A nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy and its preparation method

Abstract

The present invention relates to the field of biomedical technology, and in particular to a nano-diagnostic and therapeutic preparation for multimodal imaging and cancer combined immunotherapy and a preparation method thereof, wherein the nano-diagnostic and therapeutic preparation comprises: a hypoxia-activated probe, a vascular disruptor and a metal organic framework, wherein the hypoxia-activated probe and the vascular disruptor are jointly loaded on the metal organic framework; wherein the metal organic framework is a metal organic framework that can be degraded under acidic conditions; and the preparation method of the nano-diagnostic and therapeutic preparation comprises the following steps: preparation of the hypoxia-activated probe; preparation of the metal organic framework loaded with the hypoxia-activated probe and the vascular disruptor; and covering the surface of the metal organic framework with an immune cell membrane. The nano-diagnostic and therapeutic preparation provided by the present invention proposes a new strategy for the treatment of tumors, realizes the diagnostic and therapeutic performance of hypoxia activation and controllable drug release, and provides a new solution for realizing precise imaging-guided tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a nano-diagnostic and therapeutic preparation for multi-modal imaging and cancer combined immunotherapy and a preparation method thereof. Background Art

[0002] Although great progress has been made in the diagnosis and prognosis of cancer in the past few decades due to advances in early detection methods, surgical techniques, and advanced diagnostic and therapeutic technologies, cancer remains a major public health problem worldwide. Therefore, combining efficient comprehensive disease diagnosis and treatment strategies is crucial to improving cancer treatment outcomes and overall survival. However, most of the commonly used imaging techniques currently have inherent advantages and disadvantages and scope of application. For example, fluorescence (FL) imaging technology has high sensitivity and low price, but is limited in penetration depth and spatial resolution. Photoacoustic (PA) imaging has good spatial resolution and tissue penetration ability, but low sensitivity. Recently reported fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) can reduce tissue scattering and autofluorescence, and has improved penetration and resolution. Therefore, applying NIR-II FL / PA dual-mode imaging to tumor diagnosis is an attractive strategy. However, NIR-II FL / PA dual-mode imaging of tumors still faces challenges. First, achieving the maximum brightness of NIR-II FL and PA signals simultaneously is a difficult task because they correspond to the radiative and non-radiative channels, respectively. Second, the lack of disease-specific activation of the “always-on” NIR-II FL / PA signals hinders their role in selectively distinguishing normal and pathological tissues. Therefore, it is urgent to develop activatable probes that can achieve “light-on” NIR-II FL / PA dual-mode signals for specific pathological characteristic microenvironments to improve the sensitivity and accuracy of disease diagnosis.

[0003] Recently, phototherapy strategies such as photothermal therapy (PTT) and photodynamic therapy (PDT) have attracted much attention due to their non-invasiveness, low toxicity, high spatiotemporal specificity, and excellent clinical performance. In addition, phototherapy can also induce cancer cells to release a large number of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) through immunogenic cell death (ICD), which can promote the maturation of dendritic cells (DC) and facilitate antigen presentation to T cells. In addition to ICD, pattern recognition receptor (PRR) stimulators have also become effective tools for activating the tumor immune system. Activation of the cyclic guanosine monophosphate-adenosinemonophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway can induce the production of type I interferons and other immune mediators, enhance innate immune responses and anti-tumor immune cycles. Recent studies have found that PDT and PTT can disrupt the redox balance in cells, leading to mitochondrial and nuclear DNA damage and even subsequent leakage into the cytoplasm. A large number of studies have shown that these leaked mitochondrial and nuclear DNA can effectively activate the cGAS-STING signaling pathway, thereby stimulating anti-tumor immune responses. Although PDT and PTT are expected to activate both ICD and cGAS-STING pathways, current phototherapy usually faces some challenges. First, most of the reported photosensitizers are in a "normally on" state regardless of whether they are located in tumor lesions. Second, among various photosensitizers, organic molecules have the advantages of simple composition, clear structure, good biocompatibility, and adjustable functions. However, most organic photosensitizers suffer from poor stability, severe photodegradation, and aggregation-induced quenching effects, which hinder their widespread application.

[0004] Hypoxia is a prominent feature of the tumor microenvironment (TME). At the tumor site, microvessels exhibit structural and functional disorders, while the metabolic demands of rapidly proliferating cancer cells create an imbalance between insufficient oxygen supply and accelerated oxygen consumption. This dynamic change leads to the hypoxic condition that is prevalent in most solid tumors. Hypoxia triggers upregulation of gene expression associated with tumorigenesis, malignant progression, metastasis, and resistance to chemotherapy and radiotherapy. Therefore, selective hypoxia detection in vivo is a key tool for characterizing tumor formation and development, and also provides important information for predicting treatment response and formulating patient-specific treatment plans. Molecular probes that simultaneously have noninvasive multimodal hypoxia imaging and hypoxia-triggered immunotherapy functions as well as tunable photophysical energy conversion processes have great application prospects, but have rarely been reported.

[0005] Therefore, how to utilize the hypoxic characteristics of the tumor microenvironment to provide a diagnostic and therapeutic preparation that can not only achieve accurate and real-time tumor diagnosis through activated bioimaging, but also exert powerful anti-tumor and immunomodulatory effects through intelligent phototherapy is a technical problem that urgently needs to be solved. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a nano-diagnosis and treatment preparation for multi-modal imaging and cancer combined immunotherapy; the second object of the present invention is to provide a method for preparing a nano-diagnosis and treatment preparation for multi-modal imaging and cancer combined immunotherapy.

[0007] In order to achieve the first purpose, the technical solution adopted by the present invention is:

[0008] A nano-diagnostic and therapeutic preparation for multimodal imaging and cancer combined immunotherapy, comprising: a hypoxia-activated probe, a vascular disruptor, and a metal-organic framework, wherein the hypoxia-activated probe and the vascular disruptor are co-loaded on the metal-organic framework;

[0009] Wherein, the metal organic framework is a metal organic framework that can be degraded under acidic conditions.

[0010] Furthermore, it also includes an immune cell membrane, which is covered on the surface of the metal organic framework.

[0011] Furthermore, the hypoxia-activated probe is a molecular probe prepared based on an N-oxide structure. The molecular probe of the N-oxide structure can be converted into a corresponding amine compound under hypoxia stimulation, and the molecular structure changes from a receptor-receptor configuration to a donor-receptor type.

[0012] Furthermore, the molecular probe based on the N-oxide structure is 4,4'-(benzo[1,2-c:4,5-c']thiadiazole-4,7-diyl)bis(N,N-diethylaniline oxide) (abbreviated as BN-O).

[0013] Furthermore, the vascular damaging agent is a tubulin inhibitor.

[0014] Furthermore, the microtubule protein inhibitor is Compredin phosphate (abbreviated as CA4P).

[0015] Furthermore, the immune cell membrane is an M1 macrophage membrane.

[0016] In order to achieve the second purpose, the technical solution adopted by the present invention is:

[0017] A method for preparing a nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy, for preparing the above-mentioned nano-diagnostic and therapeutic preparation, comprising the following steps:

[0018] S100, preparing hypoxia-activated probe;

[0019] S200, using deionized water as a solvent to cause a covalent chemical reaction between 2-methylimidazole (2-MI for short), zinc nitrate hexahydrate, a hypoxia-activated probe, and a vascular disrupting agent, and centrifuging, washing, and drying the resulting solution to obtain a metal organic framework loaded with the hypoxia-activated probe and the vascular disrupting agent;

[0020] S300, covering the immune cell membrane on the surface of the metal-organic framework.

[0021] Further, the hypoxia-activated probe in S100 is a molecular probe based on an N-oxide structure;

[0022] The specific synthesis process is as follows: a halogenated N,N-diethylaniline derivative undergoes a Stiller reaction with benzobithiadiazole, followed by an oxidation reaction with m-chloroperbenzoic acid to obtain a molecular probe prepared based on an N-oxide structure.

[0023] Furthermore, in S300, the immune cell membrane is covered on the surface of the metal organic framework by co-extrusion.

[0024] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0025] The hypoxia-activated probe and vascular disruptor are integrated into an acid-responsive metal organic framework (MOF) to form a nano-diagnostic and therapeutic preparation. The MOF can be rapidly degraded under weak acid conditions (pH less than 6.5), thereby promoting the release of vascular disruptors. The released vascular disruptors can induce hypoxia in the tumor microenvironment (TME) by destroying the blood vessels in the tumor site, thereby accelerating the transformation process of the hypoxia-activated probe. After the hypoxia-activated probe loses oxygen, in addition to being able to efficiently induce PTT and PDT, the absorption spectrum red-shifts due to the reduction of the energy band gap, which will realize a "lighting-on" photoacoustic signal in the near-infrared region and generate a bright NIR-II FL signal. The nano-preparation can sensitively depict the information of the tumor in vivo through in situ activatable fluorescence. After entering the tumor tissue, the nano-diagnostic and therapeutic preparation has the ability to induce hypoxia-induced potent PDT, PTT properties and efficiently induce immunogenic death of tumor cells. Due to its excellent ROS generation ability, photothermal conversion efficiency and self-accelerated probe transformation, this nanotheranostic agent can not only inhibit the growth of primary tumors but also suppress distant tumors in 4T1 tumor-bearing mice by triggering a strong immune response.

[0026] When the metal-organic framework surface of the nano-diagnostic and therapeutic preparation is covered with immune cell membranes, especially macrophage membranes, under the mediation of the macrophage membrane, it can not only reduce the phagocytosis of monocytes, but also prolong the circulation time and enhance the targeted aggregation of tumors. This is because the α4β1 integrin on macrophages can bind to the vascular cell adhesion molecules on cancer cells, thereby improving the tumor targeting ability of nanoparticles.

[0027] The nano-diagnostic and therapeutic preparation provided by the present invention proposes a new strategy to achieve hypoxia-activated diagnostic and therapeutic performance and controllable drug release, providing a new solution for achieving precise imaging-guided tumor immunotherapy.

[0028] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the synthesis route diagram of BN and BN-O provided in Example 1 of the present invention.

[0030] Figure 2 This is the NMR carbon spectrum of BN-O provided in Example 1 of the present invention.

[0031] Figure 3 This is the high-resolution mass spectrum of BN-O provided in Example 1 of the present invention.

[0032] Figure 4Experimental Example 2 of the present invention provides a characterization diagram of the spectral properties of different photosensitizers; wherein, Figure A is the absorption spectrum of BN and BN-O in dimethyl sulfoxide (DMSO); Figure B is the absorption spectrum of BN and BN-O in DMSO; Figure C is the relationship between I / I0 and white light irradiation time, I0 and I represent the emission intensity of 2',7'-dichlorofluorescein (2',7'-Dichlorofluorescein, DCF) at 525 nm before and after light irradiation; Figure D is the relationship between temperature and illumination time of BN and BN-O molecules under 730 nm laser irradiation.

[0033] Figure 5 It is a spectral transformation characterization of the BN-O molecule under hypoxic conditions provided in Experimental Example 3 of the present invention; wherein A is a graph showing changes in the ultraviolet absorption spectrum of BN-O under hypoxic conditions, B is a graph showing changes in the fluorescence spectrum of BN-O under hypoxic conditions, C is a graph showing changes in the fluorescence spectrum of BN under hypoxic conditions, and D is a high-resolution mass spectrometry characterization graph after the response.

[0034] Figure 6 This is a diagram of the near-infrared PL / PA opening rate of BC@ZM after treatment with different active oxygen species / redox molecules / metal ions provided in Experimental Example 4 of the present invention.

[0035] Figure 7 This is the change in particle size and morphology of BC@ZM at different pH values ​​provided in Experimental Example 5 of the present invention; wherein A is the TEM image and particle size image of untreated BC@ZM, and B is the TEM image and particle size image of BC@ZM under acidic conditions.

[0036] Figure 8 This is the study on the molecular conversion properties of BC@ZM under hypoxic conditions provided in Experimental Example 6 of the present invention; wherein A is the change in PL spectrum before and after hypoxic response, B is the characterization of ROS production capacity before and after hypoxic response, C is the characterization of thermal properties before and after hypoxic response, D is the characterization of optical properties before and after hypoxic response, and I0 and I represent the emission intensity of DCF at 525 nm before and after light irradiation.

[0037] Fig. 9 This is the in vitro targeting study provided by Experimental Example 7 of the present invention; wherein A is a graph showing the uptake of BC@Z and BC@ZM by 4T1 (mouse breast cancer cells) tumor cells, B is a graph showing the uptake of BC@Z and BC@ZM by RAW264.7 (mouse mononuclear macrophage leukemia cells) macrophages, and C is a statistical graph showing the quantitative results of the uptake of BC@Z and BC@ZM by 4T1 cells and RAW264.7 cells.

[0038] Fig.10This is the in vitro cell hypoxia response study provided in Experimental Example 8 of the present invention; wherein A is a graph showing changes in near-infrared fluorescence in the first / second zone after 4T1 cells were incubated with BC@ZM for different periods of time under hypoxic conditions, B is a graph showing changes in near-infrared fluorescence in the first / second zone after 4T1 cells were incubated with BC@ZM for different periods of time under normoxic conditions, and C is a statistical graph of fluorescence values ​​at the cell level under normoxic / hypoxic conditions.

[0039] Fig.11 The ROS staining monitoring of 4T1 cells treated with different formulations for 24 hours under hypoxia / normoxia conditions provided in Experimental Example 9 of the present invention is performed, scale: 50 μm, wherein 2',7'-dichlorofluorescein (2',7'-Dichlorofluorescein, referred to as DCF) is used.

[0040] Fig.12 This is a graph showing the results of the MTT assay of the killing effects of different concentrations of BC@ZM on 4T1 tumor cells under hypoxic conditions provided in Experimental Example 9 of the present invention.

[0041] Fig.13 The live-dead staining monitoring of 4T1 cells treated in different ways for 24 hours under hypoxia / normoxia conditions is provided in Experimental Example 9 of the present invention. Scale bar: 100 μm.

[0042] Fig.14 This is the level of calreticulin externalization (etco-CRT) in 4T1 cells treated with different substances provided in Experimental Example 10 of the present invention, scale bar: 50 μm.

[0043] Fig.15 This is the efflux level of high mobility group box-1 protein (HMGB1) in 4T1 cells treated with different substances provided in Experimental Example 10 of the present invention, scale bar: 50 μm.

[0044] Fig.16 It is the ATP and cGAS-STING pathway activation level in 4T1 cells treated with different substances provided in Experimental Example 10 of the present invention; wherein A is ATP and B is the cGAS-STING pathway activation level.

[0045] Fig.17 This is a schematic diagram of the extraction and culture of dendritic cells (DCs) provided in Experimental Example 11 of the present invention, wherein the dendritic cells are derived from mouse bone marrow (BMDCs).

[0046] Fig.18 This is the result diagram of the loss of DCs activation after different drug administration provided in Experimental Example 11 of the present invention

[0047] Fig.19; It is the maturation statistics of DCs in different drug treatment groups and the statistical graph of IL-6 expression levels of DCs provided in Experimental Example 11 of the present invention; wherein A is the DCS maturation statistics graph, and B is the statistical graph of IL-6 expression levels of DCs.

[0048] Fig. 20 This is the research situation of hypoxia conversion ability provided by Experimental Example 12 of the present invention; wherein, A is the verification of hypoxia fluorescence onset at the living level, scale: 10 mm, and B is the slice characterization of hypoxia fluorescence onset of normal tissue and tumor tissue, scale: 100 μm.

[0049] Fig.21 These are the NIR-II FI results of tumor-bearing mice at different times after injection of BC@ZM nanoparticles provided in Experimental Example 12 of the present invention, scale bar: 10 mm.

[0050] Fig. 22 This is the PAI result of tumor-bearing mice at different times after injection of BC@ZM nanoparticles provided in Experimental Example 12 of the present invention, scale bar: 1 mm.

[0051] Fig.23 This is a schematic diagram of a timetable for evaluating the therapeutic effects of various intervention measures in 4T1 tumor-bearing mice provided in Experimental Example 13 of the present invention.

[0052] Fig.24 Experimental Example 13 of the present invention provides the conditions after different therapeutic interventions; wherein, A is a tumor growth curve graph (n=5), B is a tumor growth picture after different therapeutic interventions (n=5), and C is the survival rate of mice after different treatments (n=5).

[0053] Fig.25 This is the CD31 immunofluorescence staining of tumor sections after different therapeutic interventions provided in Experimental Example 13 of the present invention, Figure, scale bar: 50 μm.

[0054] Fig.26 This is a statistical graph of the fluorescence quantification results of CD31 immunofluorescence staining of tumor sections after different therapeutic interventions provided in Experimental Example 13 of the present invention.

[0055] Fig. 27 These are the pathological sections of liver / lung tissues of each group after different treatment interventions provided in Experimental Example 13 of the present invention.

[0056] Fig.28 This is a flow cytometry characterization diagram of activation of macrophages (TAMs) in tumor tissue provided in Experimental Example 13 of the present invention.

[0057] Fig.29 This is a quantitative analysis statistical graph of activation of macrophages (TAMs) in tumor tissue provided in Experimental Example 13 of the present invention.

[0058] Fig.30 This is a flow cytometry characterization diagram of cytotoxic T cell activation in tumor tissue provided in Experimental Example 13 of the present invention.

[0059] Fig.31 This is a quantitative analysis statistical chart of cytotoxic T cell activation in tumor tissue provided in Experimental Example 13 of the present invention.

[0060] Fig.32 This is a schematic diagram of the BC@ZM-mediated synergistic treatment process in the bilateral tumor model provided in Experimental Example 14 of the present invention.

[0061] Fig.33 This is the situation of bilateral tumors after different treatments provided in Experimental Example 14 of the present invention, wherein Primary is carcinoma in situ, Distant is distant cancer, A is the tumor growth curve in the bilateral tumor model, and B is the weight statistics of bilateral tumors after different treatments (n = 5).

[0062] Fig.34 This is the flow cytometry analysis result of cytotoxic T cell infiltration in bilateral tumors after different therapeutic interventions provided in Experimental Example 14 of the present invention.

[0063] Fig.35 This is a quantitative analysis statistical graph of cytotoxic T cell infiltration in tumors after different therapeutic interventions provided in Experimental Example 14 of the present invention.

[0064] Fig.36 This is a mouse organ pathological section provided in Experimental Example 15 of the present invention.

[0065] Fig.37 This is the blood test result of mice provided in Experimental Example 15 of the present invention, wherein A is the blood routine index and B is the blood biochemical index. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0067] Explanation of terms

[0068] Vascular disruptors: Targeting tumor blood vessels is an effective approach to treating tumors. Vascular disruptors specifically destroy tumor blood vessels by damaging the endothelial cells of tumor blood vessels, leading to large-scale necrosis in the center of the tumor, and have shown good anti-tumor efficacy in clinical trials.

[0069] Photoacoustic imaging (PAI) is a biological imaging technology that combines the principles of optical and ultrasonic imaging. It has the characteristics of high spatial resolution, deep penetration and molecular specificity, and is widely used in the biomedical field. The principle is to use light pulses generated by a laser to irradiate biological samples. The light energy absorbed by the tissue will cause local thermal expansion and form photoacoustic signals. These photoacoustic signals are captured by ultrasonic sensors (usually ultrasound probes) in the tissue and converted into electrical signals. By measuring the time delay and amplitude of the photoacoustic signal, the structural and functional information inside the tissue can be reconstructed. PAI has the advantages of high resolution, high penetration depth, non-invasiveness and multimodal imaging. It can also be used in fields such as tumor diagnosis, treatment detection and drug screening. It is of great significance to biomedical research and clinical diagnosis and treatment.

[0070] Near-infrared II fluorescence imaging (NIR-II FI): is a fluorescence imaging technology based on the near-infrared (NIR) light region, with a wavelength range of approximately 1000 to 1700 nanometers. In this wavelength range, biological tissues have low light scattering and absorption, and good tissue depth penetration, making it suitable for in vivo imaging. NIR-II FI usually uses fluorescent probes or fluorescent markers, such as specific binding to tumor tissue, or for marking specific cells or molecules. When these fluorescent probes or markers are excited, they emit NIR-II fluorescence signals, which can be captured and recorded by imaging equipment. Near-infrared II fluorescence imaging has a wide range of applications in the biomedical field, including tumor diagnosis, tumor microenvironment research, and tumor treatment monitoring.

[0071] Immunogenic death (ICD) is a type of regulated cell death, which usually refers to the death of tumor cells due to external stimuli. The dying cells will produce new antigenic epitopes and release cellular contents, including a variety of pro-inflammatory factors and damage-associated molecular patterns (DAMPs). The released DAMPs are bound, recognized and phagocytosed by antigen-presenting cells, and presented to T cells, thereby activating the adaptive immune response. It is a process of changing from non-immunogenicity to immunogenicity and mediating the body's anti-tumor immune response.

[0072] Tumor-associated antigens (TAAs): refer to antigen molecules present on tumor cells or normal cells, including embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., which are often used in the diagnosis of clinical tumors.

[0073] Damage-associated molecular patterns (DAMPs): Damage-associated pattern molecules are a class of substances released into the intercellular space or blood circulation after tissues or cells are stimulated by damage, hypoxia, stress and other factors. They can induce autoimmunity or immune tolerance through pattern recognition receptors such as Toll-like receptors, RIG-1-like receptors or NOD-like receptors, and play an important role in the occurrence and development of diseases such as arthritis, atherosclerosis, tumors, systemic lupus erythematosus and so on.

[0074] Dendritic cells (DCs): They are the most powerful professional antigen-presenting cells in the body. They can efficiently take up, process and present antigens. Immature DCs have strong migration ability, and mature DCs can effectively activate naive T cells. They are at the central link in initiating, regulating and maintaining immune responses.

[0075] Pattern recognition receptors (PRRs) are a class of recognition molecules that are mainly expressed on the surface of innate immune cells, distributed in a non-clonal manner, and can recognize one or more pathogen-associated molecular patterns.

[0076] The cGAS-STING pathway is an important immune system signaling pathway whose main function is to sense and respond to DNA damage or viral infection in cells. When DNA damage or viral infection occurs in cells, DNA fragments are released into the cytoplasm. These DNA fragments are recognized and bound to cGAS in the cell, activating the catalytic activity of the enzyme, thereby prompting cGAS to synthesize cytosine dimerization adenylate (cGAMP). As a secondary signaling molecule, cGAMP binds to STING, thereby triggering the activation of STING. Activated STING further initiates downstream signaling pathways, leading to the release of interferon and other inflammatory mediators. The release of these molecules can activate immune cells such as dendritic cells, thereby triggering antiviral or antitumor immune responses in cells or in the tumor microenvironment. The cGAS-STING pathway plays a key role in immune surveillance against viral infection and tumor development. Therefore, in-depth research on this pathway not only helps to better understand the immune system's response mechanism to pathogen infection, but also provides important clues for the development of new immunotherapy methods for diseases such as viral infection and cancer.

[0077] Photothermal therapy (PTT): It is a treatment method that uses light energy and heat energy to treat diseases. It is often used in tumor treatment and other medical applications. This method is based on the characteristics of photothermal agents, combined with light irradiation of specific wavelengths, to generate heat and thermally damage the target tissue to achieve the purpose of treatment. During the tumor PTT process, the patient first receives an injection or local application of a photothermal agent, which will be enriched in the tumor tissue. Subsequently, a laser or light source of a specific wavelength is used to irradiate the affected area, and the photosensitizer absorbs the light energy and converts it into heat energy, resulting in a local temperature increase. This local thermal effect can cause thermal damage, coagulation, necrosis of the target tumor tissue, or induce other therapeutic effects, such as vascular occlusion, immune response, etc. This treatment method has the advantages of strong locality, non-invasiveness, reusability, and simple operation. However, it also has some disadvantages and limitations: limited laser penetration depth, difficulty in local treatment of metastatic tumors, thermal damage to normal tissues, unstable treatment effects, and time-consuming treatment.

[0078] Photodynamic therapy (PDT): It is a widely used treatment method in the medical field. Its principle is based on the fact that photosensitizers are excited under light of a specific wavelength. In the excited state, they react with oxygen to produce a series of reactive oxygen species, such as free radicals and singlet oxygen. These reactive oxides have strong oxidizing properties and can cause strong damage to cell membranes and organelles, thereby inducing apoptosis of tumor cells or causing damage to other diseased tissues. The appropriate dosage, irradiation time and irradiation intensity can be selected according to the needs and the severity of the disease. Therefore, the advantages of PDT are its non-invasiveness, selectivity and high specificity. It only causes damage to diseased tissues and has almost no side effects on normal tissues. Like PTT, PDT is still limited by photosensitizers, light penetration depth and maximum laser exposure. Further research and improvement of PDT is still a hot research field, which is expected to bring more breakthroughs and progress to clinical treatment.

[0079] Metal-organic framework (MOF): It is a kind of porous material, which is a crystal structure formed by metal ions and organic ligands through coordination bonds. The characteristics of MOF include highly ordered structure, controllable pore size and high specific area. These frameworks show great potential in drug delivery, multifunctionality, biocompatibility and controlled release, and can be applied to many aspects of tumor treatment, including drug delivery, targeted therapy, diagnosis and monitoring, which is expected to bring new breakthroughs and progress in tumor treatment.

[0080] Tumor microenvironment (TME): refers to the surrounding microenvironment of tumor cells, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules and extracellular matrix. In solid tumors, due to the rapid growth of tumor tissue, high expansion and incomplete vascular system inside the tumor tissue, these will lead to insufficient oxygen supply in the tumor tissue, and the tumor microenvironment presents the characteristics of overall hypoxia. Due to insufficient oxygen supply, tumor cells can only metabolize energy through anaerobic glycolysis, which will cause the accumulation of lactic acid; at the same time, the ion exchange proteins on the tumor cell membrane are also continuously transporting H+ inside the cell to the outside of the cell to avoid autoacidosis. These cellular reactions also cause the pH of the tumor microenvironment to decrease to varying degrees, presenting an acidic environment as a whole. In the development of tumors and hypoxia, acidic microenvironment, a large number of tumor tissues and peripheral tissue cells will undergo apoptosis, release cell fragments and chemokines, leading to inflammatory cell infiltration and secretion of inflammatory factors. At the same time, the development of the tumor itself will also trigger the immune response of the immune system, causing inflammatory cells to gather in this area and trigger a severe inflammatory response.

[0081] Reactive oxygen species (ROS): A class of highly active oxidizing compounds, usually including superoxide anions, hydroxyl radicals, singlet oxygen, hydrogen peroxide, etc. A small amount of ROS is produced during cell metabolism and is a normal metabolite in the body. These ROS have unpaired electrons or contain active oxygen-oxygen bonds and show high reactivity. Excessive ROS can lead to increased intracellular oxidative stress, which in turn causes cell damage and tissue inflammation, and is closely related to the occurrence and development of a variety of diseases. In cancer treatment, the cytotoxicity of reactive oxygen species can effectively induce apoptosis of tumor cells.

[0082] Stille reaction, also known as Stille coupling reaction, is a cross-coupling reaction between organotin compounds and halogenated hydrocarbons without β-hydrogen under palladium catalysis. The reaction is generally carried out in a dehydrated and deoxygenated solvent and an inert environment. Equivalent amounts of Cu(I) or Mn(II) salts can improve the specificity and reaction rate of the reaction. Oxygen will oxidize the palladium catalyst and cause the organotin compound to self-couple. Tetrakis(triphenylphosphine)palladium is the most commonly used palladium catalyst, and other catalysts include: PdCl2(PPh3)2, PdCl2(MeCN)2, etc. The halogenated hydrocarbons used are generally vinyl or aryl trifluoromethanesulfonates or chlorine, bromine, or iodine hydrocarbons.

[0083] The MTT method, also known as the MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is measured at a wavelength of 490nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.

[0084] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0085] Example 1 Preparation of BN-O, the synthetic route is as follows Figure 1 shown.

[0086] 1. Preparation of N,N-diethyl-4-(tributyltinyl)aniline. The specific preparation process is as follows: 4-bromo-N,N-diethylaniline (1.37 g, 6 mmol) is dissolved in 40 ml of anhydrous tetrahydrofuran (THF). Under an argon atmosphere, the reaction is cooled to -78°C, using a dry ice-acetone mixture for 30 minutes, and then n-butyl lithium (n-BuLi) (2.5 M hexane solution, 2.4 ml, 6 mmol) is added. After stirring at -78°C for 2 hours, tributyltin chloride (1.63 ml, 6 mmol) is added, and the mixture is slowly warmed to room temperature and stirred overnight for 12 to 24 hours. Water is added to quench the reaction, and then the mixture is extracted three times with dichloromethane. The collected organic phase mixture is dried using MgSO4. After removing the solvent under reduced pressure, N,N-diethyl-4-(tributyltinyl)aniline was obtained as a colorless oil, which was directly used in the next reaction without purification.

[0087] 2. Preparation of 4,4'-(Benzo[1,2-c:4,5-c']thiadiazole-4,7-diyl)bis(N,N-diethylaniline) (BN for short), the specific preparation process is as follows: Under an argon atmosphere, N,N-diethyl-4-(tributyltinyl)aniline (1.31 g, 3 mmol), 4,7-dibromobenzo[1,2-c:4,5-c']thiadiazole (0.35 g, 1 mmol) and Pd(PPh3)4 (0.35 g, 0.3 mmol) were mixed in anhydrous THF (50 ml). The mixture was heated to reflux and stirred continuously for 24 hours. After cooling to room temperature, water was added and the mixture was extracted three times with dichloromethane. The collected organic phases were mixed, dried using MgSO4, and concentrated. The crude product was purified by silica gel column chromatography using dichloromethane / n-hexane (v / v 1:2) as the eluting solvent to give 4,4'-(benzo[1,2-c:4,5-c']thiadiazole-4,7-diyl)bis(N,N-diethylaniline) as a green solid in a yield of 73%.

[0088] 3. Preparation of BN-O. The specific preparation process is as follows: 200 mg of BN was dissolved in 10 ml of ethyl acetate, and the reaction mixture was placed in an ice-water bath. After slowly dropping 210 mg of ethyl acetate solution of p-chloroperbenzoic acid (m-CPBA), the resulting mixture was stirred for 4 hours. After concentration, it was purified on a silica gel column using methanol as an eluent. After the mixed eluent was evaporated, the product was evaporated and redissolved in dichloromethane. The silica gel was removed by suction filtration using a Buchner funnel. The organic solvent was removed by vacuum rotary evaporation to obtain BN-O as a purple-red powder with a yield of 86%.

[0089] like Figure 2 As shown, 13 C NMR (100 MHz, CDCl3) δ 152.65, 149.77, 135.00, 132.44, 121.94, 77.51, 77.20, 76.88, 67.02, 49.96, 29.65, 8.59.

[0090] like Figure 3 As shown, HRMS (MALDI-TOF, m / z): [M + H] + calcd. for C 26 H 28 N6O2S2,521.1795; found, 521.1792.

[0091] Example 2

[0092] Preparation of the nanotheranostic preparation, the specific preparation process is as follows: 10 mg of zinc nitrate hexahydrate and 20 mg of CA4P were dissolved in 5 ml of deionized water at room temperature 10-30 ° C, and then added dropwise to a 5 ml deionized water solution containing 400 mg of 2-MI and 20 mg of BN-O, while under magnetic stirring. After stirring the resulting mixture for 10 minutes, the product was collected by centrifugation (5,000 rpm) and washed three times with deionized water. The encapsulation efficiencies of CA4P and BN-O were 22.05% and 10.9%, respectively. Then, the resulting nanotheranostic preparation BC@Z (100 µL) without cell membrane coverage was combined with 0.1 mg of freshly extracted M1 macrophage membranes, followed by 20 repeated cycles of ultrasound and microextruder through a 400 nm polycarbonate filter membrane. Subsequently, the polycarbonate filter membrane was replaced with a filter membrane with a pore size of 220 nm and extruded continuously for 20 times. Finally, the nanotheranostic preparation BC@ZM with the metal-organic framework surface covered with M1 macrophage membrane was obtained by centrifugation (5,000 rpm, 15 minutes).

[0093] The BN and BN-O used in the following experimental examples are the BN and BN-O prepared in Example 1; the BC@Z and BC@ZM used in the following experimental examples are the BC@Z and BC@ZM prepared in Example 2.

[0094] In the following experimental examples, phosphate buffered saline (PBS) was the blank control group, L was the laser irradiation group, C@Z was the group loaded with CA4P nanoformulation, BC@Z was the group of nanoformulations not covered with cell membranes, BC@ZM was the group of nanoformulations covered with macrophages M1 on the surface, BC@Z+L was the group treated with BC@Z and L together, and BC@Z-M+L was the group treated with BC@ZM and L together.

[0095] Experimental Example 1 Synthesis route and characterization of photosensitizer.

[0096] The results of nuclear magnetic resonance analysis of BN and BN-O and the positional analysis of hydrogen / carbon atoms of BN and BN-O are as follows: Figure 2 As shown, Figure 3 As shown, the high-resolution mass spectrometry results of BN and BN-O also confirmed that the two molecules were accurately obtained: the mass-to-charge ratio (m / z) of BN = 488.1817, the mass spectrometry result M+1 = 489.1890, and the m / z of BN-O = 520.1715, the mass spectrometry result M+1 = 521.1792.

[0097] Experimental Example 2 Characterization of the spectral properties of different photosensitizers, such as Figure 4 shown.

[0098] Photophysical properties of BN and BN-O: In DMSO, the maximum absorption wavelengths of BN-O and BN are 538 nm and 770 nm, respectively; the maximum emission wavelengths are 650 nm and 1113 nm, respectively.

[0099] Under 730 nm laser irradiation, the warming effect and ROS-producing ability of BN and BN-O were evaluated using a thermal imager and a DCFH-DA probe. Compared with BN-O, BN showed stronger warming ability and ROS-producing ability.

[0100] Experimental Example 3 Characterization of the spectral transition of BN-O molecules under hypoxic conditions, such as Figure 5 shown.

[0101] Under hypoxic conditions, the absorption of BN-O at a wavelength of 550 nm gradually decreases with the extension of hypoxia time, and the absorption at a wavelength of 730 nm gradually increases. The color change of the solution is also consistent with that of BN and BN-O. The fluorescence spectrum of BN-O gradually decreases with the increase of hypoxia time, and the fluorescence of BN gradually increases with the increase of hypoxia time. BN-O has been completely transformed into BN.

[0102] Experimental Example 4 Monitoring of near-infrared second-zone fluorescence signal / photoacoustic signal of BC@ZM in the presence of different interfering ions / substances, such as Figure 6 shown.

[0103] Neither metal ions nor redox substances can significantly turn on the fluorescence of BC@ZM, but under hypoxic conditions, the fluorescence is significantly turned on, and similar conclusions are obtained in photoacoustic tests.

[0104] Experimental Example 5 Changes in particle size and morphology of BC@ZM at different pH values, such as Figure 7 shown.

[0105] Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) measurements showed that BC@ZM has a spherical structure with an average diameter of approximately 134 nm.

[0106] BC@ZM was introduced into a phosphate buffered saline (PBS) solution with a pH of 6.5 to simulate the weak acid conditions of the tumor microenvironment. The acid response of BC@ZM was measured, and TEM and DLS were used to study the changes in the morphology of nanoparticles (NPs). After weak acid treatment, the morphology of BC@ZM changed to an irregular broken state, indicating that BC@ZM had been degraded.

[0107] Experimental Example 6 Study on the molecular transformation properties of BC@ZM under hypoxic conditions, such as Figure 8 shown.

[0108] Under hypoxia conditions at 37 °C, BC@ZM was co-incubated with rat liver microsomes enriched with cytochrome P450 (CYP450) and reduced coenzyme II (nicotinamide adenine dinucleotidephosphate, NADPH), and the fluorescence changes of the system were detected at different time points. The fluorescence signal showed an obvious red shift from 650 nm of BN-O to about 900 nm of BN. This is because the BN-O molecules in BC@ZM respond to hypoxia, causing their fluorescence emission to shift from the near-infrared region to the long-wavelength region, with some luminescence above 1000 nm.

[0109] Photothermal and photodynamic tests were carried out on the nanosystem after hypoxia transformation. Compared with BC@ZM without hypoxia treatment, BC@ZM after hypoxia treatment had obvious ROS production ability. Compared with BC@ZM without hypoxia treatment, BC@ZM after hypoxia treatment had obvious photothermal warming effect.

[0110] Experimental Example 7 In vitro cell targeting study, such as Fig. 9 shown.

[0111] Nanoparticles generally have poor tumor targeting ability, partly due to poor in vivo circulation, which makes them easily removed by phagocytes as foreign bodies, and on the other hand, lack effective tumor targeting, thus reducing tumor aggregation. In this regard, the present invention explores the immune escape and tumor targeting ability of nanoparticles. Compared with BC@Z, M1 macrophage membrane encapsulation can greatly improve the tumor targeting ability of BC@ZM. The macrophage uptake of BC@ZM is much lower than that of BC@Z, which is because the M1 macrophage membrane encapsulation successfully reduces the phagocytosis of BC@ZM by macrophages.

[0112] Experimental Example 8 In vitro cellular hypoxia response study, such as Fig.10 shown.

[0113] Inspired by the spectral changes in response to hypoxia at the molecular level, the spectral changes in response to hypoxia were subsequently detected at the cellular level. After 4T1 cells were incubated with BC@ZM for 3 hours under hypoxic conditions, obvious NIR-I fluorescence quenching and illuminated NIR-I fluorescence were observed. However, under normoxic conditions, the fluorescence intensities of NIR-I and NIR-II did not change significantly. Scale bar: 50 μm.

[0114] Experimental Example 9 In vitro cell studies, such as Fig.11 , Fig.12 , Fig.13 shown.

[0115] When CA4P and BN-O were encapsulated into acid-responsive COF carriers, their antitumor effects were weakened under normoxic conditions, which was due to the fact that the normoxic environment restricted the transformation of BN-O molecules and thus did not efficiently turn on PDT. Under hypoxic stimulation, the BC@ZM + light irradiation group showed considerable ROS production ability, which greatly improved the tumor killing ability. In addition, with the increase of concentration, BC@ZM showed dose-dependent tumor killing ability. The results of cell live-dead staining also showed that the BC@ZM + light irradiation group could effectively kill tumor cells under hypoxic conditions.

[0116] Experimental Example 10 Characterization of ICD and cGAS-STING pathway induction, such as Fig.14 , Fig.15 , Fig.16 shown.

[0117] The present invention evaluated the calreticulin (etco-CRT) externalization, high mobility group protein B1 (HMGB1) efflux level and adenosine triphosphate (ATP) release level of 4T1 tumor cells after different treatments. As key markers in the ICD process, the "BC@Z-M+L" group cells had the highest etco-CRT externalization and HMGB1 efflux; the "BC@Z-M+L" group cells had the highest ATP release efficiency. This result indicates that the integration of PTT effect and PDT effect promotes the occurrence of strong ICD. In addition, protein expression analysis of proteins extracted from 4T1 tumor cells in different treatment groups revealed that protein phosphorylation of the cGAS-STING pathway was significantly upregulated, indicating the successful activation of the cGAS-STING pathway, such as Fig.31 As shown, it provides a basis for anti-tumor immunotherapy.

[0118] Experimental Example 11 Study on immune activation at the cellular level, such as Fig.17 , Fig.18 , Fig.19 shown.

[0119] Dying / dead tumor cells can release a large number of death-related pattern molecules or antigens, which can effectively activate DCs and thus activate downstream immunity. The activation of DCs after BC@ZM killed tumor cells was studied. The specific process is as follows: primary DCs cells from mice were extracted and plated in the lower chamber of the cell migration (transwell) chamber, and then 4T1 cells were plated in the upper chamber. After different treatment interventions, 4T1 cells and DCs cells were co-incubated. After incubation, DCs cells in the lower chamber were collected, and DCs maturation marker proteins were marked. The maturation of DCs was monitored by flow cytometry; the BC@ZM + light irradiation group showed the highest DCs maturation rate, indicating that BC@Z-M + light irradiation can effectively kill tumor cells and promote DCs maturation; at the same time, the BC@Z-M + light irradiation group showed the highest IL-6 factor level, showing strong anti-tumor immunity.

[0120] Experimental Example 12 Tumor targeting verification, such as Fig. 20 , Fig.21 , Fig. 22 shown.

[0121] First, the hypoxia transformation ability of BC@ZM at the in vivo level was investigated. When BC@ZM was injected intratumorally into tumor-bearing mice, the same dose of BC@ZM was injected subcutaneously on the contralateral side. It can be seen that as time goes on, there is no obvious change in the fluorescence of the subcutaneous injection side, while the NIR-I fluorescence of the intratumor injection side weakens and the fluorescence of NIR-II is significantly enhanced. Subsequently, frozen sections of the tumor and contralateral tissues were taken. The confocal results showed that the NIR-II fluorescence intensity in tumor tissues was higher than that in normal tissues. Immunofluorescence staining analysis of hypoxia-inducible factor 1-α (HIF-1α) found that tumor tissues had more HIF-1α expression, which is consistent with the result that the NIR-II fluorescence in tumor tissues is stronger than that in normal tissues.

[0122] After the tail vein administration of 4T1 tumor-bearing mice, the mice were subjected to fluorescence and photoacoustic imaging at different time points. The results showed that the fluorescence signal was strongest at the tumor site at 24 hours after administration. The same conclusion was obtained by photoacoustic imaging of the tumor area of ​​mice, proving the effective accumulation and hypoxia response of BC@ZM at the tumor site.

[0123] Experimental Example 13 In vivo anti-tumor effect study, such as Figures 23 to 31 shown.

[0124] The injection and irradiation procedures were repeated three times on days 0, 3, and 6, and the tumor growth and body weight were monitored every other day. Compared with the PBS group, mice treated with C@Z, BC@Z, or "BC@Z+L" all showed a certain degree of tumor inhibition, among which the tumors in the "BC@Z-M+L" group showed the most obvious inhibition. The average tumor volume on day 16 was about PBS (1222 mm 3 )、L(1149 mm 3 )、C@Z(754 mm 3 )、BC@Z(746 mm 3 )、“BC@Z+L”(460 mm 3 ) groups are “BC@Z-M+L” (123 mm 3 ) groups. In addition, the weight of isolated tumors harvested on day 16 confirmed the superior antitumor efficiency of “BC@Z-M+L” treatment compared with other interventions. These results indicate that BC@ZM synergistic delivery and targeted release of CA4P and BN-O can significantly improve mouse survival.

[0125] Immunofluorescence staining of tumor tissues revealed that vascular disruptors combined with PDT / PTT treatment can effectively inhibit the growth of new blood vessels in tumors. Pathological staining analysis of lung / liver tissues of mice in each group revealed that the "BC@Z+L" group can effectively inhibit liver / lung metastasis of cancer cells. Flow cytometry analysis of tumor-associated macrophages and tumor-infiltrating cytotoxic T cells revealed that the "BC@Z+L" group can effectively promote the anti-tumor M1 macrophages (CD80 + 86 + ) polarization and cytotoxic T cells (CD3 + 4 + 8 + ) tumor infiltration, thereby exerting an excellent anti-tumor effect, among which G1 is the PBS group, G2 is the PBS + L group, G3 is the C@Z group, G4 is the BC@Z group, G5 is the BC@Z + L group, and G6 is the BC@ZM + L group.

[0126] Experimental Example 14 Evaluation of the anti-tumor effect on bilateral tumors Figure 32 to Figure 35 shown.

[0127] Establishment of a bilateral tumor model in mice, in which 4T1 tumor cells were implanted into the right flank of female BALB / c mice on day 0. Six days after primary tumor inoculation, an equal number of tumor cells were implanted into the left flank to simulate the presence of distant tumors. In vivo tumor treatment was performed by systemic administration of different formulations on day 7, followed by local photoirradiation of the primary tumor 24 h after injection. . The treatment was repeated on the 10th and 13th days, respectively. The progression of the tumors on both sides was carefully monitored every two days to evaluate the therapeutic effect. The evaluation results showed that the growth of the primary tumor and the distal tumor in the PBS treatment group was uncontrolled. The "PBS+L" treatment had a weak inhibitory effect on both the primary tumor and the distal tumor. This may be due to the lack of photosensitizer molecules, which made the simple laser irradiation unable to inhibit tumor growth. After the "BC@Z-M+L" treatment, the growth of the primary tumor and the distal tumor was significantly delayed, resulting in a significant inhibition rate of 81% and 85%, respectively. On the 22nd day, the average weight of the distant tumor in the "BC@Z-M+L" group was 0.09 g, and the average weight of the tumor in the PBS group was 0.556 g. The tumor weight in the BC@Z-M+L group was much smaller than that in the PBS group. In addition, through the analysis of immune cell infiltration in the in situ tumor and distal tumor, it was found that the cytotoxic T cells in the in situ tumor and distal tumor of mice in the "BC@Z-M+L" group were 2.2 times and 2 times that of the PBS treatment group, respectively. The results showed that the "BC@Z-M+L" group has a strong ability to recruit immune cells, thereby exerting an excellent anti-tumor immune effect.

[0128] Experimental Example 15 Biosafety Study, such as Fig.36 and Fig.37 shown.

[0129] The hearts, livers, spleens, lungs, and kidneys of mice in the PBS and BC@ZM intravenous injection groups were paraffin-sectioned and pathologically stained. The results showed that there was no obvious organ damage in the mice treated with BC@ZM. Scale bar: 100 μm. In addition, the results of blood routine and blood biochemical analysis also showed that there were no abnormalities in the mice treated with BC@ZM, proving the safety of BC@ZM nanodiagnostic preparations.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy, characterized in that: include: A hypoxia-activated probe, a vascular disrupting agent, and a metal-organic framework, wherein the hypoxia-activated probe and the vascular disrupting agent are co-loaded on the metal-organic framework; Wherein, the metal organic framework is a metal organic framework that can be degraded under acidic conditions; Also included is an immune cell membrane, which is covered on the surface of the metal organic framework; The hypoxia-activated probe is a molecular probe prepared based on an N-oxide structure, and the molecular probe of the N-oxide structure can be converted into a corresponding amine compound under hypoxia stimulation; The molecular probe based on the N-oxide structure is 4,4'-(benzo[1,2-c:4,5-c']thiadiazole-4,7-diyl)bis( N , N -diethylaniline oxide); The vascular damaging agent is Compredin phosphate; The immune cell membrane is an M1 macrophage membrane.

2. A method for preparing a nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy, characterized in that: The method for preparing the nano-diagnostic and therapeutic preparation according to claim 1 comprises the following steps: S100, preparing hypoxia-activated probe; S200, using deionized water as a solvent to cause a covalent chemical reaction between 2-methylimidazole, zinc nitrate hexahydrate, a hypoxia-activated probe and a vascular disrupting agent, and centrifuging, washing and drying the resulting solution to obtain a metal organic framework loaded with the hypoxia-activated probe and the vascular disrupting agent; S300, covering the immune cell membrane on the surface of the metal-organic framework.

3. The method for preparing a nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy according to claim 2, characterized in that: The hypoxia-activated probe in S100 is a molecular probe based on an N-oxide structure; The specific synthesis process is as follows: a halogenated N,N-diethylaniline derivative undergoes a Stiller reaction with benzobithiadiazole, followed by an oxidation reaction with m-chloroperbenzoic acid to obtain a molecular probe prepared based on an N-oxide structure.

4. The method for preparing a nano-diagnostic and therapeutic preparation for multimodal imaging and combined cancer immunotherapy according to claim 2, characterized in that: In S300, the immune cell membrane is coated on the surface of the metal organic framework by co-extrusion.

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