Iron-copper bimetallic MOF biomimetic nanoparticles, and preparation method and application thereof

Abstract

The application discloses an iron-copper bimetallic MOF biomimetic nanoparticle and a preparation method and application thereof, the nanoparticle comprises a core layer and a shell layer, and the shell layer is coated on the outer surface of the core layer; the core layer is an iron-copper bimetallic organic framework loaded with cisplatin; and the shell layer is a hybrid membrane formed by fusing a red blood cell membrane and a cancer cell membrane. The application combines the iron-copper bimetallic MOF, cisplatin and the hybrid membrane, the Fe-Cu bimetallic MOF generates ROS and consumes GSH through Fenton / Fenton-like reaction, the sensitization chemotherapy effect is enhanced; the cisplatin induces immunogenic cell death; the red blood cell membrane in the hybrid membrane endows the nanoparticle with immune escape ability, the cancer cell membrane endows the nanoparticle with homologous targeting ability, active homing of the tumor is realized, and therefore the anti-tumor effect of the nanoparticle is improved.

Description

Technical Field

[0001] This invention relates to the fields of nanomedicine and tumor immunotherapy, and in particular to an iron-copper bimetallic MOF biomimetic nanoparticle, its preparation method, and its application. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related death worldwide, and the leading cause of cancer-related death in my country. Current clinical treatments for lung cancer mainly include surgical resection, radiotherapy, chemotherapy, and immunotherapy. In recent years, the development of nanomedicine has provided new opportunities to overcome these treatment obstacles. Among various nanoplatforms, metal-organic frameworks (MOFs) have become ideal candidate carriers for drug delivery systems. In particular, iron-copper bimetallic MOFs have been used by many researchers as nanocarriers for cisplatin, aiming to enhance anti-tumor effects through the combination of chemotherapy and chemokinetics.

[0003] However, unmodified MOF nanoparticles are easily recognized and cleared by the reticuloendothelial system (RES), making it difficult to exert a sustained anti-tumor effect; nanoparticles lack active targeting ability; after cisplatin is loaded onto the MOF carrier, the drug may be released prematurely before reaching the tumor, causing off-target toxicity to normal tissues, resulting in poor anti-tumor effect of the bimetallic MOF-loaded cisplatin system, which restricts its further clinical translation and application. Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide an iron-copper bimetallic MOF biomimetic nanoparticle to address the problems of poor tumor targeting and insufficient immune activation in existing nanodelivery systems, which lead to poor anti-tumor effects. The second objective of this invention is to provide a method for preparing the iron-copper bimetallic MOF biomimetic nanoparticle. The third objective of this invention is to provide applications of the iron-copper bimetallic MOF biomimetic nanoparticle.

[0005] Technical solution: The iron-copper bimetallic MOF biomimetic nanoparticles of the present invention include a core layer and an outer shell layer, wherein the outer shell layer covers the outer surface of the core layer; the core layer is an iron-copper bimetallic organic framework loaded with cisplatin; and the outer shell layer is a hybrid membrane formed by the fusion of red blood cell membrane and cancer cell membrane.

[0006] The iron-copper bimetallic organic framework possesses the ability to consume glutathione (GSH) and catalyze the generation of reactive oxygen species (ROS), achieving chemokinetic therapy through Fenton / Fenton-like reactions. Cisplatin, through DNA damage, generates a spatiotemporally synergistic tumor-killing effect in conjunction with chemokinetic therapy. The erythrocyte membrane endows the nanoparticles with immune escape capabilities, while the lung cancer cell membrane endows the nanoparticles with homologous targeting capabilities to achieve active tumor homing.

[0007] Preferably, in the iron-copper bimetallic organic framework, the molar ratio of iron ions to copper ions is 1 to 2:1. The molar ratio of iron ions to copper ions affects the morphology and antitumor activity of the material. When the molar ratio of iron ions to copper ions is 3:2, it exhibits the most uniform morphology and the most regular crystal structure, resulting in the strongest antitumor effect.

[0008] Preferably, the nanoparticles have a particle size of 100~300 nm.

[0009] Preferably, the cancer cell membrane is a lung cancer cell membrane.

[0010] The preparation method of the iron-copper bimetallic MOF biomimetic nanoparticles of the present invention includes the following steps:

[0011] (1) Synthesis of iron-copper bimetallic organic frameworks;

[0012] (2) The iron-copper bimetallic organic framework prepared in step (1) is added to a solvent to prepare a dispersion. Cisplatin is dissolved in the solvent to obtain a cisplatin solution. Under light-protected conditions, the cisplatin solution is slowly added to the iron-copper bimetallic organic framework dispersion. The mixture is stirred to load cisplatin into the iron-copper bimetallic organic framework. Unloaded free cisplatin is removed to obtain iron-copper bimetallic MOF nanoparticles (MPt-NPs) loaded with cisplatin.

[0013] (3) The red blood cell membrane and the cancer cell membrane are fused to form a hybrid membrane, and the hybrid membrane is wrapped around the outer surface of the iron-copper bimetallic MOF nanoparticles loaded with cisplatin prepared in step (2) to obtain the iron-copper bimetallic MOF biomimetic nanoparticles (RCMPt-NPs).

[0014] Preferably, in step (1), the iron-copper bimetallic organic framework material is synthesized by a hot solvent method.

[0015] Preferably, in step (1), the organic ligand in the synthesized iron-copper bimetallic organic framework material is 1,4-phthalic acid.

[0016] Preferably, in step (1), the iron-copper bimetallic organic framework material is prepared by completely dissolving iron salt, copper salt, and 1,4-phthalic acid in DMF. The mixture is then reacted at 90-120°C for 10-15 hours. The resulting precipitate is washed with water by centrifugation and then freeze-dried to finally obtain iron-copper bimetallic organic framework (MOF) nanoparticles.

[0017] In step (2), cisplatin is loaded onto the iron-copper bimetallic organic framework through coordination or physical adsorption.

[0018] Preferably, in step (3), the method of wrapping the hybridization membrane around the outer surface of the cisplatin-loaded nanoparticles prepared in step (2) is as follows: the cisplatin-loaded nanoparticles are dispersed in a solvent, then mixed with the hybridization membrane, first ultrasonically treated, and finally squeezed through a polycarbonate porous membrane to collect the solid.

[0019] Preferably, the method for fusing the erythrocyte membrane and the cancer cell membrane to form a hybrid membrane is as follows: extract the erythrocyte membrane and the cancer cell membrane separately, then mix the erythrocyte membrane and the cancer cell membrane, and sonicate to obtain the hybrid membrane.

[0020] The application of the iron-copper bimetallic MOF biomimetic nanoparticles described in this invention in the preparation of antitumor drugs.

[0021] Preferably, the tumor is lung cancer.

[0022] Preferably, the nanoparticles are used in combination with immune checkpoint inhibitors.

[0023] Mechanism of Invention: The iron-copper bimetallic MOF biomimetic "Trojan horse" nanoparticles (RCMPt-NPs) constructed in this invention adopt a "core-shell" structure design, with an iron-copper bimetallic organic framework (MPt-NPs) loaded with cisplatin as the active core and a hybrid membrane (RCM) formed by the fusion of erythrocyte membrane and cancer cell membrane as the biomimetic shell. Through the binding of self-markers such as CD47 on the surface of erythrocyte membranes to SIRPα on the surface of macrophages, a "don't eat me" signal is transmitted to inhibit phagocytosis and clearance; simultaneously, homologous adhesion molecules on the surface of cancer cell membranes are utilized to achieve active tumor homing. The hybrid membrane integrates these two functions into the same nanosystem. After the nanoparticles accumulate in tumor tissue and are internalized by tumor cells, the Fe-Cu bimetallic MOF initiates a cascade catalytic reaction: Fe... 2+ and Cu + They catalyze the overexpression of H2O2 in tumor cells to produce highly toxic hydroxyl radicals (•OH), while Fe... 3+ With Cu 2+ Through mutual valence state cycle (Fe) 3+ Cu + Reduced to Fe 2+ Fe 2+ Reduced Cu 2+ Cu + ) Continuous regeneration of catalytically active species significantly enhances the efficiency of the Fenton reaction; in addition, Fe 3+ and Cu 2+ It can oxidize and consume intracellular glutathione (GSH), not only weakening the antioxidant defenses of tumor cells, but also producing Fe, a reduction product. 2+ / Cu +This feedback enhances the Fenton reaction, forming a self-amplifying positive feedback loop of "GSH consumption → metal valence reduction → Fenton enhancement → ROS increase → further GSH consumption," significantly amplifying the oxidative damage effect. Simultaneously, cisplatin is released from the MOF, and through DNA damage, it exerts a spatiotemporally synergistic tumor-killing effect with chemokinetics; the depletion of GSH also interrupts the detoxification and efflux pathways of cisplatin, effectively reversing tumor drug resistance and achieving chemosensitization.

[0024] Building upon the aforementioned direct killing effect, RCMPt-NPs further activate systemic anti-tumor immune responses by multimodal synergistically inducing immunogenic cell death (ICD). This involves recruiting and activating dendritic cells (DCs), promoting their uptake, processing, and cross-presentation of tumor-associated antigens, thereby initiating tumor-specific CD8 activation. + Initial activation of T cells. At this time, combined administration of anti-PD-1 antibodies can block the PD-1 / PD-L1 signaling axis, relieving the immune exhaustion state of T cells and forming a closed loop of "activation first, then enhancement" in immunotherapy. A portion of the activated tumor-specific T cells differentiate into central memory T cells (TCM) and effector memory T cells (TEM), residing long-term in lymphoid organs and peripheral tissues. When tumor recurrence or metastasis occurs, they can rapidly proliferate and eliminate tumor cells, thereby establishing a durable immune memory effect. In summary, this invention achieves multimodal deep synergy between chemotherapy, chemokinetic therapy, and immunotherapy through a four-level cascade effect of targeted accumulation, efficient killing, immune activation, and long-term memory, providing a novel engineering platform for precision immunotherapy of lung cancer and solid tumors.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] (1) Multimodal synergistic anti-tumor effect: This invention integrates cisplatin chemotherapy, iron-copper bimetallic MOF-mediated chemokinetics (CDT) and hybrid membrane biomimetic technology into a nanoplatform. The hybrid membrane (RLCM) can not only significantly prolong the in vivo circulation time, but also enable tumor active homing, increase drug accumulation at the tumor site, generate ROS and consume GSH through Fenton-like reaction, and enhance the effect of chemotherapy. At the same time, cisplatin induces immunogenic cell death (ICD). The three synergistically amplify the anti-tumor immune response.

[0027] (2) Enhanced anti-tumor immune response: While killing tumor cells, the nanoparticles of the present invention release tumor-associated antigens, promote dendritic cell maturation and antigen presentation. When used in combination with anti-PD-1 therapy, they can significantly activate T cell-mediated anti-tumor immune response and establish a lasting immune memory effect, effectively reducing the risk of tumor recurrence and metastasis.

[0028] (3) Good biocompatibility: The present invention uses biologically derived cell membranes for biomimetic camouflage, which has good biocompatibility and reduces immunogenicity and systemic toxicity. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the preparation process and anti-tumor mechanism of the iron-copper bimetallic MOF biomimetic nanoparticles (RLMPt-NPs) of this invention.

[0030] Figure 2 Characterization of metal-organic frameworks (MOFs) with different Fe / Cu molar ratios; (A) Transmission electron microscopy (TEM) images showing the morphology of MOFs with Fe / Cu molar ratios of 1:1 (a), 2:1 (b), and 3:2 (c), scale bar = 200 nm; (B) Production of reactive oxygen species (ROS) in Lewis lung cancer cells after treatment with MOFs with different Fe / Cu molar ratios, scale bar = 50 μm; (C) Live / dead cell staining results of Lewis lung cancer cells after 24 hours of treatment with MOFs with different Fe / Cu molar ratios, using Calcein-AM (green, live cells) and propidium iodide (PI, red, dead cells), scale bar = 50 μm;

[0031] Figure 3 Fluorescence resonance energy transfer (FERT) diagrams of red blood cell membranes and Lewis lung cancer cell membranes at different ratios;

[0032] Figure 4 The images show the in vitro validation of MOF and RLMPt-NPs: (A) Transmission electron microscopy image of MOF; (B) Transmission electron microscopy image of RLMPt-NPs; (C) Average particle size distribution of MOF and RLMPt-NPs; (D) SDS-PAGE protein analysis of membrane-related products obtained during the preparation of RLMPt-NPs (I: erythrocyte membrane, II: tumor cell membrane, III: MOF, IV: RLMPt-NP).

[0033] Figure 5 (A) shows the nitrogen (N2) adsorption-desorption isotherms of MOF and RLMPt-NPs nanoparticles; (B) shows the XRD patterns of MOF and RLMPt-NPs; (C) shows the XPS spectrum of RLMPt-NPs nanoparticles.

[0034] Figure 6 The diagrams show the core function verification of RLMPt-NPs: (A) RLMPt-NPs generating •OH radicals, (B) depletion of GSH, and (C) in vitro release curves of MPt-NPs and RLMPt-NPs releasing free CDDP in PBS solutions at pH 6.5 and 7.4.

[0035] Figure 7 The following are images from in vitro cytotoxicity experiments: (A) Apoptosis of Lewis lung cancer cells after co-culturing with different drugs (I: control group, II: CDDP, III: MOF, IV: MPt-NPs, V: RLMPt-NPs); (B) Apoptosis rate of cells in each group; (C) Survival rate of Lewis lung cancer cells after different treatments.

[0036] Figure 8 (Image showing Lewis lung cancer cells stained with calcein AM (green, live cells) and PI (red, dead cells), scale bar 100 μm;)

[0037] Figure 9 Intracellular uptake of NR-labeled nanoparticles, scale bar 50 μm, Free-NR (free Nile Red), MOF-NR (NR-labeled MOF), RLM@MOF-NR (NR-labeled RLMPt-NPs).

[0038] Figure 10 The diagram shows the in vitro biochemical mechanisms. (A) A flowchart illustrating the mechanism of action of RLMPt-NPs on lung cancer cells from different sources; (B) Fluorescent images of Lewis lung cancer cells stained with DCFH-DA after treatment with various drugs; (C) Fluorescent images of γ-H2AX expression levels in Lewis lung cancer cells after treatment with various drugs; (D) Fluorescent images of mitochondrial activity in Lewis lung cancer cells; Scale bar 50 μm.

[0039] Figure 11 The following diagrams illustrate the in vivo antitumor effects of RLMPt-NPs: (A) Schematic diagram of the treatment regimen; (B) Representative photographs of LLC tumor mice after treatment; (C) Average tumor growth curve, showing the changes in tumor volume in five groups of mice under different treatment regimens; (D) Figure of weight fluctuation during treatment; (E) Survival curve of mice receiving treatment.

[0040] Figure 12 For HE, Ki-67, CD8 + Immunohistochemical and immunofluorescence staining results of T cells and γ-H2AX (scale bar = 50 μm, I: control group, II: anti-PD1, III: CDDP, IV: MPt-NPs, V: RLMPt-NPs, VI: RLMPt-NPs + anti-PD1).

[0041] Figure 13The targeting map of RLMPt-NPs is shown in (A) for fluorescence images of mice after intravenous injection of free ICG, MICG and RLMICG. The right panel shows in vitro fluorescence images of organs and tumors isolated 24 hours after injection (H: heart, Li: liver; S: spleen; Lu: lung, K: kidney, T: tumor). The iron content in tumors (B) and major organs (C) of mice after injection of RLMPt-NPs is also shown.

[0042] Figure 14 A validation diagram of the immune response induced by RLMPt-NPs: (A) Flow cytometry analysis of mature dendritic cells in the tumor; (B) CD8+ in the tumor. + (C) Flow cytometry results of T cells; (I) Flow cytometry analysis of natural killer cells (NK cells) in tumors (I: control group, II: anti-PD1, III: CDDP, IV: MPt-NPs, V: RLMPt-NPs, VI: RLMPt-NPs+anti-PD1);

[0043] Figure 15 The following is a graph illustrating the long-term immune memory effect of RLMPt-NPs combined with anti-PD-1 therapy: (A) Timeline of the tumor rechallenge experiment; (B) Representative tumor images taken after rechallenge; (C) Tumor volume growth curve after rechallenge; (D) CD8+ in tumors after different treatments. + Representative flow cytometry analysis of T cells; (E) CD8 in tumors + (F) Quantitative analysis of T cells; (G) Flow cytometry analysis of representative effector memory T cells (TEM) in tumors after different treatments;

[0044] Figure 16 For in vitro hemolysis analysis, (A) shows photomicrographs of representative erythrocyte cultures containing different drugs (scale bar = 15 μm); a: distilled water (DW, positive control); b: normal saline (NS, negative control); c: metal-organic framework (MOF); d: cisplatin (CDDP); e: platinum nanoparticles (MPt-NPs); f: RLMPt-NPs; (B) shows a photograph of hemolysis; (C) UV-Vis absorption spectra of each group; (D) hemolysis rate of each group;

[0045] Figure 17 This is an in vivo analysis chart of the systemic toxicity of various treatment regimens by detecting biochemical indicators and routine blood parameters;

[0046] Figure 18H&E staining images of major organs (heart, liver, spleen, lung and kidney) of mice after treatment, scale bar = 50 μm (I: control group, II: anti-PD1, III: CDDP, IV: MPt-NPs, V: RLMPt-NPs, VI: RLMPt-NPs+anti-PD1). Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the embodiments.

[0048] Example 1

[0049] Synthesis of iron-copper bimetallic organic frameworks: First, FeCl3·6H2O (1.62 g), CuCl2·2H2O (0.68 g), and 1,4-phthalic acid (1.50 g) were completely dissolved in 70 mL of DMF, and then the mixture was reacted at 100 °C for 12 h. The resulting precipitate was washed with water by centrifugation and then freeze-dried (-80 °C, 72 h) to finally obtain iron-copper bimetallic MOF nanoparticles (iron to copper molar ratio 3:2).

[0050] Example 2

[0051] Based on Example 1, the mass of FeCl3·6H2O was changed to 1.081 g, while other conditions remained unchanged, to obtain iron-copper bimetallic MOF nanoparticles (iron to copper molar ratio 1:1).

[0052] Example 3

[0053] Based on Example 1, the mass of FeCl3·6H2O was changed to 2.162 gg, while other conditions remained unchanged, to obtain iron-copper bimetallic MOF nanoparticles (iron to copper molar ratio 2:1).

[0054] The structures of the iron-copper bimetallic MOF nanoparticles prepared in Examples 1-3 were characterized, and the results are as follows: Figure 2 As shown in (A).

[0055] Figure 2 (A) Transmission electron microscopy (TEM) images show the morphology of iron-copper bimetallic MOF nanoparticles with Fe / Cu molar ratios of 1:1 (a), 2:1 (b), and 3:2 (c). As can be seen from the figure, the MOF with a Fe / Cu molar ratio of 3:2 has a more uniform morphology and a more regular crystal structure, while the MOFs with ratios of 2:1 and 1:1 are slightly inferior in terms of morphological uniformity.

[0056] The antitumor effects of the iron-copper bimetallic MOF nanoparticles prepared in Examples 1-3 were tested.

[0057] ROS generation assay: Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). Lewis lung cancer cells (2x10⁻¹²) were used in this assay. 5 The cells were co-cultured for 24 hours with an equal volume of MOFs prepared in Examples 1-3 at a concentration of 100 μg / ml (prepared with DMEM). After washing three times with PBS, DCFH-DA (1 mL, 10 μmol / L) was added, and the cells were cultured for another 30 minutes. Observation was performed using a fluorescence microscope (OLYMPUS, IX83, Japan). The test results are as follows: Figure 2 As shown in (B).

[0058] Live and dead cell staining: Lewis lung cancer cells (2x10⁻¹²) 5 Cells were seeded into 6-well culture plates, and an equal volume of MOF (prepared in Examples 1-3) prepared with DMEM to a concentration of 100 μg / ml was added and incubated with the cells for 24 hours. Cells were gently washed twice with PBS, and then stained for 30 minutes with Calcein-AM (1 μg / ml) and pyridine iodide (PI) (1 μg / ml) staining reagent. Viable cells were stained green fluorescence, and dead cells were stained red fluorescence. Cells were washed three times with PBS, and the cell viability was observed using a fluorescence microscope. The test results are as follows: Figure 2 (C) shown

[0059] Figure 2 (B) The production of reactive oxygen species (ROS) in Lewis lung cancer cells after treatment with MOFs of different Fe / Cu molar ratios. Scale bar = 50 μm. Figure 2 (C) Staining results of live / dead cells in Lewis lung cancer cells after 24 hours of MOF treatment with different Fe / Cu molar ratios. Figure 2 (B) It can be seen that, compared with the other two molar ratios, the MOF treatment group with a molar ratio of 3:2 showed the strongest green fluorescence signal, and its fluorescence intensity was significantly higher than that of the control group, indicating that its ROS generation ability was the best; the MOF with a molar ratio of 1:1 showed a medium intensity fluorescence signal; while the MOF with a molar ratio of 2:1 showed a relatively weak fluorescence signal. Figure 2 (C) It can be seen that, compared with other groups, the MOF group with Fe / Cu molar ratio of 3:2 showed the strongest red fluorescence (dead cells) and the weakest green fluorescence (live cells), indicating that it had the strongest anti-tumor effect.

[0060] In summary, the MOF with a Fe / Cu molar ratio of 3:2 exhibits good morphology, the best ROS generation capacity, and the strongest anti-tumor effect. Therefore, the MOF with a Fe / Cu molar ratio of 3:2 was selected for subsequent experiments.

[0061] Example 4

[0062] A method for preparing cisplatin-loaded iron-copper bimetallic MOF nanoparticles includes the following steps:

[0063] (1) Add 7 mg of MOF obtained in Example 1 to 7 mL of double-distilled water and stir at room temperature to disperse to obtain a fraction (1 mg / mL).

[0064] (2) Weigh 2 mg of cisplatin (CDDP) and add it to 2 mL of double-distilled water. Stir to dissolve and obtain cisplatin solution (1 mg / mL).

[0065] (3) Under light-protected conditions at room temperature (20~30℃), the cisplatin solution prepared in step (2) was slowly added dropwise to the dispersion prepared in step (1), with the adding rate controlled at 0.5 mL / min. After the addition was complete, the mixture was stirred for 24 hours under light-protected conditions at room temperature. After stirring, the mixture was centrifuged at 4℃ and 13,000 rpm for 30 minutes, and the precipitate was collected. The precipitate was washed three times with double-distilled water to remove unloaded free cisplatin. The final precipitate was collected, resuspended in an appropriate amount of double-distilled water, and freeze-dried to obtain cisplatin-loaded iron-copper bimetallic MOF nanoparticles (denoted as MPt-NPs).

[0066] Drug loading rate and drug loading rate methods:

[0067] High-performance liquid chromatography (HPLC) was used to determine the cisplatin loading and encapsulation efficiency in MPt-NPs. Chromatographic conditions: Column: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol-water (10:90, v / v); Detection wavelength: 210 nm; Column temperature: 25°C; Flow rate: 1.0 mL / min; Injection volume: 20 μL. Preparation of standard curve: Accurately weigh an appropriate amount of cisplatin reference standard, dissolve it in the mobile phase, and serially dilute it to prepare a series of standard solutions with gradient concentrations. Inject and measure the solutions under the above chromatographic conditions. Perform linear regression of peak area (A) against concentration (C) to plot the standard curve and calculate the regression equation. Sample determination: Accurately weigh approximately 1 mg of lyophilized MPt-NPs sample, add an appropriate amount of mobile phase, dissolve with ultrasonic assistance, and bring the volume to 10 mL. After passing through a 0.22 μm organic filter membrane, inject and measure the solution under the above chromatographic conditions, and record the peak area. Substitute the sample peak area into the standard curve regression equation to calculate the concentration of cisplatin in the sample.

[0068] Drug loading (DL%) and encapsulation efficiency (EE%) are calculated using the following formulas:

[0069] Drug loading (DL%) = (mass of cisplatin in MPt-NPs / total mass of MPt-NPs) × 100%;

[0070] Encapsulation efficiency (EE%) = (mass of cisplatin in MPt-NPs / total mass of cisplatin added) × 100%;

[0071] In this embodiment, HPLC analysis showed that the cisplatin loading in MPt-NPs was 11.4%, and the encapsulation efficiency was 80.0%.

[0072] Example 5

[0073] Preparation of hybridization membrane:

[0074] (1) Extraction of red blood cell membrane: Whole blood of C57BL / 6 mice was centrifuged at 3000 rpm (4℃) for 5 minutes to remove plasma. The resulting red blood cell precipitate was washed three times with physiological saline. Then, deionized water was added to the red blood cell precipitate and incubated at 4℃ for 1 hour to lyse the cells. The lysate was then centrifuged at 13,000 rpm for 5 minutes to remove hemoglobin and obtain red blood cell membrane. The red blood cell membrane was washed with deionized water until the supernatant was colorless. The red blood cell membrane was collected and stored at -80℃ for subsequent use.

[0075] (2) Extraction of tumor cell membranes: Lewis lung cancer cells were scraped with a cell scraper, and cell membranes were extracted from the collected cells using a membrane protein extraction kit (Beyotime, China, product number P0033). The cell pellet was collected and resuspended in 2 mL of membrane protein extraction kit solution A (kit-compatible) and 1 mM PMSF solution. The pellet was incubated on ice for 10-15 minutes, followed by three freeze-thaw cycles using liquid nitrogen. The membrane fragments were separated by differential centrifugation, and the obtained membrane fractions were stored at -80°C for subsequent use.

[0076] (3) Quantification of erythrocyte membrane and Lewis lung cancer cell membrane protein: The concentration of erythrocyte membrane prepared in step (1) and Lewis lung cancer cell membrane protein prepared in step (2) was measured using the BCA protein assay kit. The concentration of erythrocyte membrane protein was 2.0 mg / mL and the concentration of Lewis lung cancer cell membrane protein was 1.5 mg / mL.

[0077] (4) Preparation and fusion ratio optimization of hybridization membrane (RLCM): Red blood cell membrane and Lewis lung cancer cell membrane were mixed at a protein ratio of 1:1 (the specific volume was: 1.0 mL of red blood cell membrane solution and 1.33 mL of Lewis lung cancer cell membrane solution, so that the protein content of both was 2.0 mg), and PBS was added to a total volume of 4 mL.

[0078] The fusion process was studied and the optimal ratio was optimized using fluorescence resonance energy transfer (FRET) technology. Specifically, Lewis lung cancer cell membranes were stained with DiD and DiI fluorescent dyes, respectively. Then, erythrocyte membrane to Lewis lung cancer cell membrane protein ratios of 5:1, 3:1, 1:1, and 0:1 were added to DiD / DiI-stained LLC membranes. Fluorescence emission spectra in the 550-750 nm wavelength range were measured at an excitation wavelength of 525 nm. The ratio that resulted in the highest FRET efficiency was determined as the optimal fusion ratio. Results are as follows: Figure 3 As shown.

[0079] Depend on Figure 3 It was found that when DiD / DiI-labeled cancer cell membranes were fused with gradually increasing amounts of unlabeled erythrocyte membranes, the donor fluorescence (DiI) at 565 nm gradually recovered (dequenching effect), while the acceptor fluorescence (DiD) at 670 nm gradually decreased, indicating that the two membrane components underwent mutual mixing and rearrangement of membrane lipids and membrane proteins. Among these, the recovery of donor fluorescence and the decrease in acceptor fluorescence were most significant when the mass ratio of erythrocyte membrane to Lewis lung cancer cell membrane was 1:1, resulting in the largest change in FRET efficiency, indicating that the fusion efficiency of the two membranes was highest at this ratio. These synergistic changes in FRET fluorescence signals confirm the successful fusion of erythrocyte membranes and Lewis lung cancer cell membranes, rather than a simple physical mixing, and that 1:1 is the optimal fusion ratio.

[0080] After confirming the optimal ratio (1:1), mix the red blood cell membrane and Lewis lung cancer cell membrane solution according to the above volume, and sonicate in an ice bath (100 W power, 10 minutes total time) to promote membrane fusion, thus obtaining the hybridization membrane (RLCM) suspension, which is stored at 4°C for later use.

[0081] Example 6

[0082] Preparation of iron-copper bimetallic MOF biomimetic nanoparticles:

[0083] The MPt-NPs (concentration of 1 mg / mL) prepared in Example 4 were mixed with the hybridization membrane RLCM suspension (4 mL, protein concentration of 1 mg / mL) prepared in Example 5. To better encapsulate the nanoparticles in the hybridization membrane, the mixture was first subjected to ice bath sonication (power 100 W, total time 10 minutes). Then, the mixture was repeatedly extruded 10 times through a 1 μm polycarbonate porous membrane using a liposome extruder (LiposoFast-50), followed by 10 extrusions through a 400 nm polycarbonate porous membrane, and finally 10 extrusions through a 200 nm polycarbonate porous membrane. After extrusion, the product was collected by centrifugation at 12,000 rpm for 10 minutes, washed twice with PBS to remove uncoated free membrane fragments, and resuspended in PBS to obtain the hybridization membrane-coated biomimetic nanoparticles RLMPt-NPs, which were stored at 4°C for later use.

[0084] like Figure 4 As shown in the transmission electron microscope (TEM) images, the MOF nanoparticles prepared in Example 1 have a clear, star-shaped morphology and an average particle size of approximately 180 nm. Figure 4 A); Transmission electron microscopy analysis showed that the average particle size of the RLMPt-NPs nanoparticles prepared in Example 6 was approximately 200 nm (A); Figure 4 B); Dynamic light scattering (DLS) measurements revealed that the average hydrated particle size of MPt-NPs was 180 nm. After coating with an RLCM hybridization film, the average hydrated particle size of RLMPt-NPs increased to approximately 200 nm, confirming the successful preparation of RLMPt-NPs. Figure 4 C); SDS-PAGE analysis showed no significant difference in protein bands between RLMPt-NPs and the lysate from Lewis lung cancer cells. This similarity indicates that the cell membrane encapsulating RLMPt-NPs retained almost the same protein profile as the Lewis lung cancer cell membrane. Figure 4 D, I: RBCM, II: LCM, III: MOF, IV: RLMPt-NPs).

[0085] like Figure 5 As shown, the N2 adsorption-desorption isotherms indicate that the MOF (prepared in Example 1) has a specific surface area of ​​41.1406 m² / g and an average pore size of 15.8322 nm; the RLMPt-NPs (prepared in Example 6) has a specific surface area of ​​25.2960 m² / g MOF and an average pore size of 11.2916 nm. After loading platinum-based drugs and coating with hybridization films, both the specific surface area and pore size of the MOF decreased, confirming the success of drug encapsulation. Figure 5A); X-ray diffraction (XRD) results showed that the drug-loaded RLMPt-NPs retained the characteristic diffraction peaks of MOF, indicating that its core crystal structure remained intact. Figure 5 B); The elemental composition of RLMPt-NPs was confirmed by X-ray photoelectron spectroscopy (XPS), and Fe, Cu, C and O were detected. Figure 5 C).

[0086] Example 7

[0087] Core functionality verification of RLMPt-NPs:

[0088] (1) Generation of hydroxyl radicals (•OH): The generation of •OH radicals was detected using TMB. The RLMPt-NPs prepared in Example 6 were dispersed in 0.1 M sodium acetate buffer to prepare a 100 μg / mL solution. Then, TMB solution (1 mL, 0.25 mg / mL) was added to the RLMPt-NPs solution and stirred for 5 minutes. The absorbance of TMB at 370 nm and 652 nm was measured using a UV spectrophotometer. The results are as follows: Figure 6 As shown in (A).

[0089] (2) In vitro GSH consumption experiment: The consumption of GSH by different nanoparticles was detected using a DTNB probe. The simplified steps are as follows: 100 μL (1 mg / mL) of RLMPt-NPs or MPt-NPs (a solution obtained by adding PBS buffer to the drug) was mixed with 100 μL of GSH (10 mM) in 3.8 mL of distilled water. The mixture was then incubated at 37 °C for 30 minutes. Subsequently, the solution was centrifuged at 9000 rpm for 10 minutes. 600 μL of the supernatant was taken and mixed with 3 mL of DTNB solution (0.02 mg / mL). -1 The sample was mixed with Tris-HCl buffer (pH = 6.5), and the absorbance was measured at 412 nm using a UV spectrophotometer. The results are as follows: Figure 6 As shown in (B).

[0090] (3) Drug release experiment: Equal amounts of different drugs (CDDP, MPt-NPs prepared in Example 4, and RLMPt-NPs prepared in Example 6) were dissolved in PBS (pH = 6.5 and 7.4) to prepare solutions of 1 mg / mL. These solutions were placed in dialysis bags with a molecular weight (MW) of 3500 and in a shaking water bath at 100 rpm. At predetermined time intervals, 2 mL of the released PBS was drawn and replaced with an equal volume of fresh PBS. The cumulative drug release rate curve was determined and plotted using a high-performance liquid chromatograph (LC-30a, Shimadzu, Japan). The results are shown below. Figure 6 As shown in (C).

[0091] like Figure 6 As shown, the generation of hydroxyl radicals (•OH) was confirmed by the 3,3',5,5'-tetramethylbenzidine (TMB) probe. This probe was oxidized by •OH, producing a characteristic absorption peak at 652 nm. Figure 6 A); When measured using a 5,5'-dithiobis(2-nitrophenyl)thiazole (DTNB) probe, the characteristic absorption peak of GSH at 412 nm shifted, indicating GSH depletion. Figure 6 B); Under pH 7.4 conditions, free drug (CDDP) and MPt-NPs released 81.95 ± 1.34% and 76.25 ± 1.23% of cisplatin, respectively, within 24 hours, while RLMPt-NPs released only 43.72 ± 0.1% of the drug; furthermore, under acidic conditions at pH 6.5, the release rate of RLMPt-NPs increased to 56.73 ± 0.26% ( Figure 6 C).

[0092] Figure 6 In vitro experiments confirmed that RLMPt-NPs can release cisplatin in a controlled and pH-sensitive manner, while generating high concentrations of •OH and depleting GSH.

[0093] Example 8

[0094] In vitro cytotoxicity and biochemical mechanism related experiments:

[0095] (1) In vitro apoptosis experiment: Lewis lung cancer cells (2x10⁻¹⁰) were used to induce apoptosis. 5 Cells were seeded in 6-well plates and co-cultured for 24 hours with an equal volume of normal saline (NS), cisplatin (CDDP), MOF (prepared in Example 1), MPt-NPs (prepared in Example 4), and RLMPt-NPs (prepared in Example 6) (100 μg / ml, prepared with DMEM). After incubation, cells were collected and stained with Annexin V-FITC / PI for 20 minutes. Apoptosis rate was determined using flow cytometry (Beckman Coulter). The test results are as follows. Figure 7 As shown in (A) and (B).

[0096] (2) Cytotoxicity test: Lewis lung cancer cells (5x10⁻¹²) were subjected to cytotoxicity assay. 3Cells were seeded in 96-well plates, and equal volumes (100 μL) of different concentrations (1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL, prepared with DMEM) of free cisplatin, MOF, MPt-NPs, and RLMPt-NPs were added respectively. Cells were cultured for 48 hours, and cell viability was determined using the CCK-8 assay. The results are shown below. Figure 7 As shown in (C).

[0097] (3) Live and dead cell staining: Lewis lung cancer cells (2x10⁻¹²) were stained with live and dead cells. 5 Cells were seeded into 6-well culture plates, and incubated with equal volumes (100 μg / ml, prepared with DMEM) of NS, CDDP, MOF, MPt-NPs, and RLMPt-NPs for 24 hours. Cells were gently washed twice with 5% PBS, and stained for 30 minutes with Calcein-AM (1 μg / ml) and pyridine iodide (PI) (1 μg / ml) staining reagent. Viable cells were stained green fluorescence, and dead cells were stained red fluorescence. Cells were washed three times with PBS, and the cell viability was observed using a fluorescence microscope. The test results are shown below. Figure 8 As shown.

[0098] (4) Cell uptake experiment: Lewis lung cancer cells (2x10⁻¹²) were added to the cell uptake test. 5 Cells were seeded in confocal culture dishes, and free NR, NR-labeled RLMPt-NPs (RLM@MOF-NR), and NR-labeled MOF (MOF-NR) (NR: 1 μg / mL, prepared with DMEM) were added respectively. Cells were incubated at 37°C for 2 hours. Cells were washed three times with PBS, and cell uptake was observed using a confocal microscope. The test results are as follows: Figure 9 As shown.

[0099] (5) ROS generation experiment: Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). Lewis lung cancer cells (2x10⁻¹²) were used in the assay. 5 Cells were co-cultured for 24 hours with an equal volume concentration of 100 μg / ml (prepared with DMEM) of NS, CDDP, MOF, MPt-NPs, or RLMPt-NPs. After washing three times with PBS, DCFH-DA (1 mL, 10 μmol / L) was added, and the cells were cultured for another 30 minutes. Observation was performed using a fluorescence microscope (OLYMPUS, IX83, Japan). The test results are as follows: Figure 10 As shown in (B).

[0100] (6) DNA damage experiment: Lewis lung cancer cells (2x10⁻¹⁰) were subjected to DNA damage experiments. 5Cells were seeded into 6-well culture plates and treated with equal volumes (100 μg / ml, prepared with DMEM) of various drugs for 12 hours, including NS, CDDP, MOF, MPt-NPs, and RLMPt-NPs. Cells were then washed with PBS, fixed with PBS containing 4% glutaraldehyde, permeabilized with PBS containing 0.2% Tween X-100 for 10 min, blocked with 5% FBS for 60 min, and stained overnight at 4°C using Alexa Fluor 488-labeled anti-γ-H2AX antibody (1 μg / mL, 1:500 dilution). Finally, slides were mounted with DAPI mounting medium and observed under an inverted microscope (OLYMPUS, IX83, Japan). The test results are as follows: Figure 10 As shown in (C).

[0101] (7) Mitochondrial membrane potential detection: Lewis lung cancer cells (2x10⁻¹⁰) were used to detect mitochondrial membrane potential. 5 Cells were seeded into 6-well plates and treated with an equal volume (100 μg / ml, prepared with DMEM) of NS or RLMPt-NPs for 24 hours. The culture medium was then aspirated, and the cells were washed once with PBS. After washing, JC-1 dye was added, and the cells were incubated for 30 minutes. The cells were washed three times with PBS and observed using a fluorescence microscope (OLYMPUS, IX83, Japan). The test results are as follows: Figure 10 As shown in (D).

[0102] like Figure 7 As shown in AB, in Lewis lung cancer cells, the apoptosis experiment showed that the cell death rate induced by RLMPt-NPs was significantly higher than that induced by cisplatin-CDDP and MPt-NPs. Figure 7 AB).

[0103] Depend on Figure 7 As shown in Figure C, compared with the CDDP group and the MPt-NPs group, RLMPt-NPs exhibited stronger cytotoxicity, and the cytotoxicity increased with increasing concentration.

[0104] like Figure 8 The image shows calcein-AM / PI live / dead cell staining in Lewis lung cancer cells. The proportion of cell death induced by MPt-NPs (red) was higher than that induced by cisplatin (CDDP), which can be attributed to the Fenton reaction triggered by iron ions in the MOF backbone. Quantitative cytotoxicity assays confirmed that RLMPt-NPs exhibited superior cytotoxicity compared to cisplatin (CDDP) and were effectively able to kill Lewis lung cancer cells.

[0105] like Figure 9As shown, confocal laser scanning microscopy (CLSM) imaging results of NR-labeled RLMPt-NPs (RLM@MOF-NR) prepared in Example 6 after incubation showed that RLM@MOF-NR exhibited stronger red fluorescence compared to the free NR group, indicating that RLMPt-NPs can be significantly taken up by cells.

[0106] like Figure 10 As shown, the biological efficacy and mechanism of action of RLMPt-NPs on lung tumor cells from different sources were investigated. First, fluorescent staining was performed using the dichlorofluorescein diacetate (DCFH-DA) probe to visualize the generation of reactive oxygen species (ROS) in Lewis lung cancer cells. Compared with the CDDP treatment group, which only showed weak green fluorescence, the RLMPt-NPs treatment group (prepared in Example 6) showed strong green fluorescence, indicating that RLMPt-NPs significantly increased intracellular ROS levels. Figure 10 B). DNA damage detection revealed significant DNA damage in both the MPt-NPs (prepared in Example 4) group and the RLMPt-NPs group. Figure 10 C). JC-1 assay results showed that RLMPt-NPs treatment significantly exacerbated mitochondrial damage in both cell lines ( Figure 10 D). In summary, RLMPt-NPs exert their effects through a unique mechanism: this mechanism increases the production of reactive oxygen species (ROS), causing DNA damage and disrupting mitochondrial membrane potential, ultimately leading to tumor cell death.

[0107] Example 9

[0108] In vivo antitumor drug efficacy experiment:

[0109] Six- to eight-week-old female C57BL / 6 mice were subcutaneously inoculated with Lewis lung cancer cells (5 × 10⁻⁶) in the right leg. 5 (One per tumor). Wait until the tumor volume reaches approximately 50-100 mm. 3 Mice were randomly divided into the following groups (n=6): PBS control group (I: Control), anti-PD1 (II), free cisplatin group (CDDP, III), MPt-NPs group (IV), RLMPt-NPs group (V), and RLMPt-NPs+anti-PD1 combined group (VI).

[0110] Dosage regimen: CDDP, RLMPt-NPs, and MPt-NPs were administered via tail vein injection (CDDP: 2 mg / kg, diluted with saline before injection), once every 2 days for a total of 3 doses; anti-PD-1 antibody was administered intravenously (100 μg / mouse), once every 2 days for a total of 3 doses. Tumor volume and mouse weight were measured every 2 days. After the experiment, tumor tissue was collected for immunofluorescence staining, and serum was collected to detect cytokine levels (IFN-γ, TNF-α, IL-6). Test results are as follows. Figure 11 and 12 As shown.

[0111] like Figure 11 As shown, to evaluate the in vivo efficacy of RLMPt-NPs combined with anti-PD-1 immunotherapy, a subcutaneous Lewis lung cancer model was established in C57BL / 6 mice, according to... Figure 11 Treatment was administered according to the plan shown in A. Significant differences in treatment outcomes were observed between the different treatment groups. Figure 11 BC). Free cisplatin treatment only moderately inhibited tumor growth; in stark contrast, the combination of RLMPt-NPs and anti-PD-1 therapy produced the most significant anti-tumor response, with minimal tumor progression in this group of mice. Mice treated with CDDP showed significant weight loss indicating systemic toxicity, but such adverse reactions were not observed in groups IV-VI. Figure 11 D). Furthermore, the survival rate VI > V > IV, and compared to the MPt-NPs group and the RLMPt-NPs group, the combined use of RLMPt-NPs and anti-PD-1 therapy significantly improved the survival rate. Figure 11 E). By Figure 11 This enhanced efficacy demonstrates a strong synergistic effect between the nanoplatform and immunotherapy, along with superior safety and significantly improved survival rates.

[0112] like Figure 12 As shown, tumor sections from the RLMPt-NPs+anti-PD1 group exhibited the most severe histopathological damage, including extensive cavitation and widespread cytoplasmic fragmentation. Immunohistochemical analysis of the proliferation marker Ki-67 showed a significantly lower positive rate in the RLMPt-NPs+anti-PD1 group; the RLMPt-NPs+anti-PD1 group also showed more significant CD8+. + T cell infiltration; γ-H2AX staining results showed that the DNA damage was more severe in the RLMPt-NPs+anti-PD1 group compared with other groups. Figure 12 It can be seen that RLMPt-NPs+anti-PD1 has a strong therapeutic response and a potent in vivo inhibitory effect on tumor cell growth.

[0113] Example 10

[0114] RLMPt-NPs targeting validation:

[0115] In vivo imaging: In vivo imaging was performed using ICG-labeled RLMPt-NPs. To synthesize ICG-labeled RLMICG, ICG was added to an RLMPt-NPs solution (1 mg / mL) and stirred for 24 hours at room temperature in the dark. RLMICG was then collected by centrifugation and washed with double-distilled water for later use. Lewis lung cancer cells were subcutaneously transplanted into the right hind leg of 6-week-old male C57BL / 6 mice to establish a lung cancer xenograft model. Mice were randomly assigned to four groups and intravenously injected with free ICG, MICG, or RLMICG solution, respectively. Mice were imaged at predetermined time points (6, 12, and 24 hours) using a multimodal small animal in vivo imaging system (PerkinElmer, USA). Mice were euthanized 24 hours after injection, and tumors and major organs removed from the euthanized mice were imaged.

[0116] Biodistribution of Nanoparticles: Mice carrying LLC lung cancer tumors were randomly divided into two groups. Each group of mice was intravenously injected with 100 μl of MPt-NPs or RLMPt-NPs (Pt: 2 mg / kg, prepared with physiological saline before injection). Twenty-four hours later, the mice were euthanized, dissected, and the tumor tissue and major organs were weighed. The gold content in the samples was determined using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent ICPMS7800, USA). Results are as follows: Figure 13 As shown.

[0117] like Figure 13 As shown in Figure A, RLMICG gradually accumulates in tumor tissue over time, reaching its peak at 24 hours post-injection. In vitro imaging of excised major organ tissues revealed a strong fluorescence signal in the tumor tissue at this 24-hour time point.

[0118] like Figure 13 As shown in Figure B, inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that in the RLMPt-NPs group, the iron content in tumor tissue (76.023 ± 3.677 % ID / g) was significantly higher than that in the liver (29.709 ± 2.208 % ID / g) and kidney (18.515 ± 1.057 % ID / g).

[0119] like Figure 13 As shown in Figure C, similarly, the iron content in tumor tissue of the RLMPt-NPs group was significantly higher compared to all major organs examined.

[0120] Depend on Figure 13 It can be seen that RLMPt-NPs exhibit excellent active targeting ability and are mainly enriched in tumors.

[0121] 5. Validation of the immune response induced by RLMPt-NPs

[0122] To investigate immune cells in secondary tumors and the spleen, tumor tissue was harvested from mice in each treatment group in Example 9. These tissues were prepared into single-cell suspensions. Cells were stained with various antibodies (FITC anti-mouse CD3 antibody, APC anti-mouse CD4 antibody, BV711 anti-mouse CD8 antibody, APC-CY7 anti-mouse CD45 antibody, BB700 anti-mouse CD11c antibody, PE-CY7 anti-mouse CD86 antibody, PE anti-mouse CD80 antibody, and V500 anti-mouse MHCII antibody) according to the manufacturer's (BD Pharmingen) operating procedures. Finally, flow cytometry analysis was performed using a BD FACSAria (USA). Statistical analysis of all flow cytometry data was performed using FlowJo software (version 10.0). Test results are as follows: Figure 14 As shown.

[0123] like Figure 14 As shown, compared with the control group and the RLMPt-NPs group, injection of RLMPt-NPs + anti-PD1 induced dendritic cell maturation and migration to peripheral immune organs. Specifically, the proportion of mature dendritic cells within the tumor was higher in the RLMPt-NPs + anti-PD1 group than in the RLMPt-NPs group, indicating that RLMPt-NPs and anti-PD1 have a synergistic effect in promoting dendritic cell recruitment and maturation. Figure 14 A) Flow cytometry results showed that RLMPt-NPs + anti-PD1 significantly increased CD8+ in tumor tissue. + The number of T lymphocytes ( Figure 14 (B) This indicates that RLMPt-NPs + anti-PD1 have a synergistic effect in promoting dendritic cell recruitment and maturation, and synergistically amplify tumor-specific CD8. + T cell proliferation and effector function; the proportion of NK1.1 positive cells in tumor tissue of the RLMPt-NPs+anti-PD1 group was 4 times that of the control group ( Figure 14 (C) This indicates that the combined regimen simultaneously activates NK cells in the innate immune system and synergistically enhances adaptive immunity.

[0124] Depend on Figure 14It can be seen that RLMPt-NPs+anti-PD1 can significantly regulate the immune system. In addition, RLMPt-NPs+anti-PD1 enhances immunity by promoting the accumulation of NK cells.

[0125] Example 11

[0126] Verification of immune memory effect:

[0127] To evaluate the immune memory effect after RLMPt-NPs+anti-PD1 treatment, 5×10 5 Lewis lung cancer cells suspended in PBS were subcutaneously injected into the left thigh of mice that had received RLMPt-NPs+anti-PD1 treatment 20 days prior. An equivalent dose of Lewis lung cancer cells was injected into healthy mice as a control group. Tumor growth was monitored every two days. After euthanasia, the tumors were photographed for documentation, and tumor tissue was harvested for flow cytometry analysis. The results are as follows: Figure 15 As shown.

[0128] like Figure 15 As shown, in order to evaluate the immune memory effect of RLMPt-NPs combined with anti-PD-1 therapy, according to... Figure 15 A secondary tumor model was established as shown in Figure A. The results showed that the RLMPt-NPs+anti-PD1 group effectively inhibited the growth of re-inoculated tumors, while the tumors in the control group mice progressed rapidly. Figure 15 Specifically, in the RLMPt-NPs+ anti-PD1 group after vaccination, the mean tumor volume was 57.507 ± 30.422 mm. 3 The value was significantly lower than that of the control group (643.762 ± 16.319 mm). 3 ( Figure 15 C); Flow cytometry results showed that, compared with other groups, RLMPt-NPs+anti-PD1 treatment induced a sustained anti-tumor response. Specifically, CD8+ in tumor tissue of the RLMPt-NPs+anti-PD1 group was significantly higher. + The proportion of T cells reached 32.03 ± 0.9%, significantly higher than that of the control group (20.13 ± 1.5%). Figure 15 DE); more importantly, in CD8 + In the T cell subsets, the proportion of effector memory T cells in the RLMPt-NPs+anti-PD1 group reached 76.7±2.2%, significantly higher than that in the control group (22.03±2.1%). Figure 15 FG).

[0129] Depend on Figure 15Therefore, RLMPt-NPs combined with anti-PD-1 can not only induce specific long-term protection, but also effectively prevent tumor recurrence.

[0130] Example 12

[0131] Biosafety validation of RLMPt-NPs:

[0132] Hemolysis assay: 1 mL of erythrocyte suspension (2%, v / v) was mixed with 1 mL of CDDP, MOF (prepared in Example 1), MPt-NPs (prepared in Example 4), and RLMPt-NPs (prepared in Example 6) dissolved in physiological saline (100 μg / mL). Double-distilled water and physiological saline were used as positive and negative controls, respectively. All samples were incubated at 37°C for 4 hours and then centrifuged at 3000 rpm for 5 minutes. The absorbance (OD) of the supernatant at 540 nm was measured using a UV-Vis spectrophotometer, and the hemolysis rate was calculated.

[0133] Complete blood count: After the experiment in Example 9, approximately 200 μL of whole blood was collected from each group of mice using the orbital venous plexus sampling method. The blood was placed in dipotassium ethylenediaminetetraacetate (EDTA-K2) anticoagulant tubes and gently inverted to mix to prevent clotting. The following complete blood count indicators were measured using a fully automated blood cell analyzer: white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), platelet count (PLT), neutrophil percentage (NEU), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

[0134] Blood biochemistry: Approximately 500 μL of whole blood from each group of mice was collected and placed in a regular centrifuge tube without anticoagulant. The tubes were allowed to stand at room temperature for 30 minutes to allow for natural coagulation. The blood was then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant pale yellow serum was carefully aspirated and transferred to a new centrifuge tube. The following serum biochemical indicators were measured using an automated biochemical analyzer: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), urea (UREA), albumin (ALB), glucose (GLU), and total cholesterol (TC).

[0135] HE: After the experiment in Example 9, mice in each group were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were dissected and separated. Fresh tissues were fixed in 4% paraformaldehyde fixative for at least 48 hours. The fixed tissues were dehydrated with a gradient of ethanol, cleared with xylene, and then embedded in molten paraffin. After trimming the paraffin-embedded blocks, they were cut into 4-6 μm thick sections using a paraffin microtome, spread on glass slides, and baked at 60°C for 2 hours. Before staining, the sections were dewaxed with xylene, rehydrated with a gradient of ethanol, and rinsed with distilled water for 2 minutes. The sections were then stained with hematoxylin stain for 5-8 minutes, rinsed with tap water for 2 minutes, differentiated with 1% hydrochloric acid ethanol for 5-10 seconds, and rinsed with tap water for 15-30 minutes to achieve blue reversion. Subsequently, the sections were stained with eosin stain for 2-5 minutes and quickly rinsed with distilled water. The stained sections were dehydrated with a gradient of ethanol and cleared with xylene, and finally mounted with neutral resin. After mounting, the histomorphological changes of major organs such as the heart, liver, spleen, lungs, and kidneys were observed under an optical microscope.

[0136] like Figure 16 The image shown is a microscopic image of erythrocytes in the RLMPt-NPs group and a photograph of hemolysis. Figure 16 (AB) No obvious hemolysis was observed. Analysis of UV-Vis absorption spectroscopy ( Figure 16 C) all indicate that the degree of hemolysis is extremely low, and the directly measured hemolysis rate ( Figure 16 D) No obvious hemolysis was observed. RLMPt-NPs exhibited excellent blood compatibility.

[0137] like Figure 17 As shown, compared with the control group, all hematological and biochemical indicators of mice injected with RLMPt-NPs+anti-PD1 remained within the normal range; no abnormalities were found in renal and liver function indicators in the RLMPt-NPs+anti-PD1 treatment group; the RLMPt-NPs+anti-PD1 group showed good biocompatibility.

[0138] like Figure 18 As shown, the free cisplatin group exhibited varying degrees of histological damage in the heart, liver, and kidneys, with renal tubular epithelial cell vacuolar degeneration and necrosis being the most typical, consistent with the clinical dose-limiting toxicity of cisplatin. In contrast, the histological damage to all organs was significantly reduced in the RLMPt-NPs group, while the histological morphology of all organs in the RLMPt-NPs group and the RLMPt-NPs + anti-PD1 combination group was basically consistent with the PBS control group, with no obvious pathological changes. These results indicate that the biomimetic coating of the RLCM hybrid membrane effectively reduces the systemic off-target toxicity of cisplatin. RLMPt-NPs, whether used alone or in combination with anti-PD-1, did not cause significant histological damage to any major organs, confirming the good biocompatibility of this nanodelivery system. In other words, RLMPt-NPs + anti-PD1 has good biocompatibility.

Claims

1. A bimetallic iron-copper MOF biomimetic nanoparticle, characterized in that, The nanoparticles comprise a core layer and an outer shell layer, with the outer shell layer covering the outer surface of the core layer; the core layer is an iron-copper bimetallic organic framework loaded with cisplatin; and the outer shell layer is a hybrid membrane formed by the fusion of red blood cell membrane and cancer cell membrane.

2. The iron-copper bimetallic MOF biomimetic nanoparticles according to claim 1, characterized in that, In the iron-copper bimetallic organic framework, the molar ratio of iron ions to copper ions is 1~2:

1.

3. The iron-copper bimetallic MOF biomimetic nanoparticles according to claim 1, characterized in that, The nanoparticles have a particle size of 100~300 nm.

4. The iron-copper bimetallic MOF biomimetic nanoparticles according to claim 1, characterized in that, The cancer cell membrane is a lung cancer cell membrane.

5. A method for preparing iron-copper bimetallic MOF biomimetic nanoparticles according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Synthesis of iron-copper bimetallic organic frameworks; (2) The iron-copper bimetallic organic framework prepared in step (1) is added to a solvent to prepare a dispersion. Cisplatin is dissolved in the solvent to obtain a cisplatin solution. Under light-protected conditions, the cisplatin solution is slowly added to the iron-copper bimetallic organic framework dispersion. The mixture is stirred to load cisplatin into the iron-copper bimetallic organic framework. Unloaded free cisplatin is removed to obtain iron-copper bimetallic MOF nanoparticles loaded with cisplatin. (3) The red blood cell membrane and the cancer cell membrane are fused to form a hybrid membrane, and the hybrid membrane is wrapped around the outer surface of the iron-copper bimetallic MOF nanoparticles loaded with cisplatin prepared in step (2) to obtain the iron-copper bimetallic MOF biomimetic nanoparticles.

6. The method for preparing iron-copper bimetallic MOF biomimetic nanoparticles according to claim 5, characterized in that, In step (1), the iron-copper bimetallic organic framework material is synthesized using a hot solvent method.

7. The method for preparing iron-copper bimetallic MOF biomimetic nanoparticles according to claim 5, characterized in that, In step (3), the method of wrapping the hybrid membrane around the outer surface of the cisplatin-loaded nanoparticles prepared in step (2) is as follows: the cisplatin-loaded nanoparticles are dispersed in a solvent, then mixed with the hybrid membrane, first ultrasonically treated, and finally squeezed through a polycarbonate porous membrane to collect the solid.

8. The use of the iron-copper bimetallic MOF biomimetic nanoparticles according to any one of claims 1 to 4 in the preparation of antitumor drugs.

9. The application according to claim 9, characterized in that, The tumor is lung cancer.

10. The application according to claim 9, characterized in that, The nanoparticles are used in combination with immune checkpoint inhibitors.

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