Zirconium-copper bimetallic nano-material, preparation method and application of zirconium-copper bimetallic nano-material in preparation of medicine for treating breast cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a zirconium-copper bimetallic nano-material, a preparation method and application of the zirconium-copper bimetallic nano-material in preparation of medicines for treating breast cancer. The zirconium-copper bimetallic nano-material is formed by loading an oxidative stress inducer on a nano-carrier, wherein the nano carrier is an organic framework material Zr-Cu MOF (Metal Organic Framework) containing double metals of Zr and Cu, and the oxidative stress inducer is optimally Elesclomol. A product, namely ZCEH, for treating breast cancer is obtained based on coupling of the zirconium-copper bimetallic nano-material with a targeted drug such as trastuzumab. The ZCEH provides a new method for developing a nano platform with an antibody function, and realizes a treatment strategy of combined anti-tumor of trastuzumab and an oxidative stress inducer. As a safe and effective tumor treatment strategy, the ZCEH has huge clinical transformation potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a zirconium-copper bimetallic nanomaterial, a preparation method and use thereof in preparing a drug for treating breast cancer. Background Art

[0002] The incidence of breast cancer remains high worldwide. Human epidermal growth factor receptor 2 (HER2)-positive breast cancer, as a subtype of breast cancer, accounts for approximately 20-30% of all breast cancer cases. Studies have shown that conventional chemotherapy suffers from shortcomings such as poor precision, low efficiency, and severe side effects. Therefore, the development of new treatments for breast cancer and novel drugs with targeted, high efficacy, and low toxicity has become a current research hotspot. Trastuzumab (Herceptin), a cornerstone of the treatment of HER2-positive breast cancer, is currently used as a targeted drug in numerous tumors that overexpress HER2. Despite this, although a number of new targeted drugs have entered clinical trials and have initially demonstrated promising clinical application potential, further research and development is still needed due to numerous challenges, such as tumor heterogeneity, drug resistance, and the limited efficacy of single-pathway treatments. Therefore, developing effective HER2-targeted drugs based on novel mechanisms of action to enhance their effectiveness is of great significance.

[0003] Copper death, caused by the binding of copper ions to acylated proteins in the mitochondrial tricarboxylic acid (TCA) cycle, leads to protein oligomerization and loss of iron-sulfur cluster proteins, which in turn triggers protein toxic stress and cell death. It is a different form of cell death from ferroptosis and pyroptosis, which shows good tumor suppression and minimal drug resistance. Studies have shown that copper death can enhance the sensitivity of doxorubicin in the treatment of breast cancer and reduce the dosage of doxorubicin; in addition, it can also enhance the chemotherapy sensitivity of docetaxel in the treatment of prostate cancer. However, copper death is hindered in its anti-tumor application because it depends on copper ion levels and the glutathione (GSH)-rich microenvironment in the tumor. Elesclomol (esclomol, ES) is a stable copper chelator that can transport extracellular copper into cell mitochondria at a ratio of 1:1 (molar ratio). The dissociated ES is rapidly excreted and continues to bind to extracellular Cu 2+ Chelation forms a continuous copper accumulation shuttle mechanism in the cell. This mechanism allows the cytoplasm to neutralize the Cu in the mitochondria. +Cu increases in tumor cells, catalyzing the production of hydroxyl radicals and inducing copper death in cells. However, treatment with ilisimol may not increase Cu levels in tumor cells to the level required to trigger apoptosis, and its rapid metabolism in vivo and limited efficacy also limit its clinical use. Furthermore, copper death can occur in various tissue sites in the body, so avoiding copper-related toxicity in normal tissues remains a challenge. Therefore, targeted intervention in the transport and distribution of copper ions in the body and cells is a potential strategy for breast cancer treatment.

[0004] Nanomaterials have become a research focus for treating various types of tumors, leveraging the enhanced permeability and retention (EPR) effect to improve the bioavailability of loaded drugs. Among nanoparticles suitable for drug delivery, copper-based nanoparticles have emerged as a promising approach for delivering exogenous copper to the tumor microenvironment (TME). Metal-organic frameworks (MOFs) are crystalline and porous materials composed of metal clusters or metal ions interconnected by organic linkers. Their high porosity and large surface area allow for the maximum loading of small molecules, proteins, nucleotides, and other substances within MOFs. Using copper-based MOFs to load siRNA effectively silences target genes, increases copper uptake, and thus induces cell apoptosis, enhancing anti-tumor effects. Copper-based MOFs loaded with DOX have also been shown to enhance the in vivo therapeutic efficacy of prostate cancer. Furthermore, the structural size of MOFs allows for the development of intelligent synergistic drug systems combining metals and drugs tailored to specific drug delivery requirements, making them a promising new drug delivery vehicle. However, the heterogeneity of tumor cells makes it difficult to effectively and uniformly deliver drugs to tumor cells using the EPR effect alone. Consequently, the distribution and accumulation efficiency within tumor tissue vary widely, limiting therapeutic efficacy. Therefore, it is crucial to improve the active targeting of nanomaterials, achieve stable and uniform effective transport and distribution within the body and tumor cells, and induce cancer cell death to achieve synergistic effects. Summary of the Invention

[0005] The purpose of the present invention is to provide a zirconium-copper bimetallic nanomaterial, a preparation method and its use in the preparation of a drug for treating breast cancer, which can not only inhibit the proliferation of in situ tumors, but also has excellent therapeutic effects on inhibiting lung metastasis tumors, and is a HER2 + Precision therapy of breast cancer cells provides a potential new strategy.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The first aspect of the present invention provides a zirconium-copper bimetallic nanomaterial, which is composed of a nanocarrier loaded with an oxidative stress inducer; wherein the nanocarrier is an organic framework material Zr-Cu MOF containing Zr and Cu bimetallics, and the oxidative stress inducer includes Elesclomol, Disulfiram, NSC-319726 and tetrathiomolybdate.

[0008] A second aspect of the present invention provides a method for preparing a zirconium-copper bimetallic nanomaterial, the method comprising the following steps:

[0009] S1: Dissolve zirconium salt, ligand and PVP in a solvent, mix them ultrasonically, then add copper salt and dissolve them evenly. Then, obtain Zr-Cu MOF containing Zr and Cu bimetallic organic framework material through hydrothermal reaction.

[0010] S2: The obtained Zr-Cu MOF ethanol solution and the oxidative stress inducer ethanol solution are mixed and stirred, and then the precipitate is collected by centrifugation to obtain the zirconium-copper bimetallic nanomaterial Zr-Cu MOF@oxidative stress inducer.

[0011] Preferably, in step S1, the zirconium salt is ZrCl4.

[0012] Preferably, in step S1, the ligand is terephthalic acid.

[0013] Preferably, in step S1, the solvent includes N,N-dimethylformamide.

[0014] Preferably, in step S1, the copper salt is CuCl2·2H2O2, CuI2 or CuBr2.

[0015] Preferably, in step S1, the hydrothermal reaction conditions are: temperature 120° C., time 10 h.

[0016] Preferably, in step S2, the mass ratio of the Zr-Cu MOF to the oxidative stress inducer is 1:1.

[0017] Preferably, the stirring reaction conditions are: room temperature, and time 8 to 12 hours.

[0018] The third aspect of the present invention provides a use of a zirconium-copper bimetallic nanomaterial in preparing any of the following medicines:

[0019] (i) drugs for the treatment of breast cancer;

[0020] (ii) drugs for the treatment of breast cancer lung metastasis;

[0021] (iii) Drugs that increase the sensitivity of breast cancer to targeted drugs.

[0022] The fourth aspect of the present invention provides a drug for treating breast cancer, comprising the zirconium-copper bimetallic nanomaterial as described above, and a targeted drug; and the targeted drug is coupled to the zirconium-copper bimetallic nanomaterial via a coupling agent.

[0023] Preferably, the breast cancer comprises HER2 + Breast cancer.

[0024] Preferably, the targeted drug includes trastuzumab.

[0025] Preferably, the coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

[0026] A fifth aspect of the present invention provides a method for preparing a drug for treating breast cancer, the method comprising the following steps:

[0027] (1) dissolving the zirconium-copper bimetallic nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting a precipitate;

[0028] (2) Adding a targeted drug aqueous solution to the obtained precipitate, stirring and centrifuging, and taking the precipitate to obtain Zr-Cu MOF@oxidative stress inducer@targeted drug for treating breast cancer.

[0029] Preferably, the coupling agent in step (1) consists of a 4-6 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and a 4-6 mg / mL N-hydroxysuccinimide aqueous solution.

[0030] Preferably, in step (1), the stirring conditions are: room temperature, and time 20 to 25 minutes.

[0031] Preferably, in step (1), the centrifugal conditions are: rotation speed 10000-12000 rpm, time 5-10 min.

[0032] Preferably, in step (2), the stirring conditions are: room temperature, and time 8 to 12 hours.

[0033] Preferably, in step (2), the centrifugal conditions are: rotation speed 10000-12000 rpm, time 5-10 min.

[0034] By means of the above technical solution, the present invention has at least the following advantages:

[0035] The present invention constructs a bimetallic organic framework material Zr-Cu MOF containing zirconium and copper, and modifies the surface of the monoclonal antibody of Her-2 receptor, trastuzumab, with the oxidative stress inducer Elesclomol to obtain the nanomaterial Zr-CuMOF@Elesclomol@Herceptin, referred to as ZCEH, which aims to cause oxidative stress damage to HER2-positive breast cancer cells and induce copper death. The nanomaterial can disintegrate and release trastuzumab and Cu in the acidic environment of the tumor. 2+ The modification of trastuzumab can enhance the stability of ZCEH in blood circulation and its targeting to tumor cells, and reduce its toxicity to non-target cells and tissues. 2+ The ES is chelated and enters the cytoplasm and mitochondria before dissociating. The dissociated ES is rapidly excreted and continues to interact with extracellular Cu 2+ Chelation forms a continuous copper accumulation shuttle mechanism in the cell. 2+ A part of it can convert H2O2 into highly cytotoxic ·OH through the Fenton reaction under the action of GSH, causing cellular oxidative stress damage. 2+ ZCEH is reduced to Cu+ by FDX-1 and binds to acylated proteins involved in glucose metabolism, disrupting the tricarboxylic acid cycle and promoting their aggregation. This, in turn, causes the loss of iron-sulfur cluster proteins, ultimately triggering proteotoxic stress and leading to copper cell death. In vivo experiments have shown that ZCEH can significantly inhibit the proliferation of both in situ and metastatic tumors, effectively inducing copper cell death in tumor cells, providing an innovative solution for the precision treatment of HER2-positive breast cancer.

[0036] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The preparation of Zr-Cu MOF@ES@aH for inducing copper death is shown; wherein, a) is a schematic diagram of the preparation process of Zr-Cu MOF@ES@aH; b) is the biological mechanism of Zr-Cu MOF@ES@aH inducing copper death - after cell internalization, Zr-Cu MOF@ES@aH simultaneously releases copper and ES in tumor cells. The released copper binds to lipoylated dihydrolipoamide S-acetyltransferase (DLAT), thereby activating the copper transferase; at the same time, the released Cu 2+ Depletion of glutathione (GSH) in tumor cells and generation of ROS together enhance synergistic therapy against HER2-positive breast cancer.

[0038] Figure 2 Characterization of Zr-Cu MOF@ES@αH is shown; among them, a) SEM and TEM images of ZC, ZCE, ZCH and ZCEH, SEM, scale bar: 200 nm, TEM, scale bar: 100 nm; b) TEM and corresponding elemental mapping of ZCEH (scale bar: 100 nm); c) particle size and polydispersity index (PDI) of ZC, ZCE, ZCH and ZCEH; d) X-ray diffraction patterns of ZC, ZCE, ZCH and ZCEH; e) Fourier transform infrared (FTIR) spectrum of each sample; f) Zeta potential of ZC, ZCE, ZCH and ZCEH; g) XPS of ZC; h) drug release behavior of ZCE at pH 7.4 and pH 5.7; i) solubility of ZCEH at pH 5.7 and pH 7.4.

[0039] Figure 3 Elemental analysis diagram is shown.

[0040] Figure 4 Shown are SDS-PAGE gel electrophoresis images of each sample.

[0041] Figure 5 High-resolution XPS spectra of Cu 2p and Zr 3d of Zr-Cu MOF are shown.

[0042] Figure 6 The ultraviolet absorption of each sample is shown; among them, a) is the ultraviolet absorption spectrum of ES at different concentrations; b) is the drawn standard curve; c) is the ultraviolet absorption spectrum of Zr-Cu MOF and Zr-Cu MOF@ES.

[0043] Figure 7 In vitro efficacy experiments are shown; wherein, a) is a laser confocal microscope image of ZCEH changing with time (scale bar: 20 nm); b) is a laser confocal microscope image of PBS, ZC, ZCE, ZCH and ZCEH (scale bar: 20 nm); c) MTT assay of ZC; d) is an MTT assay of PBS, ZC, ZCE, ZCH and ZCEH with the addition of the copper death inhibitor UK5099; e) is a laser confocal image of live-dead cell staining of PBS, ZC, ZCE, ZCH and ZCEH with the addition of the copper death inhibitor UK5099 (scale bar: 100 nm); f) is the change of intracellular GSH / GSSG under different treatment conditions; g) is the change of hydrogen peroxide content under different treatment conditions; h) is a laser confocal image of PBS, ZC, ZCE, ZCH, ZCEH and ZCEH with the addition of the copper death inhibitor UK5099 and the addition of a copper ion probe (scale bar: 20 nm).

[0044] Figure 8A comparison of the cellular uptake capacity of different samples is shown.

[0045] Figure 9 The cytotoxicity tests of different samples are shown; wherein, a) is the cytotoxicity of different concentrations of ZC on L929 cells at different times; b) is the comparison of the cytotoxicity of different samples on BT474 cells at different times.

[0046] Figure 10 The figures show the induction of cell copper death; a) laser confocal microscopy images (scale bar: 100 nm) of reactive oxygen species (ROS) generated by PBS, ZC, ZCE, ZCH, and ZCEH with the addition of the copper death inhibitor UK5099, and b) flow cytometry graphs; c) ROS flow cytometry statistical graphs; d) laser confocal images (scale bar: 20 nm) and e) flow cytometry graphs of mitochondrial damage by PBS, ZC, ZCE, ZCH, and ZCEH; f) biological electron microscopy images (scale bar: 2 nm) of mitochondrial damage by PBS, ZC, and ZCEH; g) immunoblot images of PBS, ZC, ZCE, ZCH, ZCEH, and ZCEH with the addition of the copper death inhibitor UK5099.

[0047] Figure 11 A schematic diagram of the fluorescence intensity of the lungs of nude mice bearing lung metastasis tumors constructed in the present invention is shown.

[0048] Figure 12 The in vivo and in vitro safety is shown; a) MTT experiment on L929 using PBS, ZC, ZCE, ZCH and ZCEH; b) in vitro hemolysis experiment; c) body weight, d) blood routine and e) biochemical changes of KM mice after injection of normal saline and ZCEH; f) H&E staining analysis of important organs such as heart, liver, spleen, lung and kidney; g) in vivo imaging of mice after treatment at 0h, 3h, 12h, 24h, 72h and 144h; h) imaging of different organs at different times.

[0049] Figure 13 The therapeutic and anti-metastatic properties of ZCEH were evaluated in the BT474-LUC breast cancer model; wherein, a) is a schematic diagram of the establishment of the BT474-LUC tumor-bearing mouse model and the treatment plan; b) is the change in mouse body weight after drug injection; c) is the growth of BT474-LUC tumors in mice visualized by IVIS; d) is the tumor volume growth curve of primary tumors in mice in different treatment groups; e) is a photograph of the primary tumors removed from each group of mice; f) is the quantitative analysis of tumor weight; g) is the immunofluorescence staining of H&E, Ki67, TUNEL, DLAT, and LAS of each group of mice; h) is the WB analysis and quantitative analysis of copper-related protein expression in mouse tumor tissues; i) is the expression of key genes in tumor tissues.

[0050] Figure 14 The effects of ZCEH inhibition on lung metastasis are shown; wherein, a) is a schematic diagram of the treatment regimen in the BT474 lung metastasis model; b) is the weight change of mice in each group; c, d) show H&E staining of lung tissues of tumor-bearing mice in the ZCEH group and the Control group, respectively. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0052] Human epidermal growth factor receptor 2 (HER2)-expressing breast cancer is one of the most common subtypes of female breast cancer. Among them, HER2 as a key target has made significant progress in the treatment of HER2-positive breast cancer, but problems such as tumor heterogeneity, limited effect of single-pathway treatment, and cellular pathway signaling disorders still restrict the efficacy of anti-tumor drugs. To this end, the present invention designs and constructs an active targeted bimetallic organic framework composite drug Zr-Cu MOF@ES@αH (ZCEH), by loading Elesclomol (ES) on Zr-Cu MOF and using HER2-positive breast cancer cell-specific antibody-trastuzumab for surface functionalization modification. The purpose is to cause oxidative stress damage to HER2-positive breast cancer cells and induce copper death. The nanoplatform can disintegrate and release trastuzumab and Cu in the acidic environment of the tumor. 2+ The modification of trastuzumab can enhance the stability of ZCEH in blood circulation and its targeting to tumor cells, and reduce its toxicity to non-target cells and tissues. 2+ The ES is chelated and enters the cytoplasm and mitochondria before dissociating. The dissociated ES is rapidly excreted and continues to interact with extracellular Cu 2+ Chelation forms a continuous copper accumulation shuttle mechanism in the cell. 2+ A part of it can convert H2O2 into highly cytotoxic ·OH through the Fenton reaction under the action of GSH, causing cellular oxidative stress damage. 2+ Reduced to Cu by FDX-1 +ZCEH binds to acylated proteins associated with glucose metabolism, disrupting the tricarboxylic acid cycle and driving their aggregation. This, in turn, causes the loss of iron-sulfur cluster proteins, ultimately triggering proteotoxic stress and leading to copper death. In vivo experiments have shown that ZCEH can significantly inhibit the proliferation of both in situ and metastatic tumors, effectively inducing copper death in tumor cells, providing an innovative solution for the precision treatment of HER2-positive breast cancer.

[0053] Specifically, in one embodiment of the present invention, a zirconium-copper bimetallic nanomaterial is provided, characterized in that it is composed of a nanocarrier loaded with an oxidative stress inducer; wherein the nanocarrier is an organic framework material Zr-Cu MOF containing Zr and Cu bimetallics, and the oxidative stress inducer includes Elesclomol, Disulfiram, NSC-319726 and tetrathiomolybdate. As used herein, oxidative stress inducers are a class of drugs or compounds that can induce oxidative stress by increasing the level of intracellular reactive oxygen species (ROS), thereby selectively killing cancer cells. Cancer cells are usually at a high ROS basal level due to metabolic abnormalities (such as the Warburg effect). Further increasing ROS can break through their antioxidant defense threshold, leading to DNA damage, mitochondrial dysfunction and cell apoptosis. This type of drug is one of the important directions of anti-cancer research, especially for drug-resistant or highly metabolic tumors. This type of inducer includes but is not limited to Elesclomol, Disulfiram, NSC-319726 and tetrathiomolybdate, preferably Elesclomol. As used in this article, Zr-Cu MOF (zirconium-copper metal-organic framework) is a porous crystalline material composed of zirconium (Zr) and copper (Cu) bimetallic nodes and organic ligands. It has high stability, tunable pore structure and redox activity, and has broad application potential in catalysis, gas adsorption, sensing and environmental remediation.

[0054] In another embodiment, the present invention also provides a method for preparing a zirconium-copper bimetallic nanomaterial, comprising the following steps: S1: Dissolving a zirconium salt, a ligand, and PVP in a solvent and ultrasonically mixing them, then adding a copper salt and dissolving them uniformly. A hydrothermal reaction is performed to obtain a Zr-Cu bimetallic organic framework (MOF) material containing Zr and Cu. In this step, the zirconium salt is ZrCl4; the ligand is terephthalic acid; the solvent comprises N,N-dimethylformamide; and the copper salt is CuCl2. This reaction is a one-pot hydrothermal reaction, wherein the hydrothermal reaction conditions are: a temperature of 120°C and a reaction time of 10 hours. S2: Mixing and stirring the resulting Zr-Cu MOF ethanol solution with an oxidative stress inducer ethanol solution, followed by centrifugation to collect the precipitate, thereby obtaining the Zr-Cu bimetallic nanomaterial (Zr-Cu MOF@oxidative stress inducer). In this step, the mass ratio of Zr-Cu MOF to oxidative stress inducer is 1:1. The mixing and stirring conditions are room temperature for 8 to 12 hours.

[0055] In another embodiment, there is provided a use of a zirconium-copper bimetallic nanomaterial in the preparation of a drug for treating breast cancer; or in the preparation of a drug for treating breast cancer lung metastasis; or in the preparation of a drug for improving the sensitivity of breast cancer targeted drugs. The Zr-Cu MOF@Elesclomol@Herceptin (ZCEH) constructed by the present invention is used to improve HER2 + The sensitivity of breast cancer to trastuzumab. Studies have shown that ZCEH can not only inhibit the proliferation of in situ tumors, but also has excellent efficacy in inhibiting lung metastasis, thus providing a reference for HER2 + Precision treatment of breast cancer cells provides an application prospect.

[0056] In another embodiment, a drug for treating breast cancer is provided, comprising the aforementioned zirconium-copper bimetallic nanomaterial and a targeted drug; wherein the targeted drug is coupled to the zirconium-copper bimetallic nanomaterial via a coupling agent. The breast cancer includes HER2 + Breast cancer. Targeted drugs include trastuzumab. The coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

[0057] In another embodiment, a method for preparing a drug for treating breast cancer is provided, comprising the following steps: (1) dissolving a zirconium-copper bimetallic nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting a precipitate; the coupling agent comprises 200 μL of a 4-6 mg / mL aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 200 μL of a 4-6 mg / mL aqueous solution of N-hydroxysuccinimide. The stirring conditions in this step are: room temperature for 20-25 minutes. The centrifugation conditions are: a rotation speed of 10,000-12,000 rpm for 5-10 minutes.

[0058] (2) Adding a targeted drug aqueous solution to the resulting precipitate, stirring, centrifuging, and collecting the precipitate to obtain the Zr-Cu MOF@oxidative stress inducer@targeted drug for treating breast cancer. In this step, stirring conditions are: room temperature for 8 to 12 hours. Centrifugation conditions are: 10,000 to 12,000 rpm for 5 to 10 minutes.

[0059] This paper designs a Zr-Cu bimetallic MOF that targets HER2-positive breast cancer cells and enhances tumor killing by utilizing glutathione (GSH) depletion and mitochondrial copper overload mechanisms. Specifically, ES, which has the characteristics of efficient copper ion transport carrier, is embedded in Zr-Cu MOF by hydrothermal synthesis, and trastuzumab is coupled to the surface to construct Zr-Cu MOF@ES@αH (ZCEH, Figure 1 a) ZCEH is precisely targeted by trastuzumab and accumulates in the tumor site, where it disintegrates and releases copper ions and ES in the tumor microenvironment. 2+ Chelates to form ES-Cu complex, which is then taken up by cells. 2+ dissociates from the complex and ES is expelled to bind extracellular Cu again. 2+ , forming a continuous copper ion "shuttle" accumulation mechanism. 2+ A portion of it is reduced to Cu by GSH + The latter reacts with H2O2 through the Fenton reaction to generate highly toxic hydroxyl radicals (·OH), which leads to a significant increase in the level of reactive oxygen species (ROS) and destroys the cellular redox homeostasis. 2+ After entering the mitochondria, it is reduced to Cu by FDX-1. + , Cu +It binds to acylated proteins in the tricarboxylic acid cycle (TCA), triggering oligomerization of acylated proteins and the loss of iron-sulfur cluster proteins, thereby inducing protein toxic stress. The copper-induced imbalance in the regulation of cellular redox levels combined with the protein toxic stress mechanism significantly enhances the killing effect on tumor cells and provides a new strategy for clinical treatment. To further clarify the mechanism of action of ZCEH in inducing copper death in HER2-positive breast cancer cells (AU565), the present invention uses the copper death inhibitor UK5099 for verification experiments. The results of in vitro and in vivo studies showed that ZCEH not only exhibited significant tumor inhibitory effects, but also had excellent biosafety. In summary, ZCEH provides an innovative therapeutic strategy for the elimination of primary lesions and inhibition of metastatic lesions in HER2-positive breast cancer.

[0060] The present invention will further illustrate the principles, processes and effects of the above materials through specific embodiments below, so that those skilled in the art can understand the essence of the present invention.

[0061] The following embodiments relate to and mention:

[0062] 1. Materials:

[0063] Zirconium chloride, copper chloride, N,N-dimethylformamide (DMF), polyvinylpyrrolidone, hydrogen peroxide (3 wt %), anhydrous ethanol, rhodamine B, and Cy7 dye were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China); terephthalic acid was purchased from J&K Technology Co., Ltd. (Beijing, China); elesclomol (purity 98%), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid were purchased from Zhengzhou Alpha Chemical Co., Ltd. (Zhengzhou, China); trastuzumab for injection was purchased from Roche Pharmaceuticals Co., Ltd. (Shanghai, China); protease inhibitors, RIPA lysis buffer (strong), reactive oxygen species detection kit, hydrogen peroxide detection kit, GSH / GSSG detection kit, and Hoechst 33342 live cell staining solution (100X) and Calcein / PI cell viability and cytotoxicity assay kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); the BCA protein assay kit was purchased from Solebo Technology Co., Ltd. (Beijing, China); the copper ion fluorescent probe (GuprosGreen) and the mitochondrial membrane potential fluorescent probe (JC-1) were purchased from Dongren Chemical Technology Co., Ltd. (Shanghai, China); tubulin polyclonal antibody, DLAT, LIAS, FDX-1, and Hsp70 monoclonal antibodies were purchased from Wuhan Sanying Biological Co., Ltd. (Wuhan, China); goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Abcam Trading Co., Ltd. (Shanghai, China); RPMI 1640, FBS, streptomycin / penicillin, and other biological reagents were purchased from Gibco (New York, USA).

[0064] 2. Cell lines

[0065] AU565 and L929 cells were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China), and BT474 cells were purchased from Yiaobang Biotechnology Research Co., Ltd. (Beijing, China). AU565 cells were isolated from patient pleural effusion cells and maintained in 1640 medium. BT474 cells were derived from human ductal breast carcinoma cells and maintained in 1640 medium. L929 cells were isolated from mouse subcutaneous connective tissue and maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin (P / S). Cells were cultured at 37°C in 5% CO2.

[0066] 3. Animals

[0067] Five-week-old BALB / c female mice (≈20 g) and KM female mice (≈18 g) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were housed in a pathogen-free environment with a 12-h light / dark cycle, relative humidity (40–70%), and a temperature of 21 ± 2°C. All mice had ad libitum access to food and water. All in vivo experiments were conducted in accordance with the animal care guidelines of the National Institutes of Health and in the strict pathogen-free environment of the Central Laboratory Animal Facility of the Air Force Specialty Medical Center.

[0068] 4. Statistical analysis

[0069] Grayscale values were semiquantitatively analyzed using Image J 1.48v (National Institutes of Health, Bethesda, MD, USA). All statistical data were analyzed using GraphPad Prism (GraphPad Software 8.0.2, San Diego, CA, USA) and Origin software (Origin Lab 8.0, Northampton, MA, USA). Quantitative data are presented as mean ± standard deviation (SD) of at least three tests. One-way ANOVA was performed to assess significant differences, and a value of p < 0.05 was considered statistically significant.

[0070] Example 1: Preparation of related products

[0071] Preparation of Zr-Cu MOF: 120 mg of zirconium tetrachloride (ZrCl₄), 86.5 mg of terephthalic acid (H₂BDC), and 56.5 mg of polyvinylpyrrolidone (PVP) were dissolved in 120 mL of N,N-dimethylformamide (DMF) and sonicated for 5 minutes to fully dissolve. 50 mg of copper chloride (CuCl₂) was then added to the mixture and sonicated to form a homogeneous solution. The resulting solution was then transferred to a Teflon-lined stainless steel autoclave and placed in an oven at 120°C for 10 hours. After cooling to room temperature, the resulting reaction solution was centrifuged at 10,000 rpm for 5 minutes, the supernatant discarded, and the resulting precipitate washed three times with anhydrous ethanol to obtain the Zr-Cu bimetallic organic framework (ZC). 8 mL of anhydrous ethanol was added to the resulting ZC, sonicated to dissolve it homogeneously, and then stored at room temperature.

[0072] Preparation of Zr-Cu MOF@Elesclomol: To the prepared Zr-Cu MOF solution (3 mL, 5 mg / mL) in ethanol, add 5 mL of Elesclomol (ES) in ethanol (3 mg / mL). Mix and stir at room temperature for 12 hours. Centrifuge at 12,000 rpm for 5 minutes, and collect the supernatant and precipitate. The resulting lower precipitate is Zr-Cu MOF@Elesclomol (hereinafter referred to as ZCE). Add anhydrous ethanol to the resulting ZCE, sonicate until uniformly dissolved, and store at room temperature.

[0073] Preparation of Zr-Cu MOF@Elesclomol@Herceptin: Take a ZCE (10 mL, 3 mg / mL) aqueous solution, add EDC (200 μL, 5 mg / mL) and NHS (200 μL, 5 mg / mL) aqueous solutions respectively, stir at room temperature for 20 minutes, then centrifuge at 12000 rpm for 5 minutes to remove the supernatant, add 4 mL of deionized water and 1.5 mL of trastuzumab for injection (20 mg / mL) to the resulting precipitate, stir for 12 hours, and then centrifuge at 12000 rpm for 5 minutes to obtain Zr-Cu MOF@ES@αH (hereinafter referred to as ZCEH).

[0074] Preparation of Zr-Cu MOF@αH: Take a ZC (10 mL, 3 mg / mL) aqueous solution, add EDC (200 μL, 5 mg / mL) and NHS (200 μL, 5 mg / mL) aqueous solutions respectively, stir at room temperature for 20 minutes, then centrifuge at 12000 rpm for 5 minutes to remove the supernatant, add 4 mL of deionized water and 1.5 mL of trastuzumab for injection (20 mg / mL) to the resulting precipitate, stir for 12 hours, and then centrifuge at 12000 rpm for 5 minutes to obtain Zr-Cu MOF@αH (hereinafter referred to as ZCH).

[0075] Preparation of Zr-Cu MOF@RhB and Zr-Cu MOF@ES@αH@RhB: Rhodamine B was added to the above-obtained ZC and ZCH aqueous solutions, respectively, and stirred in the dark at room temperature for 12 h. The resulting precipitates were then centrifuged at 12,000 rpm for 5 min. The resulting precipitates were washed with deionized water to obtain Zr-Cu MOF@RhB (hereinafter referred to as ZCR) and Zr-Cu MOF@ES@αH@RhB (hereinafter referred to as ZCEHR), respectively.

[0076] Preparation of Cy7 fluorescently labeled Zr-Cu MOF@αH@Cy7: The only difference from the above method is that rhodamine B is replaced by Cy7. The rest is consistent with the above preparation method to obtain Zr-Cu MOF@αH@Cy7 (hereinafter referred to as ZCH@Cy7).

[0077] Example 2: Material structure analysis and characterization

[0078] 1. Experimental Methods

[0079] 1.1 Material analysis and characterization

[0080] In this example, the physicochemical properties of the product obtained in Example 1 were analyzed by the following methods: the morphology, size, and microstructure of the sample were examined using a scanning electron microscope (ZEISS Sigma 360, Germany) and a transmission electron microscope (Hitachi, Tokyo, Japan) at an electron gun operating voltage of 300 kV, and the elemental composition of the ZCEH was analyzed by transmission electron microscopy. The structure and particle size changes of the nanosamples were analyzed by Fourier transform infrared spectroscopy (Nicolet 380, Thermo, Waltham, Massachusetts, America) and Malvern Zetasizer Nano ZS90, UK. The crystal structure of the Zr-Cu MOF was determined using a D-MAX2200 X-ray powder diffractometer (RIGAKU, Japan). The elemental and chemical states of the Zr-Cu MOF were analyzed using an X-ray photoelectron spectrometer (Thermo Scientific K-Alpha, Waltham, Massachusetts, America).

[0081] 1.2 Acidic pH-triggered Zr-Cu MOF disintegration

[0082] Zr-Cu MOF was dispersed in PBS at two different pH values (pH 7.7 and pH 5.5) to form a 1 mg / mL mixed solution. The sample tube was then shaken in a 37°C air bath shaker. Samples were taken at 0, 1, 6, and 12 hours for electron microscopy (TEM) preparation. The morphological changes of the Zr-Cu MOF were then observed using a transmission electron microscope (Hitachi, Japan).

[0083] 1.3 Elesclomol loading rate

[0084] Drug loading experiments were performed as follows: 1. A Zr-Cu MOF ethanol solution (1.5 mg / mL, 2 mL) was stirred with anhydrous ethanol (3 mL) for 24 hours; 2. A Zr-Cu MOF ethanol solution (1.5 mg / mL, 2 mL) was stirred with an ethanol solution of Elesclomol (1 mg / mL, 3 mL). After 24 hours, the mixture was centrifuged at 12,000 rpm for 5 minutes, the supernatant was aspirated, and the volume was diluted to 200 mL with deionized water. The absorbance at 276 nm was measured using a UV spectrophotometer (UV-2401PC, Shimadzu, Japan), and the Elesclomol encapsulation efficiency and drug loading were calculated.

[0085] 2 Results and Analysis

[0086] like Figure 1 Figure 2 shows the preparation process of Zr-Cu MOF@ES@aH (ZCEH). Zr-Cu MOF (ZC) was synthesized by a hydrothermal method using zirconium tetrachloride (ZrCl4) and terephthalic acid (H2BDC) as raw materials and polyvinylpyrrolidone (PVP) as a dispersant. Next, Elesclomol (ES) was loaded into the pores of ZC by physical adsorption to obtain Zr-CuMOF@ES (ZCE). The resulting Zr-Cu MOF@ES was further carboxyl-activated and linked to the amino group of trastuzumab via an amide reaction to obtain Zr-Cu MOF@ES@αH (ZCEH).

[0087] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the obtained Zr-Cu MOF had a uniform hexahedral structure with a particle size of about 78 nm ( Figure 2 a). Loading ES and trastuzumab can increase the particle size of the nanomaterial to a certain extent, but there is no obvious change in the structure. In addition, TEM images show that trastuzumab is modified on the surface of the material in the form of flocs. Further elemental mapping analysis of ZCEH ( Figure 2 b), N, O, Zr, and Cu elements are evenly distributed in the material, and the presence of S element proves the successful loading of trastuzumab (e.g. Figure 3 Dynamic light scattering indicates that the hydrated particle size of the materials at different stages gradually increases, and the hydrated particle size of ZCEH is the largest, about 180nm ( Figure 2 c). In addition, the dispersibility of the materials in each stage is less than 0.7, indicating that the nanocomposite drug has good dispersibility and uniformity, which is consistent with the SEM and TEM phenomena. X-ray diffraction ( Figure 2 d) The results show that the materials in each stage exhibit three strong peak signals at 2θ = 7.4°, 8.6° and 25.8°, indicating that the crystal form of the materials in each stage is the same and the loading of drugs and antibodies does not change the original structure of ZC. Fourier transform infrared spectroscopy results ( Figure 2 e) Display, at 3000cm -1 ~3600cm -1 All samples show a broad absorption peak in the range of 3000 cm-1, which is mainly attributed to the abundant stretching vibrations of NH and OH bonds in the samples. Compared with ZC and ES monomers, the peak related to OH stretching vibration in ZCE sample (3000 cm-1) is larger than that in ZC and ES monomers. -1 ~3600cm -1 ) and the peak associated with OH bending vibration (1375 cm -1 and 974cm -1) shifted to higher wavenumbers and weakened or disappeared, indicating that strong hydrogen bonding occurred between ZC and ES monomers in ZCE. The absorption peak of C=O group in ZCEH shifted from 1584 cm -1 Move to 1589cm -1 And a new characteristic peak 1664cm was split -1 At the same time, the tensile vibration intensity of CO and CN was further enhanced, further indicating that the loading of trastuzumab was successful. The results of SDS-PAGE gel electrophoresis showed that ZCH, ZCEH and pure trastuzumab had the same bands at around 50KD and 25KD, representing the light chain and heavy chain of trastuzumab respectively. The total molecular weight of the monoclonal antibody was about 150KD (such as Figure 4 Compared with ZC (26.4mV), the potential value of ZCEH (31.6mV) is significantly increased ( Figure 2 f), indicating the successful loading of ES and HER2 monoclonal antibody. XPS ( Figure 2 g) The results suggest that there is Cu at 932.5eV 2+ The characteristic peaks of the copper ions were confirmed.

[0088] In addition, the UV spectrophotometer detected that ES had an UV absorption peak at 283 nm, and a standard curve was drawn based on the dependence of the absorption value and concentration (e.g. Figure 5 and 6 As shown). Calculations show that the encapsulation efficiency of Zr-Cu MOF@ES is 19.2% and the drug loading is 16.2%. Based on the acid response characteristics of ZCE, drug release experiments were carried out at pH 7.4 and pH 5.5 ( Figure 2 The results showed that in a neutral environment (pH 7.4), some ES drugs were released, with the cumulative release reaching 41.09% after 24 hours. In an acidic solution (pH 5.7), 44% was released after 2 hours of treatment, and the drug release rate reached 65.54% after 24 hours. TEM images of ZCEH decomposition at pH 7.4 and pH 5.5 showed that the structure of ZCEH gradually disintegrated under acidic conditions, while the structure and morphology remained basically unchanged under neutral conditions. Figure 2 i), indicating that the material has good stability in blood circulation and can disintegrate and release drugs after reaching the acidic environment of the tumor.

[0089] Example 3: Cell experiment

[0090] 1 Experimental Methods

[0091] 1.1 In vitro cellular uptake

[0092] 1.1.1 Rhodamine B labeling and time-gradient uptake experiments

[0093] Rhodamine B (RhB) was loaded onto the surface of a Zr-Cu MOF series by physical adsorption: 1 mg / mL of the nanomaterial was incubated with 50 μg / mL of RhB solution (PBS, pH 7.4) in the dark for 12 h with shaking. Free dye was removed by centrifugation (8000 × g, 10 min), and the supernatant was washed three times until the fluorescence signal disappeared. A reverse time-series drug delivery design was used: AU565 cells (3 × 10 cells / dish) were seeded on confocal microplates and pre-incubated for 12 h. ZCH@RhB (150 μg / mL) was then added at 0, 1, 2, and 4 h. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. The membranes were permeabilized with 0.1% Triton X-100 for 15 min, and nonspecific binding was blocked with 5% BSA for 30 min. The cytoskeleton was stained with Actin-Tracker Green and incubated in the dark for 30 min at room temperature. The nuclei were counterstained with DAPI (1 μg / mL) for 10 min. Three-dimensional imaging: Z-stack images (0.5 μm layer thickness) were collected using a laser confocal microscope.

[0094] 1.1.2 Effect of material surface modification on uptake efficiency:

[0095] Five comparison systems were set up: PBS control group, 150 μg / mL ZC, ZCE, ZCH, ZCEH, and ZCEH+UK5099 (4 μM) after continued culture for 6 hours; cell nucleus labeling: Hoechst 33342 (5 μg / mL) staining for 20 minutes, PBS washing three times; imaging was performed by laser confocal microscopy: RhB (red, 552 / 580 nm) and Hoechst (blue, 350 / 461 nm) dual-channel acquisition.

[0096] 1.2 Cell viability assay

[0097] Mouse L929 fibroblast suspension was cultured at 5×10 3Cells (100 μL) were seeded per well in a 96-well plate and pre-incubated at 37°C, 5% CO for 12 hours to allow attachment. The experimental setup included a material toxicity gradient group (Zr-Cu MOF nanomaterials) (7 concentration gradients, 0-2000 μg / mL); a material efficacy comparison group (150 μg / mL ZC, ZCE, ZCH, ZCEH, and ZCEH + UK5099 (4 μM); and a cell-specific group (n=6) to evaluate the cytotoxicity of these different nanomaterials against L929 fibroblasts and BT474 breast cancer cells (HER2-positive). After 24 hours of treatment, 12 μL of MTT working solution (5 mg / mL) was added to each well and incubated for an additional 4 hours. After discarding the culture medium, 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The cells were centrifuged at 3000 rpm for 30 minutes to remove interference from the nanomaterial. 100 μL of the supernatant was transferred to a new 96-well plate. The absorbance difference (ΔOD) at 570 nm (detection wavelength) and 650 nm (reference wavelength) was measured using a full-wavelength microplate reader.

[0098] 1.3 Cell growth inhibition assay

[0099] AU565 cells (2×10 5 After cells were seeded into 6-well plates and adhered to the wall, the cells were divided into 6 groups and each group was treated as follows:

[0100] Control group: complete culture medium;

[0101] ZC group: complete medium + ZC (150 μg / mL);

[0102] ZCE group: complete medium + ZCE (150 μg / mL);

[0103] ZCH group: complete medium + ZCH (150 μg / mL);

[0104] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0105] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0106] Note: The sample volume for each group mentioned above is 1 mL / well.

[0107] The cells in each group were treated accordingly and cultured for 24 h. The cells were then washed three times with PBS and incubated in the dark for 15 min with 1 mL of calcein AM / PI double staining solution (2 μM calcein-AM, 1.5 μM PI dissolved in phenol red-free culture medium). The cells were washed twice with PBS and imaged in multiple channels under a laser confocal microscope. Live cells were labeled with calcein-AM (excitation wavelength 488 nm, emission channel 500-550 nm, green fluorescence) and dead cells were labeled with PI (excitation wavelength 561 nm, emission channel 570-620 nm, red fluorescence). The images were scanned along the Z axis with a step of 1 μm. The confocal aperture was set to 1 Airy unit, and deconvolution was performed using Huygens software.

[0108] 1.4 Intracellular GSH / GSSG detection

[0109] AU565 cells (2×10 5 Cells were seeded into 6-well plates and adhered to the wall. The cells were then divided into 6 groups and each group was treated as follows:

[0110] Control group: complete culture medium;

[0111] ZC group: complete medium + ZC (150 μg / mL);

[0112] ZCE group: complete medium + ZCE (150 μg / mL);

[0113] ZCH group: complete medium + ZCH (150 μg / mL);

[0114] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0115] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0116] Note: The sample volume for each group mentioned above is 1 mL / well.

[0117] After the corresponding treatment, the cells in each group were cultured for 24 h, washed three times with PBS, and measured using the GSH and GSSG detection kits to obtain the GSH / GSSG ratio.

[0118] 1.5 Intracellular copper ion detection

[0119] AU565 cells (2×10 5 Cells were seeded into 6-well plates and adhered to the wall. The cells were then divided into 6 groups and each group was treated as follows:

[0120] Control group: complete culture medium;

[0121] ZC group: complete medium + ZC (150 μg / mL);

[0122] ZCE group: complete medium + ZCE (150 μg / mL);

[0123] ZCH group: complete medium + ZCH (150 μg / mL);

[0124] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0125] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0126] Note: The sample volume for each group mentioned above is 1 mL / well.

[0127] After the corresponding treatment, the cells in each group were cultured for 24 h, washed twice with serum-free medium, the supernatant was discarded, CuprosGreen Working Solution was added to the cells, and the cells were incubated for 3 h. The fluorescence was observed under a laser confocal microscope (Ex / Em: 488 / 500-600 nm).

[0128] 1.6 Intracellular ROS detection

[0129] AU565 cells in the logarithmic growth phase were obtained and a single-cell suspension of 3×10 cells / mL was prepared. The cells were inoculated into a laser confocal microscopy-specific culture dish (35 mm glass-bottom dish) and cultured for 12 h. The cells were then divided into 6 groups and each group was treated as follows:

[0130] Control group: complete culture medium;

[0131] ZC group: complete medium + ZC (150 μg / mL);

[0132] ZCE group: complete medium + ZCE (150 μg / mL);

[0133] ZCH group: complete medium + ZCH (150 μg / mL);

[0134] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0135] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0136] Note: The sample volume for each group mentioned above is 1 mL / well.

[0137] After the corresponding treatment, cells in each group were cultured for an additional 24 hours and then washed three times with PBS. 1 mL of DCFH-DA working solution was added and incubated at 37°C in the dark for 30 minutes. Nuclear staining: DAPI stain (1 μg / mL) was added at 20 minutes of incubation. The cells were washed three times with PBS and imaged in multiple channels under a laser confocal microscope. ROS detection was performed using DCF (excitation 488 nm, emission 500-540 nm, green fluorescence) and nuclear localization using DAPI (excitation 405 nm, emission 410-480 nm, blue fluorescence).

[0138] 1.7 Reactive oxygen species (ROS) flow cytometry

[0139] AU565 cells (2×10 5 Cells were seeded into 6-well plates and adhered to the wall. The cells were then divided into 6 groups and each group was treated as follows:

[0140] Control group: complete culture medium;

[0141] ZC group: complete medium + ZC (150 μg / mL);

[0142] ZCE group: complete medium + ZCE (150 μg / mL);

[0143] ZCH group: complete medium + ZCH (150 μg / mL);

[0144] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0145] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0146] Note: The sample volume for each group mentioned above is 1 mL / well.

[0147] After the corresponding treatment, the cells in each group were cultured for 24 hours and then washed twice with PBS (to remove serum interference). DCFH-DA probe working solution (final concentration 20 μM) was added, incubated at 37°C in the dark for 30 minutes, and washed twice with PBS. Using a flow cytometer, select the FITC channel (excitation wavelength 488 nm, emission wavelength 525 nm). Filter the cell suspension with a 40 μm filter before loading to avoid clogging the instrument. Detect at least 1×10 cells / sample and record the fluorescence intensity (MFI, mean fluorescence intensity). Use FlowJo to analyze the data and compare the differences in fluorescence intensity among the groups;

[0148] 1.8 Intracellular H2O2 concentration detection

[0149] AU565 cells (2×105 Cells were seeded into 6-well plates and adhered to the wall. The cells were then divided into 6 groups and each group was treated as follows:

[0150] Control group: complete culture medium;

[0151] ZC group: complete medium + ZC (150 μg / mL);

[0152] ZCE group: complete medium + ZCE (150 μg / mL);

[0153] ZCH group: complete medium + ZCH (150 μg / mL);

[0154] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0155] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0156] Note: The sample volume for each group mentioned above is 1 mL / well.

[0157] After culturing the cells in each treatment group for an additional 24 hours, the cells were harvested and homogenized with 200 μL / well of hydrogen peroxide detection lysis buffer. The cells were then centrifuged at 10,000 × g for 5 minutes at 4°C. The supernatant was collected and the absorbance at 560 nm was measured using a microplate reader (Spectramax 190, Molecular Devices, USA) according to the instructions of the hydrogen peroxide detection kit. The hydrogen peroxide concentration in the sample was calculated using a standard curve.

[0158] 1.9 Mitochondrial membrane potential detection

[0159] The transfer cell density was 2.4×10 5 AU565 (10% fetal bovine serum, 1% penicillin-streptomycin) at 50 cells / mL was cultured overnight in a laser confocal microplate. The cells were then divided into 6 groups and each group was treated as follows:

[0160] Control group: complete culture medium;

[0161] ZC group: complete medium + ZC (150 μg / mL);

[0162] ZCE group: complete medium + ZCE (150 μg / mL);

[0163] ZCH group: complete medium + ZCH (150 μg / mL);

[0164] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0165] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0166] Note: The sample volume for each group mentioned above is 1 mL / well.

[0167] After culturing the cells in each treatment group for 24 hours, JC-1 working solution (4 mol / L) was added and incubated for 30 minutes. The supernatant was removed and the cells were washed twice with PBS. After adding 200 μL of Imaging Buffer Solution, the cells were observed under a fluorescence microscope. Green: Ex = 488 nm, Em = 500-550 nm; Red: Ex = 561 nm, Em = 60-610 nm.

[0168] 2.0 Mitochondrial damage flow cytometry assay

[0169] The cells to be tested, AU565, were seeded into a culture plate, and then the cells were divided into 6 groups. Each group was treated as follows:

[0170] Control group: complete culture medium;

[0171] ZC group: complete medium + ZC (150 μg / mL);

[0172] ZCE group: complete medium + ZCE (150 μg / mL);

[0173] ZCH group: complete medium + ZCH (150 μg / mL);

[0174] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0175] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0176] Note: The sample volume for each group mentioned above is 1 mL / well.

[0177] The cells in the above groups were treated and co-cultured for 24 hours, and mitochondrial damage and morphology were detected by flow cytometry and transmission electron microscopy. The culture medium was discarded and the cells were gently washed twice with PBS (to remove serum interference). JC-1 probe working solution was added and incubated for 30 minutes. The JC-1 solution was discarded and the cells were washed twice with PBS to remove unbound dye. Using a flow cytometer, JC-1 polymer (red fluorescence): detect PE channel (Ex 488nm / Em 590nm). JC-1 monomer (green fluorescence): detect FITC channel (Ex 488nm / Em 530nm), and analyze the red / green fluorescence intensity ratio (reflecting the mitochondrial membrane potential). FlowJo was used to analyze the data and compare the differences in fluorescence intensity among the groups.

[0178] 2.1 In vitro Western blot analysis

[0179] To determine the expression levels of copper-induced death proteins DLAT, LIAS, FDX-1, and Hsp70, AU565 cells were seeded into 6-well plates and cultured for 24 hours. Subsequently, the cells were divided into 6 groups and each group was treated as follows:

[0180] Control group: complete culture medium;

[0181] ZC group: complete medium + ZC (150 μg / mL);

[0182] ZCE group: complete medium + ZCE (150 μg / mL);

[0183] ZCH group: complete medium + ZCH (150 μg / mL);

[0184] ZCEH group: complete medium + ZCEH (150 μg / mL);

[0185] ZCEH+UK5099 group: complete medium+ZCEH (150 μg / mL)+UK5099 (4 μM);

[0186] Note: The sample volume for each group mentioned above is 1 mL / well.

[0187] After the corresponding treatment, cells in each group were cultured for an additional 24 hours and rinsed three times with PBS. Cell lysis buffer was added for complete lysis, and the cells were harvested and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected. Protein concentration in the supernatant was determined by the BCA assay. Samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with skim milk in TBST for 2 hours and washing with TBST for 10 minutes, the membranes were incubated with primary antibodies (1:1000) to DLAT, LIAS, FDX-1, and Hsp70 at 4°C overnight. After washing three times with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 2 hours. ECL chemiluminescence kit (Shanghai, China) was used for visualization.

[0188] 2 Results and Analysis

[0189] 2.1 In vitro cellular uptake and cytotoxicity of Zr-Cu MOF@Elesclomol@Herceptin

[0190] Cell uptake experiments were conducted to determine whether trastuzumab could enhance the uptake of ZCEH by cells expressing HER2. HER2-positive breast cancer cells (AU565) and HER2-negative mouse fibroblasts (L929) were used for validation. Rhodamine B and the various stage materials were stirred for 12 hours using a physical adsorption method to produce fluorescence. Laser confocal microscopy was used to observe the time-dependent uptake of ZCEH. Figure 7 a), blue is the cell nucleus, green is the cytoskeleton, and red is ZCH@RhB. It can be seen that as time goes by, the uptake of ZCH by AU565 cells increases, and the red fluorescence in the cells is the highest at 4 hours. Subsequently, the uptake of materials at different stages was observed at the same time ( Figure 7 b), the results showed that the red fluorescence in ZCH cells was significantly higher than that in other groups, proving that the material with HER2 monoclonal antibody was more easily taken up by tumor cells; while for L929 cell line, the fluorescence intensity of ZCEH was significantly lower than that of AU565 cells (e.g. Figure 8 The above results show that ZCEH surface modified trastuzumab still has biological activity and can specifically bind to receptors on the surface of HER2-positive cells, thereby improving the targeting of the material and reducing its toxicity to non-HER2-positive cells. MTT cytotoxicity experiments were further conducted to study the toxicity of different concentrations of ZC on L929 cells at different times. The results showed that with the increase of ZC concentration, AU565 cells showed a concentration-dependent toxicity reaction ( Figure 7c). When the ZC concentration reached 163.1 μg / mL, the survival rate of L929 cells was above 80%. Based on this result, it can be concluded that 150 μg / mL ZC has good safety for normal cells. Next, AU565 cells were incubated with different materials for 24h, 48h and 72h respectively. Figure 7 d, Figure 9 ), the results showed that the cytotoxic effect of ZCEH was enhanced after modification with trastuzumab. After 72 hours of incubation, the growth inhibition rate of cells in the ZCEH group reached about 80%, and after the copper death inhibitor UK5099 was added to the ZCEH group, the cell survival rate increased, indicating that ZCEH can exert significant anti-tumor activity through the copper death mechanism under the targeting effect of trastuzumab. Next, calcein AM (green, living cells) and propidium iodide PI (red, dead cells) were used to observe AU565 cells after incubation of materials at various stages for 24 hours. The results showed that the ZCE group and ZCEH group had more red fluorescence ( Figure 7 e), which is consistent with the MTT results. The inhibition rates of different materials were further studied using BT474 cells (HER2 positive), which were consistent with the results of AU565 cells (e.g. Figure 9 After the addition of UK5099, the red fluorescence ratio of the ZCEH group was significantly reduced, indicating that ZCEH can target HER2-overexpressing tumor cells and induce copper cell death by loading ES and modifying trastuzumab.

[0191] 2.2 Induction of Cellular Death by Copper

[0192] Glutathione (GSH) plays a crucial role in the cellular redox state. Copper death is hindered in anti-tumor applications because it depends on the copper ion level and the glutathione (GSH)-rich microenvironment in the tumor. ZCEH releases Cu under the action of the acidic microenvironment of the tumor. 2+ It can consume GSH in TME and convert it into Cu + , producing GSSG. Compared with the ZC group, the GSH / GSSG content in the other groups was significantly reduced ( Figure 7 f). The GSH / GSSG ratio decreased from 10.3 (ZC group) to 2.1 (ZCEH group), indicating that the copper-mediated oxidative stress response was activated. After the addition of UK5099, the GSH / GSSG ratio increased, and the H2O2 content was consumed by 77% after 24 hours of incubation with ZCEH ( Figure 7 g), indicating that ES and Cu 2+ It plays a role in inhibiting and consuming GSH synthesis.

[0193] In addition, high levels of Cu +It can promote the occurrence of copper death and further detect the Cu in the cell through CuprosGreen fluorescent probe. + The probe can detect the content of Cu + Specific reaction, producing a strong fluorescence signal. Compared with the control group, the green fluorescence intensity of the material groups at each stage was enhanced ( Figure 7 h), the green fluorescence intensity of the ZCEH group was the most obvious, and the intervention of UK5099 could improve this phenomenon. This shows that ZCEH can release ES and Cu in TME. 2+ , consumes GSH to increase intracellular Cu + ion content, thereby inducing copper death in tumor cells.

[0194] H2O2 in TME can react with the Cu + Through the Fenton reaction, it is converted into highly toxic hydroxyl radicals (·OH), thereby enhancing the oxidative stress damage caused by ROS to cells. The changes in ROS content in AU565 cells were detected by flow cytometry and laser confocal microscopy. The results of flow cytometry showed that ( Figure 10 ac), compared with the PBS group (30%), the ROS generated in the ZCE and ZCEH groups were higher than 70%. It is worth noting that the addition of copper death inhibitors reduced the generation of ROS. The same phenomenon was also found under laser confocal fluorescence microscopy. The green fluorescence generated in the ZCE and ZCEH groups was significantly higher than that in the other groups. After the addition of copper death inhibitors, the cell fluorescence was weakened relative to that of ZCEH, but higher than that of ZC group. This phenomenon may be related to the transport of Cu by ES. 2+ into the cell, as ES transports Cu 2+ Entering the cell, Cu 2+ Reacts with high intracellular GSH to generate Cu + and GSSG, Cu + It reacts with H2O2 in cells to produce hydroxyl free radicals and a large amount of ROS, thereby effectively eliminating tumor cells.

[0195] Further research found that the Cu 2+ ions are reduced to Cu by FDX-1 reductase in the mitochondria. + . Subsequently, Cu + Combined with the fatty acylated proteins produced during sugar metabolism, it seriously interferes with the normal operation of the tricarboxylic acid (TCA) cycle, causing abnormal aggregation of fatty acylated proteins and downregulation of Fe-S proteins. These changes further increase the intracellular protein toxicity pressure, ultimately leading to copper death. In order to further explore the changes in mitochondria during copper death, the mitochondria of tumor cells were detected by laser confocal microscopy and flow cytometry. The experimental results showed that ( Figure 10 d,e), after the addition of JC-1 dye to the AU565 cells treated with different material groups, it can be observed that the proportion of green fluorescence in the ZCE group and the ZCEH group was higher, indicating that the mitochondrial membrane potential of these two groups of cells was significantly depolarized. After the addition of UK5099, some mitochondria returned to an aggregated state, indicating that ZCEH can induce copper death in AU565 cells. Flow cytometry analysis also obtained the same results. The proportion of mitochondrial damage in the ZCE group and the ZCEH group was 55.5% and 66.3%, respectively. After the addition of UK5099, the mitochondrial damage status was roughly the same as that of the ZC group, about 48%. The mitochondrial damage status of the Control, ZC, and ZCEH groups was further observed by biological transmission electron microscopy ( Figure 10 f) The results showed significant changes in the mitochondrial structure of the experimental group cells, with significant mitochondrial shrinkage, reduced inner membranes, and the formation of large vacuoles, which are characteristic of mitochondrial damage after copper death. Furthermore, endocytosis of the material was observed using biological transmission electron microscopy.

[0196] Combined with the above experimental results and phenomena, we further explored the expression of key proteins related to copper death. The key proteins FDX-1, DLAT, LIAS and HSP70 were detected by immunoblotting. The results showed that ( Figure 10 g) In the material group, FDX-1 expression was significantly downregulated, with DLAT and LIAS showing the same downward trend. The expression of these proteins in the ZCE group also showed a significant downward trend. However, after the addition of UK5099, FDX-1 expression was upregulated, further confirming the occurrence of copper-induced apoptosis. Furthermore, the upregulation of Hssp70 also reflects the cellular response to copper-induced proteotoxic stress. These results demonstrate that the material can enhance the occurrence of copper-induced apoptosis, thereby effectively killing tumor cells.

[0197] Example 4: Animal Experiment

[0198] 1 Experimental Methods

[0199] 1.1 Construction of tumor-bearing mouse model

[0200] 200 μL containing 5 × 10 6 / mL BT474 cells expressing luciferase (BT474-luc) resuspended in normal saline were injected subcutaneously into the right axilla of Balb / c nude mice. 3 At that time, a tumor-bearing mouse model was successfully constructed for subsequent experiments.

[0201] 1.2 In vivo targeting and in vitro organ imaging

[0202] When the tumor volume of the tumor-bearing mice was about 100 mm 3Cy7-labeled ZCH@Cy7 (25 mg / kg, 0.2 mL) was injected via the tail vein. Fluorescence images of mice were recorded using a small animal in vivo imaging system at 0, 3, 6, 12, 24, 72, and 144 hours after ZCEH@Cy7 injection. Mice were euthanized at the end of the recording, and tumors and major organs were removed for ex vivo fluorescence imaging.

[0203] 1.3 In vivo anti-tumor efficacy

[0204] After establishing the Balb / c nude tumor-bearing mouse model, 24 nude mice were randomly divided into 4 groups: Control group, ZC group, ZCE group, and ZCEH group. The ZC group, ZCE group, and ZCEH group were given intravenous injections of 25 mg / kg of ZC, ZCE, and ZCEH on days 1, 3, and 5, respectively, for anti-tumor treatment ( Figure 13 a) The control group was given 0.2 mL of normal saline. The tumor size and body weight were measured every 2 days after treatment. 3 Mice were euthanized at 4 hr, tumor tissues were harvested, and tumor weights were calculated for each group. Tumor tissues were used for DLAT and LIAS immunofluorescence staining, Western blot, H&E staining, Ki67, and Tunel staining.

[0205] 1.4 Establishment of lung metastasis-bearing mice

[0206] Inject 0.2 mL of 1×10 6 BT474-luc cells / mL were added to each Balb / c nude mouse. When the in vivo imaging of the small animal showed a fluorescent signal in the lung, the lung metastasis tumor-bearing nude mouse model was successfully constructed. 30 lung metastasis tumor-bearing nude mice were randomly divided into 5 groups - Control group and ZCEH group, and the other three groups were used as other experimental samples, with 6 mice in each group. It should be noted that the fluorescence intensity of the lungs of the constructed lung metastasis tumor-bearing nude mice was basically the same, that is, the number of lung metastasis nodules in the mice was approximately the same (such as Figure 11 The control group was injected with 0.2 mL of normal saline into the tail vein on days 1, 3, and 5, while the experimental group was injected with ZCEH (0.2 mL, 25 mg / kg) on the same days. The body weight and tumor volume of the mice were recorded and monitored every other day. After 14 days, the mice were euthanized, and lung tissues were removed for ex vivo fluorescence imaging and H&E staining to analyze the lung tumor status.

[0207] 1.5 Hemolysis test

[0208] Fresh mouse whole blood was mixed with 3.8% sodium citrate at a ratio of 9:1 (v / v), and then the fresh anticoagulated whole blood was diluted with normal saline at a ratio of 1:1 (v / v) for later use. Different concentrations of ZCEH were placed in 10 mL centrifuge tubes, and 200 μL of diluted anticoagulated whole blood and 5 mL of normal saline were added and mixed, so that the final concentration of ZCEH was 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 400 μg / mL. The normal saline group was used as a negative control, and the deionized water group was used as a positive control. Three parallel tubes were set up for each group. All centrifuge tubes were incubated at 37°C for 1 hour, centrifuged at 3000 rpm for 5 minutes, and the supernatant was aspirated. At the same time, the absorbance was measured at 545 nm using a UV-visible spectrophotometer. The hemolysis rate was calculated according to the following formula:

[0209] Hemolysis rate (%) = (average absorbance of experimental group - average absorbance of negative control group) / (average absorbance of positive control group - average absorbance of negative control group) × 100%

[0210] 1.6 In vivo toxicity evaluation of ZCEH

[0211] KM mice were used to evaluate the in vivo toxicity of ZCEH. ZCEH (0.2 mL, 25 mg / kg) was injected via the tail vein. The physiological status and body weight of the mice were observed and recorded for 14 days. At the end of the observation period, plasma and serum were collected from the mice for routine blood and biochemical examinations (n=3). The mice were euthanized, and major organs were collected, embedded in paraffin, and analyzed by H&E staining.

[0212] 2 Results and Analysis

[0213] 2.1 In vivo and in vitro biosafety

[0214] After confirming that ZCEH has an inhibitory effect on tumor cell proliferation in vitro, this example further evaluated its biocompatibility. The MTT experiment was used to detect the effects of nanomaterials at different stages on L929 cell proliferation. The results showed that ( Figure 12 a) ZCEH at 150 μg / mL had little effect on the proliferation rate of L929 cells, and the cell survival rate was above 80%. After 24 hours of ZCE treatment, the survival rate of L929 cells was 72%, but as time went on, the survival rate exceeded 80%, proving that the material had good cell compatibility at all stages. The results of in vitro hemolysis test showed that ( Figure 12 b) The hemolysis rate of ZCEH at different concentrations was less than 5%, indicating that ZCEH has good blood compatibility. The in vivo safety of ZCEH was evaluated by acute toxicity test. The body weight of mice after tail vein injection of 30.1 mg / kg ZCEH increased steadily ( Figure 12 c), blood routine ( Figure 12d) and blood biochemistry ( Figure 12 e) All indicators were within the normal range, indicating that ZCEH has good in vivo safety. In addition, the H&E staining results of important tissues such as the heart, liver, spleen, lung, and kidney of mice ( Figure 12 f) No obvious pathological changes or organ damage were observed, further demonstrating the in vivo safety of ZCEH.

[0215] The targeting ability of ZCEH in vivo was further investigated using a BT474-luc tumor-bearing mouse model. Balb / c nude mice were injected with Cy7 dye-loaded ZCEH ( Figure 12 (g, h) The in vivo targeting effect of the nanoplatform was analyzed using in vivo imaging at 0, 3, 12, 24, 72, and 144 hours after injection. The results showed that 3 hours after ZCH@Cy7 injection, the fluorescence intensity in tumor and lung tissues was highest. The fluorescence in the lungs may be due to the deposition of ZCEH in lung endothelial cells due to its particle size, which may be beneficial for inhibiting breast cancer lung metastasis. Fluorescence in the liver and kidneys suggests that ZCEH is metabolized in the liver and kidneys. Taken together, these results indicate that ZCEH can precisely target tumor tissues and be cleared through the liver and kidney metabolic pathways.

[0216] 2.2 Antitumor efficacy of Zr-Cu MOF@Elesclomol@Herceptin in vivo

[0217] This example studies the anti-tumor efficacy of nanomaterials at different stages in vivo using in situ tumors and lung metastases. First, a tumor-bearing mouse model was constructed using the BT474-luc cell line. 3 The mice were treated with the drug (0.2 mL, 25 mg / kg) on days 1, 3, and 5, and the body weight and tumor volume of the mice were observed and measured every other day ( Figure 13 a). Experimental results ( Figure 13 b) showed that the weight of mice in the ZC, ZCE, and ZCEH groups increased steadily, indicating that the nanomaterials at different stages had good biocompatibility. Compared with the control group, the ZCEH group significantly inhibited tumor growth with an inhibition rate of 66.75%. In addition, by performing in vivo imaging of the tumor on the 1st, 7th, and 13th day ( Figure 13 c) It can be observed that on the 13th day, the fluorescence intensity of the ZCEH group was significantly lower than that of the control group, which further confirmed the inhibitory effect of ZCEH on tumor growth. When the tumor volume of the model group mice reached an average of 1000 mm 3 At 3 pm, the mice were euthanized and the tumor tissues were obtained and weighed. Figure 13df) showed that the tumor weights of the ZCE and ZCEH groups were lower than those of the control group. This indicates that trastuzumab modification and ES loading improved the therapeutic effect of ZCEH. Tumor tissues were analyzed by H&E, Ki67, and TUNEL staining ( Figure 13 g) It was found that the proliferation of tumor cells in the ZCEH group was significantly inhibited, while the death of tumor cells was increased.

[0218] After DLAT and LIAS staining of tumor tissues, the fluorescence intensity of the ZCEH group was significantly lower than that of the other groups. In addition, by extracting tumor tissue proteins and verifying the expression of key proteins ( Figure 13 h,i) showed that the expression of key copper apoptosis proteins DLAT, LIAS, and FDX-1 was downregulated in the ZCEH group, while Hsp70 was upregulated, demonstrating significant copper apoptosis in the tumor tissues of the ZCEH group, consistent with the results of in vitro protein immunoblotting. In contrast, the downregulation of key protein expression in the ZCE group was not as significant as in the in vitro immunoblotting, which is somewhat correlated with its lack of tumor targeting. This result further confirms the targeting properties of the final material and its advantage in enhancing copper apoptosis.

[0219] 2.3Zr-Cu MOF@Elesclomol@Herceptin for the treatment of metastatic tumors

[0220] Because tumor metastasis is systemic and insidious, eradicating early-stage metastatic tumors is more challenging than eliminating the primary tumor. BT474-luc cells were injected into Balb / c nude mice via the tail vein to create a model that mimics clinical breast cancer lung metastasis. Three doses were administered on days 1, 3, and 5, and the mice's body weight was measured every other day. Figure 14 a). The results showed that the weight of mice increased slowly after injection of BT474 cells ( Figure 14 b), and on the 8th day, the mice showed a decrease in body weight, indicating the formation of lung metastases, while the mice in the tail vein injection of ZCEH group recovered their body weight, indicating that ZCEH inhibited the growth of metastases; after 14 days of observation, the mice were euthanized and the lung tissues were stained with H&E ( Figure 14 c, d) The results showed that there were a large number of scattered tumor metastases in the model group under HE staining observation, while only two lesions were observed in the ZCEH group, which was consistent with the conclusion of the in vivo targeting experiment, indicating that ZCEH can effectively inhibit the growth of lung metastases.

[0221] In summary, the present invention successfully designed and prepared a targeted bimetallic organic framework (MOF) nanocomposite drug - ZCEH. This composite drug uses the zirconium and copper bimetallic nanomaterial ZrCu-MOF as a carrier, is modified with trastuzumab, and loaded with the potent copper ion carrier ilisimol to obtain Zr-Cu MOF@ES@aH (abbreviated as ZCEH). ZCEH is a new type of tumor drug that can specifically destroy the homeostasis of tumor cells and induce copper cell death, thereby significantly promoting the death of HER2-positive tumor cells. Under the mediation of trastuzumab, ZCEH can target the slightly acidic environment of the tumor and decompose, and then use ilisimol to increase the Cu inside the cell. 2+ Concentrations of ZCEH lead to the generation of large amounts of ROS and copper cell death in tumor cells. In vitro and in vivo experimental results show that ZCEH can effectively kill tumor cells in both in situ and metastatic tumor tissues, revealing its potential as a targeted nanocomposite inducer of copper cell death in HER2-positive breast cancer cells. This provides new methods and ideas for the development of functional nanoplatforms and therapeutic strategies that rely on the sensitive target HER2, and has promising clinical application prospects.

[0222] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A zirconium-copper bimetallic nanomaterial, characterized in that: The invention is composed of a nanocarrier loaded with an oxidative stress inducer; wherein the nanocarrier is an organic framework material Zr-Cu MOF containing Zr and Cu bimetallics, and the oxidative stress inducer includes Elesclomol, Disulfiram, NSC-319726 and tetrathiomolybdate.

2. The method for preparing the zirconium-copper bimetallic nanomaterial according to claim 1, characterized in that: The method comprises the following steps: S1: Dissolve zirconium salt, ligand and PVP in a solvent, mix them ultrasonically, then add copper salt and dissolve them evenly. Then, obtain Zr-Cu MOF containing Zr and Cu bimetallic organic framework material through hydrothermal reaction. S2: The obtained Zr-Cu MOF ethanol solution and the oxidative stress inducer ethanol solution are mixed and stirred, and then the precipitate is collected by centrifugation to obtain the zirconium-copper bimetallic nanomaterial Zr-Cu MOF@oxidative stress inducer.

3. The preparation method according to claim 2, characterized in that In step S1, the zirconium salt is ZrCl4; The ligand is terephthalic acid; The solvent includes N,N-dimethylformamide; The copper salt is CuCl2·2H2O2, CuI2 or CuBr2; The hydrothermal reaction conditions are: temperature 120° C., time 10 h.

4. The preparation method according to claim 2, characterized in that In step S2, the mass ratio of the Zr-Cu MOF to the oxidative stress inducer is 1:1; The stirring reaction conditions are: room temperature, time 8 to 12 hours.

5. Use of the zirconium-copper bimetallic nanomaterial according to claim 1 in the preparation of any of the following drugs: (i) drugs for the treatment of breast cancer; (ii) drugs for the treatment of breast cancer lung metastasis; (iii) Drugs that increase the sensitivity of breast cancer to targeted drugs.

6. A drug for treating breast cancer, characterized in that: It comprises the zirconium-copper bimetallic nanomaterial according to claim 1, and a targeted drug; and the targeted drug is coupled to the zirconium-copper bimetallic nanomaterial through a coupling agent.

7. The drug according to claim 6, characterized in that The breast cancer includes HER2 + Breast cancer; The targeted drug includes trastuzumab.

8. The drug according to claim 6, characterized in that The coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

9. The method for preparing a drug for treating breast cancer according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: (1) dissolving the zirconium-copper bimetallic nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting a precipitate; (2) Adding a targeted drug aqueous solution to the obtained precipitate, stirring and centrifuging, and taking the precipitate to obtain Zr-Cu MOF@oxidative stress inducer@targeted drug for treating breast cancer.

10. The preparation method according to claim 9, characterized in that In step (1), the stirring conditions are: room temperature, time 20 to 25 min; the centrifugal conditions are: speed 10000 to 12000 rpm, time 5 to 10 min; In step (2), the stirring conditions are: room temperature, time 8 to 12 hours; the centrifugal conditions are: rotation speed 10000 to 12000 rpm, time 5 to 10 minutes.

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