Bionic self-assembly delivery system with remodeling system and local immune function as well as preparation method and application of bionic self-assembly delivery system

Abstract

The invention discloses a bionic self-assembly delivery system with a remodeling system and a local immune function, the bionic self-assembly delivery system comprises a core, the core contains two drugs, the two drugs can be self-assembled to form nanoparticles, one drug is a small molecule drug for regulating and controlling the immune function of the system, and the other drug is a small molecule drug for regulating and controlling the immune function of the system. The other medicine is a medicine for regulating and controlling the local immune function of tumors, and cell membranes and brain targeting molecules are coated on the surfaces of the nanoparticles, so that the brain targeting property of the nanoparticles is improved. The invention further discloses a preparation method of the bionic self-assembly delivery system with the remodeling system and the local immune function. The invention further provides application of the bionic self-assembly delivery system in preparation of drugs for treating glioma. By remodeling the system and local immune function, T cell infiltration in glioma is increased, and the response of glioma to immunotherapy is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a self-assembly delivery system, in particular to a bionic self-assembly delivery system for remodeling system and local immune function, and a preparation method and use thereof. Background Art

[0002] Malignant glioma is the most common and deadly primary brain tumor in the central nervous system. Clinical treatment is mainly surgery combined with radiotherapy and chemotherapy, but the treatment effect is limited. Immunotherapy is one of the most promising development directions in the field of tumor treatment, bringing unprecedented treatment options to a series of difficult-to-treat tumors. However, the response rate of existing immunotherapy in glioma is still low. Most studies focus on local immunosuppression, promoting T cell infiltration and enhancing the effect of immunotherapy by reshaping the tumor microenvironment, but clinical progress is far from the expected goal. Clinical studies have shown that glioma can also induce unique systemic immunosuppression, reduce immune function, and weaken the effect of immunotherapy. Therefore, it is necessary to simultaneously overcome systemic and local immunosuppression to improve the immunotherapy of glioma.

[0003] Systemic immunosuppression is an important unique clinical pathological change of glioma, which is manifested by a significant decrease in the number of peripheral blood T cells and CD4 + T cell counts even reach the level of patients with acquired immunodeficiency syndrome. First, glioma causes a large number of initial T cells to be sequestered in the bone marrow due to the lack of sphingosine-1-phosphate receptor 1, resulting in a decrease in systemic T cells; secondly, glioma induces the presence of specific non-steroidal factors in the serum to inhibit T cell proliferation and induce thymus and spleen atrophy. Thymus and spleen atrophy usually represent severe defects in immune function, suggesting that glioma weakens the body's systemic immune function. However, there is currently a lack of safe and effective treatment strategies to overcome systemic immunosuppression.

[0004] Local immunosuppression is closely related to the intrinsic factors of glioma, and is affected by different driver gene mutations and metabolic characteristics, forming a unique tumor microenvironment. Isocitrate dehydrogenase (IDH) gene mutation is the most common "gain of function" mutation in glioma, and IDH1-R132H is its main mutation form, which can cause the following effects on the local tumor: ① IDH mutants catalyze α-ketoglutarate to 2-hydroxyglutaric acid (2-HG), downregulate intratumoral IFN-γ and CXCL10 levels, reduce immune cell infiltration, and reduce local immune response levels; ② Because the generation of 2-HG consumes NADPH, it destroys intracellular redox homeostasis, and the level of reactive oxygen species (ROS) in tumor cells increases, making them susceptible to oxidative damage. Intracellular ROS levels are closely related to immunogenic cell death (ICD), which can release tumor-associated antigens and damage-associated molecular patterns (DAMP) signals, promote the infiltration and activation of immune cells, and enhance local specific anti-tumor immune responses. Therefore, ROS-based therapeutic strategies have the potential to enhance the local immune function of IDH mutant gliomas.

[0005] α-Mangostin (α-M) is a xanthone compound derived from the fruit shell of Garciniamangostana L., a plant of the family Garcinia family. It has anti-inflammatory, antibacterial, antiviral, anti-angiogenic and anti-tumor effects. In addition, a small number of studies have reported that α-M has the function of regulating immune cells and enhancing the body's immune function. Our research group has found through preliminary studies that α-M has the following biological activities: (1) α-M can significantly enhance the phagocytic function of microglia, suggesting its regulatory effect on innate immunity; (2) In vitro studies have found that α-M can significantly upregulate the ROS level of glioma cells, and its killing effect on IDH mutant glioma is stronger than that on IDH wild-type; (3) α-M can increase the number of T cells in glioma mice and enhance the immune function of tumor-bearing mice. Therefore, this paper intends to use α-M to induce tumor cells to upregulate ROS levels and inhibit tumor growth; at the same time, with the help of the immune regulatory effect of α-M, the systemic immune function of tumor-bearing mice is enhanced. However, α-M has inherent drugability defects such as poor water solubility, low oral bioavailability and short half-life, which result in low concentrations at the target site and are unable to effectively kill tumors and regulate immune function.

[0006] Photodynamic therapy (PDT) is a new type of therapy that uses laser and photosensitizer to generate a large amount of ROS for specific killing by local irradiation at a specific wavelength. It has the advantages of few side effects and high selectivity, and is increasingly used in tumor treatment. Currently, there are a number of PDT therapies for gliomas in phase I / II clinical research (NCT05363826, NCT04469699, NCT03897491, NCT04391062, NCT03048240), indicating that it has certain application potential in the treatment of gliomas.

[0007] Indocyanine green (ICG) is a tricarbocyanine near-infrared dye and is still the only FDA-approved contrast agent for clinical optical imaging and diagnosis. ICG can be used not only as a contrast agent for in vivo diagnosis, but also as a photosensitizer to achieve PDT of tumors. When tumor cells take up ICG, they can produce ROS under laser irradiation, leading to cell apoptosis. At the same time, ROS can cause oxidative stress in the endoplasmic reticulum, induce ICD in tumor cells, release DAMP signals such as calreticulin (CRT), adenosine triphosphate (ATP) and high mobility groupbox-1 protein (HMGB1), and promote the maturation of dendritic cells (DC) and T cell infiltration. However, ICG is unstable in aqueous solution and easily degraded. After intravenous injection, it quickly binds to plasma proteins and is metabolized, resulting in a very short half-life (2 to 4 minutes). In addition, the quantum yield of ICG is low, and the ROS produced alone are not sufficient to effectively kill tumors. These properties hinder the potential application of ICG in PDT. Summary of the invention

[0008] In view of the above-mentioned problems in the prior art, the present invention provides a bionic self-assembly delivery system for remodeling system and local immune function, and a preparation method and use thereof. The bionic self-assembly delivery system for remodeling system and local immune function, and a preparation method and use thereof are intended to solve the problem of poor effect of drugs in the prior art in treating gliomas.

[0009] The present invention provides a bionic self-assembly delivery system for remodeling systemic and local immune functions, the bionic self-assembly delivery system comprises a core, the core contains two drugs, the two drugs can self-assemble to form nanoparticles, one drug is a small molecule drug for regulating systemic immune function, the other drug is a drug for regulating local immune function of tumors, the surface of the nanoparticles is coated with cell membranes and brain targeting molecules. The self-assembly bionic delivery system can simultaneously overcome the systemic and local immunosuppression of gliomas, promote T cell infiltration, and improve the immunotherapy effect.

[0010] Furthermore, the small molecule drug for regulating systemic immune function is selected from natural products that have the effect of restoring systemic immune function, and the drug for regulating local immune function of tumors is a photosensitizer.

[0011] Furthermore, the small molecule drug for regulating the immune function of the system is selected from α-mangostin (α-M), and the photosensitizer is selected from the FDA-approved fluorescent probe indocyanine green (ICG).

[0012] Furthermore, the cell membrane is selected from tumor cell membrane, erythrocyte membrane, neutrophil membrane, platelet membrane or macrophage membrane.

[0013] Furthermore, brain-targeting molecules are used to increase the blood-brain barrier-crossing ability of biomimetic self-assembled nanoparticles.

[0014] Furthermore, the brain targeting molecule is selected from glucose-polyethylene glycol 2000 - Distearoylphosphatidylethanolamine (Glucose-PEG 2000 -DSPE).

[0015] The present invention also provides a method for preparing the above-mentioned bionic self-assembly delivery system, comprising the following steps:

[0016] 1) A step for preparing small molecule drugs regulating systemic immune function and drugs regulating local immune function of tumors to self-assemble into nanoparticles IM;

[0017] 2) a step for preparing tumor cell membranes;

[0018] 3) a step of coating the tumor cell membrane with IM to form nanoparticles IMM;

[0019] 4) One inserts Glucose-PEG on the surface of nanoparticle IMM 2000 -DSPE after the steps of forming the biomimetic self-assembled delivery system GIMM.

[0020] Furthermore, the small molecule drug for regulating systemic immune function is α-M, and the drug for regulating local immune function of tumors is ICG.

[0021] Specifically, in step 1), an ICG solution and an α-M solution are prepared with organic solvents respectively, and the molar ratio of ICG to α-M is 1:2 to 1:12, respectively, and the mixed solution is dropped into the aqueous phase under magnetic stirring. After the dropwise addition, stirring is continued, and the supernatant is discarded by centrifugation, and the nanoparticles are resuspended in triple distilled water to remove the unencapsulated free ICG, and the unencapsulated α-M is removed by a 0.22 μm microporous filter membrane, so as to obtain the nanoparticles IM formed by self-assembly of α-mangostin and indocyanine green.

[0022] Specifically, in step 2), the GL261-MUT cells expressing IDH1-R132H are digested and collected, washed with PBS buffer, and then resuspended in TM buffer containing protease inhibitors, hypotonic at 4°C overnight, repeatedly frozen and thawed 3 to 8 times, transferred to a Dounce homogenizer, repeatedly ground on ice, the homogenate is transferred to a centrifuge tube, centrifuged, the supernatant is separated, and centrifuged again, the supernatant is discarded to obtain a tumor cell membrane precipitate, and the cell membrane suspension is squeezed through a 400 nm polycarbonate membrane, and squeezed back and forth 10 to 20 times to obtain a tumor cell membrane.

[0023] Specifically, in step 3), the nanoparticles IM formed by self-assembly of α-M and ICG are mixed with the cell membrane, squeezed through a 200 nm polycarbonate membrane, extruded back and forth 10 to 20 times, centrifuged, the free cell membranes not encapsulated on the IM in the supernatant are discarded, and PBS is added to resuspend the precipitate to obtain the nanoparticles IMM formed after the tumor cell membrane is coated with the IM.

[0024] Specifically, in step 4), weigh Glucose-PEG 2000 -DSPE was dissolved in PBS, and then added to the nanoparticles IMM formed by tumor cell membrane-coated IM, incubated at 37°C for 1-2h, and centrifuged to remove the unencapsulated Glucose-PEG 2000 -DSPE, that is, the biomimetic self-assembly delivery system GIMM.

[0025] The present invention also provides application of the bionic self-assembly delivery system in preparing medicine for treating glioma.

[0026] The present invention also provides the use of the bionic self-assembly delivery system in combination with T cell-based immunotherapy in the treatment of glioma.

[0027] Glioma can induce unique systemic and local immunosuppression, weaken systemic and local immune functions, lead to lack of effector T cell infiltration in the tumor, and make glioma less responsive to immunotherapy. Therefore, it is necessary to construct a treatment strategy to overcome the systemic and local immunosuppression of glioma, synchronously restore systemic and local immune functions, and synergistically increase T cell infiltration in the tumor.

[0028] The present invention constructs a bionic self-assembly delivery system for reshaping systemic and local immune functions, and adopts carrier-free self-assembly technology to encapsulate two drugs, which can simultaneously overcome the systemic and local immunosuppression of gliomas and greatly promote T cell infiltration in gliomas. The core of the systemic self-assembly delivery system is nanoparticles self-assembled from natural small molecule products and photosensitizers, and tumor cell membranes and brain targeting molecules are coated on the surface of the nanoparticles. Bionics is achieved through cell membrane coating, which prolongs the long circulation of drugs in the body, and increases brain targeting with the help of glucose molecules, thereby improving the response of gliomas to immunotherapy.

[0029] The new effect of α-M discovered in the present invention is combined with the PDT effect of ICG to treat glioma through bionic self-assembly technology, which is clinically helpful to improve the patient's response to immunotherapy.

[0030] The co-delivery technology used in the present invention is mainly self-assembly and cell membrane biomimetic technology. With the help of the structural characteristics of the two drug molecules, nanoparticles are self-assembled and coated with cell membranes on the surface of the nanoparticles to prolong the circulation time of the preparation in the body and improve the targeting of tumors. At the same time, natural products and photosensitizers are efficiently delivered, thereby increasing the infiltration of T cells in gliomas.

[0031] Compared with the prior art, the technical effect of the present invention is positive and obvious. The bionic self-assembly delivery system constructed by the present invention is suitable for glioma, and the mouse orthotopic transplantation tumor model test shows that this bionic self-assembly delivery system can significantly enhance the immunotherapy effect of glioma. In summary, the delivery system constructed by the present invention has potential clinical application and transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effects of α-M on two types of glioma cells are shown; (A) Histogram of ROS levels in glioma cells detected by flow cytometry; (B) ROS levels in glioma cells after treatment with different preparations, data are expressed as mean ± SD (n = 3); (C) Effect of α-M on the survival rate of GL261-WT cells, data are expressed as mean ± SD (n = 6); (D) Effect of α-M on the survival rate of GL261-MUT cells, data are expressed as mean ± SD (n = 6).

[0033] Figure 2The effect of α-M on systemic immune function is shown; (A) Changes in thymus index of tumor-bearing mice at different α-M doses, data are expressed as mean ± SD (n = 6); (B) Thymus anatomy of tumor-bearing mice; (C) Changes in spleen index of tumor-bearing mice at different α-M doses, data are expressed as mean ± SD (n = 6); (D) Spleen anatomy of tumor-bearing mice; (E) CD8 + The number of T cells is expressed as mean ± SD (n = 4); (F) CD4 + The number of T cells, data are expressed as mean ± SD (n = 4); (G, I) CD8 + The proportion of T cells, data are expressed as mean ± SD (n = 4); (H, J) CD4 + The data of T cell ratio are expressed as mean ± SD (n = 4).

[0034] Figure 3 The formulation characterization of the biomimetic self-assembly delivery system GIMM is shown; (A) particle size distribution diagram; (B) particle size of the preparation, data are expressed as mean ± SD (n = 3); (C) surface potential of the preparation, data are expressed as mean ± SD (n = 4); (D) transmission electron microscopy image of the nanoparticles; (E) Coomassie brilliant blue staining image; (F) western blotting characterization of the expression of E-Cadherin and N-Cadherin on the cell membrane and the preparation.

[0035] Figure 4The in vitro and in vivo brain targeting evaluation of GIMM is shown; (A) Flow cytometry quantitative analysis of the uptake behavior of different preparations on GL261-MUT cells, and the data are expressed as mean ± SD (n = 3); (B) Flow cytometry quantitative analysis of the uptake behavior of different preparations on RAW264.7 cells, and the data are expressed as mean ± SD (n = 3); (C) Fluorescence microscopy qualitative observation of the uptake behavior of different preparations on GL261-MUT cells; (D) Fluorescence microscopy qualitative observation of the uptake behavior of different preparations on RAW264.7 cells; (E) Schematic diagram of the in vitro blood-brain barrier construction model; (F) Flow cytometry quantitative analysis of the uptake degree of GL261-MUT cells in the lower chamber at 4h and 8h, and the data are expressed as mean ± SD (n=3); (G) Qualitative observation of the uptake of GL261-MUT cells in the lower chamber at 4h under fluorescence microscopy; (H) Qualitative observation of the uptake of GL261-MUT cells in the lower chamber at 8h under fluorescence microscopy; (I) Pharmacokinetic behavior of different preparations in SD rats, data are expressed as mean±SD (n=3); (J) In vivo imaging of different preparations in mice with orthotopic glioma; (K) In vitro imaging; (L) Semi-quantitative results of in vitro brain imaging, data are expressed as mean±SD (n=3); (M) ICG fluorescence intensity of tumor cells in glioma, data are expressed as mean±SD (n=3); (N) Distribution of GIMM in glioma, data are expressed as mean±SD (n=3).

[0036] Figure 5 The cytotoxicity evaluation of GIMM combined with PDT on GL261-MUT cells is shown; (A, B) The effect of GIMM combined with PDT on ROS of GL261-MUT cells, the data are expressed as mean ± SD (n = 3); (C) MTT method was used to evaluate cell viability, the data are expressed as mean ± SD (n = 6); (D) Cell live-dead staining method was used to analyze GL261-MUT cells after treatment with different preparations; (E, F) Annexin V-FITC and PI staining methods were used to evaluate the degree of apoptosis of GL261-MUT cells after treatment with different preparations, the data are expressed as mean ± SD (n = 3).

[0037] Figure 6The evaluation of GIMM combined with PDT-induced immunogenic death of GL261-MUT is shown; (A) Laser confocal microscopy was used to observe the degree of CRT exposure of GL261-MUT cells; (B) Flow cytometry was used to quantitatively analyze the degree of CRT exposure of GL261-MUT cells, and the data were expressed as mean ± SD (n = 3); (C) Laser confocal microscopy was used to observe the expression of HMGB1 in GL261-MUT cells; (D) ELISA was used to analyze the content of HMGB1 in the cell culture medium, and the data were expressed as mean ± SD (n = 8); (E) ELISA was used to analyze the content of ATP in the cell culture medium, and the data were expressed as mean ± SD (n = 8); (F) Schematic diagram of in vitro DC maturation experiment; (G) Flow cytometry was used to quantitatively analyze the expression of CD11c + The data of CD80 and CD86 expression of cells are presented as mean±SD (n=3).

[0038] Figure 7 The results show that GIMM combined with PDT enhances systemic immune function; (A) Schematic diagram of the dosing regimen; (B) Immune organ index of tumor-bearing mice, data are presented as mean ± SD (n = 6); (C) Total thymic cell and T cell numbers, data are presented as mean ± SD (n = 4); (D) Total spleen cell and T cell numbers, data are presented as mean ± SD (n = 4); (E) Splenic CD8 + and CD4 + T cell ratio, data are expressed as mean ± SD (n = 4); (F) Splenic CD8 + and CD4 + The number of T cells is expressed as mean ± SD (n = 4); (G) spleen IFN + CD8 + T cell ratio, data are expressed as mean ± SD (n = 4); (H) Lymph node CD8 + and CD4 + T cell ratio, data are expressed as mean ± SD (n = 4); (I) Lymph node CD8 + and CD4 + T cell numbers, data are presented as mean ± SD (n = 4); (J) lymph node IFN + CD8 + The ratio of T cells and mature DCs, data are expressed as mean ± SD (n = 4); (K) peripheral blood CD8 + and CD4 + T cell ratio, data are expressed as mean ± SD (n = 4); (L) peripheral blood CD8 + and CD4 + T cell numbers, data are expressed as mean ± SD (n = 4); (M) peripheral blood IFN + and Trp-2+ CD8 + The proportion of T cells and mature DCs, data are expressed as mean ± SD (n = 4); (N) levels of pro-inflammatory cytokines IFN-γ and TNF-α in peripheral blood; (O) levels of anti-inflammatory cytokines TGF-β and IL-10 in peripheral blood.

[0039] Figure 8 The results show that GIMM combined with PDT enhances local immune function; (A) The ratio of myeloid-derived suppressor cells and regulatory T cells in tumors, data are presented as mean ± SD (n = 4); (B) The ratio of M1 / M2 phenotype macrophages and mature DCs in tumors, data are presented as mean ± SD (n = 4); (C) The ratio of CD8 + and CD4 + The proportion of T cells is shown as mean ± SD (n = 4); (D) CD8 + and CD4 + The absolute number of T cells, data are expressed as mean ± SD (n = 4); (E) intratumoral IFN-γ + and Trp-2 + The absolute number of CD8 T cells, data are expressed as mean ± SD (n = 4); (F) PD-1 + and TIM-3 + CD8 + The absolute number of T cells, data are expressed as mean ± SD (n = 4); (G) Laser confocal microscopy was used to observe the infiltration of different subtypes of T cells in the tumor; (H) Image J semi-quantitative analysis of the infiltration degree of different subtypes of T cells in (G), data are expressed as mean ± SD (n = 6); (I) ELISA method was used to analyze the levels of different cytokines in the tumor.

[0040] Fig. 9 The figure shows the treatment of IDH mutant glioma with GIMM combined with PDT and αPD-1; (A) Schematic diagram of the dosing regimen; (B) Bioluminescence image of tumor-bearing mice; (C) Semi-quantitative data of bioluminescence of tumor-bearing mice, data are expressed as mean ± SD (n = 6); (D) Survival curve of tumor-bearing mice; (E) Changes in body weight of tumor-bearing mice, data are expressed as mean + SD (n = 6); (F) Representative images of H&E and Ki-67 staining of the brain of tumor-bearing mice; (G) Semi-quantitative data of Ki-67 by Image J, data are expressed as mean + SD (n = 6); (H) Photo of the isolated thymus of tumor-bearing mice; (I) Photo of the isolated spleen of tumor-bearing mice.

[0041] Fig.10 The figure is a flow chart of the preparation process of the product of the present invention. DETAILED DESCRIPTION

[0042] The present invention is described in detail below by specific embodiments in conjunction with the accompanying drawings. In the specific embodiments, detailed descriptions are added to make the present invention better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other elements, materials, methods.

[0043] Preparation code description:

[0044] IM: nanoparticles formed by self-assembly of α-M and ICG;

[0045] IMM: Nanoparticles formed by tumor cell membrane coating IM

[0046] PIMM: Insertion of methoxy-terminated polyethylene glycol on the surface of IMM 2000 -Distearoylphosphatidylethanolamine followed by the formation of nanoparticles.

[0047] GIMM: Glucose-PEG is inserted into the surface of IMM 2000 - DSPE-formed nanoparticles.

[0048] The following substances without source provided are all commercially available substances.

[0049] Example 1: Effect of α-M on ROS and Cytotoxicity of Glioma Cells

[0050] Construction of glioma cell line: GL261 cells in logarithmic growth phase (Tongpai (Shanghai) Biotechnology Co., Ltd.) were obtained and cultured at 1×10 5 The cells were seeded in 6-well plates at a density of 100 cells / well, and an appropriate amount of IDH1-R132H overexpression lentivirus (NM_010497, Shanghai Jikai Gene Medical Technology Co., Ltd.) was added and mixed. At the same time, GL261 cells were added with control lentivirus (CON290) to set as GL261-WT cells. The lentivirus culture medium solution was added to the 6-well plate cells, incubated for 12 hours, and then the medium was replaced. The cells were cultured for 24 hours, and geneticin was added to select the cells to obtain GL261-WT cells (control cells) and GL261-MUT cells (expressing IDH1-R132H).

[0051] ROS level: GL261-WT (IDH wild type) and GL261-MUT (IDH1-R132H mutant) cells in the logarithmic growth phase were taken and 2×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and cultured for 24 hours. The culture medium was discarded, PBS and α-M were added respectively, and after incubation for 4 hours, the cells were rinsed 3 times with PBS, and DCFH-DA staining solution was added and incubated for 30 minutes. The cells were collected, washed 3 times with PBS, and analyzed by flow cytometry.

[0052] Cytotoxicity: GL261-WT and GL261-MUT cells in the logarithmic growth phase were collected and cultured at 5×10 3 The cells were seeded at a density of 100 μL / well in a 96-well plate and cultured for 24 hours. The supernatant was discarded and replaced with α-M solution. Six replicate wells were set up in each group and incubated for 24 hours. Then 5 mg / mL MTT solution was added and incubated in an incubator for 4 hours. The supernatant was discarded and 150 μL DMSO was added to each well. The cells were shaken at room temperature for 20 minutes to fully dissolve the generated crystalline formazan. The OD value was measured at a wavelength of 490 nm using an enzyme reader.

[0053] The results show that: Figure 1 As shown in A and B, α-M significantly increased the ROS level in GL261-WT and GL261-MUT cells; and the ROS level in GL261-MUT cells under the action of α-M was significantly higher than that in GL261-WT cells. Figure 1 C and D), IC of α-M in GL261-WT and GL261-MUT cells 50 They were 17.09 μM and 9.05 μM respectively, indicating that IDH gene mutation makes glioma cells more sensitive to the effects of α-M.

[0054] Example 2: Regulatory Effects of α-M on Systemic Immune Function

[0055] A GL261-MUT orthotopic glioma model was established, and the immunoregulatory effect of α-M was investigated. Intravenous injection was started on the 6th day after inoculation of orthotopic glioma, and a total of 5 doses were given. After the end of the administration, the body weight of the mice was weighed, and the peripheral blood, spleen and thymus of the mice were separated. The spleen and thymus were weighed, the immune organ index was calculated, and the changes of T cells in the spleen and peripheral blood of the mice were analyzed by flow cytometry.

[0056] The results show that: Figure 2 As shown in the results, glioma caused a significant decrease in the spleen index and thymus index of tumor-bearing mice, and a significant decrease in the proportion and number of T cells in the spleen and peripheral blood, while α-M significantly increased the spleen index and thymus index of tumor-bearing mice, and a significant increase in the proportion and number of T cells in the spleen and peripheral blood. In summary, α-M can significantly enhance the systemic immune function of tumor-bearing mice.

[0057] Example 3: Preparation and characterization of a biomimetic self-assembled delivery system containing α-M and ICG

[0058] like Fig.10As shown, DMSO was used to prepare ICG solution and α-M solution, and the molar ratio of ICG to α-M was 1:8. The mixed solution was slowly dripped into the aqueous phase under magnetic stirring. After the dripping, stirring was continued for 30 minutes. The supernatant was centrifuged and discarded. The nanoparticles were resuspended in triple distilled water, and centrifuged again. Repeated twice to remove the unencapsulated free ICG. Finally, the resuspended nanoparticles were added and filtered through a 0.22μm microporous membrane to remove the unencapsulated α-M, and IM was obtained. GL261-MUT cells were digested and collected, washed three times with PBS, and resuspended in 10mM hypotonic TM buffer (containing protease inhibitors) and hypotonic overnight at 4°C. Repeated freezing and thawing 5 times, transferred to a Dounce homogenizer, and repeatedly ground on ice. The homogenate was transferred to a centrifuge tube and centrifuged at 1000g and 10000 respectively. Separate the supernatant, centrifuge at 100,000 g, discard the supernatant, and obtain the GL261-MUT cell membrane precipitate. Extrude the cell membrane suspension through a 400 nm polycarbonate membrane, extruding back and forth 15 times, and determine the protein concentration of the cell membrane suspension using a test kit. Mix the IM and cell membrane, extrude through a 200 nm polycarbonate membrane, and extrude back and forth 15 times. Centrifuge at 12,000 rpm for 15 min, discard the free cell membrane that is not encapsulated on the IM, add PBS to resuspend the precipitate, and obtain IMM. Weigh the Glucose-PEG 2000 -DSPE was dissolved in PBS, 100 μL of the solution was added to IMM, incubated at 37°C for 1 h, and centrifuged to remove unencapsulated Glucose-PEG 2000 -DSPE, that is, the biomimetic self-assembly delivery system GIMM.

[0059] The preparation method of PIMM is as above: insert methoxy-terminated polyethylene glycol on the surface of IMM 2000 -Distearoylphosphatidylethanolamine was added to form nanoparticles. Dynamic light scattering was used to characterize the particle size and potential of GIMM, and transmission electron microscopy was used to observe the morphology of nanoparticles and determine the encapsulation efficiency of α-M and ICG.

[0060] Take GL261-MUT cells, GL261-MUT cell membranes, IMM and GIMM, add RIPA lysis buffer, place on ice for lysis, centrifuge at 12000rpm, 4℃ for 15min, take the supernatant, add SDS-PAGE protein loading buffer, adjust the protein concentration, mix the sample, heat at 95℃ for 10min, and store at -80℃ for use. According to the instructions of the SDS-PAGE gel preparation kit, prepare the corresponding concentrated gel and separation gel and add them to the mold. After gelation, add the sample to the lane, constant voltage mode, 60V electrophoresis for 1h, turn to 120V and continue electrophoresis for 1h. After the electrophoresis is completed, put the gel into Coomassie Brilliant Blue staining solution, shake slowly at room temperature, and stain for 1h. Take out the gel, rinse it with triple distilled water 3 times, add decolorizing solution, rinse repeatedly until the gel background is removed, and place it in a gel imager for photography. Western blotting: After electrophoresis, transfer and blocking methods, the primary antibodies added were Anti-E-Cadherin and Anti-N-Cadherin, and the other steps remained unchanged.

[0061] The results show that: Figure 3 As shown in AC, the particle sizes of IM, IMM, GIMM and cell membrane (CM) were 114.9±4.5nm, 132.6±9.0nm, 145.8±5.3nm and 279.7±38.6nm, respectively, the PDI were 0.118±0.051, 0.151±0.052, 0.156±0.033 and 0.448±0.035, respectively, and the Zeta potential were -61.8±5.1mv, -27.5±1.4mv, -32.2±3.1mv and -22.0±2.2mv, respectively. Compared with IM, the particle size of IMM increased by about 20nm, and the absolute value of Zeta potential decreased, which was close to the potential of CM. In addition, transmission electron microscopy observed that the surface of IMM and GIMM was covered with a cell membrane layer (attached Figure 3 D), indicating that the cell membrane is successfully coated on the IM surface. Figure 3 E and F show that the current preparation process can make GIMM retain membrane proteins better. High performance liquid chromatography and fluorescence spectrophotometry showed that the encapsulation efficiency of GIMM for α-M was 82.17±4.55%, and the encapsulation efficiency of ICG was 41.61±4.54%.

[0062] Example 4: Evaluation of the biomimetic self-assembly delivery system GIMM for brain targeting

[0063] Cell uptake: The tumor targeting and immune camouflage of GIMM were preliminarily evaluated by in vitro cell uptake experiments. GL261-MUT cells and RAW264.7 cells were taken at 2×10 5The cells were seeded in 6-well plates at a density of 100 cells / well and cultured for 24 hours. The supernatant was discarded and replaced with ICG-S (ICG solution), IM, IMM, PIMM and GIMM and incubated for 2 hours. The cells were collected and analyzed by flow cytometry. At the same time, GL261-MUT cells and RAW264.7 cells were seeded in 12-well plates, and ICG-S, IM, IMM, PIMM and GIMM were added and incubated for 2 hours. The cells were fixed with 4% paraformaldehyde solution, and the nuclei were stained with Hoechst and observed under a fluorescence microscope.

[0064] In vitro trans-BBB experiment: bEnd.3 cells in logarithmic growth phase were seeded in 24-well Transwell chambers at a cell number of 20,000 per well. When the transendothelial resistance reached 200 Ω·cm 2 GL261-MUT cells were seeded in the lower chamber of the Transwell, and ICG-S, IM, IMM, PIMM (mPEG 2000 -DSPE replaces Glucose-PEG 2000 -DSPE) and GIMM, incubated for 4h and 8h respectively. After the incubation, the culture medium was discarded, the GL261-MUT cells were digested and collected, and analyzed by flow cytometry. At the same time, after the incubation, the culture medium in the well plate was discarded, PBS was rinsed 3 times, 4% paraformaldehyde solution was used, and the nuclei were stained with Hoechst and observed under a fluorescence microscope.

[0065] Pharmacokinetic experiment: 15 SD rats were randomly divided into 5 groups, 3 rats in each group, and ICG-S, IM, IMM, PIMM and GIMM were injected into the tail vein respectively. The ICG injection dose was 3 mg / kg. 200 μL of blood was collected from the orbit at 1min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h and 24h after injection, placed in a heparin anticoagulation tube, and centrifuged at 4000rpm for 5min at 4°C. The supernatant plasma was taken, methanol was added, and vortexed for 5min to fully precipitate the protein. Centrifuged at 12000rpm for 10min at 4°C, the supernatant was taken, and the fluorescence intensity (λ ex =790nm,λ em =810nm). The drug concentration in plasma was calculated by the standard curve, the drug-time curve was drawn, and the pharmacokinetic parameters were calculated using DAS2.0 software.

[0066] In vivo cross-BBB experiment: Healthy 6-8 week old C57BL / 6 mice were injected with ICG-S, IM, IMM, PIMM and GIMM through the tail vein. The ICG injection dose was 1.2 mg / kg. 8 hours after the injection, the mice were anesthetized and perfused with 0.9% NaCl solution and 4% paraformaldehyde solution in sequence. The brain was dissected and isolated, and placed in a small animal in vivo imager to capture images and perform semi-quantitative analysis.

[0067] An orthotopic mouse glioma model was constructed. On the 10th day after tumor cell inoculation, ICG solution (ICG-S), IM, IMM, PIMM and GIMM were injected into the tail vein. The ICG injection dose was 1.2 mg / kg. Photos were taken with a small animal in vivo imager at 1h, 2h, 4h, 8h and 12h after injection. After 12h of shooting, the mice were anesthetized and perfused with 0.9% NaCl solution and 4% PFA in turn. The heart, liver, spleen, lung, kidney, brain, thymus and lymph nodes of the tumor-bearing mice were dissected and analyzed in vitro.

[0068] Flow cytometry was used to detect the uptake of the preparation by intratumoral cells: an orthotopic model of GL261-MUT-RFP mouse glioma was constructed. On the 10th day after inoculation of tumor cells, ICG-S, IM, IMM, PIMM and GIMM were injected into the tail vein, and the ICG injection dose was 1.2 mg / kg. The mice were anesthetized 8 hours after injection, and the heart was perfused with 0.9% NaCl solution. The brain tumor tissue was dissected and separated, and a single cell suspension was obtained through a 40μm cell sieve. The cells were collected by centrifugation at 500g for 5min. Red blood cell lysis buffer was added, lysed at room temperature for 2min, centrifuged at 500g for 5min, and washed 3 times with PBS after collecting the cells. Antibody staining was added according to the instructions. After incubation at 4°C for 40min, the cells were washed 3 times with PBS, and analyzed by flow cytometry.

[0069] The results show that: Figure 4 As shown in AD, compared with the IM group, the ICG fluorescence intensity in GL261-MUT cells in the IMM group was significantly increased; compared with the IMM group, the ICG fluorescence intensity in GL261-MUT cells in the GIMM group was increased by 2.72 and 2.26 times, respectively. Figure 4 As shown in EH, the ICG fluorescence intensity of the GIMM group in the tumor cells in the Transwell chamber was the highest. The above results show that GIMM significantly improves its ability to cross the BBB and target tumor cells after modification of tumor cell membranes and glucose molecules. In vivo pharmacokinetic studies have shown that GIMM has good long circulation and can prolong the retention time of drugs in the body ( Figure 4 I). like Figure 4 As shown in JL, GIMM has the highest ICG fluorescence intensity in the brain of tumor-bearing mice, indicating that it has good glioma targeting. Analysis of tumor cell ICG fluorescence intensity and distribution cells by flow cytometry showed that GIMM can better target in situ gliomas and selectively deliver drugs to tumor cells ( Figure 4 M and N).

[0070] Example 5: Effect of GIMM combined with PDT on tumor cell activity

[0071] ROS level assessment: GL261-MUT cells in the logarithmic growth phase were collected and 2×10 5 The cells were seeded at a density of 1000 cells / well in a 6-well plate and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM, and GIMM. The ICG concentration was 200 ng / mL and the α-M concentration was 1.68 μg / mL. After incubation for 4 h, the cells were rinsed with PBS three times and irradiated with a laser at 808 nm (0.8 W / cm 2 , 1min), and DCFH-DA staining solution was added and incubated for 30min. The cells were collected and the DCF fluorescence intensity was analyzed by flow cytometry. In addition, GL261-MUT cells were seeded in a confocal dish, treated with drugs according to the above scheme, fixed with 4% paraformaldehyde, stained with Hoechst, and observed under a laser confocal microscope.

[0072] MTT method: GL261-MUT cells in the logarithmic growth phase were taken and 5×10 3 The cells were seeded at a density of 100 / well in a 96-well plate and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM and GIMM. The ICG concentrations were adjusted to 100, 200, 400 and 500 ng / mL, and the corresponding α-M concentrations were 840, 1680, 3360 and 4200 ng / mL, respectively. The cells were incubated for 4 h, rinsed with PBS once, replaced with DMEM culture medium, and irradiated with laser at 808 nm (0.8 W / cm 2 , 1min), where laser irradiation is marked with "L", and cultured for 20h. Add 5mg / mL MTT solution and incubate in an incubator for 4h. Discard the supernatant, add 150μL DMSO, shake at room temperature for 20min to dissolve the crystallized formazan, and measure the OD value at a wavelength of 490nm using an enzyme marker.

[0073] Live / dead cell staining method: GL261-MUT cells in logarithmic growth phase were collected and 5×10 4 The cells were seeded at a density of 100 cells / well in a confocal culture dish and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM and GIMM. The ICG concentration was 500 ng / mL and the α-M concentration was 4.2 μg / mL. The cells were incubated for 4 h and irradiated with laser at 808 nm (0.8 W / cm 2 , 1 min), cultured for 20 h. The culture medium was discarded, the cells were rinsed 3 times with PBS, and the cells were observed and photographed under a laser confocal microscope according to the instructions of the Calcein / PI cell activity and cytotoxicity detection kit.

[0074] Cell apoptosis detection: GL261-MUT cells in the logarithmic growth phase were collected and 1×10 5The cells were seeded at a density of 1000 cells / well in a 24-well plate and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM, and GIMM, with an ICG concentration of 500 ng / mL and an α-M concentration of 4.2 μg / mL. The cells were incubated for 4 h and irradiated with a laser at 808 nm (0.8 W / cm 2 , 1min), and culture for 20h. Collect the supernatant. Combine the collected cells and the supernatant, centrifuge at 500g for 5min, and discard the supernatant. Adjust the cell concentration to 10 with 1× binding buffer of the kit. 6 100 μL of cell suspension was added with Annexin V-FITC and PI respectively, and incubated at room temperature in the dark for 15 min. Finally, 400 μL of binding buffer was added, the cell suspension was mixed, and the flow cytometer was used for analysis.

[0075] The results show that: Figure 5 As shown in A and B, α-M can synergize with the PDT effect of ICG-S to increase the ROS level of GL261-MUT (IM+L group), and under the targeting effect of GIMM, the ROS increase effect is most significant. Figure 5 As shown in CF, GIMM+L showed the highest cytotoxic effect and induced apoptosis of GL261-MUT cells to the greatest extent.

[0076] Example 6: GIMM combined with PDT to induce tumor cell ICD

[0077] Flow cytometry assay: GL261-MUT cells in the logarithmic growth phase were collected and 2×10 5 The cells were seeded at a density of 100 / well in a 6-well plate and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM and GIMM. The ICG concentration was 400 ng / mL and the α-M concentration was 3.36 μg / mL. After incubation for 4 h, the supernatant was discarded, the cells were rinsed with PBS 3 times, and DMEM complete culture medium was added. The cells were irradiated with laser at 808 nm (0.8 W / cm 2 , 1min), and cultured for 20h. The supernatant was discarded, the cells were collected, and FITC anti-mouse CRT antibody was added according to the instructions. After incubation at 4°C in the dark, PBS was washed three times, and the cells were analyzed by flow cytometry.

[0078] Laser confocal microscopy observation: GL261-MUT cells in the logarithmic growth phase were collected and 5×10 4The cells were seeded at a density of 100 / dish in a confocal culture dish and cultured for 24 h. The culture medium was discarded and replaced with PBS, ICG-S, α-M, IM, IMM and GIMM. The ICG concentration was 400 ng / mL and the α-M concentration was 3.36 μg / mL. The cells were incubated for 4 h, the supernatant was discarded, DMEM culture medium was added, and the cells were rinsed with PBS for 3 times. The cells were irradiated with a laser at 808 nm (0.8 W / cm 2 , 1min), and cultured for 20h. The supernatant was discarded, and the cells were rinsed with PBS three times. After fixation and blocking, FITC anti-mouse CRT antibody and AF647anti-mouse HMGB1antibody were added according to the instructions, and incubated overnight at 4°C in the dark. After incubation, the cells were rinsed with PBS three times, the nuclei were stained with Hoechst, and the cells were rinsed with PBS three times. The cells were observed and photographed under a laser confocal microscope.

[0079] ELISA analysis: After incubation according to the above method, the supernatant was removed and centrifuged at 2000 rpm for 5 min to separate the supernatant. The subsequent operation was performed according to the instructions of the ELISA kit to determine the HMGB1 concentration.

[0080] Result analysis: as attached Figure 6 As shown in AE, compared with the PBS control group, the CRT exposure on the surface of GL261 cells in the GIMM+L group increased significantly, the expression of HMGB1 in the nucleus decreased, and the levels of HMGB1 and ATP in the cell culture medium increased, indicating that GIMM combined with PDT can significantly promote CRT exposure of GL261-MUT cells and the extracellular release of HMGB1 and ATP, suggesting that it can effectively induce ICD in tumor cells. To further evaluate the activation effect of ICD on DC, in vitro DC maturation experiments showed that ( Figure 6 F and G), tumor cells treated with GIMM+L can significantly promote DCs to express CD80 and CD86, indicating that GIMM combined with PDT improves the immunogenicity of tumor cells and effectively promotes DC activation.

[0081] Example 7: GIMM combined with PDT to regulate systemic and local immune function

[0082] The mouse GL261-MUT orthotopic model was established and randomly divided into PBS, PBS+L, ICG-S+L, α-M, IM, IM+L, IMM+L and GIMM+L groups. After the administration, the mice were weighed and killed by cervical dislocation after anesthesia. The thymus and spleen were dissected and separated, and the organ weights were weighed after taking pictures. The immune organ index was calculated according to the following formula.

[0083] Immune organ index (mg / g) = immune organ mass (mg) / mouse body mass (g).

[0084] At the same time, the brain was dissected and separated, fixed with 4% paraformaldehyde for 48 hours, dehydrated with 15% sucrose solution and 30% sucrose solution, embedded in OCT and frozen, and then immunofluorescence staining was performed. After the administration, the spleen, thymus, peripheral blood and brain tumor were separated, and single cell suspension was obtained through a 70μm mesh, washed 3 times with PBS, and flow antibody staining was performed separately. After the end, it was fixed with 4% paraformaldehyde and detected on a flow cytometer. ELISA was used to detect cytokine levels in tumors and peripheral blood.

[0085] The results showed that the dosage regimen was as follows: Figure 7 As shown in A, after three treatments, the immune organ index of the GIMM+L group increased significantly, and the number of spleen cells and thymus cells increased significantly compared with the control group PBS+L. Further flow cytometry analysis showed that ( Figure 7 BM), GIMM combined with PDT can significantly increase the proportion, number and activity of T cells in spleen, lymph nodes and peripheral blood, upregulate the proportion of tumor-specific T cells, and promote DC activation. Analysis of serum cytokines showed that ( Figure 7 N and O), GIMM combined with PDT can significantly increase the levels of pro-inflammatory cytokines IFN-γ and TNF-α, while reducing the levels of anti-inflammatory cytokines TGF-β and IL-10. Analysis of tumors shows that, Figure 8 AF, intratumoral immunosuppressive cells such as myeloid-derived suppressor cells, regulatory T cells, and M2 phenotype macrophages were significantly reduced, while M1 phenotype macrophages and mature DCs were significantly increased, and CD8 + and CD4 + T cells increased significantly, and tumor-specific CD8 + T cells were significantly upregulated and their activity increased, but the expression of PD-1 and TIM-3 did not change significantly, suggesting that CD8 + There was no significant effect on the T cell exhaustion phenotype. In conclusion, GIMM combined with PDT can significantly enhance the systemic and local immune function of tumor-bearing mice.

[0086] Example 8: GIMM combined with PDT and immune checkpoint inhibitors for the treatment of IDH mutant glioma

[0087] The GL261-MUT-luc model was constructed and randomly divided into 8 groups, including PBS, temozolomide (TMZ), anti-PD-1 antibody (αPD-1), GIMM+L, and GIMM+L+αPD-1. The ICG dosage was 1.2 mg / kg, the α-M dosage was 10 mg / kg, the temozolomide (TMZ) dosage was 20 mg / kg, and the αPD-1 dosage was 5 mg / kg. The dosing schedule was as follows Fig. 9As shown in A. On days 6, 12, and 18, D-luciferin potassium solution (150 mg / kg) was intraperitoneally injected into tumor-bearing mice. Ten minutes later, the mice were anesthetized with isoflurane, and photos were taken with a small animal in vivo imager. The progression of intracranial tumors was monitored using bioluminescence technology and semi-quantitative analysis was performed. The survival status of tumor-bearing mice was continued to be observed at the end of administration, and the changes in the body weight of the mice were recorded. When the body weight loss of the mice exceeded 20% or obvious neurological dysfunction occurred, the mice were anesthetized and killed by cervical dislocation.

[0088] The results show that: Fig. 9 As shown in B and C, the brain bioluminescence intensity of mice in the GIMM+L group was significantly lower than that in the PBS group, while there was no significant difference in brain bioluminescence intensity between the αPD-1 group and the PBS group. The combination of GIMM+L and αPD-1 can further reduce the bioluminescence intensity of brain tumors, indicating that it can effectively inhibit the growth of gliomas. Combined with the survival time and body weight of mice, it can be seen that ( Fig. 9 D and E), the body weight of mice in the PBS, TMZ and αPD-1 groups decreased rapidly, the body weight of mice in the GIMM+L group remained stable during the treatment period, and decreased slightly after the treatment, while the body weight of mice in the GIMM+L+αPD-1 group showed a steady upward trend, indicating that the treatment can effectively inhibit the growth of glioma. Consistent with the results of bioluminescence data, H&E and Ki-67 staining results showed (Appendix Fig. 9 F and G), the tumor area of ​​GIMM+L+αPD-1 group was the smallest and the tumor cell proliferation activity was the weakest. Fig. 9 H and I showed that the spleen and thymus of mice in the GIMM+L+αPD-1 group were significantly larger than those in the PBS control group, indicating recovery of immune function.

Claims

1. A biomimetic self-assembly delivery system for remodeling systemic and local immune functions, characterized in that: The bionic self-assembly delivery system includes a core, which contains two drugs. The two drugs can self-assemble to form nanoparticles, one drug is a small molecule drug that regulates the systemic immune function, and the other drug is a photosensitizer, photothermal agent or sonosensitizer that regulates the local immune function of tumors. The surface of the nanoparticles is coated with a cell membrane, and the cell membrane is modified with brain targeting molecules.

2. A biomimetic self-assembly delivery system for remodeling system and local immune function as claimed in claim 1, characterized in that: The small molecule drug for regulating systemic immune function is selected from natural products having the function of restoring systemic immune function, and the drug for regulating local immune function of tumor is selected from photosensitizer.

3. A biomimetic self-assembly delivery system for remodeling system and local immune function as claimed in claim 2, characterized in that: The small molecule drug for regulating the immune function of the system is selected from α-mangostin, and the photosensitizer is selected from indocyanine green.

4. A biomimetic self-assembly delivery system for remodeling system and local immune function as claimed in claim 1, characterized in that The cell membrane is selected from tumor cell membrane, erythrocyte membrane, neutrophil membrane, platelet membrane or macrophage membrane.

5. A biomimetic self-assembly delivery system for remodeling system and local immune function as claimed in claim 1, characterized in that: The brain targeting molecule is selected from glucose-polyethylene glycol 2000 -Distearoylphosphatidylethanolamine.

6. The method for preparing the biomimetic self-assembly delivery system according to any one of claims 1 to 5, characterized in that The steps include: 1) A step for preparing nanoparticles IM formed by self-assembly of small molecule drugs regulating systemic immune function and drugs regulating local immune function of tumors; 2) a step for preparing tumor cell membranes; 3) a step of coating the tumor cell membrane with IM to form nanoparticles IMM; 4) One inserts glucose-polyethylene glycol on the surface of nanoparticle IMM 2000 - Steps for forming the biomimetic self-assembled delivery system GIMM after distearoyl phosphatidylethanolamine.

7. The method for preparing a biomimetic self-assembly delivery system according to claim 6, characterized in that: The small molecule drug for regulating the systemic immune function is α-mangostin, and the drug for regulating the local immune function of tumors is indocyanine green.

8. The method for preparing a biomimetic self-assembly delivery system according to claim 7, characterized in that: Also includes any of the following conditions, 1) In step 1), an indocyanine green solution and an α-mangostin solution are prepared with organic solvents respectively, and the indocyanine green and α-mangostin solution are mixed at a molar ratio of 1:2-1:

12. The mixed solution is dripped into the aqueous phase under magnetic stirring, and the stirring is continued after the dripping is completed. The supernatant is discarded by centrifugation, and the nanoparticles are resuspended in triple distilled water to remove the unencapsulated free indocyanine green. The unencapsulated α-mangostin is removed by a 0.22 μm microporous filter membrane, so as to obtain nanoparticles IM formed by self-assembly of α-mangostin and indocyanine green; 2) In step 2), the GL261-MUT cells expressing IDH1-R132H were digested and collected, washed with PBS buffer, and then resuspended in TM buffer containing protease inhibitors, hypotonic overnight at 4°C, repeatedly frozen and thawed 3 to 8 times, transferred to a Dounce homogenizer, repeatedly ground on ice, and the homogenate was transferred to a centrifuge tube, centrifuged, and the supernatant was separated, and centrifuged again, and the supernatant was discarded to obtain a tumor cell membrane precipitate, and the cell membrane suspension was squeezed through a 400 nm polycarbonate membrane, and squeezed back and forth 10 to 20 times to obtain a tumor cell membrane; 3) In step 3), the nanoparticles IM formed by self-assembly of α-mangostin and indocyanine green are mixed with the cell membrane, and squeezed through a 200 nm polycarbonate membrane, extruded back and forth 10 to 20 times, centrifuged, the free cell membrane not encapsulated on the IM is discarded, and PBS is added to resuspend the precipitate to obtain the nanoparticles IMM formed by the tumor cell membrane encapsulating the IM; 4) In step 4), weigh glucose-polyethylene glycol 2000 - Distearoylphosphatidylethanolamine was dissolved in PBS, and then added to the nanoparticles IMM formed by coating the tumor cell membrane with IM, incubated at 37°C for 1-2 hours, and centrifuged to remove the unencapsulated glucose-polyethylene glycol 2000 -Distearoylphosphatidylethanolamine to obtain the biomimetic self-assembly delivery system GIMM.

9. Use of the bionic self-assembly delivery system according to any one of claims 1 to 5 in the preparation of a drug for treating glioma.

10. Use of the bionic self-assembly delivery system according to any one of claims 1 to 5 in combination with T cell-based immunotherapy in the treatment of glioma.

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