Multifunctional bionic nano platform FBFO-HM-aOPN and preparation method and application thereof

Abstract

The invention discloses a multifunctional bionic nano platform FBFO (at) HM (at) aOPN as well as a preparation method and application thereof, and belongs to the technical field of nano preparations. The multifunctional bionic nano platform FBFO (at) HM (at) aOPN provided by the invention comprises a hybrid membrane and Fe3O4 (at) BiFeO3 core-shell nanoparticles, and the Fe3O4 (at) BiFeO3 core-shell nanoparticles are wrapped by the hybrid membrane; the hybrid membrane comprises an M1 type macrophage source exosome and a bacterial outer membrane vesicle, the surface of the hybrid membrane is modified with an anti-osteopontin antibody through an acid-sensitive connexon. According to the invention, through a space-time controllable catalysis-immune synergistic effect and multi-dimensional microenvironment remodeling, a new strategy with high efficiency, minimally invasive property and safety is provided for treatment of glioblastoma, and three clinical bottlenecks of hypoxia, immunosuppression and blood brain barrier are broken through.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoformulations, and particularly to a multifunctional biomimetic nano-platform FBFO@HM@aOPN, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Glioblastoma multiforme (GBM), as the most aggressive primary malignant tumor of the central nervous system, has long been restricted in treatment by its complex pathological characteristics and multiple technical bottlenecks. The tumor microenvironment of this tumor presents a high degree of heterogeneity, and is composed of an abnormal vascular network, a dense extracellular matrix (ECM), and M2-type tumor-associated macrophages (TAMs) to jointly form an immunosuppressive barrier. This unique hypoxic environment not only restricts the infiltration of immune cells, but also secretes immunosuppressive factors such as osteopontin (OPN) through TAMs, binds to the CD44 / integrin receptor to form a physical barrier (TIB), directly hinders the infiltration of lymphocytes and induces T cell exhaustion, resulting in a significant reduction in the efficacy of radiotherapy, chemotherapy, and immune checkpoint inhibitors (such as anti-PD-1 therapy).

[0004] At the level of drug delivery, the presence of the blood-brain barrier (BBB) further exacerbates the treatment difficulty. Due to the limitation of molecular characteristics, the accumulation of traditional drugs at the tumor site after penetrating the BBB is less than 30% of the effective treatment concentration, seriously affecting the treatment effect. Although photodynamic therapy (PDT), which has received much attention in recent years, kills tumors by generating reactive oxygen species (ROS), its action efficiency is limited by the hypoxic environment of the tumor microenvironment (TME) and the short lifespan characteristics of ROS, and traditional photosensitizers (such as Fe3O4) are more difficult to achieve the dual goals of effective oxygen supply and targeted killing due to problems such as the limited light absorption range in the ultraviolet region and low catalytic efficiency.

[0005] Existing treatment strategies face numerous limitations in clinical practice. Although the anti-angiogenic drug bevacizumab can transiently improve vascular abnormalities, long-term use exacerbates hypoxia, prompting TAMs to polarize towards the immunosuppressive M2 phenotype. In the field of immunotherapy, the blockade strategy targeting the OPN signaling pathway is not yet mature, and traditional nanodelivery systems using single-source biomimetic designs of erythrocyte membranes or tumor cell membranes are difficult to break through the delivery barrier of the BBB and cannot synchronously regulate the immunosuppressive microenvironment. In addition, although Fe3O4 nanoparticles with enzyme-like activity can relieve hypoxia by mimicking peroxidase (POD) and catalase (CAT), their light absorption efficiency and ROS generation ability are insufficient, and they lack the dual targeting ability for tumor cells and TAMs, making it difficult to achieve synergistic therapeutic effects. Therefore, there is an urgent need to provide a treatment strategy to solve the core problems such as low drug delivery efficiency, TME immunosuppression, and PDT hypoxia limitation in GBM treatment. Summary of the Invention

[0006] In view of this, the present invention provides a multifunctional biomimetic nanoplatform FBFO@HM@aOPN, its preparation method and application. The multifunctional biomimetic nanoplatform FBFO@HM@aOPN provided by the present invention provides a more comprehensive treatment framework for invasive solid tumors through multi-dimensional microenvironment remodeling (hypoxia improvement, matrix softening, bidirectional targeting) and ferroptosis-induced immunogenic cell death.

[0007] In the first aspect, the present invention provides a multifunctional biomimetic nanoplatform FBFO@HM@aOPN, comprising a hybrid membrane and Fe3O4@BiFeO3 core-shell nanoparticles, wherein the hybrid membrane wraps the Fe3O4@BiFeO3 core-shell nanoparticles; the hybrid membrane comprises M1 macrophage-derived exosomes and bacterial outer membrane vesicles; the surface of the hybrid membrane is modified with an anti-osteopontin antibody through an acid-sensitive linker.

[0008] In the second aspect, the present invention provides a preparation method of the above multifunctional biomimetic nanoplatform FBFO@HM@aOPN, comprising the following steps: Prepare Fe3O4@BiFeO3 core-shell nanoparticles; Prepare bacterial outer membrane vesicles and M1 macrophage-derived exosomes modified with azide groups; Mix the bacterial outer membrane vesicles with the M1 macrophage-derived exosomes modified with azide groups, sonicate, and freeze-thaw to obtain a hybrid membrane, and then mix with the Fe3O4@BiFeO3 core-shell nanoparticles and extrude to obtain Fe3O4@BiFeO3 core-shell nanoparticles FBFO@HM wrapped with the hybrid membrane; Couple the FBFO@HM with an anti-osteopontin antibody modified with an alkyne group and an acid-sensitive linker through a click reaction to obtain the multifunctional biomimetic nanoplatform FBFO@HM@aOPN.

[0009] In a third aspect, the present invention provides the use of the above-mentioned multifunctional biomimetic nanoplatform FBFO@HM@aOPN or the multifunctional biomimetic nanoplatform FBFO@HM@aOPN prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0010] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) By constructing the multifunctional biomimetic nanoplatform FBFO@HM@aOPN, the present invention realizes the precise targeted therapy and multi-dimensional microenvironment regulation of glioblastoma (GBM). Based on the inflammatory chemotaxis characteristics of M1-type exosomes (M1EVs) and the immune activation function of bacterial outer membrane vesicles (OMVs), this system efficiently penetrates the blood-brain barrier and targets and enriches in tumor lesions. At the same time, the anti-OPN antibody (aOPN) is released in a pH-sensitive linker-responsive manner, softening the tumor extracellular matrix (ECM) and reducing tissue hardness, significantly enhancing the deep penetration ability of nanoparticles in the tumor, and solving the problem that traditional drug delivery is limited by physical barriers.

[0011] (2) At the catalytic therapy level of the multifunctional biomimetic nanoplatform FBFO@HM@aOPN provided by the present invention, the dual enzyme activities of the Fe3O4@BiFeO3 (FBFO) nanozyme play a key role: its catalase (CAT) activity continuously decomposes endogenous H2O2 in the tumor to produce oxygen, relieving the hypoxic microenvironment and simultaneously enhancing the reactive oxygen species (ROS) generation efficiency of photodynamic therapy (PDT); while the peroxidase (POD) activity amplifies the ROS killing effect through a cascade reaction, and further improves the catalytic efficiency in combination with the piezoelectric effect driven by ultrasound, providing strong photodynamic killing for deep tumors.

[0012] (3) In terms of immune co-regulation of the multifunctional biomimetic nanoplatform FBFO@HM@aOPN provided by the present invention, immunogenic molecules (such as CRT, HMGB1) are released by inducing ferroptosis of tumor cells to activate antigen-presenting cells (APCs) to trigger specific anti-tumor immune responses; at the same time, endogenous signaling molecules in M1EVs drive tumor-associated macrophages (TAMs) to polarize into pro-inflammatory M1 phenotypes, enhancing their antigen-presenting ability and phagocytic activity, and jointly activating CD8 + T cells reshape the immunosuppressive microenvironment and significantly improve the response rate of immune checkpoint inhibitors (ICIs). Through spatiotemporally controllable catalytic-immune synergy and multi-dimensional microenvironment remodeling, the present invention provides a new strategy with high efficiency, minimally invasive nature and safety for GBM treatment, breaking through the three major clinical bottlenecks of hypoxia, immunosuppression and blood-brain barrier. Brief Description of the Drawings

[0013] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0014] Figure 1 In Example 1 of the present invention: A. Schematic diagram of the synthesis route of FBFO nanoparticles (NPs); B. Preparation process of constructing a prokaryotic-eukaryotic hybrid vesicle membrane (HM) by fusing bacterial outer membrane vesicles (OMVs) with M1 macrophage-derived exosomes (M1EVs); C. Schematic diagram of the preparation of anti-SPP1 (OPN) blocking antibody (aOPN) functional modification based on a pH-sensitive benzoic acid-imide bond coupling strategy. Figure 2 In Example 1 of the present invention: A. Transmission electron microscope (TEM) of FBFO nanoparticles; Scale bar: 100 nm; B. Elemental energy spectrum surface scanning imaging of FBFO nanoparticles; Scale bar: 100 nm; C. X-ray diffraction (XRD) patterns of Fe3O4 and FBFO nanoparticles; D. Ultraviolet-visible diffuse reflectance (UV-Vis: DRS) spectra of Fe3O4 and FBFO nanoparticles; E. Schematic diagram of the mechanism of action of FBFO nanoparticles. Figure 3 In Example 1 of the present invention: A. Flow cytometry quantitative analysis of the binding efficiency of azide (N3)-modified M1EVs incubated with DBCO-Cy5.5; B. Confocal laser scanning microscopy (CLSM) observation of the fluorescence labeling of azide (N3)-modified M1EVs incubated with DBCO-Cy5.5; Scale bar: 50 nm; C. CLSM imaging showing the membrane fusion co-localization of M1EVs and OMVs; Scale bar: 50 nm; D. Western Blot verification that the hybrid membrane (HMs) contains M1EVs-derived markers (TSG101, CD63, CD81, integrin α4β1, and chemokine receptor CXCR4) and OMVs markers (FtsZ). Figure 4 In Example 1 of the present invention: A. CLSM imaging showing the binding co-localization of HMs and anti-OPN antibody (aOPN); Scale bar: 100 nm; B. Homogeneous morphological characterization of FBFO@HM@aOPN nanoparticles under TEM; Scale bar: 100 nm. Figure 5In Example 1 of the present invention: A: Quantitative results of enzyme-linked immunosorbent assay (ELISA) for the release amount of aOPN under different pH conditions; B: Degradation efficiency analysis of FBFO@HM@aOPN for MB under different conditions; C: Oxygen (O2) generation kinetic curve of FBFO@HM@aOPN under different conditions; Figure 6 In the test example of the present invention: A: Comparison of the degradation performance of Fe3O4 and FBFO nanoparticles for methylene blue (MB); B: Oxygen (O2) generation curves of Fe3O4 and FBFO nanoparticles; C: Electron spin resonance (ESR) spectroscopy analysis of different experimental groups; Figure 7 In the test example of the present invention: A: Flow cytometry analysis of the internalization efficiency of Cy5.5-labeled FBFO nanoparticles in CT2A GBM cells (scale bar: 100 μm); B: Confocal laser scanning microscopy (CLSM) imaging showing nanoparticle-induced reactive oxygen species (ROS) generation in DCFH-DA-stained CT2A GBM cells (scale bar: 200 μm); Figure 8 In the test example of the present invention: A: Cell viability of CT2A GBM cells treated with different formulations under (top) 660 nm light illumination / (bottom) no light illumination conditions; B: Comparison of cell viability of CT2A GBM cells treated with different formulations in (top) normoxia / (bottom) hypoxia environments; C: Analysis of the apoptosis level of CT2A GBM cells induced by different formulations under 660 nm light illumination by flow cytometry Annexin V-FITC / PI double staining method; D: Quantitative analysis of the lipid peroxidation level of CT2A GBM cells treated with different formulations under 660 nm light illumination by malondialdehyde (MDA) detection kit; E: CLSM co-localization imaging showing the fluorescence signals of calreticulin (CALR, red) and nuclear dye Hoechst (blue) in CT2A cells treated with different formulations under 660 nm light illumination (scale bar: 50 μm); Figure 9 In the test example of the present invention: A and B are respectively for detecting the release levels of high mobility group protein B1 (HMGB1) and adenosine triphosphate (ATP) in the culture supernatant of CT2A cells treated with different formulations under 660 nm light illumination; C is a schematic diagram of the mechanism of FBFO@HM@aOPN-induced immunogenic cell death (ICD) and enhanced immunotherapy; Figure 10In the experimental examples of the present invention: A: Flow cytometry analysis of the internalization of Cy5.5-labeled FBFO nanoparticles in Raw264.7 cells; B: Confocal laser scanning microscopy (CLSM) imaging showing the generation of reactive oxygen species (ROS) induced by nanoparticles in DCFH-DA-stained Raw264.7 cells (scale bar: 200 μm); Figure 11 In the experimental examples of the present invention: A: Volcano plot showing the expression profiles of differentially expressed genes (DEGs) related to macrophage polarization in Raw264.7 cells treated with PBS and FBFO@HM@aOPN; B: Gene Ontology Biological Process (GO BP) enrichment pathway analysis of DEGs between the PBS and FBFO@HM@aOPN treatment groups; Figure 12 In the experimental examples of the present invention: A: Gene Set Enrichment Analysis (GSEA) enrichment pathway analysis of DEGs between the PBS and FBFO@HM@aOPN treatment groups; B: Flow cytometry analysis of CD80 + CD86 + (M1 marker), C: MHC I + (antigen-presenting molecule), D: H-2Kb / SIINFEKL + (antigen-specific complex), and E: CD206 + (M2 marker) cell proportions; Figure 13 In the experimental examples of the present invention: A: Construction of a bone marrow-derived macrophage (BMDM) model; Flow cytometry analysis of CD80 in BMDMs treated with different formulations under 660 nm light + CD86 + (B), MHC I + (C), H-2Kb / SIINFEKL + (D), and CD206 + cell proportions (E); Figure 14 In the experimental examples of the present invention: A: GSEA enrichment analysis further validating the functional pathway differences of DEGs between the PBS and FBFO@HM@aOPN treatment groups; B: Western Blot detection of the phosphorylation levels of the STING-IRF3 pathway and NF-κB p65 in Raw264.7 cells and BMDMs treated with different formulations under 660 nm light; C: Schematic diagram of the mechanism by which FBFO@HM@aOPN reprograms macrophages to polarize towards the M1 type; Figure 15In the test examples of the present invention: A: Schematic diagram for constructing the in vitro blood-brain barrier (BBB) model; B: Changes in the transendothelial electrical resistance (TEER, unit: Ω·cm²) values at different time points after incubation with different preparations in the in vitro BBB model; C: Calculation of the transmembrane transport rate of different preparations after incubating the in vitro BBB model for 4 hours by detecting the Cy5.5 fluorescence intensities in the apical chamber and the basolateral chamber. Figure 16 In the test examples of the present invention: A: In vivo Cy5.5 fluorescence imaging of CT2A tumor-bearing mice injected with different preparations (at different time points after injection); Cy5.5 fluorescence imaging (B) and quantitative fluorescence intensity histograms (C) of excised CT2A tumor-bearing brain tissues and major organs (heart, liver, spleen, lung, kidney). Figure 17 In the test examples of the present invention: A: Experimental design for treating the CT2A tumor model (n = 5 mice per group); B: Representative bioluminescence imaging (left) and quantitative fluorescence intensity (right) of different treatment groups, and C: Kaplan-Meier survival curve analysis of CT2A tumor-bearing mice expressing luciferase; D: H&E staining of brain tumor tissues of CT2A tumor-bearing mice in different treatment groups (scale bar: 2 mm); Confocal laser scanning microscopy (CLSM) images showing the immunofluorescence staining results of KI67 (cell proliferation marker, scale bar: 50 μm, E), CD44 (tumor stem cell marker, scale bar: 20 μm, F), and CALR (calreticulin, scale bar: 20 μm, G) in tumor tissues of different treatment groups. Figure 18 In the test examples of the present invention: A: Flow cytometry analysis of F4 / 80 + CD45 + Macrophages on the left CD86 + (M1 phenotype marker), MHC I in the middle + (antigen-presenting molecule) and CD206 on the right + (M2 phenotype marker) cell ratio changes; Flow cytometry analysis of CD45 in CT2A tumor-bearing brain tissues treated with different preparations under 660 nm light + CD3 in cells + T cells (B), NK1.1 + CD3 - NK cells (C), CD3 + CD8 + Tumor-infiltrating T lymphocytes (TILs, D), GZMB +Percentage of (granzyme B-positive) TILs (E); Flow cytometry analysis of central memory T cells (TCM, CD3 + CD44 + CD62L + , F) and effector memory T cells (TEM, CD3 + CD44 + CD62L - , G) in the spleens of CT2A tumor-bearing mice treated with different formulations under 660 nm light illumination; Figure 19 In the experimental examples of the present invention: A: Bubble plot showing the interaction network of the PD1-PDL1 signaling axis activity (released by tumor-associated macrophages TAMs and malignant cells) and its receptors (expressed by lymphocyte subsets) in the treatment group and the PBS group; B: Schematic diagram of the experimental design of the CT2A glioblastoma (GBM) postoperative recurrence challenge model; Representative bioluminescence imaging (C) and fluorescence intensity quantification (D) of CT2A tumor-bearing mice expressing luciferase in different treatment groups under 660 nm light illumination; Figure 20 In the experimental examples of the present invention: A: Kaplan-Meier survival curve analysis; B: Flow cytometry analysis of CD45 + cells in CT2A tumor-bearing brain tissues treated with different formulations under 660 nm light illumination, showing the percentages of CD3 + T cells (left), IFN-γ + (interferon γ-positive) T cells (middle), and GZMB+ TILs (right). Detailed implementation manners

[0015] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0016] The present invention provides a multifunctional biomimetic nanoplatform FBFO@HM@aOPN, which includes a hybrid membrane and Fe3O4@BiFeO3 core-shell nanoparticles, and the hybrid membrane wraps the Fe3O4@BiFeO3 core-shell nanoparticles; the hybrid membrane includes exosomes derived from M1 macrophages and bacterial outer membrane vesicles; the surface of the hybrid membrane is modified with an anti-osteopontin antibody through an acid-sensitive linker.

[0017] The above-mentioned multifunctional biomimetic nanoplatform (FBFO@HM@aOPN) of the present invention solves the core problems in the treatment of glioblastoma (GBM), such as limited drug delivery, immunosuppression in the tumor microenvironment (TME), and low efficiency of photodynamic therapy (PDT), through multi-dimensional synergistic effects. The core of this multifunctional biomimetic nanoplatform is the Fe3O4@BiFeO3 (FBFO) double-metal core-shell nanoparticle. By doping Bi elements, the light absorption range is extended from the ultraviolet region to the near-infrared region, significantly improving the separation efficiency of photo-generated carriers. At the same time, it has dual activities of peroxidase-like (POD) and catalase-like (CAT), catalyzing H2O2 to generate ROS (•OH, •O2⁻) and continuously producing oxygen, forming a self-circulating oxygen supply system, breaking through the therapeutic limitations of traditional PDT in the hypoxic microenvironment. The piezoelectric effect driven by ultrasound further enhances the electron-hole separation and catalytic efficiency, providing efficient photodynamic killing ability for deep tumors.

[0018] To address the challenges of targeted delivery and immune-suppressive microenvironment remodeling, the multifunctional biomimetic nanoplatform provided by the present invention adopts a prokaryotic-eukaryotic hybrid membrane (HM) coating technology. By fusing M1 macrophage exosomes (M1EVs) with bacterial outer membrane vesicles (OMVs), the hybrid membrane integrates the inflammatory chemotactic function of M1EVs (targeting ICAM1 / VCAM1 on the surface of tumor cells / endothelial cells through integrin α4β1) and the immune activation characteristics of OMVs (activating the TLR4 signaling pathway through LPS). This design not only enables blood-brain barrier (BBB) penetration and active tumor targeting but also drives macrophages to polarize towards the pro-inflammatory M1 phenotype, enhancing antigen presentation ability and recruiting effector T cells. In addition, the outer layer is loaded with anti-OPN antibody (aOPN) through an acid-sensitive linker, which is precisely released in the acidic environment of the TME, blocking the OPN-CD44 / integrin signaling axis, disrupting the tumor immune barrier (TIB), softening the extracellular matrix (ECM), and reversing T cell exhaustion, significantly improving the nanoparticle permeability and lymphocyte infiltration efficiency.

[0019] At the level of the cooperative anti-tumor mechanism, FBFO@HM@aOPN induces lipid peroxidation of tumor cells through ROS, triggers ferroptosis and releases damage-associated molecular patterns (DAMPs), activating antigen-presenting cells (APCs) to enhance immunogenic cell death (ICD). Meanwhile, the activation of the TLR4 / NF-κB pathway by OMVs and the secretion of CXCL9 / 10 chemokines by M1EVs act synergistically to reprogram the phenotype of TAMs and recruit cytotoxic T cells and NK cells, forming a systemic anti-tumor immune response. Combining with immune checkpoint inhibitors (such as anti-PD-1 antibodies) can further reverse T cell exhaustion and inhibit postoperative recurrence. Compared with the existing technologies (such as single membrane modification schemes, Bioact Mater. 2024 May 21;39:206-223), the dual membrane fusion design of the present invention breaks through the limitation of single targeting and realizes the synergistic effects of TME hypoxia alleviation, matrix degradation and immunosuppressive factor blockade through multi-pathway immune regulation (TLR4 / NF-κB, cGAS-STING, OPN blockade).

[0020] The core advantage of the technical solution of the present invention lies in constructing a "catalysis-delivery-immunity" trinity treatment framework. The Fe3O4@BiFeO3 nanozyme improves the oxygen circulation efficiency and ROS generation through dual enzyme cascade reactions and piezoelectric effects, overcoming the dependence of traditional photosensitizers on high-oxygen environments; the combination of the hybrid membrane and anti-OPN antibody achieves double breakthroughs in targeted penetration and matrix remodeling, solving the obstacle of dense ECM to drug penetration; while the synergistic effect of ferroptosis-induced ICD and TAM reprogramming forms a continuous anti-tumor response by activating long-term immune memory (CD44 + CD62L + T cells). This multi-modal strategy provides an innovative treatment paradigm for invasive solid tumors with deep penetration, immune microenvironment remodeling and systemic immune activation, showing significant clinical application potential.

[0021] The present invention also provides a preparation method of the above-mentioned multifunctional biomimetic nanoplatform FBFO@HM@aOPN, which includes the following steps: Prepare Fe3O4@BiFeO3 core-shell nanoparticles; Prepare bacterial outer membrane vesicles and azide-modified M1 macrophage-derived exosomes; Mix the bacterial outer membrane vesicles with azide-modified M1 macrophage-derived exosomes, ultrasonicate and freeze-thaw to obtain a hybrid membrane, and then mix it with Fe3O4@BiFeO3 core-shell nanoparticles and extrude to obtain Fe3O4@BiFeO3 core-shell nanoparticles FBFO@HM wrapped with the hybrid membrane; The FBFO@HM is coupled with the osteopontin antibody modified with alkynyl and acid-sensitive linker through click reaction to obtain the multifunctional biomimetic nanoplatform FBFO@HM@aOPN.

[0022] In the present invention, the preparation method of the Fe3O4@BiFeO3 core-shell nanoparticles is as follows: The Fe3O4 nanoparticles are dispersed in a bismuth salt solution, and a solvothermal reaction is carried out at 150~250 °C to obtain the product. The present invention does not impose special restrictions on the selection of the bismuth salt, which can be bismuth nitrate, bismuth sulfate, bismuth chloride, etc. or their hydrates. In one or more embodiments of the present invention, the bismuth salt is selected from bismuth nitrate pentahydrate. The bismuth salt solution contains a bismuth salt, a dispersant and a solvent, and the present invention does not impose special restrictions on the types of the dispersant and the solvent.

[0023] In the present invention, the preparation method of the M1 macrophage-derived exosomes modified with azide groups is as follows: RAW264.7 cells are stimulated with LPS and azido-choline in combination, cultured in an exosome-free medium, and then the supernatant is collected and the M1 macrophage-derived exosomes modified with azide groups are obtained through separation and purification. The present invention does not impose special restrictions on the above-mentioned culture method and the specific methods of separation and purification, and the commonly used methods in the art can be adopted.

[0024] In the present invention, the preparation method of the bacterial outer membrane vesicles is as follows: Escherichia coli is cultured to the logarithmic growth phase, and the bacterial outer membrane vesicles are obtained through differential centrifugation and purification. The present invention does not impose special restrictions on the specific culture method and the differential centrifugation and purification method, and the commonly used methods in the art can be adopted.

[0025] In the present invention, the preparation method of the osteopontin antibody modified with alkynyl and acid-sensitive linker is as follows: The osteopontin antibody is reacted with benzaldehyde-polyethylene glycol-activating ester (BD-PEG2000-NHS, Benzaldehyde-PEG2000-NHS), and then dibenzocyclooctene-polyethylene glycol-amine (DBCO-PEG-NH2) is added for reaction and purification to obtain the osteopontin antibody modified with alkynyl and acid-sensitive linker. The present invention does not impose special restrictions on the specific reaction time and purification method, and the conditions of the coupling reaction and the purification method commonly used in the art can be adopted.

[0026] In the present invention, the mass ratio of the hybrid membrane, the Fe3O4@BiFeO3 core-shell nanoparticles and the osteopontin antibody modified with alkynyl and acid-sensitive linker is 100 : (40~60) : (2~5).

[0027] In the present invention, the temperature of the click reaction is 2~10 °C, more preferably 3~5 °C; the reaction time is 8~15 h.

[0028] The present invention also provides the use of the multifunctional bionic nanoplatform FBFO@HM@aOPN or the multifunctional bionic nanoplatform FBFO@HM@aOPN prepared by the above preparation method in the preparation of anti-tumor drugs. Further preferably, the tumor is glioblastoma.

[0029] The technical solution of the present invention is further described below in conjunction with specific examples. The present invention has no special restrictions on the source of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used. In the following examples, BD-PEG2000-NHS represents benzaldehyde-polyethylene glycol-active ester, which was purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0030] Example 1 This example provides the preparation of FBFO@HM@aOPN nanoparticles.

[0031] 1. Synthesis of Fe3O4 nanoparticles (Fe3O4NPs) Iron oleate was dissolved in 200 mL of diphenyl ether and 50 mL of oleyl alcohol, and the mixture was stirred and heated to 200°C and reacted for 30 minutes under nitrogen protection. After the reaction, the product was centrifuged, washed with ethanol three times, and dried to obtain a brown powder.

[0032] 2. Synthesis of Fe3O4@BiFeO3 (FBFO) core-shell nanoparticles FBFO nanoparticles were prepared by solvothermal method. The preparation schematic diagram is shown in Figure 1 As shown in A, the specific method is as follows: (1) Bismuth nitrate pentahydrate (2 mmol) and polyvinylpyrrolidone (PVP, 8 μmol) were dispersed in 20 mL of ethylene glycol and magnetically stirred (600 rpm, 120 min) to form a homogeneous solution.

[0033] (2) Disperse Fe3O4 nanoparticles (2 mmol) in 20 mL of ethylene glycol and add the above solution with magnetic stirring (800 rpm, 120 min).

[0034] (3) The mixed solution was transferred to a polytetrafluoroethylene-lined stainless steel autoclave and maintained at 200°C for 20 hours. After the reaction, the product was collected and washed three times with ethanol and deionized water.

[0035] Transmission electron microscopy (TEM) showed that the average diameter of FBFO NPs was about 30 nm ( Figure 2 A in the figure), scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray spectroscopy (EDX) and energy dispersive spectroscopy (EDS) further confirmed the presence of Bi, Fe and O elements in FBFO NPs ( Figure 2in B). The X-ray diffraction (XRD) pattern ( Figure 2 in C) verified the crystal structure of FBFO NPs. The above analyses jointly confirmed the successful synthesis of FBFO NPs. The optical properties of FBFO NPs are crucial for determining their photocatalytic activity. The ultraviolet-visible / near-infrared (UV-Vis / NIR) spectrum showed that the doping of Bi with Fe3O4 NPs broadened the light absorption range of FBFO ( Figure 2 in D), improved the separation efficiency of photo-generated carriers, and significantly enhanced the photocatalytic activity of the nanomaterials.

[0036] According to Figure 2 the schematic diagram of the mechanism in E, the 660 nm laser irradiation can drive the efficient separation of hole (h + )-electron (e - ) pairs in FBFO NPs (Equation 1), and then react with water and oxygen to generate abundant hydroxyl radicals (•OH) and superoxide radicals (•O2 - ) respectively for type I photodynamic therapy (PDT) (Equations 2 and 3). Meanwhile, the transformation of Fe 2+ to Fe 3+ mimics the peroxidase (POD) activity, further promoting the conversion of H2O2 to •OH and enhancing the generation of reactive oxygen species (ROS). In addition, the generated holes (h + ) can also oxidize H2O2 to produce O2 (mimicking catalase CAT activity), alleviating the hypoxic tumor microenvironment (TME) and providing a continuous oxygen supply for PDT.

[0037] 3. Preparation of M1 macrophage-derived exosomes (M1EVs) (1) Azide-modified exosomes: RAW264.7 cells (1×10 6 ) were stimulated with 200 nM LPS and 200 μM azido-choline (AECho) for 24 hours, and then the exosome-free medium (DMEM containing exosome-depleted fetal bovine serum) was replaced and cultured for another 48 hours.

[0038] (2) Isolation and purification of exosomes: The supernatant was collected and centrifuged at 300 g (10 minutes) and 2000 g (20 minutes) in sequence to remove cells and debris; After filtration through a 0.22 μm filter membrane, extracellular vesicles were removed by centrifugation at 10,000 g (30 minutes, 4 °C); Finally, M1EVs were obtained by ultracentrifugation (100,000 g, 70 minutes, SW32 Ti rotor).

[0039] The schematic diagram of the preparation process is shown in Figure 1As shown in B of

[0040] (3)Identification: Morphology and particle size: Observed by transmission electron microscopy (TEM); Verification of azide group labeling: Detected by click chemistry reaction (DBCO-Cy5.5 fluorescence labeling) combined with flow cytometry; Concentration and particle size distribution: Nanoparticle tracking analysis (NTA, Zeta View PMX 110).

[0041] Azide-choline, as a substrate for cell synthesis of phosphatidylcholine (a key component of cell membrane), was co-incubated with Raw264.7 cells (MΦ) in this example to obtain M1 macrophage membranes modified with azide groups (N3). M1EVs were isolated from the culture supernatant of N3-modified M1 macrophages by ultracentrifugation, and flow cytometry was used to verify that the surface of M1EVs carried N3 groups ( Figure 3 as shown in A of

[0042] 4. Preparation of bacterial outer membrane vesicles (OMVs) To prepare OMVs with low endotoxin side effects, the Escherichia coli strain W3110 derived from K12 was genetically engineered.

[0043] (1)Strain: ΔmsbB / ΔpagP mutant Escherichia coli W3110 (purchased from Biosio Company).

[0044] (2)OMV extraction (differential centrifugation method): The bacteria were cultured overnight at 37 °C on LB solid medium. Single colonies were picked and inoculated into liquid LB medium. After culturing on a shaker at 37 °C for 6 hours, they were transferred and expanded at a ratio of 1:100 to the logarithmic growth phase.

[0045] Centrifugation (5,000 g, 30 minutes) was used to remove the bacteria, and the supernatant was filtered through a 0.45 μm filter membrane and concentrated using a 100 kDa ultrafiltration centrifugal tube; Ultracentrifugation (150,000 g, 3 hours, 4 °C) was performed, and the precipitate was resuspended in PBS and centrifuged twice for purification.

[0046] The schematic diagram of the preparation process is as shown in Figure 1 B of

[0047] 5. Construction and characterization of FBFO@HM@aOPN nanoparticles (1)Coating FBFO particles with a hybrid membrane (HM) (FBFO@HM): OMVs and Cy5.5-labeled M1EVs were mixed at a concentration ratio of 1:1, stirred at 37 °C for 2 hours, sonicated for 10 minutes, and subjected to 5 cycles of liquid nitrogen freezing and thawing to promote membrane fusion.

[0048] The FBFO particles and the fusion membrane were mixed at a mass ratio of 1:2 and extruded 10 times through a liposome extruder (100 nm polycarbonate filter membrane) to form FBFO@HM coated with HM. As shown in B of Figure 3 , CLSM confirmed that the surface of FBFO@HM NPs was coated with N3-modified M1EVs. The fusion characteristics of the hybrid membrane were demonstrated by dual-color fluorescence colocalization of DiO-labeled M1EVs and DiI-labeled OMVs ( Figure 3 C in Figure 3 ). Western blot analysis further showed that the hybrid membranes HMs retained the signature proteins of M1EVs (TSG101, CD63, CD81, integrin α4β1, and chemokine receptor CXCR4) and the FtsZ protein of OMVs (

[0049] (2) Preparation of antibody-conjugated (aOPN-PEG2000-DBCO): Anti-osteopontin antibody (aOPN) and BD-PEG2000-NHS (molar ratio 1:100) were reacted in PBS at 4 °C for 4 hours, and unreacted substances were removed by ultrafiltration centrifugation (10,000 g, 10 minutes); Sulfo-DBCO-PEG4-amine (molar ratio 1:100) was added and reacted in citrate buffer (pH 6.0) for 3 hours, and aOPN-PEG2000-DBCO was obtained after ultrafiltration purification. The preparation process is shown in C of Figure 1 .

[0050] The benzaldehyde-PEG2000-NHS linker (BD-PEG2000-NHS) reacted with the amino terminus of the aOPN antibody to generate aOPN-PEG2000-BD, and then formed an acid-sensitive benzylimine bond with DBCO-PEG5-NH2, and finally aOPN-PEG2000-DBCO was synthesized to achieve the controlled release of aOPN in the acidic TME at pH 6.5.

[0051] (3) Preparation and verification of FBFO@HM@aOPN: aOPN-PEG2000-DBCO and FBFO@HM (2.4 μg antibody was added per 100 μg HM) were subjected to a click reaction overnight in PBS at 4 °C, and free antibodies were removed by centrifugation (11,000 g, 20 minutes) to obtain the final product.

[0052] Fluorescence colocalization verification: CLSM observed the confocal signals of DiI-labeled HM and Cy5.5-DBCO-labeled aOPN; Morphology and particle size: TEM analysis; Antibody sustained-release experiment: Dialyze in PBS (pH 7.4 or 6.5), detect the concentration of aOPN in the dialysis solution at different time points by ELISA, and draw a standard curve to calculate the release amount.

[0053] The co-localization of Dil-labeled HM and Alexa Fluor 647-labeled aOPN ( Figure 4 A in Figure 4 ), and the core-shell spherical structure shown by TEM ( Figure 5 B in Figure 6 ) verified the successful coupling. ELISA detection of the aOPN level in the dialysis solution showed that the antibody release amount under the condition of pH 6.5 was significantly higher than that at pH 7.4 ( Figure 5 A in Figure 5 ). The ESR spectrum ( C in

[0054] ) and the MB degradation experiment ( B in ) confirmed that FBFO@HM@aOPN NPs retained the ability to produce ROS (•OH and •O2⁻) by photodynamic action, and could catalyze the decomposition of H2O2 into O2 at different pH values (

[0055] C in ). The above results indicated that FBFO@HM@aOPN NPs had enhanced photo-induced dual-enzyme activities, providing a novel biohybrid delivery system for optimizing the PDT efficacy.

[0056] 1. Evaluation of ROS and O2 generation ability The ROS and O2 generation abilities of FBFO NPs under 660 nm laser (0.5 W / cm²) irradiation were evaluated by methylene blue (MB) degradation experiment and dissolved oxygen meter detection: compared with the Fe3O4 group and other control groups, the MB absorbance of the FBFO group and the combined laser irradiation group decreased significantly, and the O2 production increased significantly ( Figure 6 A, B in Figure 6in C). In summary, FBFO NPs exhibit excellent photocatalytic ROS and O2 production capabilities, showing significant potential for PDT anti-tumor applications.

[0057] 2. Evaluation of in vitro catalytic activity and therapeutic effect To evaluate the PDT efficacy of FBFO@HM@aOPN NPs, in vitro experiments were conducted using the CT2A glioblastoma (GBM) cell line as a model. The experimental subjects were divided into seven groups (I: PBS control, II: aOPN antibody, III: FBFO, IV: FBFO@OMV, V: FBFO@M1EV, VI: FBFO@HM, VII: FBFO@HM@aOPN). Molecules such as integrin α4β1 expressed on the surface of M1EV membranes can interact with ligands of endothelial cells (highly expressing ICAM1 / VCAM1) and GBM cells, promoting the nanoparticles to cross the blood-brain barrier (BBB) and actively target tumors. The endocytosis efficiency of FBFO labeled with Cy5.5 was evaluated: the Cy5.5 fluorescence intensity (FI) of the FBFO@HM@aOPN group was the highest, and anti-ICAM-1 / VCAM1 antibodies significantly reduced its endocytosis rate ( Figure 7 in A), indicating that M1EVs endow NPs with the ability to actively target tumor cells.

[0058] The generation of intracellular ROS was detected using the DCFH-DA probe. The results showed that the green fluorescence intensity of the FBFO@HM@aOPN group was slightly higher than that of the control, while the fluorescence intensity of its combined laser irradiation group was the strongest among all groups ( Figure 7 in B), confirming that the nanoparticles target and deliver FBFO to tumor cells with the help of M1EVs, and trigger ROS bursts under laser induction, enhancing the sensitivity of cancer cells to oxidative damage. The CCK8 experiment showed that the killing rate of the FBFO@HM@aOPN combined laser irradiation group against GBM cells was significantly higher than that of other groups ( Figure 8 in A). The hypoxic microenvironment affects the malignant progression of tumors by regulating tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT). Further findings showed that the cell inhibition rate of the FBFO@HM@aOPN group under normoxic conditions was much higher than that under hypoxic conditions ( Figure 8 in B); however, even under hypoxic conditions, the combined laser irradiation group still significantly reduced the cell survival rate ( Figure 8 in C), indicating that its PDT effect can break through the limitation of the hypoxic microenvironment.

[0059] The massive accumulation of ROS (such as ·OH) can induce membrane lipid peroxidation and trigger ferroptosis of cells. By detecting the level of malondialdehyde (MDA), an early marker of ferroptosis, it was found that the treatment with FBFO@HM@aOPN significantly increased lipid peroxidation, and laser irradiation further exacerbated this phenomenon ( Figure 8in D). Morphological observation showed that cells presented ferroptosis characteristics such as swelling, blistering until rupture ( Figure 8 in E). Ferroptosis, as a type of immunogenic cell death (ICD), is accompanied by the release of damage-associated molecular patterns (DAMPs), including calreticulin (CRT) on the cell membrane surface, high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP). Experimental verification showed that the expression of CRT on the cell membrane in the FBFO@HM@aOPN group was significantly upregulated ( Figure 8 in E), and it promoted the secretion of ATP and HMGB1 ( Figure 9 in A and B), suggesting that it could release tumor antigens by inducing ICD, activate antigen-presenting cells (APCs), and further promote T cell infiltration and activation ( Figure 9 in C), laying a foundation for the combined treatment with immune checkpoint inhibitors (ICIs).

[0060] 3. FBFO@HM@aOPN NPs significantly induced the polarization of M0 and M2 phenotype macrophages into M1 type in vitro Fe3O4 NPs is a highly potential regulator for the polarization of tumor-associated macrophages (TAMs), which can activate the downstream NF-κB pathway (the key pathway for M1 type polarization) by inducing an increase in the intracellular ROS level. As highly immunogenic vesicles, the lipopolysaccharide (LPS) on the surface of OMVs can strongly stimulate macrophages to polarize into the M1 phenotype. Based on this, we systematically explored the effects of FBFO@HM@aOPN NPs on macrophage polarization and function. It is worth noting that integrin α4β1 on the surface of M1EV mediates its specific targeting to GBM cells through the interaction of ICAM1 / VCAM1 ( Figure 3 in D). To verify whether FBFO@HM@aOPN NPs could enhance the endocytosis of FBFO by macrophages through integrin α4β1-ICAM1 / VCAM1 targeting, macrophages (MΦ) were treated with Cy5.5-labeled FBFO NPs. The results showed that the Cy5.5 fluorescence intensity in the NPs group wrapped with HM was significantly enhanced, while the neutralizing antibodies against ICAM1 / VCAM1 could block this effect ( Figure 10 in A), indicating that M1EVs promoted the delivery of NPs to macrophages through targeting. In addition, the DCFH-DA probe showed that the NPs wrapped with HM triggered the strongest ROS fluorescence signal under laser irradiation ( Figure 10 in B), confirming that FBFO@HM@aOPN could efficiently deliver FBFO into macrophages and generate ROS.

[0061] Transcriptome differences in MΦ cells between the PBS group and the FBFO@HM@aOPN group were analyzed by RNA sequencing (RNA-seq), and 4,161 differentially expressed genes (DEGs) were found between the two groups, among which 1,566 genes were upregulated and 2,595 genes were downregulated. The volcano plot showed that the M1 macrophage markers (Cd86, Cd80, Tnf, Il6) were significantly upregulated, while the M2 markers (Cd206, Arg1 encoded by the Mrc1 gene) were significantly downregulated ( Figure 11 A in). Gene ontology (GO) enrichment analysis showed that the upregulated genes were mainly enriched in innate immune responses, type I (IFN-β) and type II (IFN-γ) interferon responses, positive regulation of immune effector processes, and the classical NF-κB signaling pathway; the downregulated genes were related to extracellular matrix remodeling ( Figure 11 B in). Gene set enrichment analysis (GSEA) further showed that the antigen presentation pathway and the T cell chemotaxis pathway were significantly enriched in the upregulated genes ( Figure 12 A in), suggesting that FBFO@HM@aOPN NPs promoted M1 polarization and T cell recruitment by regulating immune-related pathways.

[0062] Flow cytometry analysis showed that compared with the control group, both the FBFO@OMV and FBFO@M1EV groups significantly upregulated the M1 markers (CD80, CD86), and the FBFO@HM@aOPN group had the strongest effect ( Figure 12 B in), verifying its ability to promote M0→M1 polarization. During antigen cross-presentation, tumor antigens are taken up by APCs (such as macrophages) and presented to MHC-I molecules to activate CD8 + T cells. Flow cytometry results showed that FBFO@HM@aOPN significantly promoted the expression of MHC-I molecules in macrophages ( Figure 12 C in); using CT2A-OVA cells as a model, the MHC-I-related SIINFEKL peptide segment was significantly upregulated in stimulated macrophages ( Figure 12 D in), laying a foundation for antigen cross-presentation and T cell activation.

[0063] The reprogramming ability of FBFO@HM@aOPN on M2 macrophages was further evaluated: after inducing M2 macrophages with CT2A conditioned medium and adding different preparations for treatment, the results showed that FBFO@HM@aOPN significantly reduced the proportion of the Cd206 marker ( Figure 12 E in), confirming its effective reversal of M2→M1 polarization. To verify the universality, bone marrow-derived macrophages (BMDMs) were isolated and cultured from mouse bone marrow ( Figure 13 A in). Flow cytometry and qPCR analysis showed that FBFO@HM@aOPN significantly upregulated the expression of M1 markers and antigen presentation-related genes and downregulated CD206 (Figure 13 B, C, D, and E) in [reference] indicate that it has an efficient polarization regulation effect on the M0 / M2 phenotype.

[0064] At the mechanistic level, GSEA indicated that the upregulated genes in the FBFO@HM@aOPN treatment group were significantly enriched in the cGAS-STING-mediated cytoplasmic DNA sensing pathway ( Figure 14 A in [reference]). Western blot results showed that FBFO@HM@aOPN promoted STING phosphorylation (p-STING) and its downstream IRF3 transcriptional activity ( Figure 14 B in [reference]). The inhibition of the STING pathway in the hypoxic TME could be partially reversed by the CAT activity (producing O2) of FBFO. Meanwhile, the NF-κB and Toll-like receptor signaling pathways were significantly enriched among the upregulated genes ( Figure 11 B in [reference], Figure 14 A in [reference]), and the phosphorylation level of p65 in the FBFO@HM@aOPN treatment group increased ( Figure 14 B in [reference]). In summary, FBFO@HM@aOPN NPs drive M1 polarization by synergistically activating the cGAS-STING and NF-κB pathways ( Figure 14 C in [reference]), enhance the antigen presentation ability of TAMs, relieve TME immunosuppression, and provide a new strategy for enhancing the in vivo anti-tumor immune response.

[0065] 3. Evaluation of in vivo biodistribution, biocompatibility, and anti-tumor effect To verify the BBB penetration ability of the FBFO@HM@aOPN biohybrid delivery system, a Transwell co-culture system was used (the upper chamber was bEnd.3 endothelial cells, and the lower chamber was bone marrow-derived macrophages BMDMs and CT2A tumor cells, Figure 15 A in [reference]). The transendothelial electrical resistance (TEER) value of the bEnd.3 monolayer cells was 200 - 300 Ω·cm², indicating barrier integrity ( Figure 15 B in [reference]). Experiments showed that the BBB penetration ability of FBFO@HM@aOPN NPs was comparable to that of FBFO@M1EV NPs, while anti-ICAM1 / VCAM1 antibodies could significantly inhibit their penetration efficiency ( Figure 15 C in [reference]), confirming that its targeting penetration through the BBB was mediated by ICAM1 / VCAM1.

[0066] The dynamic distribution of the nanoformulation in vivo was then analyzed. To verify whether M1EV membrane encapsulation can enhance the accumulation of FBFO in glioma tissue, the Cy5.5-labeled FBFO system was injected into luciferase-labeled CT2A (CT2A-Luc) tumor-bearing mice, and the Cy5.5 fluorescence signal was monitored in real time. The results showed that the FBFO@HM@aOPN group showed a strong fluorescence signal in the brain 6 hours after injection and lasted until 48 hours; the FBFO@M1EV or FBFO@HM group also showed a similar trend ( Figure 16 In vitro fluorescence imaging and quantitative analysis showed that the accumulation of FBFO@HM@aOPN NPs in tumor-bearing brain tissue was significantly higher than that in other organs (heart, liver, spleen, lung, and kidney), verifying its tumor targeting properties ( Figure 16 B, C in it).

[0067] To further evaluate the anti-tumor efficacy in vivo, the animals were divided into 7 groups ( Figure 17 A in Figure 2), and tumor growth was monitored by bioluminescence imaging. The results showed that the tumor in the PBS group progressed rapidly, the monotherapy group showed a moderate inhibitory effect, and the tumor growth in the FBFO@HM@aOPN combined with laser irradiation group was significantly limited ( Figure 17 B and C in the figure are attributed to the synergistic effect of direct PDT killing and TME regulation. After the mice were killed at the end of the experiment, hematoxylin-eosin (H&E) staining of tumor tissues showed obvious tumor cell necrosis in the FBFO@HM@aOPN group, and the expression of Ki67 (proliferation marker) and CD44 (extracellular matrix marker) was significantly reduced ( Figure 17 D, E, and F in Figure 4) confirmed its ability to inhibit tumor growth and matrix remodeling.

[0068] Immunofluorescence imaging showed that the exposure of tumor cell membrane calreticulin (CALR) was significantly increased in the FBFO@HM@aOPN group ( Figure 17 G in the figure verifies its successful induction of immunogenic cell death (ICD) in vivo. In summary, FBFO@HM@aOPN NPs achieves precise delivery through efficient penetration of the BBB and active targeted accumulation, and has the dual functions of PDT killing and TME immune remodeling, showing excellent anti-tumor efficacy and safety in vivo.

[0069] 4. FBFO@HM@aOPN enhances anti-tumor immune effect in vivo At the end of anti-tumor treatment, tumor tissues were collected and the dynamics of tumor-infiltrating immune cells were analyzed by flow cytometry. The results showed that the proportion of M1 anti-tumor macrophages and the level of cross-presented antigens in TAMs in the FBFO@HM@aOPN group were significantly increased, while the proportion of tumor-promoting M2 macrophages was significantly decreased ( Figure 18in A), further confirming that it activates antigen-specific T cell immune responses by regulating the antigen presentation ability of TAMs.

[0070] To analyze the anti-tumor immune activation effect, we detected the changes in tumor-infiltrating lymphocytes. Flow cytometry results showed that the proportion of T cells (CD3 + ) in the tumor tissues of the FBFO@HM@aOPN group increased significantly ( Figure 18 in B), and was consistent with the scRNA results. The proportions of anti-tumor cytotoxic lymphocytes (including NK cells and CTLs) were both significantly upregulated ( Figure 18 in C-D). In addition, the proportion of granzyme B (GZMB)-positive CTLs was significantly increased in the FBFO@HM@aOPN group ( Figure 18 in E), confirming that this nanoplatform can efficiently activate the tumor-killing potential of CTLs.

[0071] Long-term immune memory is crucial for inhibiting tumor recurrence. By detecting the proportions of central memory T cells (TCM, CD3 + CD44 + CD62L + ) and effector memory T cells (TEM, CD3 + CD44 + CD62L - ) in the spleen, it was found that the proportions of both types of cells increased significantly after FBFO@HM@aOPN treatment ( Figure 18 in F-G), indicating that photodynamic immunotherapy can induce a persistent immune memory effect, providing key protection for preventing tumor recurrence.

[0072] 5. Inhibition of postoperative recurrence of glioblastoma by the combination therapy of FBFO@HM@aOPN and immune checkpoint blockade (ICB) In clinical practice, surgical intervention is widely regarded as the main treatment method for early-stage solid tumors. However, due to the presence of residual tumor cells after surgery, tumor recurrence occurs around the resected lesions in almost all glioblastoma (GBM) patients. Data show that FBFO@HM@aOPN can promote the transcription of interferon-stimulated genes, and interferon-γ (IFN-γ) and type I interferon (IFN-I) can upregulate the expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and monocyte-derived macrophages (MDMs), thereby enhancing the immune escape ability of tumor cells. As expected, single-cell interaction data analysis showed that during FBFO@HM@aOPN treatment, the interaction between tumor cells and MDM with T cells through the PD-L1-PD1 pathway was significantly enhanced ( Figure 19 in A). Based on this, we further explored whether the combination of FBFO@HM@aOPN and anti-PD1 inhibitor (αPD1 antibody) could delay tumor recurrence.

[0073] The surgical operation procedures and treatment regimens are shown in Figure 19 B in it. The tumor recurrence was monitored by the bioluminescence (BLI) signal of luciferase-labeled CT2A cells (Luci+). The results showed that the BLI signal in the FBFO@HM@aOPN monotherapy group was significantly reduced ( Figure 19 C, D in it). When combined with the anti-PD1 (αPD1) antibody, the tumor suppression effect was further enhanced ( Figure 19 C, D in it). In addition, the survival rate of the mice in the FBFO@HM@aOPN combined with αPD1 antibody group was significantly higher than that of the other treatment regimen groups ( Figure 20 A in it), and all the mice survived on the 100th day of treatment ( Figure 20 A in it).

[0074] Further analysis of tumor-infiltrating lymphocytes in each group found that in the FBFO@HM@aOPN monotherapy group and the combined treatment group, the proportions of T cells, IFN-γ-positive T cells, and granzyme B (GZMB)-positive cytotoxic T lymphocytes (CTLs) were all significantly increased ( Figure 20 B in it). It is worth noting that the FBFO@HM@aOPN combined with αPD1 antibody treatment group had the strongest resistance to tumor re-challenge. The above data indicate that the combined application of FBFO@HM@aOPN and ICB therapy is a highly personalized treatment strategy that can effectively prevent tumor recurrence.

[0075] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A multifunctional bionic nanoplatform FBFO@HM@aOPN, characterized in that, It includes a hybrid membrane and Fe3O4@BiFeO3 core-shell nanoparticles, and the hybrid membrane wraps the Fe3O4@BiFeO3 core-shell nanoparticles; the hybrid membrane includes M1 macrophage-derived exosomes and bacterial outer membrane vesicles; the surface of the hybrid membrane is modified with an anti-osteopontin antibody through an acid-sensitive linker.

2. The preparation method of the multifunctional bionic nanoplatform FBFO@HM@aOPN according to claim 1, characterized in that, It includes the following steps: Prepare Fe3O4@BiFeO3 core-shell nanoparticles; Prepare bacterial outer membrane vesicles and M1 macrophage-derived exosomes modified with azide groups; Mix the bacterial outer membrane vesicles with the M1 macrophage-derived exosomes modified with azide groups, perform ultrasonic treatment and freeze-thawing to obtain a hybrid membrane, and then mix it with the Fe3O4@BiFeO3 core-shell nanoparticles and extrude to obtain Fe3O4@BiFeO3 core-shell nanoparticles wrapped by the hybrid membrane, FBFO@HM; Couple the FBFO@HM with an anti-osteopontin antibody modified with an alkyne group and an acid-sensitive linker through a click reaction to obtain the multifunctional biomimetic nanoplatform FBFO@HM@aOPN.

3. The preparation method according to claim 2, characterized in that, The preparation method of the Fe3O4@BiFeO3 core-shell nanoparticles is to disperse the Fe3O4 nanoparticles in a bismuth salt solution and perform a solvothermal reaction at 150-250°C to obtain them.

4. The preparation method according to claim 2, characterized in that, The preparation method of the M1 macrophage-derived exosomes modified with azide groups is as follows: After RAW264.7 cells are stimulated with LPS and azido-choline in combination, they are cultured in an exosome-free medium, and then the supernatant is collected and the M1 macrophage-derived exosomes modified with azide groups are obtained through separation and purification.

5. The preparation method according to claim 2, characterized in that, The preparation method of the bacterial outer membrane vesicles is: Culture Escherichia coli to the logarithmic growth phase and obtain the bacterial outer membrane vesicles through differential centrifugation and purification.

6. The preparation method according to claim 2, characterized in that, The preparation method of the anti-osteopontin antibody modified with an alkyne group and an acid-sensitive linker is: React the anti-osteopontin antibody with benzaldehyde-polyethylene glycol-active ester, and then add dibenzocyclooctene-polyethylene glycol-amine, react and purify to obtain the anti-osteopontin antibody modified with an alkyne group and an acid-sensitive linker.

7. The preparation method according to claim 2, characterized in that, The mass ratio of the hybrid membrane, Fe3O4@BiFeO3 core-shell nanoparticles and the anti-osteopontin antibody modified with an alkyne group and an acid-sensitive linker is 100 : (40-60) : (2-5).

8. The preparation method according to claim 2, wherein, The temperature of the click reaction is 2-10°C, and the reaction time is 8-15 h.

9. The application of the multifunctional biomimetic nanoplatform FBFO@HM@aOPN described in claim 1 or the multifunctional biomimetic nanoplatform FBFO@HM@aOPN prepared by the preparation method described in any one of claims 2-8 in the preparation of anti-tumor drugs.

10. The application according to claim 9, characterized in that, The tumor is glioblastoma.

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