Tumor-targeted metal organic framework nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a tumor-targeted metal organic framework nano-drug delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, drugs are encapsulated in double-layer metal organic framework nanoparticles with a core-shell structure; the bacterial outer membrane vesicles are modified on the surfaces of the nanoparticles to form a nano-drug delivery system; the core layer material of the double-layer metal organic framework nanoparticles is MOF801, and the shell layer material of the double-layer metal organic framework nanoparticles is MOF199. The nano-drug delivery system can actively target a tumor site after being injected in a mouse tumor model through caudal vein, the double-layer metal organic framework structure is degraded under the co-stimulation of ATP and H < + > in a tumor microenvironment, and drugs are released to kill tumor cells. Meanwhile, Cu < 2 + > released by degradation can also catalyze a Fenton-like reaction to generate hydroxyl radicals so as to further kill tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a tumor-targeting metal-organic framework nanomedicine delivery system, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is the second leading cause of cancer-related deaths worldwide, and its incidence ranks third among all cancer types. Chemotherapy remains the primary treatment in first-line clinical care, with platinum-based drugs such as oxaliplatin and cisplatin demonstrating clear and significant therapeutic effects in colorectal cancer. However, chemotherapy drugs suffer from non-specific delivery and short retention times in the body, resulting in significant toxic side effects on normal organs and limiting their therapeutic efficacy. Therefore, there is an urgent need to develop new strategies for targeted tumor delivery and controlled release of chemotherapy drugs, effectively killing cancer cells while avoiding toxic side effects on normal tissues.

[0003] Metal-organic frameworks (MOFs) are a class of porous materials formed by the self-assembly of metal ions or metal clusters with organic ligands. As novel drug nanodelivery carriers, they exhibit unique advantages in the biomedical field. MOFs possess tunable nanopore sizes, high specific surface areas, and high porosity, effectively improving drug loading efficiency. By rationally selecting organic ligands, they can also be endowed with the ability to release drugs in response to stimuli, enabling controlled drug release at tumor sites under endogenous signals such as acid, glutathione, and ATP, or exogenous excitation such as light and ultrasound. Furthermore, the inherent metal components in MOFs not only serve as nanocarriers but can also achieve therapeutic functions by releasing metal ions. However, traditional MOF-based nanodelivery systems, relying on passive targeting through enhanced penetration and retention (EPR) effects, typically exhibit low enrichment efficiency in solid tumors.

[0004] Therefore, it is particularly urgent to develop multifunctional nanodelivery systems that combine active targeting capabilities with good biocompatibility. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a tumor-targeting metal-organic framework nanomedicine delivery system, its preparation method, and its applications.

[0006] This invention utilizes the outer membrane vesicles of *Fusobacterium nucleatum* to achieve active targeting of colorectal tumors, promoting the accumulation of the nanodelivery system in the tumor region. After entering tumor cells, the constructed nanodrug delivery system will... + Degradation occurs under dual stimulation of ATP, releasing chemotherapy drugs and Cu. 2+This allows for synergistic treatment with chemotherapy and chemokinetics, enhancing the killing effect on colorectal tumor cells.

[0007] Technical solution: The objective of this invention is achieved through the following technical solution: This invention provides a tumor-targeting metal-organic framework nanomedicine delivery system, which encapsulates drugs in bilayer metal-organic framework nanoparticles with a core-shell structure; and modifies the surface of the nanoparticles with bacterial outer membrane vesicles to form the nanomedicine delivery system.

[0008] This invention constructs core-shell structured metal-organic framework nanoparticles, which can be deployed in H... + Controlled drug release from the tumor site is achieved through dual stimulation of ATP.

[0009] Preferably, the core material of the bilayer metal-organic framework nanoparticle is MOF801, and the shell material is MOF199.

[0010] Preferably, the bacterial outer membrane vesicles are nucleated Fusobacterium outer membrane vesicles (OMVs).

[0011] Preferably, the drug is any one or more of cisplatin (CDDP), oxaliplatin, paclitaxel, or doxorubicin.

[0012] The present invention also provides a method for preparing the above-mentioned nanomedicine delivery system, comprising the following steps: (1) Preparation of core-shell bilayer metal-organic framework nanoparticles Fumaric acid, zirconium oxychloride octahydrate, and the drug were dissolved in an aqueous acetic acid solution, heated and stirred, and after the reaction was cooled to room temperature. The zirconium-based MOF encapsulating the drug was collected by centrifugation. The zirconium-based MOF was dispersed in an ethanol-water mixture, copper sulfate solution was added, stirred, and centrifuged. The MOF was redispersed in an ethanol-water mixture, and triethylamine pyromellitic acid solution was added, stirred, and centrifuged. The above steps were repeated 5-8 times. Finally, the obtained solid was washed and dried to obtain the core-shell structured bilayer metal-organic framework nanoparticles. (2) Preparation of nanomedicine delivery system The nanoparticles obtained in step (1) are combined with bacterial outer membrane vesicles by co-extrusion, centrifuged, washed, and dried to obtain the tumor-targeting metal-organic framework nanomedicine delivery system.

[0013] Preferably, in step (1), the mass ratio of fumaric acid, zirconium oxychloride octahydrate and the drug is 8~9:24~25:1.

[0014] More preferably, the mass ratio of the trans-butenedioic acid, zirconium oxychloride octahydrate, and the drug is 8.7:24.2:1.

[0015] Preferably, in step (1), the volume ratio of acetic acid to water in the acetic acid aqueous solution is 3:7.

[0016] Preferably, in step (1), the mass ratio of zirconium-based MOF, copper sulfate and triethylamine pyromellitic acid salt is 1:2~3:4~6.

[0017] More preferably, the mass ratio of the zirconium-based MOF, copper sulfate, and triethylamine pyromellitic acid salt is 1:3:6.

[0018] Preferably, in step (1), the heating and stirring temperature is 50~100℃ and the reaction time is 20~24h.

[0019] Preferably, in step (1), the volume ratio of ethanol to water in the ethanol-water mixed solution is 1:1.

[0020] Preferably, in step (1), after adding copper sulfate solution or triethylamine pyromellitic acid solution, the stirring time is 30-40 minutes each time, and the process is repeated 5-8 times.

[0021] In a preferred embodiment of the present invention, in step (1), cisplatin, fumaric acid, and zirconium oxychloride octahydrate are dissolved in an aqueous acetic acid solution, heated to 60°C and stirred for 24 hours, and centrifuged to obtain zirconium-based MOF (i.e., CM8); CM8 is redispersed in an ethanol-water mixture, copper sulfate solution is added and stirred for 30 minutes, centrifuged and redispersed in an ethanol-water mixture, then triethylamine pyromellitic acid salt solution is added and stirred for 30 minutes and centrifuged again, repeated 5 times, and the obtained solid is washed and dried to obtain CM8-1 metal-organic framework nanoparticles with a core-shell structure loaded with cisplatin.

[0022] Preferably, in step (2), the mass ratio of the core-shell bilayer metal-organic framework nanoparticles to the bacterial outer membrane vesicles is 4~5:1.

[0023] In a preferred embodiment of the present invention, in step (2), nanoparticles CM8-1 and bacterial outer membrane vesicles are mixed in a phosphate buffer solution at a mass ratio of 5:1 in pH 7.4, and a nanodrug delivery system CM8-1@OMVs is formed by co-extrusion.

[0024] This invention also provides the application of the above-mentioned tumor-targeting metal-organic framework nanomedicine delivery system in the preparation of products that inhibit tumor growth and recurrence.

[0025] The core-shell bilayer metal-organic framework material of this invention can degrade in response to low pH and high ATP conditions at tumor sites, releasing Cu. 2+ And cisplatin, the released cisplatin can inhibit tumor cell replication and promote its apoptosis, while Cu 2+It can catalyze a Fenton-like reaction to induce tumor cell death and enhance anti-cancer efficiency.

[0026] The tumor-killing mechanism of copper-azosalicylic acid nanosheets based on the catalytic Fenton reaction: . Beneficial effects

[0027] (1) This invention modifies the surface of core-shell bilayer metal-organic framework nanoparticles encapsulating chemotherapeutic drugs with Fusobacterium nucleatum outer membrane vesicles to prepare an active tumor-targeting dual-response nanodrug delivery system. The aim is to improve the targeted delivery efficiency of chemotherapeutic drugs to tumor sites and, combined with copper ion-mediated chemokinetics, achieve synergistic antitumor therapy. First, by utilizing the active targeting of Fusobacterium nucleatum outer membrane vesicles to colorectal tumors, the nanosystem is enriched in the tumor region. After reaching the tumor site, the nanodrug delivery system will... + Degradation occurs under dual stimulation of ATP, releasing chemotherapy drugs and Cu. 2+ In this process, chemotherapy drugs are efficiently concentrated at the tumor site, killing tumor cells. Simultaneously, the released copper ions react with intracellular H₂O₂ to generate hydroxyl radicals (·OH), further killing tumor cells and enhancing the anti-tumor therapeutic effect. This nanomedicine delivery system not only achieves specific delivery and controlled release of chemotherapy drugs at the tumor site but also realizes synergistic chemokinetic therapy through copper ions, providing a new strategy for combined immunotherapy of colorectal cancer.

[0028] (2) The nanomedicine delivery system provided by the present invention only responds to degradation at the tumor site to exert a therapeutic effect, without causing toxic side effects to normal tissues, and without exogenous stimulation limitation, which greatly improves the safety of chemotherapy drugs. Attached Figure Description

[0029] Figure 1 Transmission electron microscopy results of CM8 nanoparticles prepared in Example 1 ( Figure 1 a) and particle size distribution ( Figure 1 b) Figure 2 Transmission electron microscopy results of CM8-1 nanoparticles prepared in Example 1 ( Figure 2 a) and particle size distribution ( Figure 2 b) Figure 3 X-ray diffraction pattern of CM8-1 nanoparticles prepared in Example 1; Figure 4 Elemental mapping of the energy-dispersive X-ray spectra of CM8-1 nanoparticles prepared in Example 1 (Cu, Zr, C, Pt, O); Figure 5X-ray photoelectron spectrum of CM8-1 nanoparticles prepared in Example 1; Figure 6 The transmission electron microscope results are for the CM8-1@OMVs nanodelivery system prepared in Example 1; Figure 7 The drug release curve of CM8-1@OMVs prepared in Example 1 in a simulated tumor microenvironment; Figure 8 Electron paramagnetic resonance spectra of CM8-1@OMVs prepared in Example 1 generating reactive oxygen species (hydroxyl radicals) in a simulated tumor microenvironment; Figure 9 The results of the cytotoxicity assessment of CM8-1@OMVs prepared in Example 1 on mouse fibroblasts; Figure 10 The image shows the killing effect of CM8-1@OMVs prepared in Example 1 on tumor cells; Figure 11 A comparison of the cell uptake effects of CM8-1@OMVs and CM8-1@BSA prepared in Example 1; Figure 12 The CM8-1@OMVs prepared for Example 1 were used to generate hydroxyl radicals at the cellular level, enabling chemokinetic therapy (scale bar: 100 μm). Detailed Implementation

[0030] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0032] The nucleated Fusobacterium outer membrane vesicles used in the embodiments and comparative examples of this invention were obtained by the following method: Fusobacterium nucleatum (strain number: BNCC311817, purchased from Shanghai Jitai Biotechnology Co., Ltd.) was inoculated into a suitable anaerobic medium. The complete composition of this liquid thioglycolate medium included: 15.0 g / L casein (trypsin hydrolysate), 5.0 g / L yeast extract, 5.0 g / L glucose, 0.5 g / L sodium thioglycolate, 0.5 g / L L-cysteine, 2.5 g / L sodium chloride, 0.001 g / L resazurin, and 0.75 g / L agar, with a pH of 7.1 ± 0.2 (25℃). This medium lowers the redox potential through sodium thioglycolate and L-cysteine, supporting the growth of aerobic, anaerobic, and microaerobic bacteria, while resazurin indicates the presence of oxygen (pink). After the strain was cultured to the logarithmic growth phase under anaerobic conditions at 37℃, the culture medium was collected and centrifuged at 8000×g for 10 min at 4℃ to remove bacterial cells and large cell debris. The supernatant was then centrifuged at 12000×g for 30 min to further remove impurities. Subsequently, the supernatant was concentrated by ultrafiltration through a 100kDa ultrafiltration tube at 4℃. The concentrate was spread on 40% sucrose and centrifuged at 100000×g for 4 h. The outer membrane vesicle bands of Fusobacterium nucleatum corresponding to the density layer were collected. After repeated washing with pre-cooled PBS, the bands were centrifuged again at 100000×g for 2 h. The supernatant was discarded, and the precipitate was resuspended in sterile PBS and filtered through a 0.22μm filter membrane for sterilization. Finally, the obtained outer membrane vesicles were aliquoted and stored at -80℃ for later use.

[0033] Example 1: A tumor-targeting nanomedicine delivery system based on a metal-organic framework structure (1) Preparation of core-shell structured bilayer metal-organic framework nanoparticles Fumaric acid (C4H4O4, 87 mg), cisplatin (PtCl2(NH3)2, 10 mg), and zirconium oxychloride octahydrate (ZrOCl2·8H2O, 242 mg) were mixed in 10 mL of 30% (v / v) aqueous acetic acid solution, heated to 60 °C, and stirred continuously for 24 hours. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 10 min) to obtain product CDDP@MOF-801 (denoted as CM8); the product was then washed twice with deionized water and centrifuged to obtain a solid.

[0034] Next, CM8 was dissolved in 10 mL of 50% ethanol aqueous solution, and 1 mL of 0.1 M copper sulfate solution was added. After stirring for 30 min, the mixture was centrifuged. The solution was then dispersed and dissolved again in 10 mL of a 1:1 ethanol-water mixture, and 1 mL of 0.1 M triethylamine pyromellitic acid solution was added. After stirring for 30 min, the mixture was centrifuged. This process was repeated 5 times, adding either copper sulfate solution or triethylamine pyromellitic acid solution and stirring for 30 min each time. The resulting solid was washed and dried to obtain CDDP@MOF801@MOF199, designated as CM8-1, for subsequent use.

[0035] (2) Preparation of a nanomedicine delivery system modified with bacterial outer membrane vesicles 2 mg of Fusobacterium nucleatum outer membrane vesicles and 10 mg of CM8-1 prepared according to step (1) were dispersed in 10 mL of phosphate buffer solution with pH 7.4; the mixed solution was repeatedly co-extruded using a micro extruder AVESTINLF-1, and then washed three times with deionized water to finally form a bacterial outer membrane vesicle modified nanodrug delivery system CM8-1@OMVs.

[0036] The above-mentioned nanoparticles CM8 were examined by transmission electron microscopy, and the results are as follows: Figure 1 As shown, the constructed CM8 nanoparticles exhibit a spherical morphology with a size of approximately 87.78 ± 0.68 nm and good dispersibility.

[0037] The above-mentioned nanoparticles CM8-1 were examined by transmission electron microscopy, and the results are as follows: Figure 2 As shown, the constructed CM8-1 nanoparticles have a smoother and flatter surface than CM8, and their size is also increased to approximately 95±0.51 nm.

[0038] X-ray diffraction tests were performed on the above-mentioned nanoparticles CM8-1, and the results are as follows: Figure 3 As shown, CM8-1 has characteristic diffraction peaks that match those of MOF-801 and MOF-199, indicating the successful construction of the MOF@MOF bilayer structure.

[0039] Elemental mapping (Cu, Zr, Pt, C, N) experiments using energy-dispersive X-ray spectroscopy were performed on the aforementioned nanoparticles CM8-1. The results showed that Cu, Zr, and Pt were uniformly distributed within the nanoparticles, indicating the successful construction of core-shell metal-organic framework nanoparticles loaded with cisplatin. The results are as follows: Figure 4 As shown.

[0040] The chemical composition of the above-mentioned nanoparticles CM8-1 was further analyzed by X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 5As shown, the Cu 2p spectrum is decomposed into multiple peaks, among which the main peak at 934.7 eV and the satellite peaks at 940.6 eV and 944.5 eV can be attributed to Cu(II) 2p. 3 / 2 The characteristic binding energy peak at 933 eV corresponds to Cu(I) 2p 3 / 2 This indicates that both monovalent and divalent copper ions exist in CM8-1.

[0041] The above-mentioned nanomedicine delivery system CM8-1@OMVs was examined by transmission electron microscopy, and the results are as follows: Figure 6 As shown, the presence of bacterial outer membrane vesicles was observed.

[0042] Drug release was assessed using the aforementioned nanomedicine delivery system CM8-1@OMVs. CM8-1@OMVs were dissolved in phosphate buffer solutions at different concentrations (1 mg / mL): pH 7.4, pH 7.4 + ATP (1 mM), pH 6.0, and pH 6.0 + ATP (1 mM). Results are as follows: Figure 7 As shown, only under pH 6.0 + ATP conditions can the release rate of cisplatin reach 80% within 12 hours and be completely released in about 48 hours, thus achieving controllable chemotherapy drug release.

[0043] The ability of the aforementioned nanomedicine delivery system CM8-1@OMVs to generate hydroxyl radicals was verified by electron paramagnetic resonance spectroscopy. First, a phosphate buffer solution containing 100 μM H₂O₂ at pH 6.0 was prepared to simulate the tumor microenvironment. Then, groups were set up, specifically: a group simulating the tumor microenvironment (H₂O₂). + (pH 6.0) + H2O2 (100μM) group, material group (CM8-1@OMVs) and final group (CM8-1@OMVs + H + (pH 6.0) + H₂O₂ (100 μM)). Results are as follows: Figure 8 As shown, CM8-1@OMVs can generate hydroxyl radicals under tumor microenvironment conditions, thereby achieving chemokinetic therapy.

[0044] Comparative Example 1: Preparation of CM8@OMVs (1) Preparation of zirconium-based metal-organic framework nanoparticles CM8 loaded with cisplatin Fumaric acid (C4H4O4, 87 mg), cisplatin (PtCl2(NH3)2, 10 mg), and zirconium oxychloride octahydrate (ZrOCl2·8H2O, 242 mg) were mixed in 10 mL of 30% (v / v) acetic acid solution, heated to 60 °C, and stirred continuously for 24 hours. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 10 min), the product was collected, washed three times with deionized water, and dried under vacuum to obtain CDDP@MOF-801 (denoted as CM8) for subsequent use.

[0045] (2) Preparation of CM8@OMVs 2 mg of outer membrane vesicles of Fusobacterium nucleatum and 10 mg of CM8 prepared according to step (1) were dispersed in 10 ml of phosphate buffer solution at pH 7.4; the mixture was repeatedly co-extruded using a micro extruder AVESTINLF-1, and then washed three times with deionized water to obtain CM8@OMVs.

[0046] Comparative Example 2: Preparation of M8-1@OMVs (1) Preparation of core-shell structured metal-organic framework nanoparticles M8-1@OMVs Fumaric acid (C4H4O4, 87 mg) and zirconium oxychloride octahydrate (ZrOCl2·8H2O, 242 mg) were mixed in 10 mL of 30% (v / v) acetic acid solution, heated to 60 °C and stirred continuously for 24 hours. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 10 min), the product was collected, washed three times with deionized water, and dried under vacuum to obtain MOF-801.

[0047] Next, MOF801 was dissolved in 10 ml of 50% ethanol aqueous solution, and 1 ml of 0.1 M copper sulfate solution was added. After stirring for 30 min, the mixture was centrifuged. The solution was then dispersed and dissolved again in 10 ml of a 1:1 ethanol-water mixture, and 1 ml of 0.1 M triethylamine pyromellitic acid solution was added. After stirring for 30 min, the mixture was centrifuged. Copper sulfate solution or triethylamine pyromellitic acid solution was added, and the mixture was stirred for 30 min each time before centrifugation. This process was repeated for 5 cycles. The resulting solid was washed and dried to obtain MOF801@MOF199, designated M8-1, for subsequent use.

[0048] (2) Preparation of M8-1@OMVs 2 mg of outer membrane vesicles of Fusobacterium nucleatum and 10 mg of M8-1 prepared according to step (1) were dispersed in 10 ml of phosphate buffer solution at pH 7.4; the mixture was repeatedly co-extruded using a micro extruder AVESTINLF-1, and then washed three times with deionized water to obtain M8-1@OMVs.

[0049] Preparation of Comparative Example 3 CM8-1@BSA Fumaric acid (C4H4O4, 87 mg), cisplatin (PtCl2(NH3)2, 10 mg), and zirconium oxychloride octahydrate (ZrOCl2·8H2O, 242 mg) were mixed in 10 mL of 30% (v / v) acetic acid solution, heated to 60 °C, and stirred continuously for 24 hours. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 10 min) to obtain the product CDDP@MOF-801 (denoted as CM8); the product was then washed twice with deionized water and centrifuged to obtain a solid.

[0050] Next, CM8 was dissolved in 10 mL of 50% ethanol aqueous solution, and 1 mL of 0.1 M copper sulfate solution was added. After stirring for 30 min, the mixture was centrifuged. The solution was then dispersed and dissolved again in 10 mL of a 1:1 ethanol-water mixture, and 1 mL of 0.1 M triethylamine pyromellitic acid solution was added. After stirring for 30 min, the mixture was centrifuged. This process was repeated 5 times, adding either copper sulfate solution or triethylamine pyromellitic acid solution and stirring for 30 min each time. The resulting solid was washed and dried to obtain CDDP@MOF801@MOF199, designated as CM8-1, for subsequent use.

[0051] 10 mg of CM8-1 was dissolved in 10 mL of ultrapure water and stirred at room temperature. Simultaneously, 5 mL of 1 mg / mL BSA aqueous solution was added dropwise, and stirring continued for 4 hours. The solution was then washed three times with deionized water to obtain CM8-1@BSA.

[0052] Example 2 Mouse fibroblast toxicity test Mouse embryonic fibroblasts (3T3 cells, purchased from Shanghai Yaji Biotechnology) were seeded at a density of 5000 cells / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. Then, different masses of CM8-1@OMVs (the product prepared in Example 1) were added to 96-well plates containing 1640 medium solution to obtain multiple treatment conditions (12.5, 25, 50, 100, 200 μg / mL), and incubated for 24 hours at 37°C and 5% CO2. The wells containing only cells and 1640 medium solution were designated as the Control group (i.e., 0 μg / mL).

[0053] Finally, following the instructions for the CCK-8 reagent kit (Beyotime), CCK-8 was added to the wells of the plate, and cell viability was detected after co-incubation with the material. The results are as follows: Figure 9 As shown, CM8-1@OMVs at a concentration of 200 μg / mL has little effect on the activity of 3T3 cells, demonstrating good biocompatibility.

[0054] Example 3: Detection of Tumor Cell Killing Effect Mouse colorectal cancer cells (CT26 cells, purchased from Shanghai Yaji Biotechnology) were seeded at a density of 5000 cells / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. Then, the original culture medium was removed, and 1640 medium solution with pH 6.5 containing different materials was added to the 96-well plates. The specific materials and concentrations were: free cisplatin (10 μg / mL), CM8@OMVs (12.5, 25, 50, 100, 200 μg / mL, the product obtained in Comparative Example 1), M8-1@OMVs (12.5, 25, 50, 100, 200 μg / mL, the product obtained in Comparative Example 2), and CM8-1@OMVs (12.5, 25, 50, 100, 200 μg / mL, the product obtained in Example 1). The plates were incubated together at 37°C and 5% CO2 for 24 hours. Among them, well plates containing only cells and 1640 culture medium solution at pH 6.5 were set as the Control group.

[0055] Finally, following the instructions for the CCK-8 reagent kit (Beyotime), CCK-8 was added to the wells of the plate, and cell viability was detected after co-incubation with the material. The results are as follows: Figure 10 As shown, this indicates that CM8-1@OMVs has a better tumor cell killing effect.

[0056] Example 4: Validation of Active Targeting Ability of Tumor Cells Five mg each of CM8-1@OMVs and CM8-1@BSA (the product obtained in Comparative Example 3) prepared in Example 1 were dissolved in PBS solution with fluorescein isothiocyanate (FITC) (FITC concentration of 200 μg / mL). The mixture was stirred at room temperature for 2 h, centrifuged to collect the product, and washed once with deionized water to obtain FITC-labeled CM8-1@OMVs and FITC-labeled CM8-1@BSA, which were then stored at 4 °C for later use.

[0057] Mouse colorectal cancer cells CT26 (purchased from Shanghai Yaji Biotechnology) were seeded at a density of 5000 cells / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. Then, the original culture medium was removed, and 1640 medium solutions containing different materials were added to the 96-well plates: FITC-labeled CM8-1@OMVs (100 μg / mL) and FITC-labeled CM8-1@BSA (100 μg / mL). The plates were co-incubated at 37°C and 5% CO2 for 12 hours. Finally, the cells were gently washed twice with PBS to remove excess material, and DAPI dye (Beyotime Biotechnology Co., Ltd.) was added to the wells for staining. The differences in cell uptake between the two groups were observed under a confocal microscope. The results are as follows: Figure 11As shown, this indicates that CM8-1@OMVs has active tumor targeting capabilities and can be better taken up by tumor cells.

[0058] Example 5: Detection of intracellular hydroxyl radical generation in tumor cells Mouse colorectal cancer cells (CT26, purchased from Shanghai Yaji Biotechnology) were seeded at a density of 5000 cells / well in 96-well plates (containing 1640 medium solution at pH 6.5) and cultured for 24 hours at 37°C with 5% CO2. The cultured cells were then divided into the following groups: control group, M8-1@OMVs group (product obtained in Comparative Example 2), and CM8-1@OMVs group (product obtained in Example 1).

[0059] Remove the original culture medium and add 1640 culture medium solution at pH 6.5 containing different materials to 96-well plates: M8-1@OMVs (100 μg / mL) and CM8-1@OMVs (100 μg / mL).

[0060] Control group: Well plates containing only cells and 1640 medium solution at pH 6.5 were set up as the control group.

[0061] After culturing for another 12 hours according to the above groupings, the generation of hydroxyl radicals (•OH) was detected using hydroxyphenyl fluorescein (HPF, 5μM) sold by Beyotime Biotechnology Co., Ltd. The procedure was followed according to the reagent instructions (product number: Y249872). Observation was performed using a confocal microscope; green fluorescence represented a positive signal for the generation of hydroxyl radicals. Results are as follows... Figure 12 As shown, hydroxyl radicals were generated at the cellular level in both the M8-1@OMVs and CM8-1@OMVs groups in a simulated tumor microenvironment (a phosphate buffer solution containing 100 μM H2O2 at pH 6.0 to simulate the tumor microenvironment), demonstrating that the nanoparticles can be used for chemokinetic therapy.

[0062] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A tumor-targeting metal-organic framework nanomedicine delivery system, characterized in that, The drug is encapsulated in a bilayer metal-organic framework nanoparticle with a core-shell structure; bacterial outer membrane vesicles are modified on the surface of the nanoparticle to form the nanodrug delivery system.

2. The nanomedicine delivery system according to claim 1, characterized in that, The core material of the bilayer metal-organic framework nanoparticles is MOF801, and the shell material is MOF199.

3. The nanomedicine delivery system according to claim 1, characterized in that, The bacterial outer membrane vesicles are nucleated Fusobacterium outer membrane vesicles (OMVs).

4. The nanomedicine delivery system according to claim 1, characterized in that, The drug is any one or more of cisplatin (CDDP), oxaliplatin, paclitaxel, or doxorubicin.

5. A method for preparing the nanomedicine delivery system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of core-shell bilayer metal-organic framework nanoparticles Fumaric acid, zirconium oxychloride octahydrate, and the drug were dissolved in an aqueous acetic acid solution, heated and stirred, and after the reaction was cooled to room temperature. The zirconium-based MOF encapsulating the drug was collected by centrifugation. The zirconium-based MOF was dispersed in an ethanol-water mixture, copper sulfate solution was added, stirred, and centrifuged. The MOF was redispersed in an ethanol-water mixture, and triethylamine pyromellitic acid solution was added, stirred, and centrifuged. The above steps were repeated 5-8 times. Finally, the obtained solid was washed and dried to obtain the core-shell structured bilayer metal-organic framework nanoparticles. (2) Preparation of nanomedicine delivery system The nanoparticles obtained in step (1) are combined with bacterial outer membrane vesicles by co-extrusion, centrifuged, washed, and dried to obtain the tumor-targeting metal-organic framework nanomedicine delivery system.

6. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of fumaric acid, zirconium oxychloride octahydrate and the drug is 8~9:24~25:

1.

7. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of zirconium-based MOF, copper sulfate and triethylamine pyromellitic acid salt is 1:2~3:4~6.

8. The preparation method according to claim 5, characterized in that, In step (1), the heating and stirring temperature is 50~100℃ and the reaction time is 20~24h.

9. The preparation method according to claim 5, characterized in that, In step (2), the mass ratio of the core-shell bilayer metal-organic framework nanoparticles to the bacterial outer membrane vesicles is 4~5:

1.

10. The use of the tumor-targeting metal-organic framework nanomedicine delivery system according to any one of claims 1-4 in the preparation of products that inhibit tumor growth and recurrence.

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