Nanometer bionic bimetallic MOF and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a nano bionic bimetallic MOF and a preparation method and application thereof. The nano bionic bimetallic MOF has a core-shell structure and comprises an inner core and a cell membrane coating the surface of the inner core, the inner core is nano bimetal MOF, and the nano bimetal MOF contains a glucose transport inhibitor and a photosensitizer. Mn / Fe-MOF is synthesized by using a solvothermal method, a glucose transport inhibitor (BAY-876) and a photosensitizer (CyI) are loaded in the Mn / Fe-MOF, and the Mn / Fe-MOF is subjected to cell membrane bionic modification to obtain a core-shell structure which is used for enhancing tumor targeting. The hypoxia microenvironment responds to release oxygen and is actively targeted to a breast cancer tumor site, and the effect of overcoming tumor drug resistance synergistically by multiple mechanisms is verified by depending on a cell / animal model.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nano-bionic bimetallic MOF and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women, posing a serious threat to their health and lives. Current treatment options include surgery, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy. While surgical treatment is effective for patients in the early stages of the disease, it is difficult to completely remove the lesions in late-stage patients, as the tumor may have metastasized. While chemotherapy and radiotherapy kill tumor cells, they can also cause severe damage to normal tissue cells, leading to a range of side effects such as hair loss, nausea, and decreased immunity. While endocrine therapy and targeted therapy offer some specificity, they are prone to developing drug resistance, and their efficacy gradually decreases as treatment progresses.

[0003] Photodynamic therapy (PDT), an emerging cancer treatment, offers promising advantages with minimal invasiveness, high selectivity, and minimal side effects. However, the effects of PDT are significantly limited by the fact that the PDT reaction depletes oxygen in the tissue microenvironment, generating cytotoxic reactive oxygen species (ROS). This creates a generally hypoxic microenvironment within tumor tissues. Summary of the Invention

[0004] The purpose of the present invention is to provide a nano-bionic bimetallic MOF and its preparation method and application, so as to overcome the shortcomings of the existing technology, integrate multiple treatment mechanisms, and have high efficiency and low toxicity breast cancer treatment effects with precise targeting capabilities.

[0005] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides a nano-bionic bimetallic MOF having a core-shell structure, comprising an inner core and a cell membrane coated on the surface of the inner core; the inner core is a nano-bimetallic MOF, and the interior of the nano-bimetallic MOF contains a glucose transport inhibitor and a photosensitizer.

[0006] The innovative nano-bionic bimetallic MOF system constructed by the present invention for photodynamic immunotherapy of breast cancer contains multiple treatment strategies. Based on the key problem that the lack of oxygen in the tumor microenvironment seriously restricts the effectiveness of photodynamic therapy, the MOF system introduces manganese elements and utilizes its unique oxygen-releasing properties under hypoxic conditions to supplement the necessary oxygen source for the photodynamic reaction, thereby enhancing the killing efficiency of photodynamic therapy on tumor cells. At the same time, with the help of iron elements, the ferroptosis program in tumor cells occurs, effectively destroying the redox homeostasis in the cells and promoting the death of tumor cells. In addition, BAY-876 is incorporated into the system, and through its specific inhibition of the core pathway of tumor cell energy metabolism, it triggers disulfide death starvation therapy, causing tumor cells to apoptosis due to severe obstruction of energy supply. The above-mentioned multiple treatment mechanisms complement each other and work synergistically with photodynamic therapy to strike tumor cells in an all-round and deep manner.

[0007] At the same time, considering the importance of improved targeting for enhancing efficacy and reducing side effects, the cell membrane of 4T1 cells was coated on the surface of a nano-bionic bimetallic MOF. Utilizing the rich and diverse specific proteins and receptors on its surface, it can accurately recognize and specifically bind to the corresponding ligands on the surface of breast cancer cells. This allows the nanomaterial to efficiently enrich around breast cancer cells, precisely locate and target the tumor cells, significantly increase the concentration of therapeutic substances at the tumor site, and provide a guarantee for significantly improved breast cancer treatment efficacy.

[0008] In some other embodiments, the mass ratio of the nanobimetallic MOF, glucose transport inhibitor, and photosensitizer is 100:(0.5-1.5):(0.5-1.5); illustratively, the mass ratio of the nanobimetallic MOF, glucose transport inhibitor, and photosensitizer is 100:0.5:0.5, 100:0.5:1, 100:1:1, 100:1.5:1, 100:1.5:1.5, and 100:1:1.5. Preferably, the mass ratio of the nanobimetallic MOF, glucose transport inhibitor, and photosensitizer is 100:1:1. Within this ratio range, the nanobimetallic MOF, glucose transport inhibitor, and photosensitizer exhibit better synergistic effects, can enhance tumor starvation therapy and improve the efficiency of photodynamic therapy, and have shown good therapeutic effects at both the cellular and animal levels.

[0009] In some other embodiments, the cell membrane is a 4T1 cell membrane; the glucose transport inhibitor is one or more of BAY-876, S961, and quercetin; The photosensitizer is one or more of CyI, porfimer sodium and verteporfin; Preferably, the glucose transport inhibitor is BAY-876; and the photosensitizer is CyI.

[0010] In some other embodiments, the nanobimetallic MOF is a Mn-Fe bimetallic MOF; the mass ratio of Mn to Fe in the Mn-Fe bimetallic MOF is 1:(2-4); illustratively, the mass ratio of Mn to Fe in the Mn-Fe bimetallic MOF is 1:2, 1:3, or 1:4. Preferably, the mass ratio of Mn to Fe in the Mn-Fe bimetallic MOF is 1:3. The particle size of the nanobimetallic MOF is 10-100 nm. Nanobionic bimetallic MOFs use Fe as the dominant element (Fe:Mn = 3:1) to enhance the ferroptosis effect, requiring precise targeting and multi-mechanism synergy, with Mn serving as an auxiliary function to achieve efficient photodynamic therapy of tumors.

[0011] In a second aspect, the present invention provides a method for preparing the nano-bionic bimetallic MOF according to the first aspect, comprising the following steps: (1) Mixing manganese salt, iron salt and 2-hydroxyterephthalic acid and dissolving them in N,N-dimethylformamide to prepare a mixed solution; subjecting the mixed solution to a solvothermal reaction to prepare a nano bimetallic MOF; (2) dissolving the nano bimetallic MOF, glucose transport inhibitor, and photosensitizer in an ethanol solution, stirring and mixing, and then centrifuging, washing, and drying to obtain the drug-loaded bimetallic MOF; (3) The drug-loaded bimetallic MOF is dispersed in a buffer solution containing cell membranes. The cell membranes are wrapped on the surface of the drug-loaded bimetallic MOF using a membrane squeezer. After centrifugation and washing, the nano-bionic bimetallic MOF is obtained.

[0012] In some other embodiments, in step (1), the mixing mass ratio of manganese salt, iron salt and 2-hydroxyterephthalic acid is 1: (2-4): (2-3); the temperature of the solvent thermal reaction is 95-110 ° C, and the reaction time is 8-12 hours; preferably, the mixing mass ratio of manganese salt, iron salt and 2-hydroxyterephthalic acid is 1: 3: 2.5; the temperature of the solvent thermal reaction is 100 ° C, and the reaction time is 10 hours. The nano-scale particle size of the nano-bionic bimetallic MOF prepared under the reaction conditions is 100-200 nm, which can meet the nanoparticle size range for cancer treatment. In some other embodiments, in step (1), the manganese salt is one of manganese chloride, manganese nitrate and manganese sulfate; The iron salt is one of ferric chloride, ferric nitrate and ferric sulfate; After the solvent thermal reaction, a post-treatment is further performed, wherein the post-treatment is sequentially centrifuged, washed and vacuum dried, and the washing is sequentially washed with N, N-dimethylformamide and ethanol; Preferably, the manganese salt is manganese chloride, and the iron salt is ferric chloride.

[0013] In some other embodiments, in step (2), the mass ratio of the nano bimetallic MOF, the glucose transport inhibitor, and the photosensitizer is 100: (0.5-1.5): (0.5-1.5); The glucose transport inhibitor is one or more of BAY-876, S961, and quercetin; The photosensitizer is one or more of CyI, porfimer sodium and verteporfin; Stirring is 1-3h at room temperature; Preferably, the mass ratio of the nano bimetallic MOF, the glucose transport inhibitor, and the photosensitizer is 100:1:1; The glucose transport inhibitor is BAY-876; the photosensitizer is CyI; Stirring was carried out at room temperature for 2 h.

[0014] In a third aspect, the present invention provides the use of the nano-bionic bimetallic MOF described in the first aspect in the preparation of a drug for photodynamic immunotherapy of breast cancer.

[0015] In a fourth aspect, the present invention provides a drug for photodynamic immunotherapy of breast cancer, comprising the nano-bionic bimetallic MOF and a pharmaceutically acceptable salt thereof according to the first aspect, and at least one pharmaceutically acceptable excipient and / or carrier.

[0016] Beneficial effects of the present invention: (1) The nano-bionic bimetallic MOF system constructed in this invention contains multiple therapeutic strategies for synergistic efficacies. To address the problem of hypoxia in the tumor microenvironment restricting photodynamic therapy, manganese is introduced to release oxygen and supplement the oxygen source, thereby enhancing the killing efficiency of photodynamic therapy on tumor cells; iron is used to induce ferroptosis in tumor cells, disrupting the intracellular redox homeostasis; and BAY-876 is incorporated into the system to induce disulfide starvation therapy, causing tumor cells to apoptosis due to blocked energy supply. Multiple therapeutic mechanisms complement each other and work in synergy with photodynamic therapy to comprehensively and deeply attack tumor cells.

[0017] Improve targeting and ensure efficacy: The cell membrane of 4T1 cells is wrapped on the surface of the nano-bionic bimetallic MOF, and its surface-specific proteins and receptors are used to accurately identify and specifically bind to the ligands on the surface of breast cancer cells, thereby achieving efficient enrichment of nanomaterials around breast cancer cells, precise positioning and targeted action on tumor cells, increasing the concentration of therapeutic substances at the tumor site, significantly improving the treatment effect of breast cancer, and reducing side effects.

[0018] (2) The present invention synthesizes Mn / Fe-MOF using a solvothermal method and loads a glucose transport inhibitor (BAY-876) and a photosensitizer (CyI) inside it. The resulting core-shell structure, modified by cell membrane biomimetic, is used to enhance tumor targeting. The structure actively targets breast cancer tumors by releasing oxygen in response to the hypoxic microenvironment. Cell / animal models are used to verify the multi-mechanism synergistic effect of overcoming tumor drug resistance.

[0019] (3) The nano-bionic bimetallic MOF prepared by the present invention integrates multiple therapeutic mechanisms and can treat breast cancer with high efficiency and low toxicity with precise targeting capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 4T1@MFCB particle size distribution diagram in Example 1 of the present invention; Figure 2 This is an electron microscope image of the MFCB in Example 1 of the present invention; Figure 3 This is an electron microscope image of 4T1@MFCB in Example 1 of the present invention; Figure 4 The UV-visible absorption spectra of BAY-876, CyI, MOF, and 4T1@MFCB in Example 1 of the present invention are shown; Figure 5 : This is the fluorescence absorption spectrum of CyI and 4T1@MFCB in Example 1 of the present invention; Figure 6 The PBS, CyI, MOF and 4T1@MFCB solution in Example 1 of the present invention were exposed to near-infrared light (808 nm, 0.96 W / cm 2 ) Temperature change curve under irradiation; Figure 7 The PBS, CyI, 4T1@MFCB in Example 1 of the present invention and the 4T1@FCB in Comparative Example 1 are 0.96W / cm 2 Singlet oxygen production diagram produced under NIR laser irradiation; Figure 8Confocal images of intracellular ROS production in 4T1 cells after incubation with different samples in Example 2 of the present invention and Comparative Example 1; wherein, I: Control, II: 4T1@FeMOF@CB, III: 4T1@Mn-FeMOF@CB, IV: 4T1@FeMOF@CB+laser, V: 4T1@Mn-FeMOF@CB+laser, VI: 4T1@FeMOF@CB+laser, VII: 4T1@FeMOF@CB+laser; Figure 9 This is a diagram showing the oxygen replenishment status of 4T1 cells after incubation with different samples in Example 2 of the present invention; wherein, I: *Control, II: Control, III: BAY-876, IV: 4T1@MFCB, V: CyI+laser, VI: 4T1@MFCB+laser, VII: 4T1@MFCB+laser; Figure 10 Figure 2 shows the ferroptosis-induced conditions in 4T1 cells after incubation with different samples in Example 2 of the present invention and Comparative Example 1; Among them, I: Control, II: 4T1@Mn-FeMOF@CB, III: 4T1@Mn -FeMOF@CB+laser, IV: BAY-876, V: 4T1@FeMOF@CB+laser, VI: 4T1@Mn-FeMOF@CB+H2O2+laser, VII: 4T1@Mn-FeMOF@CB+H2O2+laser+DFO; Figure 11 This is a diagram showing the disulfide-induced cell death of 4T1 cells after incubation with different samples in Example 2 of the present invention; Figure 12 This is the in vivo distribution diagram of different preparations injected into the tail vein of mice in Example 3 of the present invention; Figure 13 This is a graph showing changes in tumor size in mice in each treatment group in Example 3 of the present invention; Figure 14 These are images of tumors in mice in each treatment group in Example 3 of the present invention; Figure 15 These are H&E sections of tumor tissue pathology of mice in each treatment group in Example 3 of the present invention; Figure 16 Graph showing liver and kidney indicators for each treatment group in Example 3 of the present invention. DETAILED DESCRIPTION

[0022] Those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the examples were performed under conventional conditions or manufacturer's recommended conditions. Components used without manufacturer's indication are commercially available conventional products.

[0023] Example 1 A method for preparing a nano-bionic bimetallic MOF comprises the following steps: (1) Synthesis of nanobimetallic MOF: Manganese salt (MnCl2·4H2O), iron salt (FeCl3·6H2O), and 2-hydroxyterephthalic acid (H2BDC) were dissolved in N,N-dimethylformamide (DMF) at a mass ratio of 1:3:2.5 and stirred at room temperature until clear to prepare a mixed solution. The mixed solution was transferred to a polytetrafluoroethylene-lined stainless steel reactor and reacted at 100°C for 10 h. After the reaction, the mixture was naturally cooled to room temperature to obtain the product. The product was centrifuged, washed three times with DMF, then washed three times with ethanol, and finally dried in vacuum to obtain the nanobimetallic MOF.

[0024] (2) Loading BAY-876 and photosensitizer CyI: BAY-876, CyI and bimetallic MOF were dispersed in an ethanol solution at a mass ratio of 1:1:100 and stirred at room temperature for 2 h to promote the full encapsulation of BAY-876 and CyI inside the bimetallic MOF. After centrifugation, the solution was washed three times with anhydrous ethanol and vacuum dried to obtain the drug-loaded bimetallic MOF, which was labeled as MFCB.

[0025] (3) Preparation of nano-bionic bimetallic MOF: 4T1 cells were cultured to the logarithmic growth phase, the cells were collected and lysed with cell lysis buffer, and the 4T1 cell membrane was separated and extracted by differential centrifugation. The above-mentioned drug-loaded bimetallic MOF was dispersed in a buffer solution containing 4T1 cell membrane. The 4T1 cell membrane was evenly wrapped on the surface of the bimetallic MOF using a membrane squeezer. Finally, the unbound 4T1 cell membrane fragments were removed by centrifugation and washing to obtain the final nano-bionic bimetallic MOF, which was labeled as 4T1@MFCB.

[0026] Comparative Document 1 The difference from Example 1 is that in step (1), the addition of manganese salt is omitted, and the other preparation methods are the same as Example 1. The prepared MOF is labeled as FeMOF, and the prepared final product is labeled as 4T1@FCB.

[0027] Comparative Example 2 The difference from Example 1 is that in step (1), manganese salt (MnCl2·4H2O), iron salt (FeCl3·6H2O) and 2-hydroxyterephthalic acid (H2BDC) are mixed in a mass ratio of 1:1:2.5. The other preparation methods are the same as those in Example 1.

[0028] Compared with Example 1, in Comparative Example 2, the Mn / Fe mass ratio of 1:1 cannot exert Fe dominance (Fe:Mn=3:1) to enhance the ferroptosis effect, nor can it achieve precise targeting and multi-mechanism synergy. Mn serves as an auxiliary function and cannot achieve efficient photodynamic therapy of tumors.

[0029] Comparative Example 3 Different from Example 1, in step (1), 2-hydroxyterephthalic acid (H2BDC) is replaced by terephthalic acid, and manganese salt (MnCl2·4H2O), iron salt (FeCl3·6H2O) and terephthalic acid are mixed in a mass ratio of 1:3:2.5.

[0030] Comparative Example 3: Compared to Example 1, in MOF synthesis, the carboxyl groups of terephthalic acid form a rigid framework. While this structure is more regular, its functionality is limited, lacking space for active site modification. However, the hydroxyl groups in 2-hydroxyterephthalic acid in Example 1 provide additional coordination sites, increasing the diversity of metal nodes and enhancing photocatalytic activity, while also regulating the pore size and stability of the MOF.

[0031] The nano-bionic bimetallic MOF (4T1@MFCB) prepared in Example 1 and the 4T1@FCB prepared in Comparative Example 1 were characterized: The hydrated particle size of nano-bionic bimetallic MOF (4T1@MFCB) was measured using a dynamic light scattering particle size analyzer (NanoZS, Malvern, UK). Figure 1 As shown. Figure 1 It can be seen that the hydrated particle size of 4T1@MFCB is 135.4±2.6nm.

[0032] The morphology of drug-loaded bimetallic MOF (MFCB) and 4T1@MFCB was characterized using transmission electron microscopy (JEOL, JEM-1200EX). Figure 2 and Figure 3 As shown. Figure 3 and Figure 2 By comparison, the MOF without cell membrane modification exhibits a regular crystalline morphology with clear surface lattice stripes. After cell membrane modification, the MOF surface is covered with a continuous or semi-continuous biofilm, and the overall morphology changes from "crystalline particles" to a "core-shell structure."

[0033] The UV-visible-near infrared absorption spectra of BAY-876, CyI, MOF and 4T1@MFCB were scanned at 270 nm and 780 nm using a UV spectrophotometer. Figure 4 As shown. Figure 4It can be seen that the characteristic peaks of BAY-876 (270 nm) and CyI (780 nm) appear simultaneously in the UV-visible-near-infrared absorption spectrum of 4T1@MFCB, which indicates that BAY-876 and CyI are successfully loaded into MOF without changing their properties.

[0034] To evaluate the fluorescence imaging capability of 4T1@MFCB, the fluorescence emission spectra of CyI and 4T1@MFCB in the range of 700-900 nm were scanned using a fluorescence spectrophotometer. Figure 5 As shown. Figure 5 The fluorescence spectra of CyI and 4T1@MFCB showed that both CyI and 4T1@MFCB had maximum fluorescence intensities at 810 nm, indicating that the bimetallic MOF containing CyI can be excited in the near-infrared region and can be used for real-time monitoring and imaging in vivo.

[0035] To evaluate the photothermal effect of 4T1@MFCB in vitro, PBS, MOF, CyI, and 4T1@MFCB solutions were prepared and placed in EP tubes. Laser (808 nm, 0.96 W / cm 2 ) for 5 min, and use a thermocouple thermometer to measure the temperature in the well every 30 s and record the results. Figure 6 As shown. Figure 6 It can be seen that after 5 minutes of laser irradiation, the temperature of CyI and 4T1@MFCB has rapidly increased to above 52°C. In contrast, after 5 minutes of laser irradiation, the temperature of PBS and MOF has almost no increase, indicating that PBS and MOF have no photothermal effect under laser irradiation.

[0036] The photosensitizer CyI generates cytotoxic singlet oxygen ( 1 O2). SOSG fluorescent probe as a 1 O2 fluorescence detection probe, can be used with 1 O2 reacts and produces green fluorescence. Therefore, the fluorescence intensity of SOSG fluorescent probe is used as an indicator to investigate the photodynamic effect of 4T1@MFCB. The results are as follows Figure 7 As shown, Figure 7 *P<0.05, ***P<0.001. Figure 7 It can be seen that under the laser (808 nm, 0.96 W / cm 2 After 5 minutes of irradiation, the fluorescence intensities of 4T1@MFCB+GSH and 4T1@MFCB+H2O2 were significantly higher than those of the other three groups. This indicates that the manganese in 4T1@MFCB not only undergoes a redox reaction with glutathione, reducing singlet oxygen consumption, but also decomposes H2O2, increasing the photodynamic singlet oxygen yield.

[0037] Example 2 The specific steps of in vitro biological evaluation of nano-bionic bimetallic MOF are: 1) Detection of the photodynamic effect of nano-bionic bimetallic MOF; 2) Detection of the oxygen replenishment effect of nano-bionic bimetallic MOF; 3) Detection of ferroptosis effects of nano-bionic bimetallic MOFs; 4) Detection of the disulfide death effect of nano-bionic bimetallic MOF.

[0038] The following is a detailed in vitro biological evaluation of the nano-bionic bimetallic MOF: 1) Photodynamic effect detection of nano-bionic bimetallic MOF: In this example, the DCFH-DA fluorescent probe was used as an evaluation indicator to detect ROS in cells to evaluate the photodynamic effect of 4T1@MFCB. 4T1 cells were incubated with different samples for 6 h, and then DCFH-DA dye was added and incubated for 20 min. The cells were illuminated by NIR laser (808 nm, 0.96 W / cm 2 ) irradiated for 5 minutes, incubated for 20 minutes, then discarded the DCFH-DA staining solution, and stained with DAPI for 10 minutes before imaging using a laser confocal microscope (Ex = 480 nm, Em = 525 nm). 4T1@FeMOF@CB+laser and 4T1@Mn-FeMOF@CB+laser were treated with 1mM sodium dithionite for 12 hours in advance to simulate an oxygen-deficient environment. Figure 8 Shown, scale bar is 20 μm.

[0039] Depend on Figure 8 It can be seen that only a small amount of ROS was generated in the cells of the Control, 4T1@FeMOF@CB and 4T1@Mn-FeMOF@CB groups. 2 After 5 minutes of laser irradiation, distinct green fluorescence was observed in the 4T1@FeMOF@CB+laser and 4T1@Mn-FeMOF@CB+laser groups, demonstrating the excellent photodynamic effect of CyI. For 4T1@FeMOF@CB, the fluorescence intensity decreased significantly in the hypoxic group, whereas for 4T1@Mn-FeMOF@CB, there was no significant difference in fluorescence between the hypoxic and normoxic groups. These results suggest that hypoxia can affect ROS generation during PDT, further confirming that 4T1@Mn-FeMOF@CB overcomes the limitation of hypoxia on ROS generation by catalyzing O₂ through Mn.

[0040] 2) Detection of oxygen replenishment effect of nano-bionic bimetallic MOF: This example uses ROS green TMH2O2Probe and Image-iT TM Green hypoxia reagent was used to detect intracellular H2O2 and hypoxia levels. 4T1 cells were cultured at 1×10 5 The cells were seeded at a density of 100 cells / mL in a confocal microplate. After incubation for 12 hours, the cells in the hypoxia group were treated with 1 mM sodium dithionite for 12 hours to simulate a hypoxic environment. The cells were treated with RPMI 1640, BAY-876, 4T1@MFCB, CyI, 4T1@FCB, and 4T1@MFCB for 6 hours, then the solution was discarded and the cells were washed with PBS. The CyI, 4T1@FCB, and 4T1@MFCB groups were irradiated with an 808 nm laser for 5 minutes, and 5 μL of ROS green was added. TM After incubation with H2O2Probe for 20 min, the cells were observed and photographed using a confocal microscope (Ex=490 nm, Em=514 nm). TM Hypoxia detection was performed with green hypoxia reagent (5 μg / mL) and incubated for 15 min. The cells were observed and photographed under a confocal microscope (Ex = 488 nm, Em = 525 nm). Figure 9 shown.

[0041] Depend on Figure 9 Compared to the control group, the 4T1@MFCB group exhibited weaker green fluorescence, likely due to the 4T1@MFCB consuming intracellular H2O2 through the Fenton reaction while simultaneously catalyzing H2O2 through Mn, further reducing intracellular H2O2 levels. Furthermore, the CyI+laser group exhibited significantly increased red fluorescence, indicating that PDT exacerbates intracellular hypoxia. The 4T1@MFCB+laser group exhibited similar red fluorescence to the control group, confirming that 4T1@MFCB, through Mn-catalyzed H2O2 production, alleviates the hypoxic environment in tumor cells.

[0042] 3) Detection of ferroptosis effect of nano-bionic bimetallic MOF: In this example, immunofluorescence staining was used to detect the expression level of GPX4 in cells. GPX4 is the fourth member of the glutathione peroxidase (CPX4) family containing selenium and has the ability to scavenge lipid peroxides (LPO). GSH is a cofactor for CPX4 to catalyze the production of lipoic acid from LPO. Lack of GSH will lead to cysteine ​​deficiency, causing CPX4 inactivation and the occurrence of ferroptosis. Therefore, CPX4, as a core regulator of ferroptosis, is considered a "star molecule" in ferroptosis research. 4T1 cells were cultured at 1×10 5The cells were inoculated at a density of 1000 / mL in confocal culture dishes. After incubation for 12 h, the original culture medium was removed and RPMI 1640, 4T1@Mn-FeMOF@CB, BAY-876, and 4T1@Mn-FeMOF@CB + H2O2 were added to each dish for 6 h. The laser group was irradiated with 808 nm, 0.96 W / cm 2 Laser irradiation for 5 minutes. Discard the original culture medium, fix with 4% paraformaldehyde for 20 minutes, wash with PBS, add CPX4 antibody, incubate at room temperature for 1 hour, discard the antibody, wash with PBS, add FITC-labeled secondary antibody to the dish, incubate for 1 hour, and then perform DAPI staining. Observe and take pictures with laser confocal microscope. The experimental results are shown in Figure 2. Figure 10 shown.

[0043] Depend on Figure 10 Compared with the control group, the 4T1@Mn-FeMOF@CB group exhibited weak green fluorescence, indicating reduced GPX4 activity. This suggests that PDT and the Fenton reaction can reduce intracellular GSH, leading to reduced GPX4 activity. Simultaneously, the fluorescence intensity in the BAY-876 group decreased slightly, indicating that inhibition of glucose transporter 1 (GLUT1) limits glycolysis, impacting cellular energy metabolism and triggering a series of cellular stress responses, leading to ferroptosis. Compared with the other groups, the 4T1@Mn-FeMOF@CB+H2O2+laser group exhibited the lowest fluorescence, indicating that the triple effects of BAY-876, PDT, and the Fenton reaction enhance intracellular ferroptosis. Adding deferoxamine (DFO) to reduce iron levels inhibits the Fenton reaction, affecting intracellular GSH levels and, in turn, increasing CPX4 activity.

[0044] 4) Detection of disulfide death effect of nano-bionic bimetallic MOF: In this example, the glucose transporter GLUT1 inhibitor BAY-876 can effectively inhibit the glucose uptake of cells, thereby causing a large amount of accumulation of disulfide bonds, resulting in abnormal cross-linking of disulfide bonds between actin and cytoskeletal proteins, cytoskeleton contraction, and ultimately leading to the collapse of the actin network and cell death. Immunofluorescence is used to specifically label and visualize intracellular actin fibers (F-actin) to study the structure, function and related pathophysiological processes of cells. The cells were cultured in a confocal culture dish. After the cells grew to an appropriate density, they were fixed with 4% paraformaldehyde for 20 minutes and then washed three times with PBS. The cells were treated with 0.1% Triton X-100 for 15 minutes to permeabilize the cell membrane and facilitate the entry of the dye into the cells. They were then washed with PBS, and F-actin dye was added. The staining was performed according to the concentration and time required by the instructions. The cells were incubated in the dark for 30 minutes and observed and photographed under a confocal microscope. The results are shown in the figure below. Figure 11 shown.

[0045] Depend on Figure 11 As can be seen, in the control group, the cytoskeleton structure composed of actin fibers can be clearly seen, and the cell morphology is normal. However, in the experimental group containing BAY-876, the cells showed morphological changes such as shrinkage and membrane bubbles. The cell edges were no longer smooth, and the overall cell morphology became irregular, confirming the disulfide cell death caused by glucose starvation caused by BAY-876.

[0046] Example 3 The in vivo activity evaluation of nano-bionic bimetallic MOF is carried out in the following steps: 1) Construction of a subcutaneous breast cancer model in BALB / c mice; 2) Distribution studies in mice; 3) Investigation of in vivo therapeutic effects.

[0047] The in vivo activity of the nano-bionic bimetallic MOF is evaluated through a specific detection process: 1) Construction of a subcutaneous breast cancer model in BALB / c mice: In this example, a subcutaneous ectopic breast cancer model was established in BALB / c mice. 4T1 tumor cells were placed in a centrifuge tube and centrifuged at 1200 rpm for 5 min. After washing three times with sterile saline, the cells were diluted with sterile saline to a density of 1×10 7 200 μL of cell suspension was inoculated into the right axilla of BALB / c mice. The right axilla of the mice was observed for 2-3 days. If a clear bulge appeared, it indicated that the tumor was successfully implanted. When the subcutaneous tumor volume of the mouse reached about 100-150 mm 3 Then, subsequent experiments can be carried out.

[0048] 2) Distribution experiment in mice: In this example, the successfully modeled mice were randomly divided into CyI group, MFCB group and 4T1@MFCB group. After injection of different preparations through the tail vein, imaging analysis was performed using a small animal in vivo imaging device at predetermined time points. The results are shown in the figure below. Figure 12 As shown. Figure 12 It can be seen that compared with the free CyI group, obvious fluorescence signals can be observed in the MFCB group and the 4T1@MFCB group within a short period of time, and the fluorescence of 4T1@MFCB in the tumor can be maintained for up to 60 hours, indicating that 4T1@MFCB can effectively accumulate in the inflammatory site of the foot, which is due to the homologous targeting of the 4T1 cell membrane to the tumor site.

[0049] 3) In vivo therapeutic effect investigation: In this example, the successfully modeled mice were randomly divided into the Saline group, the BAY-876 group, the 4T1@MFCB group, the 4T1@FCB+laser group, the MFCB+laser group, and the 4T1@MFCB+laser group, with 5 mice in each group. The first dose was administered on the 8th day after modeling, and the treatment was continued every 7 days for a total of three treatments. The tumor volume of the mice was continuously observed and measured during the treatment cycle. The results are shown in the figure below. Figure 13 As shown. Figure 13 Compared with the saline group, tumor growth was significantly inhibited in the 4T1@MFCB, 4T1@FCB+laser, MFCB+laser, and 4T1@MFCB+laser groups. The tumor inhibition effect of 4T1@MFCB+laser was superior to that of MFCB+laser, attributed to the active targeting effect of 4T1. Compared with the 4T1@FCB+laser group, the 4T1@MFCB+laser group demonstrated superior tumor inhibition, attributed to the enhanced PDT efficiency of Mn. The tumor growth curves confirm that the combined PDT / ferroptosis / disulfide apoptosis therapy has the best tumor inhibition effect in tumor-bearing mice. Figure 14 The following are photos of the tumor tissues of mice in each group after treatment. Figure 13 Tumor growth curve Figure 1 This further demonstrated that the 4T1@MFCB+laser group had a good tumor inhibitory effect on tumor-bearing mice.

[0050] This example also further evaluated the therapeutic effect of 4T1@MFCB on tumor-bearing mice from a histological perspective through H&E staining sections. The results are as follows: Figure 15 As shown. Figure 15 The Saline group showed no significant tissue damage, while the 4T1@MFCB+laser group showed significant nuclear shrinkage and nuclear lysis. H&E staining further confirmed the anti-tumor effect of the 4T1@MFCB+laser group.

[0051] Compared with other organs, 4T1@MFCB accumulates more in the liver and kidneys. Therefore, this example also observes the safety of 4T1@MFCB in vivo. Blood was collected from the orbits of BALB / c mice in the Saline group and the 4T1@MFCB+laser group. The blood was kept at 4°C overnight and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and processed, and then an automatic biochemical analyzer was used to detect liver and kidney function, including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and blood creatinine (CREA). The experimental results are as follows. Figure 16 As shown. Figure 16 It can be seen that there was no significant difference between the two groups, and the values ​​were all within the normal range of healthy mouse parameters, indicating that 4T1@MFCB did not cause liver and kidney burden or induce liver and kidney toxicity.

[0052] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A nano-bionic bimetallic MOF, characterized in that: The invention has a core-shell structure, comprising a core and a cell membrane coated on the surface of the core; the core is a nano bimetallic MOF, and the interior of the nano bimetallic MOF contains a glucose transport inhibitor and a photosensitizer.

2. The nano-bionic bimetallic MOF according to claim 1, characterized in that The mass ratio of the nano bimetallic MOF, glucose transport inhibitor and photosensitizer is 100: (0.5-1.5): (0.5-1.5); Preferably, the mass ratio of the nano bimetallic MOF, the glucose transport inhibitor and the photosensitizer is 100:1:

1.

3. The nano-bionic bimetallic MOF according to claim 1, characterized in that The cell membrane is a 4T1 cell membrane; The glucose transport inhibitor is one or more of BAY-876, S961 and quercetin; The photosensitizer is one or more of CyI, porfimer sodium and verteporfin; Preferably, the glucose transport inhibitor is BAY-876; and the photosensitizer is CyI.

4. The nano-bionic bimetallic MOF according to claim 1, characterized in that The nano bimetallic MOF is a Mn-Fe bimetallic MOF; the mass ratio of Mn to Fe in the Mn-Fe bimetallic MOF is 1:(2-4); the particle size of the nano biomimetic bimetallic MOF is 100-200 nm; Preferably, the mass ratio of Mn to Fe in the Mn-Fe bimetallic MOF is 1:

3.

5. A method for preparing the nano-bionic bimetallic MOF according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Mixing manganese salt, iron salt and 2-hydroxyterephthalic acid and dissolving them in N,N-dimethylformamide to prepare a mixed solution; subjecting the mixed solution to a solvothermal reaction to prepare a nano bimetallic MOF; (2) dissolving the nano bimetallic MOF, glucose transport inhibitor, and photosensitizer in an ethanol solution and stirring and mixing, and then centrifuging, washing, and drying to obtain the drug-loaded bimetallic MOF; (3) The drug-loaded bimetallic MOF is dispersed in a buffer solution containing cell membranes, and the cell membranes are wrapped on the surface of the drug-loaded bimetallic MOF using a membrane squeezer. After centrifugation and washing, the nano-bionic bimetallic MOF is obtained.

6. The method for preparing the nano-bionic bimetallic MOF according to claim 5, characterized in that: In step (1), the mixing mass ratio of the manganese salt, the iron salt and the 2-hydroxyterephthalic acid is 1:(2-4):(2-3); The temperature of the solvent thermal reaction is 95-110°C and the reaction time is 8-12h; Preferably, the mixing mass ratio of manganese salt, iron salt and 2-hydroxyterephthalic acid is 1:3:2.5; The temperature of the solvent thermal reaction is 100° C., and the reaction time is 10 h.

7. The method for preparing the nano-bionic bimetallic MOF according to claim 5, characterized in that: In step (1), the manganese salt is one of manganese chloride, manganese nitrate and manganese sulfate; The iron salt is one of ferric chloride, ferric nitrate and ferric sulfate; After the solvent thermal reaction, a post-treatment is further performed, wherein the post-treatment is sequentially centrifuged, washed and vacuum dried, and the washing is sequentially washed with N, N-dimethylformamide and ethanol; Preferably, the manganese salt is manganese chloride, and the iron salt is ferric chloride.

8. The method for preparing the nano-bionic bimetallic MOF according to claim 5, characterized in that: In step (2), the mass ratio of the nano bimetallic MOF, glucose transport inhibitor and photosensitizer is 100: (0.5-1.5): (0.5-1.5); The glucose transport inhibitor is one or more of BAY-876, S961 and quercetin; The photosensitizer is one or more of CyI, porfimer sodium and verteporfin; The stirring is performed at room temperature for 1-3 hours; Preferably, the mass ratio of the nano bimetallic MOF, glucose transport inhibitor and photosensitizer is 100:1:1; The glucose transport inhibitor is BAY-876; the photosensitizer is CyI; The stirring was carried out at room temperature for 2 h.

9. Use of the nano-bionic bimetallic MOF according to any one of claims 1 to 4 in preparing a drug for photodynamic immunotherapy of breast cancer.

10. A drug for photodynamic immunotherapy of breast cancer, characterized in that: The invention comprises the nano-bionic bimetallic MOF and a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and / or at least one pharmaceutically acceptable excipient and / or carrier.

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