Iron-based nano material, preparation method and application of iron-based 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 an iron-based nano material, a preparation method and application of the iron-based nano material in preparation of medicines for treating breast cancer. The iron-based nano material is formed by loading a ferroptosis inducer on a nano carrier; the nano carrier is an iron-based metal organic framework material Fe-MOF (Metal Organic Framework). A product FEH for treating breast cancer is obtained based on coupling of the iron-based nano-material provided by the invention with a targeted drug such as trastuzumab. The FEH provides a new method for developing a nano platform with an antibody function, and realizes a treatment strategy of trastuzumab and ferroptosis combined anti-tumor. As a safe and effective tumor treatment strategy, the EFH has huge clinical transformation potential.

Description

Technical Field

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

[0002] Breast cancer is the most common malignant tumor in women worldwide, ranking first in the incidence of female malignant tumors, and its incidence rate is increasing year by year. Among them, human epidermal growth factor receptor 2 (HER2) positive breast cancer accounts for 20-30% of the annual incidence of breast cancer due to its characteristics of strong invasiveness, high malignancy, and easy recurrence and metastasis in the early stage. At present, a variety of new targeted drugs such as monoclonal antibodies, tyrosine kinase inhibitors, and antibody-drug conjugates have initially shown certain clinical application potential. For example, trastuzumab (Herceptin) Herceptin) as HER2 + Targeted breast cancer drugs have been used in numerous HER2-overexpressing tumors, but there are still challenges to address, including tumor heterogeneity, drug resistance, and adverse drug reactions. Drug resistance, in particular, has garnered considerable attention. Therefore, developing new approaches to mitigate resistance to HER2-targeted drugs and enhance their sensitivity in treating breast cancer is crucial.

[0003] Ferroptosis, a protein toxic stress induced by enhanced iron-dependent lipid peroxidation and inhibition of iron ion transporters, is a new form of cell death that plays an important role in cancer treatment. Studies have shown that ferroptosis can reverse tumor chemotherapy resistance and improve chemotherapy effects. For example, ferroptosis can enhance the sensitivity of cisplatin in the treatment of esophageal cancer, increasing the inhibition rate by 17%. In addition, it can also improve the resistance of doxorubicin to the treatment of breast cancer. However, ferroptosis can occur in various tissue sites in the body, so its application in the clinical local treatment of breast cancer to avoid iron-related toxicity in normal tissues is still facing challenges. In particular, the dynamic changes in iron ion levels in cancer cells make the induction of ferroptosis more difficult. Erastin, as a highly efficient ferroptosis inducer, inhibits System Xc - , leading to glutathione (GSH) depletion and decreased glutathione peroxidase 4 (GPX4) activity, triggering lipid peroxidation and inducing ferroptosis. Studies have shown that erastin can enhance the sensitivity of glioblastoma cells to temozolomide. However, its poor metabolic stability and solubility in vivo limit its clinical use.

[0004] A promising approach to improve HER2 +Nanoparticles, an innovative approach to targeting drug sensitivity to enhance cancer therapy, can leverage the enhanced permeability and retention (EPR) effect to improve tumor targeting of therapeutic drugs, enabling more precise drug delivery. Among nanoparticles suitable for drug delivery, iron-based nanoparticles have emerged as a promising approach for delivering exogenous iron to the tumor microenvironment (TME). Metal-organic frameworks (MOFs) hold great promise in biomedicine due to their robust biodegradability, excellent biocompatibility, and ability to adsorb drugs, enhance their bioavailability, and protect them from degradation. Numerous studies have demonstrated that Fe-MOFs can release iron ions under specific conditions, increasing local iron levels in tumors. Therefore, Fe-MOFs (MIL-101), with their highly ordered structure, high surface area, and large pore volume, and the ability to selectively adsorb and release functional molecules on their surface, were selected as carriers for the delivery of erastin, improving its stability, solubility, and bioavailability. Furthermore, erastin and MIL-101 synergize to enhance the ferroptotic effect. It is worth noting that ferroptosis occurs not only in cancer cells but also in normal cells. + The targeting ability of breast cancer cells is crucial to achieve their effective transport and distribution in the body and tumor cells to exert synergistic effects. Summary of the Invention

[0005] The purpose of the present invention is to provide an iron-based nanomaterial, a preparation method and its use in the preparation of a drug for treating breast cancer, which can improve the sensitivity of HER2 therapeutic targeted drugs by inducing ferroptosis in HER2+ breast cancer cells by targeting them.

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

[0007] The first aspect of the present invention provides an iron-based nanomaterial, which is composed of a nanocarrier loaded with a ferroptosis inducer; the nanocarrier is an iron-based metal organic framework material Fe-MOF.

[0008] Preferably, the ferroptosis inducers include Erastin, Erastin derivatives, RSL3, Sorafenib, Artesunate, FIN56 and BSO.

[0009] A second aspect of the present invention provides a method for preparing an iron-based nanomaterial, the method comprising the following steps:

[0010] S1: terephthalic acid and trivalent iron salt are mixed in N,N-dimethylformamide solution, ultrasonically dispersed and then hydrothermally reacted. After the reaction, the mixture is washed and stored in ethanol to obtain Fe-MOF ethanol solution;

[0011] S2: The obtained Fe-MOF ethanol solution and the ferroptosis inducer ethanol solution are stirred to react. After the reaction is completed, the solution is washed and stored in ethanol to obtain the Fe-MOF@ferroptosis inducer ethanol solution.

[0012] Preferably, in step S1, the trivalent iron salt is FeCl3·6H2O or Fe(NO)3·9H2O.

[0013] Preferably, in step S1, the hydrothermal reaction conditions are: temperature 120° C., time 10 h.

[0014] Preferably, in step S2, the mass ratio of the Fe-MOF to the ferroptosis inducer is 1:1.

[0015] Preferably, in step S2, the stirring reaction condition is: time 10 to 15 hours.

[0016] The third aspect of the present invention provides an application of an iron-based nanomaterial in the preparation of any of the following drugs:

[0017] (i) drugs for the treatment of breast cancer;

[0018] (ii) drugs for the treatment of breast cancer lung metastasis;

[0019] (iii) Drugs that increase the sensitivity of breast cancer to targeted drugs.

[0020] The fourth aspect of the present invention provides a drug for treating breast cancer, comprising the iron-based nanomaterial as described above, and a targeted drug; and the targeted drug is coupled to the iron-based nanomaterial via a coupling agent.

[0021] Preferably, the breast cancer comprises HER2 + Breast cancer.

[0022] Preferably, the targeted drug includes trastuzumab.

[0023] Preferably, the coupling agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0024] A fifth aspect of the present invention provides a method for preparing a drug for treating breast cancer, the method comprising the following steps:

[0025] (1) dissolving the iron-based nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting a precipitate;

[0026] (2) Water and targeted drugs are added to the obtained precipitate, and then the precipitate is stirred and centrifuged to obtain Fe-MOF@ferroptosis inducer@targeted drug for treating breast cancer.

[0027] Preferably, the coupling agent in step (1) consists of a 5 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and a 5 mg / mL N-hydroxysuccinimide aqueous solution.

[0028] Preferably, in step (1), the stirring conditions are: room temperature, and time 20 min.

[0029] Preferably, in step (1), the centrifugal conditions are: rotation speed 10000-12000 rpm, time 3-5 min.

[0030] Preferably, in step (2), the stirring conditions are: room temperature, and time 10 to 15 hours.

[0031] Preferably, in step (2), the centrifugal conditions are: rotation speed 10000-12000 rpm, time 3-5 min.

[0032] By means of the above technical solution, the present invention has at least the following advantages:

[0033] This invention uses Fe-MOF as a core carrier, loading the ferroptosis inducer Erastin to produce Fe-MOF@Erastin. Trastuzumab, an active targeting antibody, is then coupled to the surface of Fe-MOF@Erastin, resulting in the Fe-MOF@Erastin@Herceptin nanoplatform, or FEH. This FEH precisely targets breast cancer cells through surface-modified trastuzumab, and disintegrates in the tumor microenvironment to release iron ions and erastin. The released iron ions react with endogenous H2O2 to produce ·OH through a Fenton reaction; erastin blocks Xc - The reverse transport protein activity of the system is inhibited by inhibiting the synthesis of intracellular GSH to inactivate GPX4, thereby accelerating cell ferroptosis. + The self-cascade platform of ROS increase and GPX4 inactivation in breast cancer cells triggers an effective ferroptosis strategy. In addition, ferroptosis inhibitor (Ferrostatin-1, Fer-1) was also applied to further study the role of FEH in inducing HER2 + The mechanism of action of FEH in ferroptosis of breast cancer cells (SK-BR-3) was investigated. The results of in vitro and in vivo experiments showed that FEH of the present invention exhibited good inhibitory effect on breast cancer cell proliferation and biosafety. In summary, the nano-platform FEH established by the present invention is a promising candidate for enhancing HER2 + Sensitivity of breast cancer targeted drugs and HER2 inhibition + Breast cancer metastasis provides a promising and innovative approach for HER2+ Intravenous infusion treatment of breast cancer, and provide new ideas and drug development directions for subsequent research on precise synergistic treatment of breast cancer.

[0034] 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

[0035] Figure 1 The preparation and anti-tumor mechanism of FEH of the present invention are shown; wherein, a) is a schematic diagram of the FEH preparation process; b) is the biological mechanism of FEH-induced cell ferroptosis.

[0036] Figure 2 The physicochemical characteristics of FEH are shown; wherein, a) SEM and TEM images of different nanoparticles; b) SEM-EDS elemental mapping image of FEH; c) hydrodynamic size; d) Zeta potential of FEH; e) FTIR spectrum of FEH; f) XRD patterns of different nanoparticles; gh) XPS spectra revealing all elements of Fe-MOF; i) continuous H2O2 catalytic ability of Fe-MOF monitored by TMB determination; j) releasability of FEH in PBS at different pH values; k) acid-responsive degradation of Fe-MOF; all data are expressed as Mean±SD (n=3).

[0037] Figure 3 The loading conditions of Erastin are shown: wherein, a) is the UV characteristic absorption curve of Erastin; b) is the standard curve of Erastin.

[0038] Figure 4 Figure 3 shows the in vitro efficacy study of FEH; ab) are fluorescence images of SK-BR-3 and L929 cells after incubation with RhB-labeled FR and FER for 6 h; c) is the cytotoxicity of different concentrations of Fe-MOF on L929 cells; d) is the cytotoxicity of different preparations on SK-BR-3 cells; e) is the live / dead fluorescence microscopy image of SK-BR-3 cells after different treatments: live cells are stained green with calcein (AM) and dead cells are stained red with propidium iodide (PI); f) is the intracellular GSH level in SK-BR-3 cells treated with different preparations; g) is the Fe 2+ Representative confocal fluorescence microscopy images of; ** p<0.01, *** p < 0.001 compared with the control group; ## p<0.01 compared with the FEH group; all data are expressed as Mean±SD (n=5).

[0039] Figure 5 The toxicity assay of Fe-MOF at different concentrations on L929 cells at different times is shown.

[0040] Figure 6 Shown is the toxicity assay of the ferroptosis inhibitor Fer-1 on SK-BR-3 cells.

[0041] Figure 7 The effect of different samples on the GSH / GSSG ratio is shown.

[0042] Figure 8 The in vitro anti-tumor mechanism of FEH is shown; wherein, a) is the intracellular H2O2 level in SK-BR-3 cells treated with different preparations; bd) is the CLSM images and flow cytometry analysis of DCFH-DA-stained SK-BR-3 cells exposed to different treatments; e) is the intracellular MDA level in SK-BR-3 cells treated with different preparations; f) is the appropriate red-green fluorescence intensity ratio of JC-1-stained SK-BR-3 observed using CLSM; g) is the mitochondrial morphological changes of tumor cells after 24 hours of different treatments observed using TEM; h) is the Western blot analysis results of GPX4 and SLC7A11 expression in SK-BR-3 cells after different treatments; ** p<0.01, *** p < 0.001 compared with the control group; ### p<0.001 compared with the FEH group; all data are expressed as Mean±SD (n=5).

[0043] Figure 9 Shown are the changes in intracellular ROS fluorescence intensity in different treatment groups.

[0044] Figure 10 Shown are the changes in malondialdehyde, a metabolite of intracellular lipid peroxides in different treatment groups.

[0045] Figure 11 Shown are the changes in GPX4 and SLC7A11 proteins in cells of different treatment groups.

[0046] Figure 12 The in vitro and in vivo safety and targeting of FEH are shown; wherein, a) is the viability of L929 cells treated with different materials (n=5); bc) are photos of hemolysis assays after treatment with different concentrations of FEH; d) is the change in body weight during the 14-day observation period; e) is the change in biochemical parameters after FEH treatment; f) is the change in blood cell levels after FEH treatment; g) is the H&E staining image of major organs after FEH treatment; hi) is the intravenous imaging of tumor-bearing mice using different preparations and ex vivo imaging including key organs.

[0047] Figure 13 Figure 2 shows the in vivo anti-tumor efficacy of FEH; wherein, a) is a schematic diagram of the establishment and treatment of the BT474 tumor-bearing mouse model; b) is imaging of tumor-bearing mice after different anti-tumor treatments; c) is the body weight change during the 14-day observation period; de) is the tumor volume change; f) is the tumor weight on day 14; g) is H&E, Ki67, TUNEL staining, and GPX4 and SLC7A11 protein immunofluorescence images of tumor tissues after different treatments; h) is the expression of GPX4 and SLC7A11 in tumor tissues after different treatments; * p<0.05, ** p<0.01, *** p<0.001 compared with the control group.

[0048] Figure 14 Shown are the GPX4 and SLC7A11 protein levels in mice of different treatment groups.

[0049] Figure 15 The effects of FEH 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 of mice in each group of the lung metastasis model; c, d) are lung tumor imaging and signal intensity statistics; e, f) are H&E staining of lung tissues of tumor-bearing mice; g) is the number of nodules in lung tissues of tumor-bearing mice.

[0050] Figure 16 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

[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] Her2-positive breast cancer is a subtype of breast cancer with high invasiveness and poor prognosis. In recent years, significant progress has been made in the treatment of Her2 targets, but many factors such as tumor tissue heterogeneity and dysregulation of cell signaling pathways have led to the problem of trastuzumab resistance. Therefore, innovative treatment strategies are urgently needed to improve the quality of life and prognosis of patients. In the scheme of the present invention, a multifunctional nanoplatform FEH was designed and prepared, which was loaded with Erastin on the mesoporous Fe-MOF and Her2 +The FEH platform gradually releases trastuzumab, erastin, and Fe in the tumor microenvironment. 3+ The modification of trastuzumab can enhance its targeting to tumor cells and reduce its toxicity to non-targeted cells and tissues. 3+ Consumes glutathione through reduction reaction and reduces itself to Fe 2+ , further enhancing the catalytic effect of H2O2, thereby triggering the Fenton reaction to produce a large amount of ROS. In the cascade reaction, Fe 2+ Increased ROS can damage mitochondrial function and structure. In addition, Erastin inhibits Xc - The synergistic effect of the three further promotes lipid peroxidation and downregulation of glutathione peroxidase 4 in breast cancer cells, inducing ferroptosis. The above results show that the FEH constructed by the present invention can significantly improve the tumor microenvironment, and the mechanism of Her2 + Precision therapy of breast cancer cells provides a potential new strategy.

[0053] Specifically, in one embodiment of the present invention, an iron-based nanomaterial Fe-MOF@ferroptosis inducer is provided. The material is composed of a nanocarrier loaded with a ferroptosis inducer. Furthermore, the nanocarrier is an iron-based metal-organic framework material Fe-MOF. The ferroptosis inducer can be any suitable inducer in the art, including but not limited to erastin, erastin derivatives such as PE and Imidazole Ketone Erastin, RSL3, sorafenib, artesunate, FIN56, BSO, etc., preferably erastin.

[0054] As used herein, Fe-MOF (Iron-based Metal-Organic Frameworks) is a type of porous crystalline material formed by self-assembly of iron ions (Fe2+ or Fe3+) and organic ligands through coordination bonds. This type of material has a high specific surface area, a controllable pore structure, and abundant active sites, and is widely used in catalysis, gas adsorption, drug delivery, and environmental remediation. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show that Fe-MOF has good dispersion and regular morphology, and is an octahedral nanoparticle. The present invention uses Fe-MOF as a carrier, and obtains an iron-based nanomaterial Fe-MOF@iron death inducer by loading the iron death inducer on the voids of Fe-MOF.

[0055] In one embodiment of the present invention, a method for preparing an iron-based nanomaterial is provided, comprising the following steps:

[0056] S1: Terephthalic acid and a ferric salt are mixed in an N,N-dimethylformamide solution, ultrasonically dispersed, and then hydrothermally reacted. After the reaction, the mixture is washed and stored in ethanol to obtain an Fe-MOF ethanol solution. The ferric salt is either FeCl₃·6H₂O or Fe(NO)₃·9H₂O. The hydrothermal reaction conditions are: temperature 120°C, time 10 hours. In this step, using iron ions as the central metal node and terephthalic acid as the organic ligand, the iron salt and organic ligand are hydrothermally heated in a solvent to form a crystalline Fe-MOF with a highly ordered microporous or mesoporous structure. The pore size and shape can be controlled by the ligand and synthesis conditions. Furthermore, Fe-MOF can be used as a drug carrier, loading drug molecules through its pore structure and achieving controlled release.

[0057] S2: The resulting Fe-MOF ethanol solution and the ferroptosis inducer ethanol solution are stirred and reacted for 12 hours. After the reaction, the solution is washed and stored in ethanol to obtain a Fe-MOF@ferroptosis inducer ethanol solution. The concentration of the Fe-MOF ethanol solution is 3.75 mg / mL, and the concentration of the ferroptosis inducer ethanol solution is 3 mg / mL. During this process, it is necessary to ensure that the mass ratio of Fe-MOF to ferroptosis inducer is 1:1. In this step, the ferroptosis inducer, such as Erastin, is loaded into the pores of the Fe-MOF by physical adsorption, ultimately obtaining Fe-MOF@Erastin.

[0058] In one embodiment of the present invention, there is provided an application of an iron-based 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 present invention is based on the fact that the clinical use of trastuzumab to treat advanced HER2-positive breast cancer is not effective, and advanced HER2-positive breast cancer is prone to the risk of cancer cell metastasis. In this application, by using "mesoporous Fe-MOF loaded with iron death inducer Erastin, and Her2 + FEH was obtained by modifying trastuzumab, a breast cancer cell-specific antibody, to improve the sensitivity of trastuzumab, a breast cancer targeted drug, to treat metastatic HER2-positive breast cancer. It also has a significant therapeutic effect on breast cancer lung metastases, indicating that FEH can significantly improve the sensitivity of trastuzumab to treat breast cancer, thereby improving HER2 + Lung invasion of breast cancer and metastases.

[0059] Based on the shortcomings of erastin, such as its poor metabolic stability and solubility in the body, which limit its clinical use, the present invention provides an iron-based nanomaterial Fe-MOF@Erastin. By loading erastin on the surface voids of Fe-MOF, the metabolic stability of erastin in the body can be achieved and its solubility can be increased, thereby improving the clinical effect of erastin.

[0060] On the other hand, the iron-based nanomaterial Fe-MOF@Erastin obtained by the present invention can also be further coupled with disease treatment drugs to achieve a synergistic treatment effect. To this end, the present invention also provides a drug for treating breast cancer, comprising an iron-based nanomaterial and a targeted drug; and the targeted drug is coupled to the iron-based nanomaterial via a coupling agent. Preferably, the breast cancer is HER2 + For breast cancer, the targeted drug is trastuzumab.

[0061] like Figure 1 As shown in the figure, the drug uses Fe-MOF (MIL-101) as the core carrier, firstly loads the ferroptosis inducer Erastin to obtain Fe-MOF@Erastin, and then couples the active targeting antibody - trastuzumab on the surface of the Fe-MOF@Erastin carrier to obtain the Fe-MOF@Erastin@Herceptin nanoplatform, referred to as FEH ( Figure 1 a).

[0062] Specifically, the preparation process of Fe-MOF@Erastin@Herceptin is as follows: (1) dissolving the iron-based nanomaterial Fe-MOF@Erastin in water, then adding 200 μL of a coupling agent, a 5 mg / mL aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and a 200 μL, 5 mg / mL aqueous solution of N-hydroxysuccinimide. After stirring at room temperature for 20 minutes, the mixture is centrifuged at 10,000-12,000 rpm for 3-5 minutes to obtain a precipitate. (2) adding water and trastuzumab to the resulting precipitate, stirring at room temperature for 10-15 hours, and then centrifuging at 10,000-12,000 rpm for 3-5 minutes to obtain Fe-MOF@Erastin@Herceptin, a drug for treating breast cancer, namely FEH.

[0063] The FEH is precisely targeted to breast cancer cells through surface-modified trastuzumab, and disintegrates in the tumor microenvironment to release iron ions and Erastin. The iron ions react with endogenous H2O2 to produce ·OH through Fenton reaction. On the other hand, Erastin blocks Xc -The reverse transport protein activity of the system inactivates GPX4 by inhibiting the synthesis of intracellular GSH, thereby accelerating cell ferroptosis. + A self-cascading platform of ROS accumulation and GPX4 inactivation in breast cancer cells triggers an effective ferroptosis strategy ( Figure 1 b).

[0064] The present invention further illustrates the selection principle, process and effect of the above-mentioned biomarkers through a specific example below, so that those skilled in the art can understand the essence of the present invention.

[0065] The following embodiments relate to and mention:

[0066] 1. Materials:

[0067] Ferrous chloride hexahydrate, N,N-dimethylformamide (DMF), methylene blue, 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); erastin (98% purity), N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Alpha Chemical Co., Ltd. (Zhengzhou, China); trastuzumab for injection was purchased from Roche Pharmaceuticals Co., Ltd. (Shanghai, China); Fer-1 (ferrostatin-1) was purchased from Selleck Biotechnology Co., Ltd. (Texas, USA); protease inhibitor (PMSF) was purchased from Bimake Biotechnology Co., Ltd. (Texas, USA); RIPA lysis buffer (strong), BCA protein detection kit, reactive oxygen species detection kit, hydrogen peroxide detection kit, lipid oxidation (MDA) detection kit, Hoechst 33342 live cell staining solution (100X) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); 30% gel preparation solution (29:1 Acr-Bis) was purchased from Prilai Gene Technology Co., Ltd. (Beijing, China); Cell Counting Kit-8 (CCK-8), Calcein-AM / PI cell double staining kit, GSSG / GSH Quantification Kit II, intracellular ferrous ion fluorescent probe (FerroOrange), and mitochondrial membrane potential fluorescent probe (JC-1) were purchased from Dongren Chemical Technology Co., Ltd. (Shanghai, China); Iron Assay Kit was purchased from Sigma-Aldrich (St. Louis, USA); Actin polyclonal antibody was purchased from Sevier Biotechnology Co., Ltd. (Wuhan, China); GPX4 monoclonal antibody was purchased from Boster Biotechnology Co., Ltd. (Wuhan, China); SLC7A11 monoclonal antibody was purchased from Abimat Biopharmaceutical Co., Ltd. (Shanghai, China); goat anti-rabbit IgG was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China); DMEM and RPMI were used in the culture medium. 1640, FBS, streptomycin / penicillin and other biological related reagents were purchased from Gibco (New York, USA).

[0068] 2. Cell lines:

[0069] SK-BR-3 and L929 cells were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). SK-BR-3 cells were isolated from patient pleural effusions and maintained in DMEM medium, while 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.

[0070] 3. Animals

[0071] 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.

[0072] 4. Statistical analysis

[0073] Grayscale values were semi-quantitatively 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, Santiago, CA, USA) and Origin software (OriginLab8.0, 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.

[0074] Example 1: Preparation of related products

[0075] Preparation of Fe-MOF: 42 mg of terephthalic acid and 135 mg of FeCl3·6H2O were dissolved in 120 mL of DMF and mixed by ultrasonication. The mixture was then reacted at 120°C for 10 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 5 min. The resulting precipitate was washed with ethanol and stored in ethanol at room temperature to obtain an Fe-MOF (hereinafter referred to as F) ethanol solution.

[0076] Preparation of Fe-MOF@Erastin: An ethanolic solution of Erastin (5 mL, 3 mg / mL) was slowly added to an ethanolic solution of Fe-MOF (4 mL, 3.75 mg / mL) and stirred for 12 hours. After stirring, the mixture was centrifuged at 12,000 rpm for 5 minutes. The resulting precipitate was washed with ethanol and stored in ethanol to obtain an ethanolic solution of Fe-MOF@Erastin (hereinafter referred to as FE).

[0077] Preparation of Fe-MOF@Erastin@Herceptin: The resulting FE ethanol solution (4 mL, 3.75 mg / mL) was centrifuged at 12,000 rpm for 5 minutes to remove the ethanol. The FE was then sonicated in deionized water. An aqueous solution of EDC (200 μL, 5 mg / mL) and NHS (200 μL, 5 mg / mL) was added, stirred at room temperature for 20 minutes, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and the precipitate was collected. To the resulting precipitate, 4 mL of deionized water and 8 mL of trastuzumab for injection (2 mg / mL) were added. After stirring for 12 hours, the mixture was centrifuged at 12,000 rpm for 5 minutes to obtain Fe-MOF@Erastin@Herceptin (hereinafter referred to as FEH).

[0078] Preparation of Fe-MOF@Herceptin: The resulting F ethanol solution (4 mL, 3.75 mg / mL) was centrifuged at 12,000 rpm for 5 minutes to remove the ethanol. The F was then sonicated in deionized water. An aqueous solution of EDC (200 μL, 5 mg / mL) and NHS (200 μL, 5 mg / mL) was added, stirred at room temperature for 20 minutes, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and the precipitate was collected. To the resulting precipitate, 4 mL of deionized water and 8 mL of trastuzumab for injection (2 mg / mL) were added. After stirring for 12 hours, the solution was centrifuged at 12,000 rpm for 5 minutes to obtain Fe-MOF@Herceptin (hereinafter referred to as FH).

[0079] Preparation of Fe-MOF@RhB and Fe-MOF@Herceptin@RhB: Rhodamine B (RhB) was added to Fe-MOF and FH solutions, respectively, and stirred in the dark at room temperature for 12 h. After stirring, the solution was centrifuged at 12,000 rpm for 5 min. The resulting precipitate was washed with deionized water to obtain RhB-labeled Fe-MOF@RhB (hereinafter referred to as FR) and RhB-labeled Fe-MOF@Herceptin@RhB (hereinafter referred to as FHR), respectively.

[0080] Preparation of Fe-MOF@Herceptin@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 Cy7 fluorescently labeled Fe-MOF@Herceptin@Cy7 (hereinafter referred to as FH@Cy7).

[0081] Example 2: Material structure analysis and characterization

[0082] 1. Experimental Methods

[0083] 1.1 Material analysis and characterization

[0084] The morphology, size, and microstructure of the samples were examined using scanning electron microscopy (ZEISS Sigma 360, Germany) and transmission electron microscopy (Hitachi, Tokyo, Japan) at an electron gun operating voltage of 300 kV. The elemental composition of the FeH was analyzed by transmission electron microscopy. The structure and particle size of the nanoplatforms were analyzed using a Fourier transform infrared spectrometer (Nicolet 380, Thermo, Waltham, Massachusetts, America) and a Malvern Zetasizer Nano ZS90, UK. The crystal structure of the Fe-MOF nanoplatforms was determined using a D-MAX2200 X-ray powder diffractometer (RIGAKU, Japan). The elemental and chemical states of the Fe-MOF were analyzed using an X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha, Waltham, Massachusetts, America).

[0085] 1.2 Erastin's Load

[0086] Drug loading experiments were performed as follows: 1. A Fe-MOF ethanol solution (1.5 mg / mL, 2 mL) was stirred with anhydrous ethanol (3 mL) for 24 hours; 2. A Fe-MOF ethanol solution (1.5 mg / mL, 2 mL) was stirred with an Erastin ethanol solution (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 encapsulation efficiency and drug loading were calculated.

[0087] 1.3 Measurement of ROS generation in Fe-MOF solution

[0088] To detect ROS production using the colorimetric 3,3',5,5'-tetramethylbenzidine (TMB) assay, 20 μL of Fe-MOF aqueous solution (Fe: 9 μg) was first mixed with 980 μL of PBS buffer. TMB (25 μL, 0.5 mM) and 10 μL of 3% hydrogen peroxide were then added. The mixture was incubated for 15 minutes, and the supernatant was aspirated to serve as the experimental group (Fe-MOF + H2O2 + TMB). A PBS buffer solution without Fe-MOF (PBS + H2O2 + TMB) served as a control. Finally, the TMB absorbance peaks in the experimental and control samples were measured at a wavelength of 652 nm using a UV-visible spectrometer (UV-2401PC, Shimadzu, Japan) to reveal ROS production.

[0089] 1.4 Detection of iron ion content released by degradation

[0090] 1. FEH (4 mg) was dispersed in 2 mL of PBS (pH 7.4) and released by shaking in a constant-temperature water bath at 37°C. 2. FEH (4 mg) was dispersed in 2 mL of PBS (pH 5.5) and released by shaking in a constant-temperature water bath at 37°C. At predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24, 28, and 32 h), the supernatant was collected by centrifugation and diluted to 10 mL. The iron content in the supernatant at each time point was measured using an iron ion detection kit (BC4355, Solebol, Beijing, China) and a microplate reader (Spectramax 190, Molecular Devices, USA).

[0091] 1.5 Fe-MOF disintegration triggered by acidic pH

[0092] Fe-MOF was dispersed in two PBS solutions of different pH values (pH = 7.4 and pH = 5.5) to form a 1 mg / mL mixed solution. The sample tube was then shaken in a 37°C constant-temperature air bath shaker. Samples were taken at 0, 1, 6, and 12 hours for electron microscopy. Transmission electron microscopy (TEM) was then used to observe the morphological changes of the Fe-MOF.

[0093] 2 Results and Analysis

[0094] like Figure 2 As shown, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show (see Figure 2 a) Fe-MOF (F) has good dispersion and regular morphology, and is an octahedral nanoparticle with a particle size of about 180nm. The morphology of FE, FH, and FEH after loading with Erastin and / or modifying with trastuzumab is still octahedral. In addition, in the TEM image of FEH, it can be found that trastuzumab is attached to the surface of the material in the form of flocs. Further elemental mapping analysis of FEH was performed (see Figure 2 b), the results show that the Fe element is evenly dispersed in the Fe-MOF skeleton, and the S element, P element and N element are evenly deposited on the MOF surface, indicating that the FEH surface is successfully modified with trastuzumab. Dynamic light scattering (DLS) results show that compared with Fe-MOF (182.6nm), the effect of Erastin loading on the particle size of the nanomaterial is negligible, but the particle size of FH (256.8nm) and FEH (278.2nm) modified with trastuzumab increases to a certain extent, which is consistent with the TEM image results (see Figure 2c). Zeta potential results showed that the surface charge of Fe-MOF (F) was 20 mV, while the surface charge of Erastin-loaded FE (17.6 mV) and trastuzumab-modified FEH (13 mV) decreased to varying degrees, indicating that Erastin was successfully loaded and trastuzumab was successfully modified (see Figure 2 d). The sample was analyzed by infrared spectroscopy. The Fe-MOF (F) peak at 3368 cm -1 The peak at 1698 cm is considered to be the stretching vibration of OH. -1 The peak at 547 cm is the stretching vibration of C=O. -1 The peak at 3201cm is the vibration of Fe-O. The infrared characteristic peak of FE after loading Erastin is similar to the characteristic peak of MOF, with only the change in peak intensity. This may be because the inducer changes the electronic environment of the MOF surface and increases the vibration frequency of the functional groups on the MOF surface, which also indicates that Erastin is bound by physical forces. -1 A broad and red-shifted peak appeared at 1689cm, indicating the formation of hydrogen bonds, and the peak was significantly red-shifted. In the FEH infrared results, the peak intensity of the characteristic peak decreased, and the peak at 1689cm -1 The characteristic peak of C=O at the position disappears, which may be related to the formation of Schiff bond. The nitrogen in the amino group replaces the oxygen of C=O to form C=N (see Figure 2 e). Powder X-ray diffraction (PXRD) pattern (see Figure 2 f) shows that the PXRD spectra of F, FE, FH and FEH have not changed, indicating that the loaded compounds have not affected the original crystal characteristics of Fe-MOF. X-ray photoelectron spectroscopy (XPS) results show that Fe-MOF has no other obvious impurity peaks, indicating that the material has high purity (see Figure 2 g, h). In addition, Fe 2+ The area is smaller than Fe 3+ area, indicating that most of the Fe in Fe-MOF is Fe 3+ Fe 3+ Can react inside cells to generate Fe 2+ , thereby reacting with H2O2 to produce the Fenton reaction. The ability of Fe-MOF to induce the Fenton reaction to produce ROS was further tested by TMB experiments (see Figure 2 i) The results showed that compared with PBS solution, Fe-MOF produced a redox reaction in the environment with the addition of H2O2, causing the solution (TMB+H2O2) to turn blue-green, indicating that Fe-MOF produced a large amount of hydroxyl radicals in the environment with the presence of H2O2.

[0095] The therapeutic effect of the drug at the tumor site depends on the specific release of the preparation in the acidic microenvironment of the tumor (pH 5.5). In this example, based on the characteristic ultraviolet absorption peak of Erastin at 276nm, a standard curve of Erastin was drawn, and the encapsulation efficiency was calculated to be 22.1% and the drug loading rate was 18.1% (as shown in Figure 2). Figure 3 The stability and pH-acid-responsive degradation characteristics of Fe-MOF and FEH at pH 7.4 and pH 5.5 were further tested by TEM and microplate reader. The results showed that FEH was stable in PBS solution at pH 7.4, with a cumulative release of about 100.45 mg / mL of total iron in the supernatant within 32 h, while the cumulative release of total iron in an acidic environment (pH 5.5) was about 126.31 mg / mL (see Figure 2 j), indicating that FEH can be better decomposed and released in the acidic environment inside tumor cells. TEM images show (see Figure 2 k) The Fe-MOF exhibited no significant morphological changes within 12 hours at pH 7.4. However, in an acidic environment (pH 5.5), the Fe-MOF structure disintegrated to varying degrees over time, with significant structural disintegration observed after 6 hours and complete structural collapse after 12 hours. These results demonstrate that FEH exhibits excellent stability in the blood and exhibits acid-responsive properties when targeted to tumor tissue, disintegrating and releasing the drug, thereby increasing the local concentration of the drug.

[0096] Example 3: Cell experiment

[0097] 1. Experimental Methods

[0098] 1.1 In vitro cellular uptake

[0099] First, SK-BR-3 and L929 cells (1×10 5 Cells (100 μg / well) were seeded in 24-well plates and cultured for 24 hours. The cells were then incubated with complete medium, FR (diluted to 100 μg / mL in complete medium, 1 mL added to each well), and FHR (diluted to 100 μg / mL in complete medium, 1 mL added to each well) for 6 hours. Following incubation, the cells were stained with Hoechst 33342. Fluorescence microscopy (Leica, Germany) was used to observe the uptake of Hoechst 33342 (blue): λex = 346 nm; λem = 460 nm; RhB (red): λex = 488 nm; λem = 525 nm.

[0100] 1.2 Cell viability assay

[0101] L929 cells (1.5×10 4Cells (100 cells / well) were seeded into 96-well plates and cultured for 24 hours. Different concentrations of Fe-MOF (600, 400, 200, 100, 60, 40, and 0 μg / mL) were then added to the plates and incubated with the cells for 24, 48, and 72 hours. At the cutoff time, the original liquid was discarded, and 600 μL of CCK-8 solution was diluted with 6 mL of fresh culture medium. Then, 100 μL of this mixture was added to each well. The cells were incubated at 37°C for 2 hours, and the absorbance at 450 nm was recorded using a microplate reader (Spectramax 190, Molecular Devices, USA).

[0102] 1.3 Cell growth inhibition assay

[0103] SK-BR-3 (1.5×10 4 Cells were seeded in 96-well plates and cultured for 24 h. The cells were then divided into 6 groups and each group was treated as follows:

[0104] Control group: complete culture medium;

[0105] Group F: complete medium + F (100 μg / mL);

[0106] FE group: complete medium + FE (100 μg / mL);

[0107] FH group: complete medium + FH (100 μg / mL);

[0108] FEH group: complete medium + FEH (100 μg / mL);

[0109] FEH+Fer-1 group: complete medium+FEH+Fer-1 (100 μg / mL).

[0110] Note: The sample volume for each group mentioned above is 1 mL / well.

[0111] The cells in the above groups were treated and incubated for 24 h, 48 h, and 72 h. CCK-8 was diluted with complete culture medium (10:1) to obtain the diluted CCK-8 working solution. At the cutoff time point, the original liquid in the well plate was discarded, and the diluted CCK-8 working solution (100 μL / well) was added and the cells were incubated at 37 ° C for 2 h. The absorbance at 450 nm was recorded by microplate reader (Spectramax 190, Molecular Devices, USA). SK-BR-3 cells (2×10 5Cells / well) were inoculated into 6-well plates, and then incubated using the above treatment method. After the incubation, 1 mL of a mixed staining solution of 2 μM Calcein-AM and 4.5 μM PI was added to the wells. After incubation for 15 minutes, the cell staining results were observed using a fluorescence microscope (Leica, Germany).

[0112] 1.4 Intracellular GSH / GSSG detection

[0113] SK-BR-3 cells were plated at 2 × 10 5 Cells were seeded in 6-well plates at a density of 100 cells / well. When the cells reached 70-80% confluence, they were divided into 6 groups and each group was treated as follows:

[0114] Control group: complete culture medium;

[0115] Group F: complete medium + F (100 μg / mL);

[0116] FE group: complete medium + FE (100 μg / mL);

[0117] FH group: complete medium + FH (100 μg / mL);

[0118] FEH group: complete medium + FEH (100 μg / mL);

[0119] FEH+Fer-1 group: complete medium+FEH+Fer-1 (100 μg / mL).

[0120] Note: The sample volume for each group mentioned above is 1 mL / well.

[0121] The cells in each group were treated accordingly and incubated for 24 h. The cells were then washed three times with PBS and measured according to the instructions of the GSSG / GSH Quantification Kit II. The GSH / GSSG ratio was calculated.

[0122] 1.5 Detection of intracellular ferrous ions

[0123] FerroOrange was used to detect the intracellular ferrous ion content. The specific operation was as follows: SK-BR-3 cells (2×10 5 Cells (100 cells / dish) were seeded in a laser confocal microscopy dish and incubated with 100 μg / mL of F, FE, FH, FEH, and FEH+Fer-1 for 24 h, respectively. They were then stained with 0.5 μM FerroOrange for 30 min, washed, and observed under a laser confocal scanning microscope (Dragonfly 200, Andor, UK).

[0124] 1.6 Intracellular ROS detection

[0125] DCFH-DA was used to detect the total ROS content in cells. The specific operation was as follows: first, SK-BR-3 cells (2×10 5 Cells were seeded into 6-well plates (cells per well). When the cells reached 70-80% confluence, they were divided into 6 groups and each group was treated as follows:

[0126] Control group: complete culture medium;

[0127] Group F: complete medium + F (100 μg / mL);

[0128] FE group: complete medium + FE (100 μg / mL);

[0129] FH group: complete medium + FH (100 μg / mL);

[0130] FEH group: complete medium + FEH (100 μg / mL);

[0131] FEH+Fer-1 group: complete medium+FEH+Fer-1 (100 μg / mL).

[0132] Note: The sample volume for each group mentioned above is 1 mL / well.

[0133] After treatment, cells in each group were incubated for 24 hours. After incubation, the cells were washed three times with PBS and stained with DCFH-DA (final concentration 10 μM) for 20 minutes. After staining, the cells were washed three times with PBS and fluorescence images were observed using a laser confocal scanning microscope (Dragonfly 200, Andor, UK). Finally, the cells were digested and ROS levels were measured using a flow cytometer (AttuneNxT, Thermo Fisher Scientific, MA, USA).

[0134] 1.7 Intracellular H2O2 concentration detection

[0135] SK-BR-3 cells (2×10 5Cells were seeded (100 μg / mL) in 6-well plates and cultured in complete medium for 24 hours. The cells were then divided into five groups: F, FE, FH, and FEH groups were incubated for 24 hours with F (100 μg / mL), FE (100 μg / mL), FH (100 μg / mL), and FEH (100 μg / mL), respectively. The control group received no treatment. After incubation, the cells were digested, harvested, and homogenized with 200 μL / well of hydrogen peroxide detection 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.

[0136] 1.8 Detection of intracellular MDA content

[0137] SK-BR-3 cells were plated at 2 × 10 5 Cells were seeded in 6-well plates at a density of 100 cells / well. When the cells reached 70-80% confluence, they were divided into 6 groups and each group was treated as follows:

[0138] Control group: only complete culture medium was added;

[0139] Group F: complete medium + F (100 μg / mL);

[0140] FE group: complete medium + FE (100 μg / mL);

[0141] FH group: complete medium + FH (100 μg / mL);

[0142] FEH group: complete medium + FEH (100 μg / mL);

[0143] FEH+Fer-1 group: complete medium+FEH+Fer-1 (100 μg / mL).

[0144] Note: The sample volume for each group mentioned above is 1 mL / well.

[0145] The cells in each group were treated accordingly and incubated for 24 h. After incubation, the cells were washed three times with PBS and measured according to the instructions of the lipid oxidation (MDA) detection kit, and the MDA concentration was calculated.

[0146] 1.9 Mitochondrial membrane potential detection

[0147] SK-BR-3 cells (2×10 5Cells (100 μg / mL) were seeded in confocal microplates and cultured for 24 hours. The cells were then divided into six groups: F, FE, FH, FEH, and FEH+Fer-1 groups, respectively, and incubated for 24 hours with F, FE, FH, FEH, and FEH+Fer-1 (100 μg / mL). The control group received no treatment. After incubation, the cells were washed twice with PBS and JC-1 working solution (4 μM, 100 μL) diluted in PBS was added. The cells were incubated in a 37°C, 5% CO2 incubator for 30 minutes. The supernatant was removed and the cells were washed twice with 200 μL of HBSS. 200 μL of Imaging Buffer Solution was then added, and the cells were observed for red and green staining using a confocal laser scanning microscope (Dragonfly 200, Andor, UK). Fluorescence parameters: Green: Ex = 488 nm, Em = 500-550 nm; Red: Ex = 561 nm, Em = 560-610 nm.

[0148] 2.0 Cell Transmission Electron Microscopy

[0149] SK-BR-3 cells (2×10 5 Cells were seeded in 6-well plates (100 μg / mL) and divided into six groups: F, FE, FH, FEH, and FEH+Fer-1 groups were treated with F, FE, FH, FEH, and FEH+Fer-1 (100 μg / mL) for 24 hours, respectively. The control group received no treatment. After incubation, the cells were washed three times with PBS, and samples were prepared for transmission electron microscopy. The mitochondrial structure of SK-BR-3 cells was observed using a transmission electron microscope (Hitachi, Japan).

[0150] 2.1 In vitro Western blot analysis

[0151] To measure the expression levels of the ferroptosis-related proteins GPX4 and SLC7A11, SK-BR-3 cells were seeded in six-well plates for 24 hours. Subsequently, the cells were divided into six groups: F, FE, FH, FEH, and FEH+Fer-1 groups, each treated with F (100 μg / mL), FE (100 μg / mL), FH (100 μg / mL), FEH (100 μg / mL), and FEH+Fer-1 (100 μg / mL), respectively, for 24 hours. The control group received no treatment. After incubation, the cells were rinsed three times with PBS. Cells were fully lysed with cell lysis buffer, harvested, and centrifuged at 4°C, 12,000 rpm, for 10 minutes. The supernatant was collected. Protein concentration in the supernatant was determined using the BCA assay. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with skim milk in TBST for 2 h and washing with TBST for 10 min, the membrane was incubated with primary antibodies against GPX4 and SLC7A11 (1:1000) overnight at 4°C. After washing three times with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The membrane was visualized using a ChemiScope 6100 (Shanghai, China) with a chemiluminescent substrate (34577, Therom, Massachusetts, USA).

[0152] 2. Results and Analysis

[0153] 2.1 In vitro cellular uptake and cytotoxicity of Fe-MOF@Erastin@Herceptin

[0154] Cell uptake experiments were performed to determine whether the enhanced cellular uptake of FEH was mediated by trastuzumab-dependent endocytosis. + Breast cancer cells (SK-BR-3) and Her2 - Mouse fibroblasts (L929) were used for verification. Figure 1 The route shown in a prepared the nanomaterials Fe-MOF@RhB(FR) and FH@RhB(FHR) labeled with rhodamine B (RhB), and then co-cultured with the two cells for 6 hours for fluorescence imaging. Fluorescence images show ( Figure 4 a), Compared with FHR, there is no difference in the uptake of FR in the two cells, and no obvious red fluorescence ( Figure 4 a, b). It is worth noting that no significant uptake of FHR was observed in L929 cells, while in SK-BR-3 cells, the red fluorescence intensity of the FHR group was significantly enhanced, indicating that the modification of trastuzumab enhanced the effect of FHR on HER2. + Cell targeting to improve HER2 -Cell toxicity. The cell viability assay (CCK-8 method) was used to study the toxicity of different concentrations of Fe-MOF on L929 cells at 24h, 48h, and 72h. When the Fe-MOF concentration reached 118.49 μg / mL (such as Figure 5 As shown), the cell activities were all above 80% ( Figure 4 c). Therefore, it can be determined that 100 μg / mL Fe-MOF 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. In addition, this example also studies the dosage of ferroptosis inhibitor Fer-1 on SK-BR-3 cells. The results show that when the Fer-1 concentration is less than 10 μmol / L, the cell viability is above 80% (such as Figure 6 Therefore, 10 μmol / L concentration of Fer-1 was selected for subsequent intervention experiments. Based on the above safety studies, the effectiveness of the nanomaterials at different stages of 100 μg / mL was subsequently verified. The results showed that the SK-BR-3 cell viability in the Fe-MOF group was above 80% ( Figure 4 d), compared with the Fe-MOF group, due to the effects of trastuzumab targeting cells and Erastin inducing ferroptosis, the cell viability of the FH (76%) and FE (65%) groups showed a gradual downward trend. It is worth noting that FEH (42%) has the strongest inhibitory effect in vitro, and after incubating SK-BR-3 cells for 48h and 72h, SK-BR-3 cells are still in a low viability state. However, after the addition of Fer-1, the cell viability of the FEH group was significantly increased, indicating that Erastin and iron ions can effectively exert anti-tumor effects in tumor cells under the targeting effect of trastuzumab, and this anti-tumor effect can be inhibited by Fer-1. In addition, it was calculated by BCA protein quantification that 100μg / mL FEH contains approximately 0.043mg of trastuzumab. As Figure 4 As shown in d, the relative content of HER2 has a lower killing effect on cells than FH, FE, and FEH, indicating that Fe-MOF and Erastin enhance the killing effect of trastuzumab, and also indicate that ferroptosis can enhance the drug sensitivity of trastuzumab to HER2+ breast cancer cells. In order to further verify the killing effect of FEH on SK-BR-3 cells, calcein AM (green, live cells) and propidium iodide PI (red, dead cells) were used to co-stain cells treated with different materials ( Figure 4e) The results of fluorescence microscopy observation were consistent with those of CCK-8. The proportion of red fluorescence in the FEH group was the highest. After the addition of Fer-1, the proportion of red fluorescence in the FEH group was significantly reduced, indicating that FEH can target Her2-overexpressing tumor cells and induce ferroptosis by loading Erastin and modifying trastuzumab.

[0155] The GSH / GSSG ratio in the tumor microenvironment (TME) plays a crucial role in the cellular redox state. 3+ Consumes GSH in the TME and converts it into Fe 2+ , producing GSSG(2Fe 3+ +2GSH→2Fe 2+ +GSSG+2H + The results showed that compared with the Fe-MOF group, the GSH / GSSG ratios in the other groups were significantly reduced (e.g. Figure 7 The GSH / GSSG ratio in the FEH group was the lowest, only 0.41 times that of the control group ( Figure 4 f), after the intervention of Fer-1, this ratio increased significantly ( Figure 4 f), indicating that Erastin inhibits the synthesis of glutathione (GSH). In addition, high levels of ferrous ions promote the occurrence of ferroptosis, further through the iron probe to Fe 2+ The probe can produce Fe 2+ The results showed that ( Figure 4 g) Compared with the control group, the red fluorescence of cells in the F, FH, FE, and FEH groups gradually increased, and the intervention of Fer-1 could improve this phenomenon, indicating that FEH can release Fe in the TME. 3+ and Erastin, consume GSH, increase intracellular Fe 2+ The content of ferroptosis was induced in SK-BR-3 cells.

[0156] 2.2Fe-MOF@Erastin@Herceptin induces ferroptosis in vitro

[0157] H2O2 in TME can react with FeH to disintegrate and transform 2+ Through the Fenton reaction, it is converted into highly toxic hydroxyl radicals (·OH), which in turn enhance the oxidative stress damage caused by ROS to cells (Fe 2+ +H2O2→Fe 3+ +·OH+OH -In order to study the ability of FEH to induce ferroptosis, the changes of important ferroptosis parameters such as H2O2, ROS, and MDA in SK-BR-3 cells were detected by ELISA kit, flow cytometry and laser confocal microscopy. The results showed that ( Figure 8 a), the content of intracellular H2O2 decreased to varying degrees after treatment with materials at different stages. Flow cytometry results showed that compared with the Control group, the ROS fluorescence signals in the F, FH, FE and FEH groups increased ( Figure 8 b, c). Among them, the ROS fluorescence intensity in the tumor cells of the FEH group was the strongest compared with that of the other groups, but the ROS content in the Fer-1 intervention group was reduced (e.g. Figure 9 The same results were observed under laser confocal microscopy. Only weak ROS green fluorescence was found in the Control group, F, FH and FE groups. This may be because most of the ROS in the F and FE groups were not taken up by cells. The green fluorescence of the FEH-treated group was significantly enhanced ( Figure 8 d), indicating that the modification of trastuzumab makes FEH easily taken up by cells, and produces a large amount of ROS by consuming H2O2 in tumor cells and undergoing Fenton reaction.

[0158] In Fe 2+ Under conditions of elevated concentrations, lipid peroxidation in cells is significantly aggravated, severely damaging cell membranes and other cell components, leading to ferroptosis. Therefore, the ELISA method was used to detect changes in malondialdehyde (MDA), a metabolite of lipid peroxides in cells of different treatment groups. The results showed that ( Figure 8 e, Figure 10 ), compared with the MDA content of the Control group (0.52μmol / mg), the different stage materials can lead to different degrees of increase in MDA content, and the MDA content of the FEH group (1.94μmol / mg) is the highest, but the addition of Fer-1 can reverse this result. This shows that FEH converts Fe in TME 2+ It can aggravate the lipid peroxidation reaction of tumor cells and lead to cell ferroptosis.

[0159] Mitochondria are the energy center of cells and are very sensitive to ROS due to their membrane structure and internal environment. The effect of FEH on the mitochondrial membrane potential of SK-BR-3 cells was detected by tetraethylbenzimidazole carbon cyanide iodide (JC-1). JC-1 aggregates formed by high mitochondrial membrane potential exhibit red fluorescence; when mitochondria are depolarized, JC-1 monomers that are not connected to the mitochondrial inner membrane exist in the cytoplasm and exhibit green fluorescence. Compared with other treatment groups, the green fluorescence of SK-BR-3 cells after FEH treatment was significantly enhanced, indicating that the mitochondrial membrane potential has undergone a significant depolarization ( Figure 8f). TEM was used to further observe the damage of mitochondrial structure. The images showed that after FEH treatment, the number of mitochondria in cells decreased, the volume decreased, the mitochondrial membrane density increased, and the cristae disappeared ( Figure 8 g), which is precisely the characteristic of mitochondrial damage induced by ferroptosis. Importantly, Fer-1 can improve the damage to mitochondrial structure.

[0160] Solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) are key factors in the ferroptosis process. SLC7A11 indirectly affects the activity of GPX4 by regulating GSH synthesis, while GPX4 can directly convert lipid peroxides into alcohols that are harmless to cells, thereby protecting cells from lipid peroxidation damage and maintaining cell homeostasis. To further explore the killing mechanism of FEH on tumors, the levels of ferroptosis-related marker proteins GPX4 and SLC7A11 were detected by Western blot. The results showed that ( Figure 8 h), compared with the control group, the GPX4 and SLC7A11 protein levels in SK-BR-3 cells were significantly downregulated after nanomaterials were treated at different stages. In addition, compared with FH (e.g. Figure 11 The protein levels of GPX4 and SLC7A11 in the FEH group were significantly downregulated, indicating that Erastin played an inhibitory role in Xc - The FEH+Fer-1 group showed increased expression of GPX4 and SLC7A11 compared to the FEH group, indicating that the addition of Fer-1 inhibited erastin-induced ferroptosis.

[0161] Example 3: Animal testing

[0162] 1. Experimental Methods

[0163] 1.1 Construction of tumor-bearing mouse model

[0164] 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.

[0165] 1.2 In vivo targeting and in vitro organ imaging

[0166] When the tumor volume of the tumor-bearing mice was about 100 mm 3At 4 hr post-injection, mice were injected with Cy7-labeled FH@Cy7 (20 mg / kg, 0.2 mL) via the tail vein. After respiratory anesthesia, fluorescence images of the mice were recorded using a small animal in vivo imaging system at 0, 3, 6, 12, 24, 72, and 144 h after FH@Cy7 injection. After recording, the mice were euthanized, and tumors and major organs were removed for ex vivo fluorescence imaging.

[0167] 1.3 In vivo anti-tumor efficacy

[0168] After establishing the Balb / c nude tumor-bearing mouse model, 24 model mice were randomly divided into 4 groups: Control group, F group, FH group, and FEH group. The F group, FH group, and FEH group were injected with 20 mg / kg of Fe-MOF, FH, and FEH into the tail vein on the 1st, 3rd, and 5th day, 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 SLC7A11 and GPX4 immunofluorescence staining, Western blotting, H&E staining, Ki67 staining, and Tunel staining.

[0169] 1.4 Establishment of lung metastasis-bearing mice

[0170] 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 FEH 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 30 lung metastasis tumor-bearing nude mice constructed was basically the same, that is, the number of lung metastasis nodules in the mice was approximately the same (such as Figure 16 The control group received a tail vein injection of 0.2 mL of normal saline on days 1, 3, and 5, while the FEH group received a tail vein injection of 0.2 mL of 20 mg / kg FEH on the same days. Body weight and tumor volume were recorded every other day. After 14 days, the mice were euthanized, and lung tissue was harvested for ex vivo fluorescence imaging and counted for the number of lung nodules. Finally, lung tissue was stained with H&E for analysis.

[0171] 1.5 Hemolysis test

[0172] 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 FEH 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 FEH 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 r / min 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:

[0173] 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%

[0174] 1.6 In vivo toxicity evaluation of Fe-MOF@Erastin@Herceptin

[0175] KM mice were used to evaluate the in vivo toxicity of FEH. 0.2 mL of FEH (24 mg / kg) was injected via the tail vein, and the physiological status and body weight of the mice were observed and recorded for 14 days. At the end of the observation period, the mice were euthanized, and plasma and serum were collected for routine blood and biochemical examinations (n = 3). Major organs were collected, embedded in paraffin sections, and H&E staining was performed according to the H&E staining protocol.

[0176] 2. Results and Analysis

[0177] 2.1 Biosafety and in vivo distribution of Fe-MOF@Erastin@Herceptin

[0178] In terms of biosafety assessment, this example first tested the cytotoxicity of 100 μg / mL Fe-MOF, FH, FE, and FEH to L929 cells using a CCK-8 assay. Figure 12 As shown in a, the cell viability of the material groups at each stage was above 80% within 72 hours, indicating that the materials at each stage had good in vitro biosafety for normal cells. The in vitro hemolysis results showed that the hemolysis rates of FEH at different concentrations were all below 5%, indicating that FEH had good blood compatibility ( Figure 12 b, c). Acute toxicity experiments showed that after tail vein injection of 23.9 mg / kg FEH, the body weight of mice increased steadily within 14 days ( Figure 12 d), blood routine and biochemical indicators are normal ( Figure 12 e, f), H&E images of major organs (heart, liver, spleen, lung, kidney) ( Figure 12 g) No obvious pathological changes or organ damage were observed, indicating that FEH treatment has good biosafety.

[0179] The tumor targeting ability of FEH in vivo was further studied using a BT474 tumor-bearing mouse model. Seven BT474 tumor-bearing mice were intravenously injected with Cy7-labeled FH (FH@Cy7), and the fluorescence images of the mice at different time points (0, 3, 6, 12, 24, 72, and 144 h) were recorded using a small animal in vivo imaging system. Figure 12 h), after FH@Cy7 was injected through the tail vein, it was targeted to the tumor site in the blood circulation and accumulated rapidly. Some FEH remained in the lung endothelial cells for metabolism due to its small particle size, which was beneficial to inhibit the tendency of breast cancer to metastasize to the lung. After 3 hours, it was mainly retained in the tumor site. After 6 hours, there was a clear fluorescent signal in the liver and kidney. After 6 days, it was basically metabolized, indicating that FH can be metabolized through the liver and kidney. The mice were euthanized at different times after injection (3, 6, 12, 24, 72, 144 hours), and the tumors and major organs (heart, liver, spleen, lungs, and kidneys) were collected for in vitro fluorescence imaging. The fluorescence image of the tissue showed ( Figure 12 i) Different levels of fluorescence signals appeared in the tumor, liver, and kidney at different time points, which were consistent with the fluorescence imaging results of the animals. These results indicate that FH has good tumor targeting properties.

[0180] 2.2 In vivo antitumor efficacy of Fe-MOF@Erastin@Herceptin

[0181] Subsequently, the effectiveness of different materials (PBS, F, FH, and FEH) in inducing ferroptosis in tumor cells in vivo was evaluated using a BT474 unilateral tumor-bearing mouse model. 3 Dosing is done when the volume Figure 13 a). The results of in vivo imaging of small animals showed that the tumors in the group injected with normal saline (Control group) grew rapidly, while the injection of materials at different stages delayed the growth of the tumors ( Figure 13 b). The body weight of mice in each group increased steadily within 14 days, indicating that FEH has good biocompatibility ( Figure 13 c). Compared with the saline group, both FH and FEH significantly inhibited tumor growth. The BT474 tumor inhibition rate in the FH group was 45.36%, and the tumor inhibition rate in the FEH group was 64.94% ( Figure 13 d,e), the tumor weight of the FEH group was significantly lower than that of the saline group, F and FH groups ( Figure 13f). The above results indicate that the modification of trastuzumab and the loading of erastin improve the therapeutic efficiency of FEH. The anti-tumor effect was further verified by hematoxylin and eosin (H&E), Ki67 and terminal deoxynucleotidyl transferase dUTP end labeling. The tumor tissues of mice in the FEH group showed obvious necrosis, and the cells showed nuclear atrophy (H&E images). The number of Ki67-positive nuclei was low, and the number of TUNEL-positive nuclei was high ( Figure 13 g).

[0182] In addition, the expression changes of ferroptosis-related proteins GPX4 and SLC7A11 were verified by immunofluorescence and Western blot. The results showed that ( Figure 13 g, h) The expression of both proteins was downregulated in the different material groups, which was consistent with the in vitro experimental results, indicating that FEH can efficiently deliver Erastin into the body. Compared with the F group, the expression of GPX4 and SLC7A11 in the FH and FEH groups was significantly downregulated (e.g. Figure 14 This indicates that the loading of trastuzumab improves the in vivo targeting of Fe-MOF, and Erastin still plays an inhibitory role in Xc in tumor-bearing mice. - The above results indicate that FEH can inhibit tumor growth by inducing ferroptosis through synergistic effects, which is beneficial for HER2 + It provides an effective strategy for the treatment of tumors.

[0183] 2.3 Fe-MOF@Erastin@Herceptin for the treatment of metastatic tumors

[0184] Because tumor metastasis is systemic and occult, eradicating early-stage metastatic tumors is more challenging than eliminating primary tumors. In this example, a BT474 lung metastasis model was established by injecting BT474 cells expressing luciferase (BT474-luc) into the tail vein of Balb / c nude mice. Figure 15 The anti-tumor treatment was carried out according to the a scheme to study the anti-metastatic activity of FEH. Based on the expression of luciferase, the location of cancer cells can be monitored by IVIS. The results showed that the weight of both groups of mice decreased on the 8th day ( Figure 15 b), indicating that metastatic tumor formation inhibited the growth of mice; however, after 10 days of treatment, the weight of mice in the Control group continued to decline, while the weight of mice in the FEH group stabilized, indicating that FEH inhibited the growth of metastatic tumors. In addition, the imaging results of lung tissue showed that the fluorescence intensity of the FEH group was significantly lower than that of the Control group ( Figure 15 c, d). Hematoxylin and eosin (H&E) staining ( Figure 15Eg) The results showed that the number of nodules in the control group (8±8) was much higher than that in the FEH group (2±2), which was consistent with the in vivo targeting conclusion, indicating that FEH can significantly improve HER2 + Lung invasion in breast cancer.

[0185] In summary, the present invention designs and synthesizes a novel iron metal framework nanoplatform FEH. The FEH is further modified with Erastin and trastuzumab to target HER2. + Specific targeting ability of breast cancer. In the acidic environment of the tumor, the platform disintegrates to release iron ions and Erastin, on the one hand, by inhibiting Xc - On the other hand, FEH can stimulate the production of a large amount of reactive oxygen species (ROS) by consuming GSH, leading to an imbalance in the antioxidant system in cells, further promoting lipid peroxidation and GPX4 downregulation in breast cancer cells, and ultimately triggering ferroptosis. + In both unilateral and metastatic tumor models, FEH significantly inhibited tumor growth. In summary, FEH provides a novel approach for developing antibody-enabled nanoplatforms, enabling a combined anti-tumor strategy of trastuzumab and ferroptosis. This approach has significant potential for clinical translation as a safe and effective tumor treatment strategy.

[0186] 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. An iron-based nanomaterial, characterized in that: The invention is composed of a nanocarrier loaded with an iron death inducer; the nanocarrier is an iron-based metal organic framework material Fe-MOF, and the iron death inducer includes erastin, erastin derivatives, RSL3, sorafenib, artesunate, FIN56 and BSO.

2. The method for preparing the iron-based nanomaterial according to claim 1, wherein The method comprises the following steps: S1: terephthalic acid and trivalent iron salt are mixed in N,N-dimethylformamide solution, ultrasonically dispersed and then subjected to hydrothermal reaction. After the reaction, the mixture is washed and stored in ethanol to obtain Fe-MOF ethanol solution; S2: The obtained Fe-MOF ethanol solution and the ferroptosis inducer ethanol solution are stirred to react. After the reaction is completed, the solution is washed and stored in ethanol to obtain the Fe-MOF@ferroptosis inducer ethanol solution.

3. The preparation method according to claim 2, characterized in that In step S1, the trivalent 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 Fe-MOF to the ferroptosis inducer is 1:1; The stirring reaction conditions are: time 10 to 15 hours.

5. Use of the iron-based 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: The invention comprises the iron-based nanomaterial according to claim 1, and a targeted drug; wherein the targeted drug is coupled to the iron-based nanomaterial via 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 iron-based nanomaterial in water, then adding a coupling agent, stirring, centrifuging, and collecting the precipitate; (2) Water and targeted drugs are added to the obtained precipitate, and then the precipitate is stirred and centrifuged to obtain Fe-MOF@ferroptosis 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 min; the centrifugation conditions are: speed 10000-12000 rpm, time 3-5 min; In step (2), the stirring conditions are: room temperature, time 12 hours; the centrifugal conditions are: rotation speed 10000-12000 rpm, time 3-5 minutes.

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