microenvironment, the bone marrow-targeting effect of small-diameter nanoparticles offers a new strategy for precise, low-toxicity, and highly efficient leukemia treatment. Leukemia lesions are primarily located in the bone marrow cavity. The bone marrow contains unique physiological barriers, such as the sinusoidal endothelial barrier and the bone marrow microenvironment barrier, preventing larger nanoparticles from entering the bone marrow parenchyma. However, small-diameter nanoparticles, due to their smaller size, can directly pass through the sinusoidal endothelial gaps and actively infiltrate the bone marrow parenchyma. They also more easily penetrate the dense network of the bone marrow microenvironment, reaching the area where leukemia cells are located, significantly increasing drug concentration in the bone marrow. Furthermore, by surface modification, such as with ligands or antibodies like CXCR4 and CD44, they can precisely identify and bind to lesion cells, achieving active targeting. Targeted enrichment significantly increases drug concentration in the bone marrow region while reducing free drug concentration in peripheral blood, thereby mitigating damage to other organs and improving treatment tolerance.

[0003] The microvessels in bone marrow are predominantly sinusoidal endothelium, with intercellular spaces of 20–100 nm, serving as the primary pathway for nanomedicines to penetrate the bone marrow. Simultaneously, nanomedicines must match this intercellular space range to achieve effective penetration. Macrophages in the bone marrow preferentially engulf nanoparticles that are too large or too small. Target cells in the bone marrow exhibit a size preference for nanoparticle uptake; nanoparticles must be able to attach to the cell surface and enter the cell via endocytosis or remain in the surrounding microenvironment. Nanoparticles smaller than 5 nm easily cross the glomerular filtration barrier and are excreted directly in the urine, failing to reach the bone marrow. Nanoparticles with a size of 5–10 nm, due to their larger specific surface area, readily adsorb opsonin proteins in the blood, leading to rapid recognition and phagocytosis by macrophages in the bone marrow. Nanoparticles larger than 100 nm cannot pass through the sinusoidal endothelial space of the bone marrow and are blocked in the vascular lumen. They can only enter the bone marrow through active phagocytosis by endothelial cells. However, because the phagocytic capacity of bone marrow endothelial cells is much weaker than that of liver macrophages, most nanoparticles are eventually cleared by the mononuclear phagocytic system (MPS) of the liver and spleen. Therefore, to achieve better bone marrow targeting, the particle size of nanoparticles should be controlled between 10 and 100 nm during preparation. Nanoparticles within this size range can efficiently pass through the sinusoidal endothelial space of the bone marrow without relying on active transport and can directly penetrate into the bone marrow parenchyma. They also have a moderate specific surface area and a low MPS phagocytic rate. Furthermore, their particle size is larger than the glomerular filtration threshold (5 nm), which can avoid renal excretion and prolong the circulation time in vivo. In particular, nanoparticles of 30–80 nm can be efficiently taken up by bone marrow target cells through clathrin-mediated endocytosis or caveolin-mediated endocytosis, and their surface can be loaded with sufficient homing ligands. Currently, most nanoparticles used for leukemia treatment have a particle size of around 200 nm, which cannot achieve good bone marrow targeting.

[0004] Epigenetic modifications can regulate the evolution of leukemia cells through DNA methylation, histone modifications, and non-coding RNA. They promote leukemia by inhibiting tumor suppressor genes and activating key oncogenes that drive leukemia development and progression. Abnormal DNA methylation and histone modifications are common in acute myeloid leukemia (AML) cells; therefore, epigenetic therapy for leukemia can reverse abnormal gene expression and restore normal cell function. DNA methylation inhibitors such as azacitidine and decitabine reverse abnormal methylation by inhibiting the activity of DNA methyltransferases (DNMTs), restoring tumor suppressor gene expression, and inducing apoptosis in leukemia cells. These drugs have become first-line treatments for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), significantly improving complete remission rates and overall survival. Protein arginine methyltransferases (PRMTs) are a class of enzymes that catalyze the methylation of arginine residues in proteins. They participate in key biological processes such as cell proliferation, differentiation, and apoptosis by regulating chromatin structure, transcription factor activity, and the function of signaling pathway proteins. In leukemia, abnormal expression or dysregulation of PRMT activity is one of the important mechanisms driving disease occurrence, progression, and drug resistance. For example, PRMT1 can methylate arginine residues on p53, weakening its transcriptional activity and allowing leukemia cells to escape apoptosis.

[0005] Immune escape by leukemia cells is the primary cause of immunotherapy failure, and epigenetic silencing is the core mechanism by which they reduce immunogenicity and evade immune recognition. Epigenetic regulation can enhance the ability of the immune system to recognize leukemia cells by restoring tumor-associated antigen expression, upregulating the expression of immune recognition-related molecules (such as MHC molecules, co-stimulatory molecules CD80, CD86, etc.), and inducing immunogenic cell death. Anti-PD-L1 antibodies, as a core type of immune checkpoint inhibitor, can relieve leukemia immunosuppression and activate the anti-leukemia immune response by blocking the PD-L1 / PD-1 signaling pathway. Enhancing the efficacy of anti-PD-L1 antibodies can reduce the suppression of effector T cells by Tregs and MDSCs and restore the killing activity of exhausted T cells, thereby breaking the immune tolerance induced by leukemia cells and allowing the immune system to re-recognize leukemia cells.

[0006] In nature, inorganic arsenic exists in two valence states (As). Ⅲ And As ⅤArsenic valence states can interconvert under specific conditions. Existing research has confirmed that arsenic valence states can interconvert in specific microenvironments. In mammals, inorganic arsenic is mainly detoxified through methylation into monomethylarsenic and dimethylarsenic. This biochemical process uses S-adenosylmethionine (SAM) as a methyl donor. When SAM is consumed in large quantities, S-adenosyl-L-homocysteine ​​(SAH) accumulates, regulating the activity of DNMTs through feedback inhibition. This biochemical process is generally associated with arsenic valence state transitions. Although arsenic compounds have potential as inorganic therapeutic agents, they exhibit low loading efficiency and poor stability in organic nanocarriers. Summary of the Invention

[0007] To address the aforementioned limitations of existing technologies, this invention provides an arsenic in-situ grown metal-organic framework material, its preparation method, and its applications. This invention avoids excessively large nanoparticle sizes in carrier preparation, drug introduction, and cell membrane encapsulation, keeping the particle size within a range suitable for passive bone marrow targeting. Furthermore, the encapsulation with homologous cancer cell membranes endows the nanoparticles with active targeting capabilities, thus solving the problem of insufficient drug targeting in leukemia treatment.

[0008] This invention is achieved through the following technical solution: An arsenic in-situ grown metal-organic framework material is formed by ligand exchange between an arsenic compound and a metal-organic framework material, thereby forming arsenic oxide nanoparticles in situ on the surface of the metal-organic framework material; the arsenic compound is selected from any one or more of sodium arsenite, disodium arsenate heptahydrate, and arsenic trioxide; the metal-organic framework material is selected from any one of ZIF-8, ZIF-67, IRMOF-8, and Fe-MOF, with ZIF-8 being preferred.

[0009] Furthermore, the loading of the arsenic compound is 2.12% to 5.20%.

[0010] The preparation method of the arsenic in-situ grown metal-organic framework material is as follows: the metal-organic framework material and the arsenic compound are thoroughly mixed in a solvent (such as water), centrifuged, and the resulting precipitate is the arsenic in-situ grown metal-organic framework material.

[0011] Furthermore, the metal-organic framework material ZIF-8 is prepared by a stirring method as follows: 1800 mg of zinc nitrate hexahydrate is added to 84 mL of methanol, denoted as solution A; 4200 mg of 2-methylimidazole is added to 84 mL of methanol, denoted as solution B; solution B is added dropwise to solution A, the mixture is stirred to react, centrifuged, and the precipitate is collected, which is ZIF-8.

[0012] A biomimetic nanoparticle with in-situ arsenic growth is formed by encapsulating an arsenic in-situ grown metal-organic framework material with a cell membrane. The cell membrane is selected from erythrocyte membrane, macrophage membrane, platelet membrane, leukocyte membrane, tumor cell membrane, and mesenchymal stem cell membrane (bone marrow mesenchymal stem cell, adipose mesenchymal stem cell, or embryonic mesenchymal stem cell), preferably homologous C1498 cell membrane.

[0013] Furthermore, the weight ratio of the cell membrane to the arsenic in situ grown metal-organic framework material is 1:(1-5), preferably 1:(2-4).

[0014] Furthermore, the arsenic in-situ grown biomimetic nanoparticles have a particle size of 40–100 nm, preferably 50–80 nm.

[0015] The preparation method of the arsenic in-situ grown biomimetic nanoparticles is as follows: the cell membrane and the arsenic in-situ grown metal-organic framework material are thoroughly mixed in a solvent (water), centrifuged, and the resulting precipitate is the arsenic in-situ grown biomimetic nanoparticles.

[0016] The application of the arsenic in-situ grown metal-organic framework material in the preparation of drugs for treating cancer. The cancer is preferably a type of cancer corresponding to a homologous cell membrane.

[0017] The application of the arsenic-in-situ grown biomimetic nanoparticles in the preparation of drugs for treating cancer. The cancer is preferably a type of cancer corresponding to a homologous cell membrane.

[0018] Furthermore, in specific applications, it is used in combination with anti-tumor drugs. The anti-tumor drugs are PD-L1 inhibitors or PD-1 inhibitors.

[0019] A pharmaceutical formulation whose active ingredient is the above-mentioned arsenic in-situ grown metal-organic framework material, or the above-mentioned arsenic in-situ grown biomimetic nanoparticles.

[0020] Furthermore, the active ingredient in the pharmaceutical preparation also includes an anti-tumor drug. The anti-tumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

[0021] The above-mentioned pharmaceutical preparation is used in the preparation of drugs for treating cancer. The cancer referred to is a type of cancer corresponding to a homologous cell membrane.

[0022] The arsenic in-situ grown metal-organic framework material of this invention is a biomimetic nanozyme with both enzyme-like activity and dual epigenetic regulatory functions. It possesses peroxidase (POD) and glutathione oxidase (GSHOx)-like activities, generating reactive oxygen species (ROS) and depleting glutathione (GSH), thereby disrupting the redox balance in mouse acute myeloid leukemia cells (C1498 cells), placing them under oxidative stress, and enhancing the sensitivity of this nanoplatform in regulating DNA methylation and histone methylation. Simultaneously, this nanoplatform effectively activates the cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene-stimulating (cGAS-STING) pathway by downregulating DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) and protein arginine methyltransferases (PRMT1, PRMT5, PRMT6), thereby inducing dendritic cell maturation, macrophage polarization, and further promoting T cell proliferation, achieving a significant immunomodulatory effect.

[0023] This invention utilizes ZIF-8 as a carrier material for in-situ arsenic growth. The preparation process is simple and low-cost, achieving successful loading of arsenic compounds. The resulting nanoparticles exhibit uniform morphology and small particle size. Coating with C1498m imparts good bone marrow targeting, effectively addressing the issue of insufficient targeting by arsenic compounds. Furthermore, this invention is the first to discover that the arsenic-loaded nanoplatform possesses POD-like and GSHOx enzyme activities, thereby causing redox imbalance in C1498 cells and enhancing the sensitivity of arsenic compounds to regulate DNA methylation and protein arginine methylation.

[0024] This invention fully utilizes the inherent properties of arsenic compounds, which combine catalytic therapy and epigenetic regulation for synergistic immunotherapy. Specifically, As / ZIF-8 not only achieves catalytic therapy through POD-like and GSHOx enzyme activities, but also enhances the sensitivity of C1498 cells to epigenetic regulation by placing them under oxidative stress. This further leads to mtDNA release, activating the cGAS-STING immune pathway, thereby triggering this pathway and downstream cascade reactions, restarting the immune response, and exerting its immunotherapeutic effect. Therefore, there is no need to co-load with other drugs, simplifying the preparation process and avoiding the potential toxic side effects of other drugs. This invention integrates arsenic salts into the ZIF-8 metal-organic framework, representing a promising strategy for constructing a synergistic therapeutic platform that can enhance dual epigenetic regulation, thereby enhancing the efficacy of tumor immunotherapy.

[0025] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0026] Figure 1Electron micrographs of ZIF-8, As / ZIF-8, and As / ZIF-8@M are shown. In the images, a, b, and c represent ZIF-8, As / ZIF-8, and As / ZIF-8@M, respectively, with the red arrow indicating the C1498 cell membrane. D and e are electron micrographs of As / ZIF-8 with a loading of 5.20%.

[0027] Figure 2 HR-TEM image of As / ZIF-8.

[0028] Figure 3 Infrared spectra of ZIF-8, NaAsO2, As / ZIF-8, and As / ZIF-8@M.

[0029] Figure 4 XPS spectra of ZIF-8, As / ZIF-8, and As / ZIF-8@M, where (a): XPS spectra; b: magnified view of the As position in As / ZIF-8; c: magnified view of the As position in As / ZIF-8@M; d: magnified view of the P position in As / ZIF-8@M.

[0030] Figure 5 : XRD patterns of ZIF-8, As / ZIF-8, As / ZIF-8@M.

[0031] Figure 6 STEM-EDS spectra of As / ZIF-8@M.

[0032] Figure 7 : Zeta potential diagram of ZIF-8, As / ZIF-8, As / ZIF-8@M.

[0033] Figure 8 SDS-PAGE electrophoresis images of different nanoparticles and whole-cell lysates.

[0034] Figure 9 Immunoblot images of cell membrane markers in different nanoparticles and whole-cell lysates.

[0035] Figure 10Characterization based on synchrotron radiation, where: a: As K-edge XANES spectra of As / ZIF-8 and reference samples; b: As K-edge FT-EXAFS spectra of As / ZIF-8 and reference samples; c: EXAFS fitting results of As K-edge in R space for As / ZIF-8; d: Zn K-edge XANES spectra of As / ZIF-8 and reference samples; e: Zn K-edge FT-EXAFS spectra of As / ZIF-8 and reference samples; f: EXAFS fitting results of Zn K-edge in R space for As / ZIF-8; g: As K-edge WT-EXAFS spectra of As / ZIF-8; h: Zn K-edge WT-EXAFS spectra of As / ZIF-8; i: As K-edge WT-EXAFS spectra of NaAsO2.

[0036] Figure 11 : Ultraviolet spectra of each group of solutions.

[0037] Figure 12 Results of POD-like enzyme activity assay of As / ZIF-8, where a: effect of reaction time; b: effect of H2O2 concentration; c: effect of formulation concentration; d: effect of pH; e: TMB dependence; f: electron spin resonance spectrum.

[0038] Figure 13 TEM observation of the morphology of As / ZIF-8 at pH 4.5 and determination of its POD-like enzyme activity at different times under pH 4.5 conditions. a: Morphology of As / ZIF-8 at pH 4.5; b: Corresponding magnified view, with blue arrows indicating the decomposed ZIF-8 framework and red boxes highlighting the released arsenic oxide nanoparticles; c: UV-Vis absorption changes of the TMB solution after incubation of As / ZIF-8 for different times at pH 4.5.

[0039] Figure 14 Michaelis-Menten equation fitting plot for As / ZIF-8 (Mean±SD) n =3).

[0040] Figure 15 The results of GSHOx-like enzyme activity assays using As / ZIF-8 are shown. The left figure shows the effect of reaction time, and the right figure shows the effect of formulation concentration.

[0041] Figure 16 : A schematic diagram of the catalytic reaction mechanism of POD at As atomic sites, where purple, blue-violet, blue, gray, red and orange spheres represent arsenic, zinc, nitrogen, carbon, oxygen and hydrogen atoms, respectively.

[0042] Figure 17: Free energy diagram of arsenic active sites similar to peroxidase-catalyzed reactions.

[0043] Figure 18 Comparative analysis of the catalytic activity of enzymes (POD, GSHOx) before and after C1498m modification, including: a) comparison of UV-Vis absorption spectra of TMB and H2O2 solutions, with As / ZIF-8@M and As / ZIF-8 added to pH 4.5 solutions respectively; b) comparison of UV-Vis absorption spectra of As / ZIF-8@M and As / ZIF-8 at pH 6.5 in GSH depletion detection.

[0044] Figure 19 : RhB-labeled As / ZIF-8 endocytosis and lysosomal localization at different time points.

[0045] Figure 20 : RhB-labeled As / ZIF-8 endocytosis and mitochondrial localization at different time points.

[0046] Figure 21 : RhB-labeled As / ZIF-8@M endocytosis and lysosomal localization at different time points.

[0047] Figure 22 : RhB-labeled As / ZIF-8@M endocytosis and mitochondrial localization at different time points.

[0048] Figure 23 Quantitative analysis of the average fluorescence intensity of RhB at different time points ( n = 3). Statistical significance was calculated using Student's t-test: p <0.001, p <0.0001.

[0049] Figure 24 Quantitative assessment results of cellular uptake of As and Zn in NaAsO2, As / ZIF-8, and As / ZIF-8@M, where a: As; b: Zn.

[0050] Figure 25 The effects of different concentrations of NaAsO2, As / ZIF-8, and As / ZIF-8@M on the cell viability of C1498 cells (n=3) were investigated. One-way ANOVA was used to test for statistical significance. p <0.05, p <0.01, p <0.001.

[0051] Figure 26 Flow cytometry results of NaAsO2, As / ZIF-8, and As / ZIF-8@M-induced apoptosis in C1498 cells ( n =3).

[0052] Figure 27 YP1 / PI co-staining results of C1498 cells induced by NaAsO2, As / ZIF-8, and As / ZIF-8@M.

[0053] Figure 28 The results of DCF fluorescence intensity detection and semi-quantitative analysis of C1498 cells are presented, including: a) fluorescence image (ROS level) detected by CLSM; b) semi-quantitative analysis after CLSM detection (n=3); c) ROS level detected by flow cytometry; and d) semi-quantitative analysis after flow cytometry detection (n=3). One-way ANOVA was used to test for statistical significance. p <0.05, p <0.01, p <0.001.

[0054] Figure 29 Results of HPF fluorescence intensity detection and semi-quantitative analysis of C1498 cells, where a: fluorescence image (·OH level) detected by CLSM; b: semi-quantitative analysis after CLSM detection (…). n =3); c: HPF level detected by flow cytometry; d: Semi-quantitative analysis after flow cytometry detection ( n =3). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001.

[0055] Figure 30 : Detection results and semi-quantitative analysis results of GSH levels, where a: fluorescence image under CLSM (GSH level); b: semi-quantitative analysis ( n =3); c: GSH level detected by the total GSH detection kit ( n =3). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001.

[0056] Figure 31 Where, a: fluorescence image detected by CLSM (MMP level); b: semi-quantitative analysis of fluorescence (MMP level) detected by CLSM (where a: fluorescence image detected by CLSM); b: semi-quantitative analysis of fluorescence (MMP level) after CLSM detection (where b: fluorescence image detected by CLSM). n =3); c: Semi-quantitative analysis after flow cytometry detection ( =3); n =3); d: MMP level detected by flow cytometry ( n =3). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001.

[0057] Figure 32 Intracellular As in C1498 cells after treatment with NaAsO2, As / ZIF-8, and As / ZIF-8@M Ⅲ Total (a, b, d) and As Ⅴ Total (c, e) n =3). Where, a: As after NaAsO2 treatment. Ⅲ Total amount; b: As / ZIF-8 processed As Ⅲ Total amount; c: As / ZIF-8 processed As Ⅴ Total amount; d: As / ZIF-8@M processed As Ⅲ Total amount; e: As / ZIF-8@M processed As Ⅴ Total amount.

[0058] Figure 33 Intracellular As after treatment of C1498 cells with As / ZIF-8, As / ZIF-8@M and antioxidants Ⅲ Total and As Ⅴ Total ( n =3). Among them, a: As / ZIF-8 and As treated with antioxidants. Ⅲ Total amount; b: As / ZIF-8 and As treated with antioxidants Ⅴ Total amount; c: As / ZIF-8@M and As after antioxidant treatment Ⅲ Total amount; d: As / ZIF-8@M and As after antioxidant treatment Ⅴ Total amount. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0059] Figure 34 AO staining fluorescence images and semi-quantitative analysis of C1498 cells, where a: fluorescence image; b: semi-quantitative analysis (…). n =3). One-way ANOVA was used to test statistical significance. p <0.001, p <0.0001.

[0060] Figure 35 Results of SAM content detection in C1498 cells ( n =3). One-way ANOVA was used to test statistical significance. p <0.001, p <0.0001.

[0061] Figure 36 Analysis results of mRNA levels of DNMT1, DNMT3A, DNMT3B, PRMT1, PRMT5, and PRMT6 in C1498 cells after different treatments. n =3), where a: statistical analysis results of DNMT1; b: statistical analysis results of DNMT3A; c: statistical analysis results of DNMT3B; d: statistical analysis results of PRMT1; e: statistical analysis results of PRMT5; f: statistical analysis results of PRMT6. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0062] Figure 37 Analysis results of protein levels of DNMT1, DNMT3A, DNMT3B, PRMT1, PRMT5, and PRMT6 in C1498 cells after different treatments. n =3), where a: Western blot analysis results; b: statistical analysis results of DNMT1; c: statistical analysis results of DNMT3A; d: statistical analysis results of DNMT3B; e: statistical analysis results of PRMT1; f: statistical analysis results of PRMT5; g: statistical analysis results of PRMT6. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0063] Figure 38 Analysis results of intracellular DNMT1 and PRMT1 protein levels in C1498 cells after treatment with As / ZIF-8@M and antioxidants ( n =3), where a: Western blot analysis results; b: statistical analysis results of DNMT1; c: statistical analysis results of PRMT1. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.0001.

[0064] Figure 39 Analysis results of cGAS, p-STING, p-TBK1 / TBK1, p-IRF3 / IRF3, and IFN-β in C1498 cells after different treatments ( n =3), where a: Western blot analysis results; b: statistical analysis results of cGAS; c: statistical analysis results of p-STING; d: statistical analysis results of p-TBK1 / TBK1; e: statistical analysis results of p-IRF3 / IRF3; f: statistical analysis results of IFN-β. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0065] Figure 40 Analysis results of cGAS, p-STING, p-TBK1 / TBK1, p-IRF3 / IRF3, and IFN-β in C1498 cells after different treatments ( n =3), where a: Western blot analysis results; b: statistical analysis results of cGAS; c: statistical analysis results of p-STING; d: statistical analysis results of p-TBK1; e: statistical analysis results of p-IRF3; f: statistical analysis results of IFN-β. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0066] Figure 41 The results of measuring the levels of IFN-β, IFN-γ, IL-6, and TNF-α in the supernatant of C1498 cells after different treatments. n =3), where a: IFN-β; b: IFN-γ; c: IL-6; d: TNF-α. One-way ANOVA was used to test statistical significance. p <0.01, p <0.001, p <0.0001.

[0067] Figure 42 Results of ICD marker assay in C1498 cells after different treatments ( n =3). Where, a: Western blot analysis results; b: CRT statistical analysis results; c: HMGB1 statistical analysis results; d: CRT immunofluorescence image; e: CRT immunofluorescence semi-quantitative analysis; f: HMGB1 immunofluorescence image; g: HMGB1 immunofluorescence semi-quantitative analysis; h: ATP content in supernatant; m: HMGB1 content in supernatant. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0068] Figure 43 Results of in vivo targeting of As / ZIF-8@M. Where: a: Real-time in vivo fluorescence imaging at different time points; b: Fluorescence images of ex vivo tissues and organs at 24 h; c: Fluorescence intensity of ex vivo tissues (…). n =3). d: Quantitative analysis of As in various tissues and organs of mice at 6 h ( n =3); e: Quantitative analysis of As in various tissues and organs of mice at 12 h ( n =3); f: Quantitative analysis of As in various tissues and organs of mice at 24 h ( n =3). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0069] Figure 44 Results of peripheral blood and spleen analysis in mice of each group, where a: number of white blood cells in peripheral blood ( n =5); b: Spleen image; c: Leukemia suppression rate ( n =5); d: Spleen weight ( n =5). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0070] Figure 45 H&E and Giemsa staining results of major organs (spleen, bone marrow, peripheral blood, lungs) of mice in each group.

[0071] Figure 46 Analysis results of Bmi-1, c-MYC, cGAS, p-STING, p-TBK1, p-IRF3, and IFN-β in the bone marrow of mice in each group ( n =3), where a: Western blot analysis results; b: statistical analysis results of Bmi-1; c: statistical analysis results of c-MYC; d: statistical analysis results of DNMT1; e: statistical analysis results of PRMT1; f: statistical analysis results of cGAS; g: statistical analysis results of p-STING; h: statistical analysis results of p-TBK1; i: statistical analysis results of p-IRF3; j: statistical analysis results of IFN-β. One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0072] Figure 47 Results of detection of ATP, HMGB-1 and CRT expression levels in bone marrow of mice in each group ( n=5), where a: ATP; b: HMGB1; c: CRT. One-way ANOVA was used to test for statistical significance. p <0.05, p <0.01, p <0.001.

[0073] Figure 48 Mature dendritic cells (CD80) in the spleen of mice in each group + CD86 + The percentage of CD11c + Cells as gating ( n =5). One-way ANOVA was used to test statistical significance. p <0.05, p <0.001, p <0.0001.

[0074] Figure 49 CD86 in tumor-associated macrophages on the surface of bone marrow-derived macrophages in each group of mice + and CD206 + The results of expression level measurement ( n =5). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.0001.

[0075] Figure 50 CD4 in the spleen of mice in each group + CD8 + The percentage of T cells. One-way ANOVA was used to test for statistical significance. p <0.05, p <0.01, p <0.0001.

[0076] Figure 51 Effector memory T cells (CD44) in the spleen of mice in each group + CD62L - The percentage of ) in CD8 + T cells act as gaters ( n=5). One-way ANOVA was used to test statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0077] Figure 52 The expression levels of IFN-β, IL-6, TNF-α and IFN-γ in the serum and bone marrow of mice in each group ( n =5), where a: serum IFN-β level; b: serum IFN-γ level; c: serum IL-6 level; d: serum TNF-α level; e: bone marrow IFN-β level; f: bone marrow IFN-γ level; g: bone marrow IL-6 level; h: bone marrow TNF-α level. One-way ANOVA was used to test for statistical significance. p <0.05, p <0.01, p <0.001, p <0.0001.

[0078] Figure 53 : Body weight change curves of mice in each group (n=3).

[0079] Figure 54 H&E staining images of the major organs (heart, liver, spleen, lungs and kidneys) of mice in each group (scale bar: 200 μm).

[0080] Figure 55 Hematological and blood biochemical results of mice (n=3), where a: white blood cells (WBC); b: red blood cells (RBC); c: mean corpuscular hemoglobin concentration (MCHC); d: mean corpuscular hemoglobin volume (MCH); e: mean corpuscular volume (MCV); f: hemoglobin (HGB); g: hematocrit (HCT); h: platelet distribution width (PDW); i: direct bilirubin (D-bil); j: glucose (Glu); k: mean platelet volume (MPV); l: monocyte count (Mon); m: albumin (ALB); n: aspartate aminotransferase / alanine aminotransferase ratio (AST / ALT); o: total protein (TP), as a liver function indicator; p: blood urea nitrogen (BUN), as a kidney function indicator. Detailed Implementation

[0081] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0082] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0083] Example 1: Preparation of arsenic in-situ grown metal-organic framework materials and biomimetic nanoparticles The steps are as follows: (1) Preparation of metal-organic framework material ZIF-8: Accurately weigh 1800 mg of zinc nitrate hexahydrate, add it to 84 mL of methanol, sonicate to dissolve it, mix it evenly and record it as solution A; accurately weigh 4200 mg of 2-methylimidazole, add it to 84 mL of methanol, sonicate to dissolve it, mix it evenly and record it as solution B; under magnetic stirring at 1200 rpm, add solution B dropwise to solution A, stir for 15 min, centrifuge in a high-speed refrigerated centrifuge (12000 rpm, 10 min), collect the precipitate, wash it 3 times with methanol to obtain ZIF-8; dry it in a vacuum drying oven (60℃, 24 h).

[0084] (2) Preparation of arsenic in-situ grown metal-organic framework material (nanozyme As / ZIF-8): The above-mentioned undried ZIF-8 was resuspended in 24 mL of methanol; 20 mL of methanol resuspension was mixed with 10 mL of NaAsO2 solution (concentration 60 mg / mL), stirred at 1200 rpm for 15 min, centrifuged in a high-speed refrigerated centrifuge (12000 rpm, 10 min), the precipitate was collected, washed three times with water, and As / ZIF-8 was obtained. It was then dried in a vacuum drying oven (60℃, 24 h). Eight parallel experiments were conducted to obtain eight samples. The NaAsO2 loadings were 2.12%, 2.23%, 2.40%, 2.67%, 2.82%, 2.92%, 3.22%, and 5.20%, respectively. The As / ZIF-8 with a loading of 2.40% was used as a representative for the next step of experiments and testing.

[0085] (3) Extraction of C1498 cell membrane: The cell membrane was extracted from C1498 cells (C1498 cells are a tumor cell line derived from mice, which is widely used in scientific research fields such as oncology, immunology and pharmacology; it is available by routine purchase). The membrane was extracted using the membrane protein extraction kit A (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) in the cell membrane protein and cytoplasmic protein extraction kit. The cell membrane fragments were washed three times with pre-cooled PBS, lyophilized, and stored at -80℃.

[0086] (4) Preparation of biomimetic nanoparticles (biomimetic nanoenzyme As / ZIF-8@M): C1498 cell membranes were ultrasonically disrupted in an ice bath (200 W, 2 s, 2 s) for 4 min to prepare an aqueous solution with a concentration of 2 mg / mL; As / ZIF-8 was prepared into an aqueous solution with a concentration of 2 mg / mL; the C1498 cell membrane solution and the As / ZIF-8 solution were mixed at a volume ratio of 1:4, ultrasonicated in an ice bath (200 W, 1 s, 2 s) for 10 min, centrifuged in a high-speed refrigerated centrifuge (14000 rpm, 10 min), the precipitate was collected, and washed three times with pre-cooled PBS to remove cell membrane fragments and uncoated As / ZIF-8; the precipitate was freeze-dried in a freeze dryer to obtain As / ZIF-8@M. It was stored at -80℃; the mass ratio of C1498 cell membrane to As / ZIF-8 was found to be 1:4.

[0087] Experiment 1: In-situ growth of arsenic-metal-organic framework materials and characterization of biomimetic nanoparticles (1) Morphological observation by transmission electron microscopy (TEM) Take appropriate amounts of ZIF-8, As / ZIF-8, and As / ZIF-8@M (prepared in Example 1), add them to a methanol solution, disperse them evenly by ultrasonication, drop the sample onto a copper grid, dry the copper grid, and record the TEM image.

[0088] Electron micrographs of ZIF-8, As / ZIF-8, and As / ZIF-8@M are shown below. Figure 1 As shown in the figure. The results show that ZIF-8 exhibits a regular dodecahedral structure, while As / ZIF-8 forms small protrusions on the surface and its overall morphology becomes rounded and no longer regular. A thin film is clearly visible on the surface of As / ZIF-8@M. The particle size of all three formulations does not exceed 100 nm.

[0089] Meanwhile, the morphology of As / ZIF-8 with a loading of 5.20% was observed using transmission electron microscopy, such as... Figure 1 As shown, the morphology of the highly loaded NaAsO2 nanoparticles clearly shows the presence of irregular nanoparticles and even plate-like structures. It is evident that if the loading of NaAsO2 continues to increase, it will affect the morphology of the As / ZIF-8 phase, transforming it into irregular nanoparticles, or even causing them to coalesce into plates.

[0090] (2) High-resolution transmission electron microscopy (HR-TEM) analysis Take an appropriate amount of As / ZIF-8 in a methanol solution, disperse it evenly by ultrasonication, drop the sample onto a copper grid, dry the copper grid, and record the TEM image.

[0091] The HR-TEM diagram of As / ZIF-8 is shown below. Figure 2 As shown in the figure. The results show that lattice fringes (around 0.310 nm) and large-sized metal nanoparticles can be observed at the protrusions on the As / ZIF-8 surface, indicating that the loading of NaAsO2 is not just a simple electrostatic adsorption, but rather the growth of As nanoparticles on the ZIF-8 surface.

[0092] (3) FT-IR spectral analysis Take appropriate amounts of ZIF-8, NaAsO2, As / ZIF-8, and As / ZIF-8@M sample powders, disperse them evenly in a sample trough under dry conditions, press them into thin sheets using the handle, and heat them at 4000–4000 cm⁻¹. -1 The Fourier transform infrared spectrum was measured within the specified range, and the characteristic vibrational peaks in the spectrum were observed.

[0093] The infrared spectra of ZIF-8, NaAsO2, As / ZIF-8, and As / ZIF-8@M are as follows: Figure 3 As shown. The results show that the infrared spectrum of As / ZIF-8 increased by 874.50 cm⁻¹ compared to ZIF-8. -1 631.06 cm -1 538.22 cm -1 515.53 cm -1 455.70 cm -1 The vibrational peaks at the location may be due to the symmetric and asymmetric modes of As(OH)₂ and the stretching vibrations of As-O. This result indicates successful As loading and also shows that As is not simply trapped into the pores of blank ZIF-8, but rather undergoes ligand exchange. The infrared spectrum of As / ZIF-8@M shows a peak at 3183.04 cm⁻¹. -1 1652.26 cm -1 1093.18 cm -1 839.43 cm -1 A new vibrational peak appeared at the location, possibly due to infrared absorption from the cell membrane, while As / ZIF-8 showed a peak at 839.43 cm⁻¹. -1 631.06 cm -1The absorption peak at the location was not reflected in the As / ZIF-8@M infrared spectrum, indicating that the C1498 cell membrane was successfully coated, masking part of the absorption of the As / ZIF-8 group.

[0094] (4) X-ray photoelectron spectroscopy (XPS) analysis Take appropriate amounts of ZIF-8, As / ZIF-8, and As / ZIF-8@M, grind them thoroughly with a mortar and pestle, compress them into tablets, and then place them in an XPS analyzer to analyze their elemental composition.

[0095] XPS spectra of ZIF-8, As / ZIF-8, and As / ZIF-8@M are as follows: Figure 4 As shown in the figure. The results show that As is assigned to both As / ZIF-8 and As / ZIF-8@M, while P is assigned to As / ZIF-8@M.

[0096] (5) X-ray diffraction pattern analysis (XRD) Weigh 20 mg of ZIF-8, As / ZIF-8, and As / ZIF-8@M powder, pass them through a 300-mesh sieve, and place them in the sample cell. Mount the sample on the sample stage with the sample surface facing upwards, aligning the sample center with the center line of the stage. Set the instrument parameters to Cu-K. α ( Figure 5 =1.5406Å), high voltage 40 kV, tube current 40 mA, scan rate 2° / min.

[0097] XRD patterns of ZIF-8, As / ZIF-8, and As / ZIF-8@M are as follows: Figure 6 As shown in the figure. The results show that the XRD characteristic peaks of As / ZIF-8@M and As / ZIF-8 correspond highly with the characteristic peaks of ZIF-8, indicating that the crystal structure of ZIF-8 did not change significantly after loading As and coating.

[0098] (6) Analysis by double aberration-corrected transmission electron microscopy (AC-HAADF-STEM) Take an appropriate amount of As / ZIF-8@M in methanol solution, disperse it evenly by ultrasonication, drop the sample onto a copper grid, dry the copper grid, and record the STEM-EDS spectrum.

[0099] STEM-EDS spectra of As / ZIF-8@M are as follows Figure 7 As shown in the figure. The results show that C, N, Zn, and P elements are uniformly distributed in As / ZIF-8@M, while O and As elements show a high degree of overlap and are concentrated at the surface protrusions of As / ZIF-8@M. Therefore, it is speculated that As exists in As / ZIF-8@M in the form of As oxide, rather than in the form of single As atoms, and that As oxide nanoparticles are formed on the ZIF-8 surface.

[0100] (7) CHN-Elemental Analysis Appropriate amounts of ZIF-8 and As / ZIF-8 were catalytically oxidized and decomposed in a high-temperature oxygen environment. Different component gases, detected according to the CHNS mode, were separated by a special adsorption column, and then the corresponding gases were detected separately using a thermal conductivity detector. Helium was used as both the carrier gas and the purge gas. The CHN- elemental analysis results of ZIF-8 and As / ZIF-8 are shown in Table 1.

[0101] Table 1. CHN elemental analysis results of ZIF-8 and As / ZIF-8 The results showed that the proportion of CHN elements in ZIF-8 was almost the same as that in theoretical ZIF-8 [Zn(C4N2H5)2], but the proportion of each element decreased after As was loaded [Zn(C4N2H5]. 1.8 This indicates that some linkers in the ZIF-8 framework were exchanged for arsenite anions. This demonstrates that As was not simply trapped within the pores of blank ZIF-8, but rather that ligand exchange occurred.

[0102] (8) Zeta potential analysis Take an appropriate amount of ZIF-8, As / ZIF-8, and As / ZIF-8@M in a methanol solution, disperse them evenly by ultrasonication, and then add them to a zeta potential measuring dish. Keep the outer surface of the measuring dish dry, place it in the instrument's measuring chamber, select the refractive index and viscosity of the dispersion medium, set the measuring temperature to 25℃, and measure the zeta potential of the sample.

[0103] The zeta potential diagrams of ZIF-8, As / ZIF-8 and As / ZIF-8@M are as follows: Figure 8 As shown in the figure. The results show that the zeta potential of ZIF-8 changed from 37.73±0.12 mV to -30.67±0.47 mV, which is mainly attributed to the negatively charged AsO. 2- And the encapsulation effect of the C1498 cell membrane.

[0104] (9) Protein retention of As / ZIF-8@M The retention of As / ZIF-8@M membrane proteins was detected using SDS-PAGE. First, the protein concentrations of C1498 cell lysates, C1498 cell membranes, As / ZIF-8@M, and As / ZIF-8 were determined using the BCA method. Protein samples were then mixed with 5× loading buffer and boiled at 100℃ for 5 min to denature the proteins. The protein loading volume was adjusted to 40 μg. After electrophoresis, the gel was stained with Coomassie Brilliant Blue overnight. After washing off the stain, protein bands were observed and photographed. SDS-PAGE electrophoresis images of different nanoparticles and whole-cell lysates are shown below. Figure 9 As shown, the results indicated that after Coomassie Brilliant Blue staining, obvious protein bands appeared in each group, and the protein band of As / ZIF-8@M was consistent with the protein bands of C1498m and C1498 cell lysates.

[0105] Repeat the above steps, blocking and cutting the membrane after transfer, and incubating it overnight at 4°C with CD44 (1:1000) and CXCR4 (1:1000) primary antibodies. Wash three times with TBST, add the corresponding secondary antibody, and incubate at room temperature for 1 h. Wash four times with TBST, develop in chromogenic reagent for 30 s, and develop using an ECL chemical imaging system. The results are as follows. Figure 10 As shown in the figure. The results indicate that As / ZIF-8@M retains the C1498m-specific proteins CD44 and CXCR4. These results suggest that the membrane coating process did not significantly affect the biological characteristics of C1498m.

[0106] (10) Characterization based on synchrotron radiation Extended X-ray Absorption Fine Structure (EXAFS) measurements of the sample (As / ZIF-8) at the As K-side and Zn K-side were performed using a Si(111) crystal monochromator on the BL14W beamline of the Shanghai Synchrotron Radiation Facility (SSRF).

[0107] The results are as follows Figure 10 As shown. The results show that for the As / ZIF-8 sample, the leading and trailing peaks of its K XANES spectrum are completely consistent with those of the NaAsO2 sample. Figure 10 a) indicates that As exists in the trivalent form in ZIF-8. Furthermore, the coordination structure of the arsenic atoms was analyzed using Fourier transform extended X-ray absorption fine structure (FT-EXAFS), as shown in [the image]. Figure 10 As shown in b, As / ZIF-8 exhibits a main peak at approximately 1.77 Å, corresponding to the As-O bond, which is consistent with the EDS prediction. Compared to other samples, As / ZIF-8 shows an As-Zn bond at 3.28 Å. To reveal the quantitative structural parameters of As atoms, least squares EXAFS fitting was performed on NaAsO2 and As / ZIF-8, as shown in... Figure 10As shown in c, the fitting results indicate that in As / ZIF-8, each As atom is on average coordinated with 2.6 ± 0.1 O atoms, with a bond length of 1.77 Å; and 1.1 ± 0.3 Zn atoms, with a bond length of 3.28 Å. In NaAsO2, each As atom is on average coordinated with 2.9 ± 0.2 O atoms, with a bond length of 1.77 Å. Figure 10 As shown in Figure d, the energy edge profile of Zn in As / ZIF-8 lies between that of Zn foil and ZnO, indicating that the average oxidation state of zinc atoms is intermediate between that of Zn. 0 and Zn 2+ The coordination structure of Zn atoms was analyzed using Fourier transform extended X-ray absorption fine structure (FT-EXAFS), as shown in the figure. Figure 10 As shown in Figure e, As / ZIF-8 exhibits Zn-N bonds at approximately 2.07 Å and Zn-As bonds at 3.28 Å. To reveal the quantitative structural parameters of the Zn atom, least squares EXAFS fitting was performed on As / ZIF-8, as shown in Figure e. Figure 11 As shown in f, the fitting results indicate that each Zn atom in As / ZIF-8 is coordinated with an average of 4.3 ± 0.3 N atoms, with a bond length of 2.07 Å. To further verify the local coordination environment of As and Zn atoms in the above nanozyme, As K-edge and Zn K-edge wavelet transforms of EXAFS (WT-EXAFS) were used as a supplement to FT-EXAFS. The WT contour plot of As / ZIF-8 only shows one maximum intensity value. At R = 4.45 Å⁻¹ and k = 1.77 Å, the As-O bond was designated as the characteristic peak, while the intensity peaks at R = 7.55 Å⁻¹ and k = 3.28 Å were attributed to the As-Zn bond, which further confirms the presence of As-Zn sites at the As K-edge. In summary, As-O and As-Zn bonds are formed in As / ZIF-8, forming a bidentate binuclear complex.

[0108] (11) Characterization of POD-like enzyme activity To detect the POD-like activity of As / ZIF-8 nanoparticles, TMB was used as the substrate, and the experiment was conducted in an environment containing hydrogen peroxide. At pH 4.5, the UV full-wavelength scans of TMB, TMB+H₂O₂, NaAsO₂+TMB+H₂O₂, ZIF-8+TMB+H₂O₂, As / ZIF-8+TMB, and As / ZIF-8+TMB+H₂O₂ were performed to eliminate the influence of the solutions themselves on the POD-like enzyme activity of the formulation groups. The UV spectra of each solution are shown below. Figure 12 As shown, the results indicate that ZIF-8 and NaAsO2 themselves do not possess POD-like enzyme activity, but when As atoms are loaded, As / ZIF-8 acquires POD-like enzyme activity, confirming that As / ZIF-8 does not possess OXD-like enzyme activity.

[0109] As / ZIF-8 nanoparticles (1 mg / mL), H₂O₂ (10 mM), and TMB (0.2 mg / mL) were added to a quartz cuvette and placed at room temperature. The mixture was then mixed with 4 mL of acetate-sodium acetate solution (pH 4.0, 4.5, 5.0, 6.5, 7.4). The absorbance of the colorimetric reaction was recorded using a UV-Vis spectrophotometer after specific reaction times. Simultaneously, steady-state kinetic analysis was performed at room temperature using acetate-sodium acetate solution (pH 4.5) with As / ZIF-8 (1 mg / mL) as a catalyst in systems containing TMB (0.2 mg / mL) and different concentrations of H₂O₂ (0.25, 0.5, 0.75, 1, 2.5, 5, 10 mM). The concentration-dependent analysis of formulations was performed using an acetate-sodium acetate solution (pH 4.5) at room temperature, with different concentrations of As / ZIF-8 (0.2, 0.4, 0.8, 1, 1.2, 1.6 mg / mL) as catalysts, in systems containing TMB (0.2 mg / mL) and H2O2 (10 mM). The concentration-dependent analysis of TMB was also performed using an acetate-sodium acetate solution (pH 4.5) at room temperature, with As / ZIF-8 (1 mg / mL) as catalysts, in systems containing different concentrations of TMB (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg / mL) and H2O2 (10 mM). To detect ·OH, As / ZIF-8 or As / ZIF-8@M (containing the same amount of As) was added to an aqueous reaction system containing 10 mM H2O2 (HAc-NaAc buffer, pH 4.5), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a scavenging agent to capture ·OH. The results of the As / ZIF-8 POD-like enzyme activity assay are shown below. Figure 12 As shown, the results indicate that after the addition of As / ZIF-8, the absorption peaks of H2O2 and TMB in the mixed solution at 652 nm gradually increase over time. Figure 12 a), and exhibits H2O2 concentration dependence ( Figure 12 b) Dose-dependent ( Figure 12 c) TMB concentration dependence ( Figure 12 e). Similar to other reported nanozymes, As / ZIF-8 exhibits almost no significant POD-like activity near neutral pH, but its catalytic activity gradually increases as the solution pH decreases. Figure 12d). This result indicates that these nanozymes not only respond to acidic lysosomes (pH 4.5–5.5), but also that the lower lysosomal pH and greater number of lysosomes in leukemia cells compared to normal cells further enhance the generation of hydroxyl radicals (•OH). Since DMPO can capture •OH in aqueous solution and convert it to DMPO-OH, a 1:2:2:1 four-line characteristic signal attributable to •OH is observed in the electron paramagnetic resonance (ESR) spectrum. Figure 13 f) Further verification of the POD-like activity of As / ZIF-8 was conducted, and As / ZIF-8@M showed a weaker •OH signal peak compared to As / ZIF-8. This may be because the coating of the C1498 cell membrane masks some of the surface active sites of As / ZIF-8.

[0110] Furthermore, TEM observation revealed that the ZIF-8 framework dissociates at pH 4.5, thereby promoting the release of arsenic oxide nanoparticles (e.g., Figure 13 (As shown in a and b). This allows more surface active sites to contact H2O2, resulting in stronger POD-like activity under acidic conditions. To verify the stability of the released arsenic oxide nanoparticles, As / ZIF-8 were incubated at pH 4.5 for different times (0.5, 1, 2, 4, 8, 24 h), and their ability to generate ox-TMB was then tested. The results showed that As / ZIF-8 exhibited similar POD-like enzyme activity at different incubation times (e.g., ...). Figure 14 (As shown in c). Therefore, it is preliminarily speculated that acidic conditions promote the release of arsenic oxide nanoparticles, thereby forming a stronger POD-like activity, and the catalytic ability of the released arsenic oxide nanoparticles is relatively stable.

[0111] (12) Analysis of Michaelis constant For each H₂O₂ concentration, the initial reaction rate was calculated based on the change in absorbance using the Bessel-Lambert law (ε: the molar absorptivity of TMB oxidation is 39000 M). -1 cm -1 Then, using different concentrations of H2O2 and their initial reaction rates, the Michaelis constant was calculated by fitting the data with the Michaelis constant. K m ) and maximum reaction rate ( V max ).

[0112] The Michaelis-Menten equation fitting plot for As / ZIF-8 is shown below. Figure 15 As shown. The results show that, under pH 4.5 conditions and with H2O2 as the reaction substrate, the TMB colorimetric reaction of As / ZIF-8 exhibits typical Michaelis kinetics. The Michaelis constant of As / ZIF-8 was determined to be...K m ) and maximum reaction rate ( V max The values ​​were 0.45107 mM and 9.34 × 10⁻⁶ mM, respectively. -9 μM·s -1 lower K m The values ​​indicate that As / ZIF-8 has a high affinity for H2O2.

[0113] (13) Characterization of GSHox enzyme activity GSH consumption was assessed using a modified Ellman zymography method. 40 μL of GSH aqueous solution (10 mM) was mixed with 3920 μL of nanoparticle PBS solution (pH 6.5) and incubated for different times in the dark at room temperature. 40 μL of GSH aqueous solution (10 mM) was then mixed with different concentrations (0.2, 0.4, 0.8, 1, 1.2, 1.6 mg / mL) of nanoparticle PBS solution (pH 6.5) and incubated for a certain time in the dark at room temperature. The supernatant was then collected using a 0.22 μm filter and reacted with 40 μL of DTNBDMSO solution for 5 min. Finally, the absorbance of the color reaction was recorded using a UV-Vis spectrophotometer.

[0114] The results are as follows Figure 16 As shown in the figure, the absorption peak of DTNB at 412 nm gradually decreased over time, confirming that As / ZIF-8 possesses GSH oxidase-like activity. Furthermore, As / ZIF-8 exhibited concentration-dependent activity within a specific time period. This confirms that As / ZIF-8 nanoparticles possess GSH oxidase-like activity.

[0115] (14) Density Functional Theory (DFT) Calculation To elucidate the simulated catalytic performance of As / ZIF-8, the potential catalytic mechanism of the As site was calculated using density functional theory (DFT). After structural optimization of the model to give it chemical meaning, the catalytic performance of introducing As / ZIF-8 was illustrated using POD-like activity as an example.

[0116] The results are as follows Figure 17 , Figure 18 As shown in the figure. The results indicate that the adsorption of H2O2 molecules at the active site is the initial step (i) of the POD-like catalytic process. In this step, the As sites possess a certain adsorption capacity for H2O2 molecules. Subsequently, the adsorbed H2O2 molecules uniformly dissociate, generating hydroxyl groups (OH groups) attached to the active site. The first step involves the adsorption of hydroxyl radicals (OH) and reactive ·OH (ii), which is the rate-limiting step. In this step, the energy barrier at the As site is negative (-0.15 eV), indicating that the As site has a low barrier, which favors the formation of ·OH. Subsequently, the adsorbed hydroxyl radicals (OH) react with the reactive ·OH radicals. Under weakly acidic conditions, it can absorb and react with hydroxide ions (H+) to generate water molecules adsorbed on the active site (iii). Finally, after desorption of water molecules (iv), the active site returns to its initial state (v). During this process, the Gibbs free energy of the As active site shows a significant decrease. The As site has good substrate adsorption capacity, reduces the energy barrier of the reaction intermediate, and promotes the rate-limiting step, which is why it has good enzyme-like activity.

[0117] (15) As / ZIF-8@M enzyme activity retention The catalytic activities of POD-like and GSHox enzymes were compared between As / ZIF-8 and As / ZIF-8@M (equal in As content) under the same conditions. Results are as follows: Figure 19 As shown in the figure. The results indicate that although the C1498m coating can cover the active sites of metal atoms, the enzyme-like catalytic activity of As / ZIF-8 is not significantly weakened, consistent with the results of the ESR test. In addition, the enhanced dispersibility of As / ZIF-8@M after C1498m coating can improve the contact efficiency between the active sites and the reaction substrate in solution, thereby partially offsetting the decrease in catalytic activity caused by the covering of active sites.

[0118] In summary, due to the introduction of As ions, As / ZIF-8 exhibits multi-enzyme mimicry catalytic properties not found in NaAsO2 and ZIF-8. These two superior enzyme mimicry activities endow As / ZIF-8 with great potential for disrupting redox homeostasis in leukemia and achieving highly efficient cancer catalytic therapy.

[0119] Experiment 2: Investigation of Cell Targeting C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates (containing 2 mL of DMEM medium), followed by co-incubation with RhB-labeled As / ZIF-8 and As / ZIF-8@M (0.3 μg / mL as As). Control group (0 h) and four time points (0.5, 1, 2, and 4 h) were established. After washing cells several times with PBS, lysosomes were labeled with Lyso-tracker Green (30 min), mitochondria were labeled with MitoTracker Green FM (30 min), and nuclear cells were labeled with Hoechst 33342 after one wash (10 min). Finally, fluorescence signals were observed using a laser confocal microscope. The results are shown below.Figure 20 , Figure 21 , Figure 22 , Figure 23 As shown. Quantitative analysis of the average fluorescence intensity of RhB at different time points was performed using ImageJ. n =3), statistical significance was calculated using Student's t-test: p <0.001, p <0.0001. The result is as follows: Figure 24 As shown.

[0120] The results showed that the intensity of red fluorescence gradually increased with incubation time, indicating that As / ZIF-8 and As / ZIF-8@M have efficient cellular uptake characteristics, and the cellular uptake capacity of As / ZIF-8@M is significantly higher than that of As / ZIF-8. This may be because the modification of C1498m increases the affinity of the nano-formulation to C1498 cells. Red and green fluorescence signals showed a co-localization trend at all time points, with a more significant co-localization trend at 4 h. This further validates the lysosomal and mitochondrial localization characteristics of As / ZIF-8 and As / ZIF-8@M.

[0121] Cell uptake quantification: C1498 cells were loaded at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As) for 0.5, 1, 2, and 4 h, respectively. Cells were then collected and digested using a microwave digester. After acid removal, the volume was adjusted to 10 mL with 2% nitric acid in a volumetric flask. Using Ge as an internal standard, As concentrations were analyzed by ICP-MS. 75 and Zn 66 The content of these substances was determined, and the average cellular uptake was calculated. The cellular uptake of As and Zn in NaAsO2, As / ZIF-8, and As / ZIF-8@M was quantitatively assessed by ICP-MS. n =3), the result is as follows Figure 25 As shown in the figure. The results showed that the uptake by C1498 cells was time-dependent, and the uptake capacity for As / ZIF-8@M was the strongest, consistent with the results of confocal microscopy.

[0122] Experiment 3: In vitro efficacy and mechanism study of As / ZIF-8@M (1) Evaluation of cytotoxicity using the CCK8 assay C1498 cells were fed at a rate of 1×10 4100 μL of each of the following solutions was seeded into 96-well plates, and different concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μg / mL as As) of NaAsO2, As / ZIF-8, and As / ZIF-8@M were added respectively. The plates were incubated at 37°C under 5% CO2 for 48 h. Cell viability was calculated using the standard CCK-8 assay.

[0123] The effects of different concentrations of NaAsO2, As / ZIF-8, and As / ZIF-8@M on the cell viability of C1498 cells are as follows: Figure 26 As shown, concentration-dependent cytotoxicity was observed in C1498 cells during treatment with NaAsO2, As / ZIF-8, and As / ZIF-8@M. At the same concentration, As / ZIF-8@M was found to be more cytotoxic than NaAsO2 and As / ZIF-8, which may be related to the fact that the nano-formulation modified with C1498m can be better taken up by cells.

[0124] (2) Annexin V-FITC / PI double staining method for evaluating cell apoptosis C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As). Single-cell suspensions were collected and analyzed by flow cytometry using the Annexin V-FITC apoptosis detection kit.

[0125] The results are as follows Figure 27 As shown in the figure. The results showed that the group treated with As / ZIF-8@M exhibited a higher overall apoptosis rate than the groups treated with NaAsO2 and As / ZIF-8, including 71% late apoptosis and 25% early apoptosis.

[0126] (3) Detection of apoptosis and necrosis C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As). Single-cell suspensions were collected and stained using the YO-PRO-1 / PI cell apoptosis and necrosis detection kit. Intracellular red and green fluorescence was then observed using an inverted fluorescence microscope.

[0127] The results are as follows Figure 28As shown, the nuclei of apoptotic cells exhibited green fluorescence after YO-PRO-1 labeling, while the nuclei of necrotic cells showed overlapping orange fluorescence after YO-PRO-1 and PI double staining. The results indicated a significant increase in the proportion of apoptotic and necrotic cells in the As / ZIF-8@M group.

[0128] (4) Effect of As / ZIF-8@M on intracellular reactive oxygen species (ROS) levels in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As) for 48 hours. Single-cell suspensions were collected, washed multiple times with PBS, and then co-incubated with the DCFH-DA probe in DMEM basal medium (37°C, 30 min). DCF fluorescence intensity was detected by flow cytometry, and semi-quantitative green fluorescence analysis was performed using ImageJ.

[0129] After the above co-incubation, it was then co-incubated with Hoechst 33342 (37℃, 10 min). The DCF fluorescence intensity was detected using CLSM, and semi-quantitative analysis of green fluorescence was performed using ImageJ.

[0130] The results are as follows Figure 29 As shown in the figure. The results showed that only a small amount of green fluorescence was detected in cells treated with NaAsO2 and As / ZIF-8. After treatment with As / ZIF-8@M, the fluorescence intensity increased significantly, indicating that As / ZIF-8@M can significantly increase the production of ROS in C1498 cells.

[0131] (5) Effect of As / ZIF-8@M on intracellular ·OH content in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As). Single-cell suspensions were collected. After washing multiple times with PBS, cells were co-incubated with HPF probes in DMEM basal medium (37°C, 30 min). HPF fluorescence intensity was detected by flow cytometry, and semi-quantitative analysis of green fluorescence was performed using ImageJ.

[0132] After the above co-incubation, it was further co-incubated with Hoechst 33342 (37℃, 10 min). HPF fluorescence intensity was detected using CLSM, and semi-quantitative analysis of green fluorescence was performed using ImageJ.

[0133] The results are as follows Figure 30 As shown in the figure. The results showed that only a small amount of green fluorescence was detected in cells treated with NaAsO2 and As / ZIF-8. After treatment with As / ZIF-8@M, the fluorescence intensity increased significantly, indicating that As / ZIF-8@M can significantly increase the generation of ·OH in C1498 cells.

[0134] (6) Effect of As / ZIF-8@M on glutathione (GSH) content in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As). Single-cell suspensions were collected. After washing several times with PBS, total intracellular GSH was detected using a total GSH assay kit.

[0135] After collecting the above single-cell suspension, Thiol Trackerrm in DMEM basal medium was added. TM The Violet probe was co-incubated with cells (37℃, 30 min) to detect GSH fluorescence intensity, and ImageJ was used for semi-quantitative analysis of green fluorescence.

[0136] The results are as follows Figure 31 As shown in the figure. The results indicated that the decrease in fluorescence intensity confirmed the GSH-consuming capacity of As / ZIF-8@M. Furthermore, the reduction in intracellular GSH content observed using a GSH detection kit further demonstrates that As / ZIF-8@M still exhibits GSHox-like enzyme activity in C1498 cells, possessing the ability to deplete intracellular GSH.

[0137] (7) Effect of As / ZIF-8@M on mitochondrial membrane potential (MMP) in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As). Single-cell suspensions were collected. After washing with PBS multiple times, the cells were stained using a mitochondrial membrane potential assay kit (JC-1), and the results were analyzed using flow cytometry and CLSM, with semi-quantitative red-green fluorescence analysis performed using ImageJ.

[0138] The results are as follows Figure 32 As shown in the figure. The results showed that the mitochondrial membrane potential of C1498 cells treated with As / ZIF-8@M decreased significantly, the red fluorescence almost disappeared, and the green fluorescence was significantly enhanced.

[0139] (8) Effect of As / ZIF-8@M on the intracellular As valence state in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As) for 4, 8, 12, 24, and 48 h, respectively. Cells were collected and digested using a microwave digester. After acid removal, the volume was adjusted to 5 mL with water, filtered through a 0.22 μm filter, and the As valence state was determined by HPLC-ICP-MS.

[0140] The results are as follows Figure 33 As shown. The results indicate that As was not detected inside C1498 cells treated with NaAsO2 at different time points. Ⅴ C1498 processed with As / ZIF-8 and As / ZIF-8@M can detect As at different times. Ⅴ And over time, As Ⅴ The total content showed an increasing trend, and the As / ZIF-8@M treated cells As Ⅴ The overall content was higher than that of cells treated with As / ZIF-8.

[0141] To demonstrate that the oxidation state transition of arsenic is caused by intracellular reactive oxygen species, antioxidants were added to systems containing As / ZIF-8 or As / ZIF-8@M. After culturing for 4 h, cells were collected and digested using a microwave digester. After acid removal, the volume was adjusted to 5 mL with water, filtered through a 0.22 μm filter, and the valence state of As was determined using HPLC-ICP-MS.

[0142] The results are as follows Figure 34 As shown. The results indicate that after the addition of antioxidants, AsⅢ The concentration of As increases, while Ⅴ The concentration decreased. The changes were most significant in the group that added XJB-5-131, likely because XJB-5-131 is a reactive oxygen species scavenger specifically targeting mitochondria. This confirms that the oxidation state transition of As is caused by intracellular reactive oxygen species.

[0143] (9) Effects of As / ZIF-8@M on intracellular lysosomes in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all at 0.3 μg / mL as As). Single-cell suspensions were collected. After washing with PBS multiple times, the cells were co-incubated with acridine orange (AO) at 37°C for 5 min and observed using a fluorescence inverted microscope. Red fluorescence semi-quantitative analysis was performed using ImageJ.

[0144] The results are as follows Figure 35 As shown in the figure. The results showed that, compared with the control group, the red fluorescence signal in C1498 cells treated with As / ZIF-8 and As / ZIF-8@M was significantly reduced, which proved that excessive ROS caused lysosomal damage.

[0145] (10) Effects of As / ZIF-8@M on intracellular S-adenosylmethionine (SAM) in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and treated for 48 hours with either 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As). Cells were collected. After multiple washes with PBS, cells were lysed using cell lysis buffer. Subsequently, the SAM content was detected using a mouse SAM enzyme-linked immunosorbent assay kit.

[0146] The results are as follows Figure 36 As shown in the figure. The results showed that the SAM content in C1498 cells decreased to some extent after treatment with NaAsO2, As / ZIF-8, and As / ZIF-8@M, with the most significant decrease in the As / ZIF-8@M group.

[0147] (11) Effects of As / ZIF-8@M on intracellular epigenetics in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1,000 cells per well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, As / ZIF-8@M, all 0.3 μg / mL as As) for 48 h.

[0148] Cells were collected, lysed with TriQuick Reagent, and RNA was extracted. The mRNA expression levels of DNMT1, DNMT3A, DNMT3B, PRMT1, PRMT5, and PRMT6 in each group of cells were measured using RT-PCR. Results are as follows: Figure 37 As shown in the figure. The results showed that As / ZIF-8@M significantly reduced the mRNA levels of DNMTs and PRMTs, demonstrating a good epigenetic regulatory effect.

[0149] Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature them, then subjected to SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature with shaking for 1.5 h. Antibodies for DNMT1, DNMT3A, DNMT3B, PRMT1, PRMT5, and PRMT6 (1:750) were added, and the mixture was incubated overnight, followed by 4 washes with TBST. Cells were then incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The mixture was then developed in a chromogenic reagent for 30 s, and developed using an ECL chemical imaging system for imaging and photography.

[0150] The results are as follows Figure 38 As shown in the figure. The results indicate that As / ZIF-8@M significantly reduced DNA methylation and PRMTs histone methylation levels, demonstrating a good epigenetic regulatory effect.

[0151] To verify the relationship between reactive oxygen species generated by As / ZIF-8@M and epigenetic regulation, As / ZIF-8@M cells were co-cultured with antioxidants, and total protein was quantified. Protein samples were boiled for 5 min to denature them, followed by SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature for 1.5 h. DNMT1 and PRMT1 antibodies (1:750) were added, and the cells were incubated overnight, followed by 4 washes with TBST. Then, cells were incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The cells were then developed in a chromogenic agent for 30 s, and developed using an ECL chemical imaging system for imaging and photography.

[0152] The results are as follows Figure 39 As shown in the figure. The results showed that the protein expression levels of DNMT1 and PRMT1 in C1498 cells increased after the addition of antioxidants, which proved that the reactive oxygen species generated by As / ZIF-8@M enhanced their epigenetic regulatory role.

[0153] (12) Effects of As / ZIF-8@M on cGAS-STING pathway-related proteins in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells / well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As) for 48 h. Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, then subjected to SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature with shaking for 1.5 h. cGAS, p-STING, p-TBK1, TBK1, p-IRF3, IRF3, and IFN-β antibody (1:700) were added, and the cells were incubated overnight, followed by 4 washes with TBST. Cells were then incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The cells were developed in chromogenic reagent for 30 s, and then visualized and photographed using an ECL chemical imaging system.

[0154] The results are as follows Figure 40 As shown in the figure. The results showed that As / ZIF-8@M significantly increased the expression of cGAS and IFN-β proteins, and significantly increased the ratios of p-STING, p-TBK1 / TBK1, and p-IRF3 / IRF3, indicating that the phosphorylation levels of STING, TBK1, and IRF3 proteins were significantly increased. This suggests that As / ZIF-8@M can effectively activate the cGAS-STING pathway and has an immune-activating effect.

[0155] To illustrate the effect of epigenetic regulation on cGAS-STING pathway activation, siRNA technology was used to inhibit the expression of DNMT1 or PRMT1 genes in C1498 cells. C1498 cells with knocked-down genes were co-cultured with As / ZIF-8@M, and cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, followed by SDS-PAGE (10%) gel electrophoresis, transferred to a PVDF membrane, and blocked with Blotto at room temperature for 1.5 h. cGAS, p-STING, p-TBK1, p-IRF3, and IFN-β antibody (1:700) were added, and the mixture was incubated overnight, followed by 4 washes with TBST. The mixture was then incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The mixture was developed in a chromogenic agent for 30 s, and then developed and photographed using an ECL chemical imaging system.

[0156] The results are as follows Figure 41As shown in the figure. The results showed that after gene knockdown, the expression levels of cGAS, p-STING, p-TBK1, p-IRF3, and IFN-β proteins increased, demonstrating that epigenetic regulation can activate the cGAS-STING pathway. Furthermore, compared with the group that only underwent gene knockout, the protein expression levels of cGAS, p-STING, p-TBK1, p-IRF3, and IFN-β in the As / ZIF-8@M treatment group did not show any significant changes.

[0157] (13) Effects of As / ZIF-8@M on intracellular cytokines in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As) for 48 h. Cell supernatants were collected. The levels of IFN-β, IFN-γ, IL-6, and TNF-α were measured using ELISA.

[0158] The results are as follows Figure 42 As shown in the figure. The results showed that As / ZIF-8@M significantly increased the expression of IFN-β, IFN-γ, IL-6, and TNF-α, indicating that As / ZIF-8@M has a certain immune-activating effect.

[0159] (14) Effects of As / ZIF-8@M on intracellular ICD markers in C1498 cells C1498 cells were planted at a rate of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 g / mL in 6-well plates and treated with 2 mL of blank medium or medium containing different drugs (NaAsO2, As / ZIF-8, and As / ZIF-8@M, all 0.3 μg / mL as As) for 48 h. Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, then subjected to SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature with shaking for 1.5 h. CRT and HMGB1 antibody (1:700) were added, and the cells were incubated overnight, followed by 4 washes with TBST. Cells were then incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The cells were developed in a chromogenic agent for 30 s, visualized using an ECL chemical imaging system, and photographed. Cells were collected, washed with PBS, and fixed in 4% paraformaldehyde solution for 10 min (room temperature). Next, cells were blocked with 5% BSA for 3 h (room temperature), treated overnight with anti-CRT antibody (1:400) (4°C), washed with PBS, and then treated with FITC-labeled goat anti-rabbit IgG antibody (1:300) for 3 h (room temperature). After staining with Hoechst 33342, the cells were observed under a confocal microscope. Cells were collected, washed with PBS, and fixed in 4% paraformaldehyde solution for 10 min (room temperature). Next, cells were permeabilized in 0.1% Triton X-100 for 10 min (room temperature), blocked with 5% BSA for 3 h (room temperature), treated overnight with anti-HMGB1 antibody (1:400) (4°C), washed with PBS, and then treated with FITC-labeled goat anti-rabbit IgG antibody (1:300) for 3 h (room temperature). After staining with Hoechst 33342, the cells were observed under a confocal microscope. Cell supernatant was collected. ATP and HMGB1 levels were measured using ELISA.

[0160] The results are as follows Figure 43 As shown in the figure. The results showed that As / ZIF-8@M significantly increased the expression of ATP, CRT, and HMGB1, indicating that As / ZIF-8@M has a certain ability to induce immunogenic cell death in C1498 cells.

[0161] Experiment 4. In vivo animal targeting evaluation of As / ZIF-8@M Establishment of the C1498 mouse model: 0.5 × 10 6 One C1498 cell was injected into male C57BL / 6 mice (20±2 g) via tail vein injection, and white blood cell (WBC) counts were routinely monitored. After blood collection from the tail vein, 2 μL of blood was taken, diluted with 38 μL of Turk blood diluent, and white blood cell counts were performed under a microscope. When the white blood cell count reached 100 × 10⁶ cells / mL, the count was recorded. 9 When / L is reached, the modeling is confirmed to be successful.

[0162] RhB-labeled nanoparticles (As / ZIF-8 and As / ZIF-8@M, both 0.2 mg / kg as As) were injected into tumor-bearing mice via the tail vein. Fluorescence imaging was performed at 0, 6, 12, and 24 h using a BioLight GelView 6000M system. After 24 h, major organs and the femur were harvested for biodistribution studies.

[0163] The results are as follows Figure 44 As shown in the figure, the accumulation of nanoparticles in mouse bone marrow gradually increased over time, with both formulations reaching a peak at 12 h and decreasing at 24 h. Furthermore, the bone marrow accumulation capacity of As / ZIF-8@M was stronger than that of As / ZIF-8 at all time points. Fluorescence imaging of isolated mouse organs at 24 h revealed that the accumulation of As / ZIF-8@M in major organs of C1498 mice was significantly lower than that of As / ZIF-8, indicating a stronger bone marrow targeting ability.

[0164] Experiment 5. In vivo pharmacodynamic evaluation of As / ZIF-8@M (I) Establishment of the C1498 mouse model: 0.5×10 6 One C1498 cell was injected into male C57BL / 6 mice (20±2 g) via tail vein injection, and white blood cell (WBC) counts were routinely monitored. After blood collection from the tail vein, 2 μL of blood sample was diluted with 38 μL of Turk blood diluent. Finally, white blood cell counts were performed under a microscope, and the count was set at 100 × 10⁶ cells / mL. 9 When / L is reached, the modeling is confirmed to be successful.

[0165] (II) Animal grouping and administration: C1498 mice were randomly divided into 6 groups (n = 5), namely Saline group, NaAsO2 group, anti-PD-L1 group, As / ZIF-8 group, As / ZIF-8@M group, and As / ZIF-8@M+anti-PD-L1 group (all were 0.2 mg / kg for As and 1 mg / kg for anti-PD-L1). The nanomedicine was injected into the tail vein every three days, and anti-PD-L1 was injected into the peritoneum every other day for 2 weeks.

[0166] (III) In vivo efficacy indicators: WBC counts were performed every 5 days, using the methods described above, to assess the treatment effect. All mice were euthanized on day 30, and peripheral blood, major organs, and femurs were collected for H&E and Giemsa staining.

[0167] The results are as follows Figure 47 , 45As shown in the figure. Results showed that compared to the Saline group, WBC counts and LGI significantly decreased in all treatment groups. The WBC counts and LGIs in the anti-PD-L1 and As / ZIF-8 groups were comparable, at 67.88%±4.86% and 71.20%±5.29%, respectively. The LGI in the As / ZIF-8@M group was 85.13%±2.04%, consistent with expectations. The As / ZIF-8@M+anti-PD-L1 group exhibited the strongest anti-leukemia activity, with an LGI as high as 94.30%±1.35%. Mice in all groups were sacrificed on day 30, and spleen weight was collected. The Saline group showed splenomegaly due to leukemia cell infiltration, and all treatment groups showed a trend towards reversing splenomegaly. The As / ZIF-8@M+anti-PD-L1 group showed the most significant activity in reversing splenomegaly. Furthermore, H&E staining and Giemsa staining demonstrated a significant reduction in leukemia cell infiltration in the spleen after treatment with each drug group. Giemsa staining of bone marrow and peripheral blood showed a significant reduction in the number of leukemia cells in these areas after treatment with each drug group. H&E staining of the lungs revealed that treatment with the As / ZIF-8@M+anti-PD-L1 group effectively alleviated leukemia cell infiltration in the lungs. Analysis of the protein expression of Bmi-1, c-MYC, DNMT1, and PRMT1 in bone marrow cells from each group showed a decrease in the expression levels of all four proteins after drug administration, indicating that the activity of leukemia stem cells may be inhibited after treatment with each drug group, and that they still play an epigenetic regulatory role in vivo.

[0168] (iv) The in vivo immune activation effect of As / ZIF-8@M (1) Activation of cGAS-STING pathway: The expression of cGAS, p-STING, p-TBK1, p-IRF3 and IFN-β in mouse bone marrow cells was detected by WB technology.

[0169] (2) ICD marker analysis: Mice were euthanized, and femoral bone samples were obtained. Bone marrow samples were then isolated for ICD marker analysis. After repeated freeze-thaw cycles, the tissues were homogenized in PBS solution and centrifuged at 2500 rpm / min for 20 min. The supernatant was collected for the detection and analysis of ATP, HMGB-1, and CRT.

[0170] The results are as follows Figure 46 As shown in the figure, the results indicated that the levels of ATP, CRT, and HMGB1 in the bone marrow supernatant of all treatment groups showed an increasing trend, with the As / ZIF-8@M+anti-PD-L1 group showing the most significant increase. These elevated levels of mouse ICD markers further confirm the activation of ICD in vivo.

[0171] (3) In vivo immune cell analysis: Mice were euthanized, and spleens and bone marrow were collected and homogenized to obtain single-cell suspensions. Cells were stained with PE-anti-CD11c, FITC-anti-CD80, and PerCP / Cy5.5-anti-CD86 antibodies (BioLegend) to identify mature dendritic cells (DCs). For macrophage phenotype detection, M1 macrophages were identified using FITC-anti-F4 / 80 and PE-anti-CD86 antibodies, and M2 macrophages were identified using FITC-anti-F4 / 80 and PE-anti-CD206 antibodies. CD4+ T cells were identified using PE-anti-CD3 and FITC-anti-CD4 antibodies, and CD8+ T cells were identified using PE-anti-CD3 and FITC-anti-CD8 antibodies. T cells in the spleen were stained with PerCP / Cy5.5-anti-CD62L and APC-anti-CD44 antibodies to distinguish between central memory T cells and effector memory T cells.

[0172] The results of cGAS-STING pathway-related protein detection in mouse bone marrow cells of each group are as follows: Figure 48 As shown in the figure, the protein expression levels of cGAS, p-STING, p-TBK1, p-IRF3, and IFN-β all increased, indicating that each treatment modality could activate the cGAS-STING pathway. Among them, the effect of As / ZIF-8@M+anti-PD-L1 was the most significant, indicating that As / ZIF-8@M can enhance the immune activation ability of anti-PD-L1 antibodies.

[0173] The results of the detection of mature dendritic cells in the spleen of mice in each group are as follows: Figure 49 As shown in the figure. The results indicate that the enhancement of leukemia cell antigenicity directly triggers the maturation of dendritic cells (DCs), which is mediated by the co-stimulatory molecule CD80. + and CD86 + The upregulation was evident. Among them, the As / ZIF-8@M+anti-PD-L1 group showed the most significant stimulation of dendritic cells in the spleen (16.66%±2.05%), which may be beneficial for the presentation of antigens to tumor-specific T cells.

[0174] Flow cytometry was used to quantitatively measure tumor-associated macrophages (TAMs; F4 / 80) on the surface of bone marrow-derived macrophages (BMDMs) in each group of mice. + CD86 + and CD206 + The level of expression, the results are as followsFigure 50 As shown in the figure. The results showed that, compared with the control group, the proportion of M1 macrophages in the anti-PD-L1 group increased from 28.38%±5.09% to 44.54%±2.42%, while the proportion of M2 macrophages decreased from 32.62%±1.45% to 20.36%±2.06%. The polarization effect of As / ZIF-8@M and As / ZIF-8@M+anti-PD-L1 groups on macrophages was significantly stronger than that of the anti-PD-L1 group. The proportions of M1 macrophages were 52.68%±3.57% and 61.78%±6.12%, respectively, and the proportions of M2 macrophages were 16.38%±2.09% and 11.88%±1.30%, respectively, indicating that As / ZIF-8@M can effectively enhance the anti-PD-L1 reversal of the immunosuppressive leukemia microenvironment.

[0175] Flow cytometry analysis was used to determine the CD4 levels in the spleens of mice in each group. + CD8 + The percentage of T cells, results as follows Figure 51 As shown. The results showed that in mice treated with As / ZIF-8@M+anti-PD-L1, CD4 + and CD8 + The proportion of T cells was significantly increased. In mice treated with As / ZIF-8@M, CD4+... + and CD8 + The T-cell levels were also higher in all treatment groups than in the other treatment groups.

[0176] Measurement of effector memory T cells (CD44) in the spleen of mice in each group + CD62L - The percentage of ) is as follows: Figure 52 As shown. The results showed that, compared with the Saline group, the As / ZIF-8@M+anti-PD-L1 treatment group had significantly higher levels of effector memory T cells (CD8+) in the spleen of mice. + T cell, CD44 + CD62L - The number of TEM cells increased significantly, and the As / ZIF-8@M treatment group also had significantly higher numbers of TEM cells than other treatment groups. This indicates that this therapy successfully elicited a strong immune memory effect.

[0177] (4) Analysis of in vivo inflammatory factors: Mice were euthanized, and bone marrow cells and peripheral blood were collected. The levels of IFN-β, IL-6, TNF-α and IFN-γ were detected using an ELISA kit.

[0178] The expression levels of IFN-β, IL-6, TNF-α, and IFN-γ in the bone marrow and serum of each group of mice are as follows: Figure 53As shown in the figure. The results showed that mice treated with As / ZIF-8@M+anti-PD-L1 exhibited the highest levels of IFN-β, IFN-γ, IL-6, and TNF-α, indicating that As / ZIF-8@M enhanced the in vivo anti-tumor immune response by synergistically supporting anti-PD-L1.

[0179] (v) In vivo safety study of As / ZIF-8@M (1) Changes in mouse body weight: During the drug administration period, the status of tumor-bearing mice in each group was observed daily and their body weight was recorded every 5 days. At the end of the treatment, a curve showing the changes in mouse body weight was plotted, such as... Figure 54 As shown.

[0180] (2) Organ and tissue morphology examination: Major organ tissues (heart, liver, spleen, lung, and kidney) of mice in each group were collected, fixed with 4% paraformaldehyde, and then subjected to paraffin embedding, sectioning, and H&E staining. The morphological changes of the tissue sections in each group were observed and photographed for analysis using an inverted fluorescence microscope. The results are as follows: Figure 55 As shown.

[0181] (3) Blood routine, liver and kidney function tests: C57 mice were intravenously injected with physiological saline (control group) or As / ZIF-8@M (Mean±SD, n=3). Blood was collected from the eyeballs for hematological and blood biochemical tests.

[0182] Hematological parameters include: white blood cells (WBC), red blood cells (RBC), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin level (MCH), mean corpuscular volume (MCV), hemoglobin (HGB), hematocrit (HCT), platelet distribution width (PDW), direct bilirubin (D-bil), glucose (Glu), mean platelet volume (MPV), and monocyte count (Mon). Blood biochemistry parameters include: albumin (ALB), aspartate aminotransferase / alanine aminotransferase ratio (AST / ALT), total protein (TP), and as indicators of liver function; blood urea nitrogen (BUN) is used as an indicator of kidney function.

[0183] The results are as follows ​As shown in the results, H&E staining of the livers of mice in the NaAsO2 group revealed vacuoles, indicating that NaAsO2 may have a toxic effect on the liver. However, no significant abnormalities were observed in the livers of mice treated with the nanodelivery agent, suggesting that As / ZIF-8@M can alleviate NaAsO2-induced liver toxicity. No significant pathological abnormalities were observed in the heart, spleen, lungs, or kidneys, indicating that As / ZIF-8@M has excellent biocompatibility. Furthermore, hematological and serum biochemical analyses showed that mice in the NaAsO2 group exhibited some degree of liver toxicity, while the liver toxicity observed in the other treatment groups was significantly lower, further confirming the low biotoxicity of As / ZIF-8@M.

[0184] Conclusion: All the above results confirm that As / ZIF-8@M has good biocompatibility in vivo.

[0185] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An arsenic in-situ grown metal-organic framework material, characterized in that: arsenic oxide nanoparticles are formed in situ on the surface of the metal-organic framework material by ligand exchange between an arsenic compound and the metal-organic framework material; wherein the arsenic compound is selected from any one or more of sodium arsenite, disodium arsenate heptahydrate, and arsenic trioxide; and wherein the metal-organic framework material is selected from any one of ZIF-8, ZIF-67, IRMOF-8, and Fe-MOF.

2. The arsenic in-situ grown metal-organic framework material according to claim 1, characterized in that: the loading of the arsenic compound is 2.12% to 5.20%.

3. The method for preparing the arsenic in-situ grown metal-organic framework material according to claim 1 or 2, characterized in that: The metal-organic framework material is thoroughly mixed with arsenic compounds in a solvent, centrifuged, and the resulting precipitate is the arsenic in-situ grown metal-organic framework material.

4. The method for preparing arsenic in-situ grown metal-organic framework materials according to claim 3, characterized in that, The metal-organic framework material is ZIF-8, which is prepared by the following method: 1800 mg of zinc nitrate hexahydrate is added to 84 mL of methanol, and this is denoted as solution A; 4200 mg of 2-methylimidazole is added to 84 mL of methanol, and this is denoted as solution B; solution B is added dropwise to solution A, the mixture is stirred and reacted, centrifuged, and the precipitate is collected, which is ZIF-8.

5. A biomimetic nanoparticle grown in situ from arsenic, characterized in that: It is made by coating the arsenic in situ growth-metal-organic framework material of claim 1 or 2 with a cell membrane, wherein the cell membrane is selected from erythrocyte membrane, macrophage membrane, platelet membrane, leukocyte membrane, tumor cell membrane, and mesenchymal stem cell membrane.

6. The arsenic-in-situ grown biomimetic nanoparticles according to claim 5, characterized in that: The cell membrane is a homologous C1498 cell membrane.

7. The arsenic-in-situ grown biomimetic nanoparticles according to claim 5, characterized in that: The weight ratio of the cell membrane to the arsenic in situ grown metal-organic framework material is 1:(1-5).

8. The arsenic-in-situ grown biomimetic nanoparticles according to claim 5, characterized in that: The arsenic-in-situ grown biomimetic nanoparticles have a particle size of 40–100 nm.

9. The method for preparing arsenic-in-situ grown biomimetic nanoparticles according to any one of claims 5 to 9, characterized in that: The cell membrane and the arsenic in situ grown metal-organic framework material are thoroughly mixed in a solvent and centrifuged. The resulting precipitate is the arsenic in situ grown biomimetic nanoparticle.

10. The use of the arsenic in-situ grown metal-organic framework material according to claim 1 or 2 in the preparation of drugs for treating cancer.

11. The use of the arsenic-in-situ grown biomimetic nanoparticles according to any one of claims 5 to 9 in the preparation of drugs for treating cancer.

12. The application according to claim 10 or 11, characterized in that: The cancer referred to is the type of cancer corresponding to the treatment of homologous cell membranes.

13. The application according to claim 10 or 11, characterized in that: In specific applications, it is used in combination with anti-tumor drugs.

14. The application according to claim 13, characterized in that: The anti-tumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

15. A pharmaceutical preparation, characterized in that: The active ingredient is the arsenic in-situ grown metal-organic framework material as described in claim 1 or 2, or the arsenic in-situ grown biomimetic nanoparticles as described in any one of claims 5 to 9.

16. The pharmaceutical preparation according to claim 15, characterized in that: The active ingredients also include anti-tumor drugs.

17. The pharmaceutical preparation according to claim 16, characterized in that: The anti-tumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

18. Use of the pharmaceutical preparation according to any one of claims 15 to 17 in the preparation of a medicament for treating cancer.

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