Zirconium-iron bimetallic nano material, preparation method and application of zirconium-iron 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 ferrozirconium bimetallic nano-material, a preparation method and application of the ferrozirconium bimetallic nano-material in preparation of medicines for treating breast cancer. The ferrozirconium bimetallic nano material is formed by loading a CDK4 / 6 inhibitor on a nano carrier; wherein the nano carrier is an organic framework material Zr-Fe MOF containing double metals of Zr and Fe, and the CDK4 / 6 inhibitor is preferably ribociclib. A product ZFRH for treating breast cancer is obtained based on coupling of the ferrozirconium bimetallic nano-material provided by the invention with a targeted drug such as trastuzumab. The ZFRH 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 a CDK4 / 6 inhibitor. As a safe and effective tumor treatment strategy, the ZFRH 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-iron bimetallic nanomaterial, a preparation method and use thereof in preparing a drug for treating breast cancer. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is a molecule widely expressed on the surface of breast cancer cells. It plays a crucial role in promoting tumor cell proliferation, enhancing tumor invasion, and promoting tumor angiogenesis. Upon binding to its corresponding ligand, the HER2 protein undergoes homodimerization or heterodimerization, activating downstream signaling pathways, promoting cell division and growth, and inhibiting apoptosis. Currently, trastuzumab, a monoclonal antibody against the HER2 receptor, is the recommended first-line treatment for HER2-positive breast cancer. However, challenges such as tumor heterogeneity, drug resistance, and adverse drug reactions remain to be addressed.

[0003] The HER2 and estrogen receptor (ER) pathways play a crucial role in the treatment of breast cancer. HER2 overexpression can activate the PI3K-AKT-mTOR signaling pathway and downregulate the ER pathway, thereby reducing tumor cell sensitivity to endocrine therapy. Clinical evidence suggests that ER pathway activation can evade HER2 inhibition and promote anti-apoptotic effects in tumor cells. This is because ER can activate the PI3K-AKT-mTOR signaling pathway in a ligand-dependent manner, which in turn activates HER2 downstream signaling pathways. Therefore, simultaneous inhibition of ER and HER2 signaling can restore tumor sensitivity to HER2 monoclonal antibodies by inhibiting tumor cell proliferation. Cyclin-dependent kinases (CDK4 / 6), downstream molecules of ER signaling, are key regulators of cell cycle progression from G1 to S phase. CDK4 / 6 catalyzes phosphorylation of specific serine / threonine (Ser / Thr) sites on the retinoblastoma protein (RB), leading to RB phosphorylation and dissociation of the transcription factor E2F to which RB binds, thereby initiating transcription of downstream molecules and promoting tumor cell proliferation. Therefore, in theory, CDK4 / 6 inhibitors and trastuzumab can achieve synergistic inhibition of HER2 by inhibiting the activation of G1 / S-specific cyclin D1 (Cyclin D1) and CDK4 / 6 complex and downregulating Rb phosphorylation levels. + / HR +The proliferation of breast cancer. However, CDK4 / 6 inhibitors have certain blood toxicity, poor biocompatibility and targeting. It is difficult to achieve the best therapeutic effect by using trastuzumab and CDK4 / 6 inhibitors alone. Therefore, a drug carrier with good biocompatibility is developed to synergistically enhance the therapeutic effect by virtue of the interaction between trastuzumab and CDK4 / 6 inhibitors, in order to achieve HER2 + Targeted therapy for breast cancer is of great significance.

[0004] MOFs are a class of crystalline porous materials with periodic network structures, based on inorganic metal centers, such as metal ions or metal clusters, which self-assemble with organic ligands. Their high porosity and large surface area allow for the maximum loading of small molecule drugs, proteins, nucleotides, and other substances onto MOFs, making them widely used in medical fields such as cancer chemotherapy, immunotherapy, and radiotherapy. The size of MOFs can be tailored to meet diverse drug delivery requirements. Nanoscale MOFs, due to their enhanced permeability and retention (EPR) effect, accumulate in tumor tissue and disintegrate at acidic pH to release drugs, thereby improving drug bioavailability and reducing systemic toxicity. The diverse array of organic ligand groups allows for functional modification of MOF surfaces. For example, surface-attached PEG can improve the hydrophilicity of the material. Wan X et al. coated MOF nanoparticles with cancer cell membranes, resulting in the material possessing homologous targeting and immune evasion capabilities. MOFs formed with zirconium as the inorganic metal ion exhibit high biocompatibility, low toxicity, and pH-responsive release. To achieve a synergistic killing effect, new metals can be doped into the original MOF to unleash the effects of both metals. Iron ions can generate reactive oxygen species (ROS) through the Fenton reaction, promoting tumor cell death. In the tumor microenvironment, the acidic pH and presence of H2O2 provide favorable conditions for metal ions to undergo the Fenton reaction, thereby triggering cell death. Summary of the Invention

[0005] The purpose of the present invention is to provide a zirconium-iron 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 + / ER + 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-iron bimetallic nanomaterial, which is composed of a nanocarrier loaded with a CDK4 / 6 inhibitor; wherein the nanocarrier is an organic framework material Zr-Fe MOF containing Zr and Fe bimetallics, and the CDK4 / 6 inhibitors include ribociclib, palbociclib, abemaciclib and darcil.

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

[0009] S1: Dissolve zirconium salt, ligand and PVP in a solvent, mix them by ultrasonication, then add iron salt and dissolve them evenly, and obtain Zr-Fe MOF containing Zr and Fe bimetallic organic framework material through hydrothermal reaction;

[0010] S2: The obtained Zr-Fe MOF ethanol solution and the CDK4 / 6 inhibitor ethanol solution are mixed and stirred, and then the precipitate is collected by centrifugation to obtain the zirconium-iron bimetallic nanomaterial Zr-Fe MOF@CDK4 / 6 inhibitor.

[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 iron salt is FeCl3·6H2O or Fe(NO)3·9H2O.

[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-Fe MOF ethanol to the CDK4 / 6 inhibitor 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-iron 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 aforementioned zirconium-iron bimetallic nanomaterial and a targeted drug; and the targeted drug is coupled to the zirconium-iron bimetallic nanomaterial via a coupling agent.

[0023] Preferably, the breast cancer comprises HER2 + / ER + 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-iron 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 a drug Zr-Fe MOF@CDK4 / 6 inhibitor@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-Fe MOF containing zirconium and iron, loads the CDK4 / 6 inhibitor Ribociclib, and modifies the surface with the monoclonal antibody for the Her-2 receptor, trastuzumab, to obtain the nanomaterial Zr-FeMOF@Ribociclib@Herceptin. This nanomaterial can disintegrate and release trastuzumab, ribociclib, and Fe in the acidic environment of the tumor. 3+ Trastuzumab can not only enhance its targeting effect on tumor cells and reduce its toxicity to normal tissues, but also inhibit the activation of G1 / S-specific cyclin D1 (Cyclin D1) and CDK4 / 6 complex through the PI3K-AKT-mTOR pathway, thereby reducing the level of Rb phosphorylation. The CDK4 / 6 inhibitor Ribociclib inhibits Rb protein phosphorylation by inhibiting CDK4 / 6 activity, thereby inhibiting tumor replication and keeping cells in the G1 phase. In addition, Fe 3+ The Fenton reaction can convert H2O2 into more cytotoxic ·OH, causing oxidative stress damage to cells. The three further inhibit HER2 by inhibiting cell proliferation and disrupting cell homeostasis. + / HR + Breast cancer proliferation. In vivo experiments have shown that ZFRH can not only inhibit the proliferation of in situ tumors, but also has excellent efficacy in inhibiting lung metastasis tumors, which is HER2 + / ER + Precision therapy of breast cancer cells provides a potential new strategy.

[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 A schematic diagram of ZFRH synthesis and its mechanism of targeting tumor cells and exerting anti-tumor effects is shown; wherein, a) is the synthesis route of ZFRH; b) is a diagram of the mechanism by which nanomaterials target tumor cells and synergistically inhibit cell proliferation.

[0038] Figure 2The preparation and characterization of ZFRH are shown; wherein, a) TEM and SEM images of ZF, ZFR, ZFH, and ZFRH; b) EDS image showing the elemental mapping distribution of nitrogen (green), oxygen (red), zirconium (orange), phosphorus (cyan), sulfur (yellow), zinc (yellow), and iron (purple), scale bar: 100 nm; c) N2 adsorption / desorption isotherm of ZF; d) XRD pattern comparison of ZF, ZFR, ZFH, and ZFRH; e) DLS measurement of hydrodynamic size (diameter) and f) Zeta potential of ZF, ZFR, ZFH, and ZFRH; all data are expressed as SD ± mean (n = 3); g) FTIR spectra of ZF, ZFR, ZFH, and ZFRH; h) XPS spectrum of ZF; i) Fe 2p high-resolution XPS spectrum; j) UV-visible absorption spectrum of ZF-oxidized TMB; k) TEM image of degradation of ZF nanomaterials after treatment at different pH values; l) Ribociclib release curve of ZF in PBS at different pH values.

[0039] Figure 3 This is the characteristic UV absorption curve of Ribociclib and the corresponding standard curve.

[0040] Figure 4 The in vitro efficacy study of ZFRH is shown; wherein, a) is the cell viability of BT474 cells at different concentrations after 24 h, 48 h, and 72 h; b) is the cell viability of BT474 cells at different concentrations after 24 h, 48 h, and 72 h; c) is the cell viability of L929 cells at different concentrations after 24 h, 48 h, and 72 h; d) is the cell viability of L929 cells at 24 h, 48 h, and 72 h after different treatments; e) is the fluorescence microscopic image of BT474 cells after different treatments; live cells were stained green with calcein-acetoxymethyl (AM), and dead cells were stained red with propidium iodide (PI); laser: 808 nm, 1.25 W cm -2 ,600; f) is the confocal image of BT474 cells after incubation with ZFRhB and ZFRhB@H, respectively; g) is the confocal image of L929 cells after incubation with ZFRhB and ZFRhB@H, respectively, scale bar: 100 μm.

[0041] Figure 5 The cellular uptake of the different materials is shown.

[0042] Figure 6The in vitro anti-tumor mechanism study of ZFRH is shown; among them, a) is confocal microscopy imaging and bc) are flow cytometry analysis to observe the intracellular ROS levels of BT474 cells after different treatments (PBS, ZF, ZFH, ZFR, ZFRH) for 6 hours; d) is Western blot analysis of Rb, p-Rb, CDK6, GSDMD, N-GSDMD, and Caspase-1 protein expression under different treatments; eh) is the expression of key proteins p-RB, CDK6 / β, Casepase-1, and N-GSDMD in the pyroptosis pathway; all data are expressed as SD±mean.

[0043] Figure 7 The in vivo safety and targeting of ZFRH are shown; a) Representative images showing the degree of hemolysis; b) Weight changes of healthy mice (n=3 mice) treated with PBS and ZFRH, respectively; c) Biochemical analysis of mouse serum treated with PBS and ZFRH (n=3); d) Routine blood test of serum of mice treated with PBS and ZFRH (n=3); e) H&E stained histological sections of different organs (heart, liver, spleen, lung, kidney) of mice with different treatments; the scale bar represents 100 μm; f) Biodistribution of ZFRH@Cy7 in mice after intravenous injection at different time points; g) Visualization of major organs and tumors (S, spleen; H, heart; L, lung; K, kidney; Li, liver; T, tumor) of mice after ZFRH@Cy7 injection.

[0044] Figure 8 The in vivo anti-tumor properties of ZFRH are shown; wherein, a) is a schematic diagram of the establishment and treatment of the BT474 tumor-bearing mouse model; b) is a FL image of BT474 tumor-bearing mice after intravenous injection of ZF, ZFR, and ZFRH; c) is the average weight of mice in each group after various treatments; de) is the relative growth rate of primary tumor volume in BT474 tumor-bearing mice after different treatments (PBS, ZF, ZFR, ZFRH, n=5); f) is the tumors extracted from mice after different treatments and g) is their weight analysis; h) is the expression of different proteins (P-Rb, CDK6, N-GSDMD) in tumor tissues after various treatments detected by Western blotting; i) is the immunofluorescence assay, H&E staining images, and Ki67 immunohistochemical analysis of TUNEL protein in tumor tissues after different treatments, scale bar = 50 μm, ** p < 0.01 and * ** p<0.001.

[0045] Figure 9The process of lung metastasis inhibition is shown; among them, a) is the establishment and treatment plan of BT474 lung metastasis model mice; b) is the weight change of lung metastasis mice; c) is the FL image of the lungs of BT474 lung metastasis mice after intravenous injection of PBS and ZFRH; d) is the quantification of the fluorescence intensity of the metastatic lung; e) is the H&E staining of the lung tissue of the control group mice; f) is the H&E staining of the lung tissue of the ZFRH group mice; g) is the corresponding quantitative analysis of the lung metastatic nodules in each group.

[0046] Figure 10 A schematic diagram of the fluorescence intensity of the lungs of nude mice bearing lung metastasis tumors constructed in the present invention is shown. DETAILED DESCRIPTION

[0047] 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.

[0048] Monoclonal antibodies in clinical HER2 + / ER + It has important application prospects in the treatment of breast cancer. However, the crosstalk between anti-tumor drug mechanism pathways, limited therapeutic effect of a single pathway mechanism, and poor drug targeting have seriously restricted the therapeutic effect of anti-tumor drugs. Figure 1 As shown in the figure, the present invention designs a MOF containing Zr and Fe bimetallics, uses Zr-Fe MOF as the core carrier, loads the CDK4 / 6 inhibitor Ribociclib and connects trastuzumab on the surface, thereby obtaining Zr-Fe MOF@Ribociclib@Herceptin (ZFRH, Figure 1 a) To improve HER2 + Breast cancer is sensitive to trastuzumab. When ZFRH enters the blood, due to the targeting effect of trastuzumab and the EPR effect, the material is enriched in the tumor tissue. Under the action of the acidic microenvironment of the tumor, ZFRH disintegrates and releases trastuzumab and Fe 3+ Trastuzumab not only improves the stability and dispersibility of the material in the blood, but also inhibits the activity of Cylinder D1 protein by inhibiting the PI3K-AKT pathway. At the same time, ribociclib blocks the binding of CDK4 / 6 and Cylinder D1 protein, and combined with trastuzumab inhibits the phosphorylation of RB protein, thereby inhibiting cell transcription activity. Due to the crosstalk between the two, the sensitivity of the two pathways can be increased, inhibiting the proliferation of tumor cells. In addition, free Fe3+ In an acidic environment, it reacts with H2O2 to generate more toxic hydroxyl radicals, which damage the cell membrane and organelles after oxidative stress, further achieving HER2 + Effective killing of breast cancer cells. In vivo and in vitro experimental results show that ZFRH has effective tumor inhibitory effect and good biosafety. In summary, ZFRH is a promising candidate for inhibiting HER2 + It provides a promising and innovative pathway for the growth and metastasis of breast cancer cells, and provides new ideas and drug development directions for subsequent research on precise synergistic treatment of breast cancer.

[0049] Specifically, in one embodiment of the present invention, a zirconium-iron bimetallic nanomaterial is provided, which is composed of a nanocarrier loaded with a CDK4 / 6 inhibitor; wherein the nanocarrier is an organic framework material Zr-FeMOF containing a Zr and Fe bimetallic, and the CDK4 / 6 inhibitor includes Ribociclib. As used herein, CDK4 / 6 inhibitors are a class of anti-tumor drugs that target key cell cycle regulatory proteins (cyclin-dependent kinases 4 and 6), inhibiting tumor cell proliferation by blocking the cell cycle process, and are currently mainly used for hormone receptor-positive (HR-positive) tumors. + ), human epidermal growth factor receptor 2 negative (HER2 - ) for the treatment of advanced or metastatic breast cancer. Such inhibitors include, but are not limited to, palbociclib, ribociclib, abemaciclib, or darcil, with ribociclib being preferred. As used herein, Zr-Fe MOF (zirconium-iron metal-organic framework) is a porous crystalline material composed of bimetallic nodes of zirconium (Zr) and iron (Fe) and organic ligands. It combines high stability, tunable pore structure, and redox activity, and has broad application potential in catalysis, gas adsorption, sensing, and environmental remediation.

[0050] In another embodiment, the present invention also provides a method for preparing a zirconium-iron bimetallic nanomaterial, comprising the following steps: S1: Dissolving a zirconium salt, a ligand, and PVP in a solvent and ultrasonically mixing them, then adding an iron salt and dissolving them uniformly. A hydrothermal reaction is performed to obtain a Zr-Fe MOF bimetallic organic framework material (Zr-Fe MOF). In this step, the zirconium salt is ZrCl4; the ligand is terephthalic acid; the solvent comprises N,N-dimethylformamide; and the iron salt is FeCl3·6H2O or Fe(NO)3·9H2O. This reaction is a one-pot hydrothermal reaction, wherein the hydrothermal reaction conditions are: temperature 120°C, time 10 hours. S2: Mixing the resulting Zr-Fe MOF ethanol solution with an ethanol solution of a CDK4 / 6 inhibitor, stirring, and then centrifuging to collect the precipitate, thereby obtaining the Zr-Fe MOF@CDK4 / 6 inhibitor bimetallic nanomaterial. In this step, the mass ratio of Zr-Fe MOF to CDK4 / 6 inhibitor is 1:1. The mixing and stirring conditions are room temperature and the time is 8 to 12 hours.

[0051] In another embodiment, there is provided a use of a zirconium-iron bimetallic nanomaterial in preparing a drug for treating breast cancer; or in preparing a drug for treating breast cancer lung metastasis; or in preparing a drug for improving the sensitivity of breast cancer targeted drugs. The Zr-Fe MOF@Ribociclib@Herceptin (ZFRH) constructed by the present invention is used to improve HER2 + The sensitivity of breast cancer to trastuzumab. Studies have shown that this ZFRH 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 + / ER + Precision treatment of breast cancer cells provides an application prospect.

[0052] In another embodiment, a drug for treating breast cancer is provided, comprising the aforementioned zirconium-iron bimetallic nanomaterial and a targeted drug; wherein the targeted drug is coupled to the zirconium-iron bimetallic nanomaterial via a coupling agent. The breast cancer includes HER2 + / ER + Breast cancer. Targeted drugs include trastuzumab. The coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

[0053] In another embodiment, a method for preparing a drug for treating breast cancer is provided, comprising the following steps: (1) dissolving the zirconium-iron bimetallic nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting a precipitate; the coupling agent comprises 400 μL of a 4-6 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and 400 μL of a 4-6 mg / mL N-hydroxysuccinimide aqueous solution. The stirring conditions in this step are: room temperature for 20-25 minutes. The centrifugation conditions are: a speed of 10,000-12,000 rpm for 5-10 minutes.

[0054] (2) Adding a targeted drug aqueous solution to the resulting precipitate, stirring, centrifuging, and collecting the precipitate to obtain a Zr-Fe MOF@CDK4 / 6 inhibitor@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.

[0055] To address the issues of crosstalk between anti-tumor pathways and poor drug targeting, the present invention utilizes a metal-organic framework containing zirconium and iron bimetallic elements to host the drug ribociclib and simultaneously surface-modify trastuzumab, creating a highly targeted, multi-mechanism synergistic tumor treatment nanoplatform, ZFRH. Further research demonstrates that the ZFRH rapidly targets tumor tissues, and the released ribociclib reduces Rb phosphorylation by inhibiting CDK4 / 6 activity. Simultaneously, trastuzumab inhibits CycD1 by inhibiting the PI3K-AKT-mTOR pathway, thereby reducing CycD1 binding to the CDK4 / 6 complex. This synergistic effect of ribociclib on Rb phosphorylation inhibits tumor cell proliferation. The released iron ions in the tumor microenvironment effectively generate ROS through the Fenton reaction, which damages and kills tumor cells through oxidative stress. Furthermore, it was unexpectedly discovered that this nanoplatform can trigger pyroptosis by activating Casepase-1. Animal experiments demonstrate that this material not only exhibits excellent therapeutic efficacy against in situ tumors but also effectively inhibits lung metastasis. In summary, this study provides a novel approach for enhancing material targeting and multi-mechanism synergistic treatment of breast cancer, which has great potential for clinical translation as a safe and effective tumor treatment strategy.

[0056] The present invention will further illustrate the principles, processes and effects of the above materials through a specific embodiment below, so that those skilled in the art can understand the essence of the present invention.

[0057] The following embodiments relate to and mention:

[0058] 1. Materials:

[0059] Zirconium chloride, N,N-dimethylformamide (DMF), polyvinylpyrrolidone (PVP), ferric chloride hexahydrate, anhydrous ethanol, ribociclib, tetramethylbenzidine dihydrochloride (TMB HCL), hydrogen peroxide (3 wt %), 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); 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Thermo Fisher Scientific (California, USA); dimethyl sulfoxide was purchased from Coolbo Technology Co., Ltd. (Beijing, China); N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Alpha Chemical Co., Ltd. (Zhengzhou, China); and trastuzumab for injection was purchased from Roche Pharmaceuticals Co., Ltd. ( Shanghai, China); protease inhibitor cocktail (universal, 100×), RIPA lysis buffer (strong), BCA protein assay kit, reactive oxygen species assay kit, Hoechst 33342 live cell staining solution (100×), and Calcein / PI cell viability and cytotoxicity assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); protein precast gel 4%-20% 15-wells was purchased from Boyi Biotechnology Co., Ltd. (Changzhou, China); β-Actin polyclonal antibody was purchased from Biosen Biotechnology Co., Ltd. (Beijing, China); Rb (D20) monoclonal antibody and CDK6 (D4S8S) monoclonal antibody were purchased from Cell Biology (Changzhou, China). Signaling Technology (Massachusetts, USA); Rb (phospho S780) monoclonal antibody, GSDMD (EPR19829) monoclonal antibody, cleaved N-terminal GSDMD monoclonal antibody, and caspase-monoclonal antibody were purchased from abcam (Cambridge, UK); goat anti-rabbit IgG was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China); RPMI 1640, FBS, streptomycin / penicillin, and other biological-related reagents were purchased from Gibco (New York, USA).

[0060] 2. Cell lines

[0061] BT474 and L929 cells were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). BT474 cells were isolated from invasive ductal breast carcinoma tissue from a 60-year-old Caucasian woman and maintained in RPMI 1640 medium. L929 cells were isolated from subcutaneous connective tissue of mice 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.

[0062] 3. Animals

[0063] Five-week-old BALB / c female mice (≈20 g) and KM 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.

[0064] 4. Statistical analysis

[0065] All statistical data were analyzed using GraphPad Prism (GraphPad Software 8.0.2, Santiago, CA, USA) and Origin software (OriginLab 8.0, Northampton, MA, USA). Quantitative data are presented as mean ± standard deviation (SD) of at least three tests. One-way ANOVA was used to assess significant differences, and values ​​of p < 0.05 were considered statistically significant.

[0066] Example 1: Preparation of related products

[0067] Preparation of Zr-Fe MOF: 120 mg of ZrCl₄, 86.5 mg of terephthalic acid, and 67.5 mg of PVP were dissolved in 120 mL of DMF and mixed by ultrasonication to obtain a mixed solution. 50 mg of FeCl₃·6H₂O was then dissolved in the resulting mixed solution and placed in a reactor to react at 120°C for 10 hours. After the reaction, the resulting solution was centrifuged at 10,000 rpm for 5 minutes, and the bottom precipitate was washed three times with anhydrous ethanol to obtain the Zr-Fe MOF (hereinafter referred to as ZF), a bimetallic organic framework containing Zr and Fe.

[0068] Preparation of Zr-Fe MOF@Ribociclib: To a 3 mL, 10 mg / mL ethanol solution of Zr-Fe MOF was added a 10 mL, 3 mg / mL ethanol solution of Ribociclib. The mixture was stirred at room temperature for 12 hours. The mixture was then centrifuged at 12,000 rpm for 5 minutes, and the supernatant and precipitate were collected. The lower precipitate was washed and centrifuged three times, and the supernatant was collected. After collecting all the supernatants, the volume was fixed and UV-visible spectroscopy (Agilent Cary 5000, Agilent Technologies, USA) was performed to analyze the drug loading. The resulting lower precipitate is Zr-Fe MOF@Ribociclib (hereinafter referred to as ZFR).

[0069] Preparation of Zr-Fe MOF@Ribociclib@Herceptin: To a 10 mL, 3 mg / mL aqueous solution of ZFR (10 mL, 3 mg / mL), NHS (400 μL, 5 mg / mL) and EDC (400 μL, 5 mg / mL) were added, respectively. After reacting for 20 minutes, the mixture was centrifuged at 12,000 rpm for 5 minutes, the supernatant removed, and the precipitate washed three times with deionized water. To the resulting precipitate was added an aqueous solution of trastuzumab (1.5 mL, 20 mg / mL). After stirring for 12 hours, the mixture was centrifuged at 12,000 rpm for 5 minutes, the supernatant removed, and the precipitate washed three times with deionized water to obtain Zr-Fe MOF@Ribociclib@Herceptin (hereinafter referred to as ZFRH).

[0070] Preparation of Zr-Fe MOF@Herceptin: To a 10 mL, 3 mg / mL aqueous solution of ZF was added NHS (400 μL, 5 mg / mL) and EDC (400 μL, 5 mg / mL), respectively. After reacting for 20 minutes, the mixture was centrifuged at 12,000 rpm for 5 minutes, the supernatant removed, and the precipitate washed three times with deionized water. To the resulting precipitate was added an aqueous solution of trastuzumab (1.5 mL, 20 mg / mL). After stirring for 12 hours, the mixture was centrifuged at 12,000 rpm for 5 minutes, the supernatant removed, and the precipitate washed three times with deionized water to obtain Zr-Fe MOF@Herceptin (hereinafter referred to as ZFH).

[0071] Preparation of Zr-Fe MOF@RhB and Zr-Fe MOF@Herceptin@RhB: ZF aqueous solution and Zr-Fe MOF@Herceptin (ZFH) aqueous solution were magnetically stirred with rhodamine B (RhB) aqueous solution at room temperature for 12 h, followed by centrifugation at 12000 rpm for 5 min. The resulting precipitates were washed three times with deionized water and dissolved in PBS (pH=7.4) solution to obtain Zr-FeMOF@RhB (hereinafter referred to as ZF@RhB) and Zr-Fe MOF@Herceptin@RhB (hereinafter referred to as ZFH@RhB), respectively.

[0072] Preparation of Zr-Fe MOF@Cy7 and Zr-Fe MOF@Cy7@Herceptin: ZF aqueous solution and ZFH aqueous solution were magnetically stirred with cyanine dye (Cy7) aqueous solution at room temperature for 12 hours, and then centrifuged at 12000 rpm for 5 minutes. The resulting precipitates were washed three times with deionized water and dissolved in PBS (pH = 7.4) solution to obtain Zr-Fe MOF@Cy7 (hereinafter referred to as ZF@Cy7) and Zr-Fe MOF@Cy7@Herceptin (hereinafter referred to as ZF@H@Cy7), respectively.

[0073] Example 2: Material structure analysis and characterization

[0074] 1. Experimental Methods

[0075] 1.1 Material analysis and characterization

[0076] In this example, the physicochemical properties of the product obtained in Example 1 were analyzed by the following methods, specifically including: characterizing the morphology of the sample by transmission electron microscopy (HT7700, Hitachi, Japan) and scanning electron microscopy (SU8600, Hitachi, Japan). The hydrodynamic particle size distribution and zeta potential of the sample were measured using a particle size analyzer (Nano ZS90, Malvern, UK). Elemental mapping and energy spectrum scanning were performed on the sample using a high-resolution transmission electron microscope (JEM-F200, JEOL Ltd., Japan). The functional groups of the sample were detected and analyzed using Fourier transform infrared spectroscopy (Nicolet iS20, ThermoFisher Scientific, USA). The crystal structure of the metal-organic composite sample was detected and analyzed using an X-ray diffractometer (XRD) (Ultima IV, Rigaku, Japan). The specific surface area and porosity of the sample were measured using a fully automatic specific surface and porosity analyzer (BET) (ASAP 2460, Micromeritics, USA). UV absorbance of the samples was measured using a UV-visible spectrophotometer. OD values ​​were measured using a microplate reader (Synergy H1, BioTek, USA). Interactions between the samples and cells were characterized and analyzed using a laser confocal microscope (FV4000, Olympus, Japan). Cellular fluorescence was quantified using flow cytometry (FACSAria III, Becton Dickinson, USA). Protein levels were characterized by immunoblotting. Tissue sections and cells were observed using a fluorescence microscope (Axio Scope A1, Zeiss, USA).

[0077] 1.2 Fenton reaction of Zr-Fe MOF

[0078] The generation of ·OH was detected using a TMB colorimetric reaction. Experiments were conducted in PBS (pH 7.4). Three groups were tested: PBS, ZrMOF (100 μg / mL), and Zr-Fe MOF (100 μg / mL). TMB (20 μL, 10 mg / mL) and H2O2 (10 μL, 10 mM) were added to each solution. The mixture was allowed to stand at room temperature for 10 minutes, and the supernatant was centrifuged and evaluated for ·OH generation using UV-visible spectroscopy.

[0079] 1.3 Drug Release Study of Zr-Fe MOF@Ribociclib

[0080] The drug release behavior of ZFRs was studied in two groups: PBS (pH 5.5) and PBS (pH 7.4), with triplicate samples per group. The PBS solution contained 1% Triton X-100. All test groups were placed in a 37°C incubator and centrifuged at specific time points. The supernatant was analyzed by UV-Vis spectrophotometry, and the original solution was added to the lower layer for further analysis. Time intervals of 0.5, 1, 2, 4, 6, 8, 12, and 24 hours were used to calculate drug release based on UV-Vis absorption peaks. Release rate-release time curves were then plotted to evaluate the controlled release behavior of the ZFRs.

[0081] 1.4 Degradation characteristics of Zr-Fe MOF

[0082] ZF (4 mg) was added to 4 mL of PBS at different pH values ​​(pH 5.7, pH 7.4) and placed in a 37°C constant-temperature air-shaking bath. At different time points, 200 μL of the sample was centrifuged and washed with ethanol. TEM samples were then prepared to investigate the in vitro degradation of ZF. The time intervals were 0, 6, 12, and 24 hours.

[0083] 2 Results and Analysis

[0084] like Figure 2 As shown, material synthesis and physical characterization were carried out in this embodiment. Zr-Fe MOF (ZF) was synthesized by a one-pot hydrothermal reaction using zirconium chloride, ferric chloride and terephthalic acid as raw materials and PVP as a dispersant. The CDK4 / 6 inhibitor Ribociclib was then loaded into the MOF pores by a physical adsorption method of charge attraction to generate Zr-Fe MOF@Ribociclib (ZFR). The carboxyl group in ZFR was then activated, and the activated carboxyl group and the amino group in trastuzumab underwent a decarboxylation reaction. Trastuzumab was modified on the MOF surface in the form of an amide bond to obtain Zr-Fe MOF@Ribociclib@Trastuzumab (ZFRH). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observed that ZF was a regular morphology, uniformly sized cube with a particle size of about 150 nm ( Figure 2 a). Loading Ribociclib did not significantly change the size and morphology of the material. In the transmission electron microscopy image of ZFRH, it can be seen that trastuzumab is modified on the surface of the material in the form of flocs. ZFRH was then subjected to elemental analysis ( Figure 2 b), Zr and Fe elements are evenly dispersed in the material, and P and S are the characteristic elements of protein in the synthesis process, which shows that the modification of trastuzumab is successful. Nitrogen adsorption experiment test ( Figure 2c) The results showed that ZF has an ideal specific surface area (1,063.2220 m 2 / g) and adsorption pore size (4.7803nm), indicating that the high specific surface area and appropriate pore size of ZF are conducive to drug loading. Powder X-ray diffraction (PXRD) shows that the crystal structure of materials in each stage is the same, indicating that the loading of drugs and antibodies does not change the original structure of MOF, and the crystal structure is good ( Figure 2 d). Dynamic light scattering (DLS) indicates that drug loading has little effect on particle size, and antibody modification slightly increases the hydrated particle size to about 190 nm ( Figure 2 e), which is consistent with the SEM and TEM phenomena. The reversal of the zeta potential from the positive charge of ZF (6.4667mV) to the negative charge of ZFRH (-4.99mV) indicates that the loading of drugs and antibodies is successful ( Figure 2 f). Fourier transform infrared spectroscopy (FTIR) shows that ZFH and ZFRH have a higher relative density than ZF and ZFR at 3100-3500 cm -1 The vibration peak at indicates the presence of an amide bond, further indicating that the loading of trastuzumab was successful ( Figure 2 g). X-ray single-emission spectroscopy (XPS) test shows that ZF contains C, N, O, Zr, Fe elements ( Figure 2 h), and most of the Fe is Fe 3+ Valence exists ( Figure 2 i), Fe 3+ Can react to generate Fe in breast cancer cells 2+ , thereby reacting with H2O2 to produce Fenton reaction. In order to verify the ability of Fe ions in Zr-FeMOF to produce ROS by Fenton reaction, a TMB colorimetric experiment was carried out ( Figure 2 j). Hydroxyl radicals (·OH) can cause TMB (3,3',5,5'-tetramethylbenzidine) to undergo a color reaction, changing from colorless to blue or yellow. After centrifuging the solution, the supernatant was taken for ultraviolet spectrophotometry monitoring, and a characteristic absorption peak was observed at 652nm. The results showed that the supernatants of PBS+TMB+H2O2 and Zr MOF+TMB+H2O2 were colorless and transparent, with no obvious ultraviolet absorption peak at 652nm. The supernatant of the Zr-Fe MOF+TMB+H2O2 group turned blue, and a clear ultraviolet absorption peak was observed at 652nm, proving that Zr-Fe MOF has the property of generating hydroxyl radicals due to the presence of Fe ions. Based on the characteristic ultraviolet absorption peak of ribociclib at 347nm, a standard curve of ribociclib was drawn (such as Figure 3 The encapsulation efficiency was calculated to be 19.9% ​​and the drug loading efficiency was 16.6%. The pH response characteristics of ZF were then investigated ( Figure 2k), ZF was dispersed in PBS with pH = 5.7 and pH = 7.4 respectively, and samples were taken at different treatment times for TEM observation. It was found that ZF can exist stably in neutral solution (pH = 7.4) and still maintain its original structure after 24h treatment. However, in acidic solution (pH = 5.7), ZF disintegrated with time, and the original cubic structure disappeared after 24h treatment. This shows that ZF is an acid-responsive release material and has time dependence. Based on the acid-responsive characteristics, drug release experiments were further carried out ( Figure 2 Results showed that in a neutral solution (pH = 7.4), minimal ribociclib release occurred, reaching a cumulative release of 42.7% after 24 hours. In an acidic solution (pH = 5.7), 44.0% of the drug was released after just 4 hours of treatment, and the drug release rate reached 70.8% after 24 hours. This suggests that the material maintains good stability in the bloodstream and can disintegrate and release the drug upon reaching the acidic environment of the tumor.

[0085] Example 3: Cell experiment

[0086] 1 Experimental methods

[0087] 1.1 Cytotoxicity study of Zr-FeMOF

[0088] Mouse epithelial fibroblasts (L929) and human HER2 + / HR + Breast cancer cells (BT474) were used as normal and tumor cell models for cytotoxicity studies. L929 cells were seeded and incubated to an appropriate density. Different concentrations of Zr-Fe MOF (final concentrations of 50, 100, 200, and 400 μg / mL) were then incubated with L929 cells for 24, 48, and 72 hours. A blank medium control was used. BT474 cells were treated with the same treatments, and cell viability was assessed using the MTT assay.

[0089] 1.2 In vitro targeting study of Zr-FeMOF

[0090] ZF@RhB (100 μg / mL) and ZFH@RhB (100 μg / mL) were incubated with BT474 cells and L929 cells for 6 h, respectively. After incubation, the cells were washed three times with PBS to remove the materials not taken up by the cells. Then, nuclear dye (DAPI) was used for co-localization and the in vitro cellular targeting of the materials was observed under a laser confocal microscope.

[0091] 1.3 Cellular ROS Study of Zr-FeMOF@R@H

[0092] The ROS at the cellular level was studied using laser confocal microscopy and flow cytometry. BT474 cells were first plated and incubated to an appropriate concentration. The cells were then divided into five groups: Control, ZF, ZFH, ZFR, and ZFRH. Each group of cells was incubated with blank culture medium, ZF, ZFH, ZFR, and ZFRH (100 μg / mL) for 6 hours. After incubation, all cells were washed three times with PBS, and culture medium containing a reactive oxygen species detection reagent was added. After a further 30 minutes of incubation, the cells were washed three times with PBS. The production of ROS fluorescence in each group was observed using a laser confocal microscope. After the same treatment, the cells were digested and centrifuged, 500 μL of PBS was added, and the ROS fluorescence production in each group was quantified.

[0093] 1.4ZFRH tumor therapy at the cellular level in vitro

[0094] BT474 and L929 cells were plated and cultured to the appropriate concentration and then divided into five groups: Control, ZF, ZFH, ZFR, and ZFRH. Cells in each group were treated with blank culture medium, ZF, ZFH, ZFR, and ZFRH (at a concentration of 100 μg / mL), respectively, and cultured in a 37°C incubator for 24, 48, and 72 hours, respectively. Finally, an MTT assay was performed to calculate cell viability and assess tumor cell inhibition.

[0095] 2 Results and Analysis

[0096] Before further verifying the in vitro efficacy of ZFRH, the cytotoxicity of ZFRH was studied by using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BT474 and L929 cells were treated with different concentrations of ZFRH for 24 h, 48 h, and 72 h ( Figure 4 a), when ZF reached 100 μg / mL, the activity of BT474 and L929 cells was above 80%. In addition, when the two cells were treated with the same concentration and time, the relative proliferation rate of L929 cells was higher than that of BT474 cells ( Figure 4 c), indicating that ZF has good safety for normal cells, and the damage to cell activity is concentration-dependent and time-dependent, that is, with the increase of concentration and the extension of treatment time, the cell activity gradually decreases. Based on the above safety studies, the effectiveness of ZFRH was subsequently verified. BT474 cells were treated with 100 μg / mL of ZF, ZFH, ZFR, and ZFRH for 24 hours, 48 ​​hours, and 72 hours, respectively. Figure 4b) The results showed that the cell activity of the ZFRH group was significantly reduced after 72 hours, and the cell activity was only 53%, which may be related to the killing mechanism of Ribociclib and Trastuzumab. It blocks the cell activation pathway, inhibits the DNA replication of tumor cells, and stops the cell cycle in the G1 phase. Compared with acute toxic drugs that destroy cell membrane structure and cause DNA damage, ZFRH requires an acidic environment and a longer duration of action. L929 cells were then treated with the same conditions ( Figure 4 c and 4d), and found that at the same concentration and time, the viability of L929 cells was much higher than that of BT474 cells, indicating that each material has good safety for normal cells, indicating that ZFRH loaded with trastuzumab and CDK4 / 6 inhibitors cannot effectively kill cells with negative HER2 expression. The above results show that ZFRH can significantly inhibit the growth of HER2-positive tumors and has excellent selectivity, that is, targeted therapeutic effects. In order to further verify the killing effect of ZFRH on BT474, the cells treated with different materials were co-stained with calcein AM (green, live cells) and propidium iodide PI (red, dead cells) ( Figure 4 e), The results of fluorescence microscopy observation were consistent with those of MTT. The proportion of red fluorescence in the ZFRH group was the highest, indicating the highest cell death.

[0097] The targeting of the bimetallic nanomaterials was then investigated through cell uptake experiments. ZF@RhB is ZF loaded with the fluorescent dye Rhodamine B (RhB), and ZFH@RhB is ZFH modified with trastuzumab loaded with Rhodamine B. ZF@RhB and ZFH@RhB loaded with fluorescent dyes were co-cultured with BT474 cells for 3h, 6h, and 9h. The cell nuclei were stained with Hoechst 33342 and the uptake of the materials by different cells was observed. It was found that the blue fluorescence and red fluorescence overlapped the most after 6h of treatment, indicating that the uptake rate of the cells was the highest after 6h of treatment (e.g. Figure 5 Then, the fluorescent dye-loaded ZF@RhB and ZFH@RhB were co-cultured with BT474 cells for 6 h, and the cell nuclei were stained with Hoechst 33342, and the uptake of the materials by different cells was observed by laser confocal microscopy ( Figure 4 f) Compared with ZF@RhB and ZFH@RhB modified with monoclonal antibody, BT474 cells treated with ZF@RhB showed stronger red fluorescence around the blue nuclei. This indicates that more nanomaterials were taken up by cells. Under the same conditions, uptake experiments were conducted using L929 cells ( Figure 4g) Results showed that despite the antibody modification on the ZFH@RhB surface, the fluorescence intensity was significantly reduced compared to BT474 cells, and the red fluorescence intensity was not significantly different from that of ZF@RhB. These results further demonstrate that ZFH-modified trastuzumab is still biologically active, specifically binding to receptors on the surface of HER2-positive cells, and that the attachment of the monoclonal antibody enhances the targeting of the material.

[0098] According to what is known in the art, compared with normal tissues, the tumor microenvironment (TME) is characterized by low pH, high hydrogen peroxide and high reduced glutathione (GSH). GSH can react with reactive oxygen species (ROS) that mediate oxidative stress damage in cells, reduce the cellular ROS content, and thus prevent cell death. ZFRH with acid-responsive properties releases Fe 3+ It can consume the GSH content in TME and generate Fe under the action of redox reaction. 2+ and oxidized glutathione (GSSG), thereby increasing ROS content (2Fe 3+ +2GSH→2Fe 2+ +GSSG+2H + ). At the same time, hydrogen peroxide in TME can react with Fe 2+ The reaction converts into highly toxic hydroxyl radicals (·OH), which in turn enhances the oxidative stress damage caused by ROS to cells (Fe2 + +H2O2→Fe 3+ +·OH+OH - BT474 cells and cells in the control group, 100 μg / mL ZF, ZFH, ZFR, and ZFRH groups were stained with 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe as a fluorescent ROS indicator and then observed by laser confocal microscopy ( Figure 6 The results showed that the control group had the weakest green fluorescence. After ZF treatment, cell fluorescence was significantly enhanced compared to the control group, with the ZFRH group showing the strongest green fluorescence. Subsequently, flow cytometry was used to quantitatively analyze the ROS production in the bimetallic nanomaterials (6b, 6c). The results showed that the ZF group had an approximately 3.6-fold greater ROS-producing capacity than the control group, while ZFRH had the strongest ROS-producing capacity, at 4.2-fold that of the control group. According to relevant literature reports, trastuzumab and ribociclib also promote ROS production in cells to a certain extent, further enhancing the ROS-producing capacity of ZFRH.

[0099] To further verify the tumor killing mechanism, Western blot experiments were also performed ( Figure 6d) Regarding the CDK6 pathway, results showed that ZFR loaded with ribociclib significantly downregulated CDK6 expression compared to the control and ZF groups. Trastuzumab-modified ZFH inhibited CycD1 activity by inhibiting the PI3K-AKT-mTOR pathway, leading to a compensatory upregulation of CDK6 expression. Ribociclib loading in ZFRH further downregulated CDK6 expression compared to the ZFH group, but increased CDK6 expression compared to the ZFR group. Furthermore, neither trastuzumab modification nor ribociclib loading significantly affected RB total protein expression. However, RB phosphorylation was significantly inhibited compared to the control and ZF groups, and the ZFH, ZFR, and ZFRH groups, with the ZFRH group showing the strongest inhibitory effect. These results suggest that trastuzumab and ribociclib in ZFRH synergistically inhibit RB phosphorylation, thereby arresting tumor cell cycle progression and arresting tumors in the G1 phase, ultimately achieving tumorigenicity.

[0100] According to relevant literature reports, trastuzumab can increase the expression of NLRP3 inflammasome. The activation of NLRP3 inflammasome triggers the activation of Casepase-1, and the activated Casepase-1 can cleave Gasdermin D (GSDMD) to generate N-GSDMD. Therefore, this example further explored the expression of key proteins p-RB, CDK6 / β, Casepase-1, and N-GSDMD in the pyroptosis pathway ( Figure 6 eh), the results showed that Casepase-1 and N-GSDMD were upregulated in the ZFH and ZFRH groups, and the expression level in the ZFRH group was the highest, indicating that ZFRH can not only kill tumor cells by inhibiting RB phosphorylation and inhibiting the cell cycle, but also promote the occurrence of cell pyroptosis.

[0101] Example 4: Animal Experiment

[0102] 1 Experimental methods

[0103] 1.1 Study on the biosafety of ZFRH

[0104] Take fresh mouse whole blood and mix it with 3.8% sodium citrate at a ratio of 9:1 (v / v), then dilute the fresh anticoagulated whole blood with normal saline at a ratio of 1:1 (v / v) for later use. Mix 200 μL of diluted anticoagulated whole blood with 5 mL of normal saline, then add 1 mL of diluted whole blood to 1 mL of PBS solution containing different concentrations of ZFRH (concentrations are 50, 100, 150, 200, 400 μg / mL). Use PBS and deionized water as negative and positive controls, respectively. After standing at room temperature for 3 hours, centrifuge and take the supernatant. Use a microplate reader to monitor the absorbance value at 570 nm, and calculate the hemolysis rate according to the following formula:

[0105] 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%

[0106] 1.2 In vivo acute toxicity study of ZFRH

[0107] KM female mice were randomly divided into two groups: a control group and a ZFRH group. 0.2 mL of normal saline or ZFRH (31.5 mg / kg) was injected into the tail vein of each mouse. Following injection, behavioral observations and body weights were recorded. Fourteen days later, mice were euthanized, and blood samples were collected from each group for routine blood tests and biochemical analysis. Major organ tissues (heart, liver, spleen, lung, and kidney) were also collected for histopathological analysis.

[0108] 1.3 Western blotting

[0109] BT474 cells were seeded and incubated to the appropriate concentration. The cells were then divided into five groups: control, ZF, ZFH, ZFR, and ZFRH. Each group was treated with a different material: blank control, ZF, ZFH, ZFR, or ZFRH (100 μg / mL). The cells were incubated at 37°C for 24 hours. After the incubation period, the supernatant was removed, the cells were digested, washed three times with PBS, and RIPA lysis buffer and phosphatase and protease inhibitors were added. The cells were disrupted by sonication and incubated on ice for 30 minutes. After denaturation with SDS-PAGE sample buffer, the cells were subjected to SDS-PAGE on a 4%-20% polyacrylamide gel. Proteins were separated and transferred to a PVDF membrane, which was blocked with 5% skim milk. After washing with TBST, the membranes were incubated with a specific primary antibody (1:1000 dilution) for 12 hours at 4°C, followed by incubation with an appropriate catalase-conjugated secondary antibody (1:1000 dilution) for 1 hour at 37°C. After washing with TBST again, the cells were exposed to light using ECL super-sensitive luminescent liquid and photographed under the CCD exposure of a multifunctional imaging system.

[0110] 1.4 ZFH targeting studies in vivo

[0111] 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, the BT474 tumor-bearing mouse model was successfully constructed for subsequent experiments.

[0112] The in vivo targeted distribution and tumor accumulation of Zr-FeMOF@H were investigated in a BT474 tumor-bearing nude mouse model. ZFH@Cy7 (0.2 mL, 30 mg / kg) was injected into the tail vein. Fluorescence images of the mice were recorded using a small animal in vivo imaging system at different time points (0, 3, 6, 12, 24, 72, and 144 hours). The mice were then euthanized. Major organ tissues (heart, liver, spleen, lung, kidney, and tumor) were harvested and fluorescently imaged using a small animal in vivo imaging system. The in vivo tumor targeting of Zr-FeMOF@H was investigated based on fluorescence images of the tumor site and the corresponding mean fluorescence intensity (MFI).

[0113] 1.5ZFRH in vivo therapeutic experiments

[0114] The BT474-Luc tumor-bearing nude mouse model was used for the ZFRH in vivo treatment experiment. The tumor-bearing nude mice were randomly divided into 4 groups (blank control group, ZF group, ZFR group, ZFRH group, 6 mice in each group, and the tumor volume was about 100mm 3 ), the ZF group, ZFR group, and ZFRH group were injected with 0.2 mL of ZF, ZFR, and ZFRH (30 mg / kg) via the tail vein on days 1, 3, and 5, respectively, while the control group was injected with an equal amount of normal saline. After injection, the body weight of the mice and the size of the mouse tumor (i.e., the length and width of the tumor) were recorded, and the tumor inhibition rate of the different groups was analyzed based on the relationship between tumor volume and time. In vivo imaging of tumor tissue was performed on days 1 and 13. After 14 days, the mice were euthanized, and tumor tissue was taken for photography and stained with HE, Ki67 immunohistochemistry, and Tunel immunofluorescence. Sections were observed and analyzed under a microscope.

[0115] 1.6ZFRH lung metastasis treatment experiment

[0116] BT474-Luc cells were used to construct a lung metastasis model in nude mice. When the in vivo imaging of the small animals showed a fluorescent signal in the lungs, the lung metastasis model was successfully constructed. 30 lung metastasis nude mice were randomly divided into 5 groups - Control group and FEH group. 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 nude mice was basically the same, that is, the number of lung metastasis nodules in the mice was approximately the same (such as Figure 10 On days 0, 2, and 4, mice were injected with 0.2 mL of ZFRH (30 mg / kg) via the tail vein. The control group received an equal amount of saline. Body weights were recorded. After 14 days, mice were euthanized, and lung tissues were collected for fluorescence intensity analysis. Hematoxylin and eosin staining was performed to determine the number of lung nodules and statistical analysis was performed.

[0117] 2 Results and Analysis

[0118] 2.1ZFRH distribution in vivo

[0119] Further research was conducted on the safety and targeting of ZFRH in vivo. The results of in vitro hemolysis test showed that the hemolysis rate of ZFRH at different concentrations was less than 5%, indicating that ZFRH has good blood compatibility ( Figure 7 a). Acute toxicity experiments showed that the mice had normal body weight ( Figure 7 b), blood routine ( Figure 7 c) and liver function, kidney function ( Figure 7 d) There was no statistical difference between the control group and the heart, liver, spleen, lung and kidney pathological tissues. HE staining showed no abnormal changes ( Figure 7 e), indicating that ZFRH has good in vivo biosafety. Subsequently, the tumor targeting of ZFRH was verified in a HER2-positive breast cancer mouse model. Seven BT474 tumor-bearing mice were intravenously injected with Cy7-labeled ZFH (ZFH@Cy7). The fluorescence images of the tumor site and the corresponding mean fluorescence intensity (MFI) showed that ZFH@Cy7 accumulated rapidly in the tumor and lung sites after injection, indicating that ZFH can inhibit the growth of unilateral tumors and lung metastases. After 3 hours, ZFH accumulated significantly in the tumor site ( Figure 7 fg), and gradually metabolized by the liver and kidneys over time. These results indicate that ZFRH has good tumor targeting properties.

[0120] 2.2 Antitumor efficacy of ZFRH in vivo

[0121] In view of the good therapeutic effect of in vitro experiments, this example further established a unilateral tumor model to evaluate the anti-tumor effect of ZFRH in vivo. The fluorescent BT474 tissue tumor mass was inoculated into the subcutaneous tissue of mice. 3 After the volume was measured, the mice were randomly divided into 4 groups: Control group, ZF group, ZFR group and ZFRH group. The mice in the above groups were injected with normal saline, ZF, ZFR, ZFRH (0.2 mL, 30 mg / kg) through the tail vein on the 1st, 3rd and 5th day, respectively ( Figure 8 a). In vivo imaging results of small animals showed that at 13 days, the fluorescence intensity of the ZFR group and ZFRH group was significantly weaker than that of the model group, indicating that both groups could inhibit tumor growth ( Figure 8 b). The weight of mice increased steadily, indicating that the above materials have good biocompatibility ( Figure 8 c). In addition, the tumors in the group injected with normal saline grew rapidly, but the growth of the tumors was delayed after the injection of ZF, indicating that ZF has a moderate therapeutic effect. Compared with the normal saline group, both ZFR and ZFRH significantly inhibited tumor growth. Among them, the BT474 tumor inhibition rate in the ZFR group was 58.33%, and the BT474 tumor inhibition rate in the ZFRH group was 66.66% ( Figure 8 dg). The above results show that the modification of HER2 monoclonal antibody significantly improved the therapeutic efficiency of ZFRH. Subsequently, the tumor pathological tissues were stained with hematoxylin and eosin (H&E), Ki67 immunohistochemistry, and TdT-mediated dUTP nick end labeling (TUNEL) immunofluorescence staining and observed. It was found that after treatment with ZFRH group mice, the tumors showed a certain degree of necrosis, with the most significant nuclear atrophy (H&E image), the least number of Ki67-positive nuclei, and the most number of TUNEL-positive nuclei ( Figure 8 i), which proved that ZFRH can significantly inhibit tumor tissue proliferation and promote cell apoptosis. Western blot results ( Figure 8 h) showed that ZFRH could significantly inhibit the phosphorylation of Rb protein and the activity of CDK6, and cleave GSDMD into N-GSDMD in tumor tissues, thereby promoting the occurrence of pyroptosis in tumor tissues. This result was consistent with the aforementioned in vitro Western blot experimental results.

[0122] Metastasis to vital organs is considered to be the main factor for the high mortality rate of breast cancer, and the lungs are one of the main target organs for breast cancer metastasis. Therefore, this example verifies the anti-tumor effect of ZFRH on breast cancer lung metastasis by constructing a BT474 lung metastasis model. The body weight of mice decreased on the 8th day ( Figure 9 b), indicating that metastatic tumor formation inhibited the growth of mice. After 10 days of treatment, the weight of mice in the ZFRH group stabilized, indicating that ZFRH inhibited the growth of metastatic tumors. The results of small animal live imaging showed that the fluorescence intensity of lung tissue in the ZFRH group was significantly lower than that in the Control group ( Figure 9 cd). Lung tissues of lung metastasis model mice were taken for pathological sectioning and stained with hematoxylin and eosin (H&E) ( Figure 9 ef), the Control group had 26 nodules, while the ZFRH group had 1 nodule, and the number of nodules was significantly lower than that in the Control group. These results indicate that ZFRH also has a good therapeutic effect on breast cancer lung metastasis.

[0123] 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-iron bimetallic nanomaterial, characterized in that: It is composed of a nanocarrier loaded with a CDK4 / 6 inhibitor; wherein the nanocarrier is an organic framework material Zr-Fe MOF containing Zr and Fe bimetallics, and the CDK4 / 6 inhibitors include ribociclib, palbociclib, abemaciclib and darcil.

2. The method for preparing the zirconium-iron bimetallic nanomaterial according to claim 1, wherein: The method comprises the following steps: S1: Dissolve zirconium salt, ligand and PVP in a solvent, mix them by ultrasonication, then add iron salt and dissolve them evenly, and obtain Zr-Fe MOF containing Zr and Fe bimetallic organic framework material through hydrothermal reaction; S2: The obtained Zr-Fe MOF ethanol solution and the CDK4 / 6 inhibitor ethanol solution are mixed and stirred, and then the precipitate is collected by centrifugation to obtain the zirconium-iron bimetallic nanomaterial Zr-Fe MOF@CDK4 / 6 inhibitor.

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 iron salt is FeCl3·6H2O or Fe(NO)3·9H2O; 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-Fe MOF to the CDK4 / 6 inhibitor is 1:1; The stirring reaction conditions are: room temperature, time 8 to 12 hours.

5. Use of the zirconium-iron 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-iron bimetallic nanomaterial according to claim 1, and a targeted drug; and the targeted drug is coupled to the zirconium-iron bimetallic nanomaterial through a coupling agent.

7. The drug according to claim 6, characterized in that The breast cancer includes HER2 + / ER + 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-iron 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 a drug Zr-Fe MOF@CDK4 / 6 inhibitor@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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