A biomimetic self-assembly delivery system for remodeling system and local immune function, and its preparation method and use

Abstract

The present invention discloses a biomimetic self-assembly delivery system for remodeling systemic and local immune functions. The biomimetic self-assembly delivery system comprises a core containing two drugs that can self-assemble into nanoparticles. One drug is a small molecule drug that regulates systemic immune function, and the other drug is a drug that regulates local tumor immune function. The nanoparticles are coated with cell membranes and brain-targeting molecules to increase their brain targeting. The present invention also discloses a method for preparing the biomimetic self-assembly delivery system for remodeling systemic and local immune functions. The present invention also provides the use of the biomimetic self-assembly delivery system in the preparation of drugs for treating gliomas. The present invention increases T cell infiltration in gliomas by remodeling systemic and local immune functions, thereby improving the glioma's response to immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a self-assembly delivery system, specifically 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 therapeutic 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 immunotherapies in gliomas 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 gliomas 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 gliomas.

[0003] Systemic immunosuppression is an important clinical pathological change unique to glioma, which is manifested by a significant decrease in the number of peripheral blood T cells and CD4 + T cell counts can even reach the level of patients with acquired immunodeficiency syndrome. First, gliomas cause a large number of naive T cells to be sequestered in the bone marrow due to the loss of sphingosine-1-phosphate receptor 1, resulting in a decrease in systemic T cells. Second, gliomas induce 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 gliomas weaken the body's systemic immune function. However, there is currently a lack of safe and effective treatment strategies to overcome systemic immunosuppression.

[0004] Local immune suppression is closely related to intrinsic factors of glioma, influenced by different driver gene mutations and metabolic characteristics, forming a unique tumor microenvironment. Isocitrate dehydrogenase (IDH) gene mutations are the most common "gain-of-function" mutations in gliomas, with IDH1-R132H being the predominant mutation. This mutation can have the following local effects on the tumor: ① IDH mutants catalyze α-ketoglutarate to 2-hydroxyglutaric acid (2-HG), downregulating intratumoral IFN-γ and CXCL10 levels, reducing immune cell infiltration, and diminishing local immune responses. ② 2-HG production consumes NADPH, disrupting intracellular redox homeostasis and increasing levels of reactive oxygen species (ROS) in tumor cells, making them susceptible to oxidative damage. Intracellular ROS levels are closely associated with immunogenic cell death (ICD), which can release tumor-associated antigens and damage-associated molecular pattern (DAMP) signals, promoting the infiltration and activation of immune cells and enhancing local specific anti-tumor immune responses. Therefore, ROS-based therapeutic strategies have the potential to enhance local immune function in IDH-mutant gliomas.

[0005] α-Mangostin (α-M) is a xanthone compound derived from the fruit shell of Garcinia mangostana L., a plant of the Garcinia genus of the family Garciniaceae. 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 has a stronger killing effect on IDH mutant glioma than on IDH wild-type glioma; (3) α-M can increase the number of T cells in glioma-bearing mice and enhance the immune function of tumor-bearing mice. Therefore, this study 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 can be enhanced. However, α-M has inherent drugability defects such as poor water solubility, low oral bioavailability and short half-life, which result in low concentration at the target site and are unable to effectively kill tumors and regulate immune function.

[0006] Photodynamic therapy (PDT) is a novel treatment that uses lasers and photosensitizers to generate large amounts of ROS for specific cell destruction through localized irradiation at specific wavelengths. With advantages such as minimal side effects and high selectivity, it is increasingly being used in tumor treatment. Currently, several PDT therapies for gliomas are in Phase I / II clinical trials (NCT05363826, NCT04469699, NCT03897491, NCT04391062, and NCT03048240), demonstrating its potential for treatment.

[0007] Indocyanine green (ICG) is a tricarbocyanine near-infrared dye and remains 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 for 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 induce oxidative stress in the endoplasmic reticulum, inducing ICD in tumor cells and releasing DAMP signals such as calreticulin (CRT), adenosine triphosphate (ATP), and high mobility groupbox-1 protein (HMGB1), promoting dendritic cell (DC) maturation 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 response to the above-mentioned problems in the prior art, the present invention provides a bionic self-assembly delivery system for remodeling the system and local immune function, as well as a preparation method and use thereof. The bionic self-assembly delivery system for remodeling the system and local immune function, as well as a preparation method and use thereof, is intended to solve the problem of poor effect of drugs in the prior art in treating gliomas.

[0009] The present invention provides a biomimetic self-assembly delivery system for remodeling systemic and local immune functions. The system comprises a core containing two drugs that can self-assemble into nanoparticles: one small molecule drug that regulates systemic immune function, and the other a drug that regulates local tumor immune function. The nanoparticles are coated with a cell membrane and a brain-targeting molecule. This self-assembly biomimetic delivery system can simultaneously overcome both systemic and local immunosuppression in gliomas, promote T cell infiltration, and improve immunotherapy efficacy.

[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, red blood cell membrane, neutrophil membrane, platelet membrane or macrophage membrane.

[0013] Furthermore, brain-targeting molecules are used to enhance 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 biomimetic self-assembly delivery system, comprising the following steps:

[0016] 1) a step for preparing small molecule drugs that regulate systemic immune function and drugs that regulate local tumor immune function, and self-assembling them 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) Inserting Glucose-PEG on the surface of nanoparticle IMM 2000 -DSPE after the formation of the biomimetic self-assembly delivery system GIMM steps.

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

[0021] Specifically, in step 1), an ICG solution and an α-M solution are prepared using organic solvents, respectively, and the mixture is mixed at a molar ratio of ICG to α-M of 1:2 to 1:12. Under magnetic stirring, the mixed solution is added dropwise to the aqueous phase. After the addition is complete, stirring is continued, the supernatant is discarded by centrifugation, and triple-distilled water is added to resuspend the nanoparticles to remove unencapsulated free ICG. The unencapsulated α-M is removed by a 0.22 μm microporous filter membrane, thereby obtaining nanoparticles IM formed by self-assembly of α-mangostin and indocyanine green.

[0022] Specifically, 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. The cells were hypotonic at 4°C overnight, 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. The tube was centrifuged to separate the supernatant, and then centrifuged again. The supernatant was discarded to obtain a tumor cell membrane precipitate. 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.

[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, and squeezed back and forth 10 to 20 times. The mixture is centrifuged, and the free cell membranes not encapsulated on the IM are discarded from the supernatant. 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 coating IM, incubated at 37℃ for 1-2h, and centrifuged to remove the unencapsulated Glucose-PEG 2000 -DSPE, namely the biomimetic self-assembly delivery system GIMM.

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

[0026] The present invention also provides the use of the above-mentioned 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, weakening both systemic and local immune function, leading to a lack of effector T cell infiltration within the tumor and a low response to immunotherapy. Therefore, it is necessary to develop therapeutic strategies that overcome the systemic and local immunosuppression of glioma, simultaneously restore systemic and local immune function, and synergistically enhance T cell infiltration within the tumor.

[0028] The present invention constructs a bionic self-assembly delivery system that reshapes systemic and local immune functions. It uses 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. Its core 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 biomimetic self-assembly technology, which helps to improve patients' response to immunotherapy clinically.

[0030] The co-delivery technology employed in this invention primarily involves self-assembly and cell membrane biomimetic techniques. Leveraging the structural characteristics of the two drug molecules, the nanoparticles are self-assembled and coated with a cell membrane, extending the drug's circulation time in the body and improving tumor targeting. This allows for efficient simultaneous delivery of the natural product and photosensitizer, thereby increasing T cell infiltration in gliomas.

[0031] Compared with existing technologies, the present invention offers significant and positive technical benefits. The biomimetic self-assembly delivery system constructed in this invention is suitable for treating gliomas. Experiments in an orthotopic transplanted tumor model in mice have shown that this biomimetic self-assembly delivery system can significantly enhance the immunotherapy effect of gliomas. In summary, the delivery system constructed in this invention has potential clinical applications and translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effects of α-M on two glioma cell lines 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 effects of α-M on systemic immune function are 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 T cells in peripheral blood at different α-M doses + The number of T cells is expressed as mean ± SD (n = 4); (G, I) CD8 T cells in peripheral blood at different α-M doses + 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 4Figure 3 shows the in vitro and in vivo brain targeting evaluation of GIMM; (A) Flow cytometry quantitative analysis of the uptake behavior of different preparations on GL261-MUT cells, data are expressed as mean ± SD (n = 3); (B) Flow cytometry quantitative analysis of the uptake behavior of different preparations on RAW264.7 cells, 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, data are expressed as mean ± SD (n=3); (G) Qualitative observation of the uptake of GL261-MUT cells in the lower chamber at 4 h by fluorescence microscopy; (H) Qualitative observation of the uptake of GL261-MUT cells in the lower chamber at 8 h by 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 figures show the evaluation of cytotoxicity of GIMM combined with PDT on GL261-MUT cells; (A, B) Effects of GIMM combined with PDT on ROS in GL261-MUT cells, data are expressed as mean ± SD (n = 3); (C) MTT method was used to evaluate cell viability, data are expressed as mean ± SD (n = 6); (D) 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, data are expressed as mean ± SD (n = 3).

[0037] Figure 6Figure 4: Evaluation of immunogenic death of GL261-MUT induced by GIMM combined with PDT; (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 cell culture medium, and the data were expressed as mean ± SD (n = 8); (E) ELISA was used to analyze the content of ATP in 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 expression of CD80 and CD86 in cells was shown as mean ± SD (n = 3).

[0038] Figure 7 Figure 3: GIMM combined with PDT enhances systemic immune function; (A) Schematic diagram of the administration regimen; (B) Immune organ index of tumor-bearing mice, data are presented as mean ± SD (n = 6); (C) Total thymic cell and T cell counts, data are presented as mean ± SD (n = 4); (D) Total spleen cell and T cell counts, data are presented as mean ± SD (n = 4); (E) Splenic CD8 + and CD4 + The proportion of T cells is 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 expressed 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 counts, data are expressed as mean ± SD (n = 4); (M) peripheral blood IFN + and Trp-2+ CD8 + The proportions of T cells and mature DCs are expressed as mean ± SD (n = 4); (N) levels of proinflammatory 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 showed that GIMM combined with PDT enhanced 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 expressed as mean ± SD (n = 4); (D) CD8 + and CD4 + The absolute number of T cells is expressed as mean ± SD (n = 4); (E) IFN-γ in tumor + and Trp-2 + The absolute number of CD8 T cells is expressed as mean ± SD (n = 4); (F) PD-1 + and TIM-3 + CD8 + Absolute number of T cells, data are expressed as mean ± SD (n = 4); (G) Laser confocal microscopy observation of the infiltration of different subtypes of T cells in the tumor; (H) Image J semiquantitative analysis of the infiltration degree of different subtypes of T cells in (G), data are expressed as mean ± SD (n = 6); (I) ELISA analysis of different cytokine levels in the tumor.

[0040] Figure 9 Figure 3 shows the treatment of IDH-mutant glioma with GIMM combined with PDT and αPD-1; (A) Schematic diagram of the dosing regimen; (B) Bioluminescence images of tumor-bearing mice; (C) Semi-quantitative bioluminescence data of tumor-bearing mice, data are expressed as mean ± SD (n = 6); (D) Survival curve of tumor-bearing mice; (E) Body weight changes 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) Photographs of isolated thymus of tumor-bearing mice; (I) Photographs of isolated spleen of tumor-bearing mice.

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

[0042] The present invention will be described in detail below by way of specific embodiments in conjunction with the accompanying drawings. In the specific embodiments, detailed descriptions are provided to facilitate a better understanding of the present invention. However, those skilled in the art will readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other elements, materials, or 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: Methoxy-terminated polyethylene glycol inserted onto the surface of IMM 2000 -Distearoylphosphatidylethanolamine followed by the formation of nanoparticles.

[0047] GIMM: Glucose-PEG inserted into the IMM surface 2000 -Nanoparticles formed after DSPE.

[0048] The following substances without source provided are 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 Cells were seeded in 6-well plates at a density of 100 cells / well. An appropriate amount of IDH1-R132H overexpressing lentivirus (NM_010497, Shanghai GeneCare Gene Medical Technology Co., Ltd.) was added and mixed. GL261 cells were also plated with a control lentivirus (CON290) to create GL261-WT cells. The lentivirus-containing culture medium was added to the 6-well plates and incubated for 12 hours. The medium was then replaced and cultured for another 24 hours. Geneticin was then added to select the cells, yielding 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 cultured at 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and allowed to adhere for 24 hours. The culture medium was discarded, and PBS and α-M were added, respectively. After incubation for 4 hours, the cells were rinsed three times with PBS and incubated with DCFH-DA staining solution for 30 minutes. The cells were harvested, washed three 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 Cells were seeded at a density of 100 μL / well in a 96-well plate and allowed to adhere 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. Subsequently, 5 mg / mL MTT solution was added and the cells were incubated in an incubator for 4 hours. The supernatant was discarded and 150 μL of DMSO was added to each well. The cells were shaken at room temperature for 20 minutes to fully dissolve the resulting crystallized formazan. The OD value was measured at 490 nm using a microplate reader.

[0053] The results show that: Figure 1 As shown in A and B, α-M significantly increased the ROS levels in GL261-WT and GL261-MUT cells; and the ROS levels in GL261-MUT cells under the action of α-M were significantly higher than those 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 to investigate the immunomodulatory effects of α-M. Five intravenous injections were administered starting on day 6 after orthotopic glioma inoculation. Following administration, mice were weighed, and peripheral blood, spleen, and thymus were isolated. Spleen and thymus were weighed, immune organ indices were calculated, and T cell levels in the spleen and peripheral blood were analyzed by flow cytometry.

[0056] The results show that: Figure 2 As shown, glioma-induced significant decreases in the spleen index and thymus index of tumor-bearing mice, as well as a significant decrease in the proportion and number of T cells in the spleen and peripheral blood. However, α-M significantly increased the spleen index and thymus index of tumor-bearing mice, and significantly increased 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-assembly delivery system containing α-M and ICG

[0058] like Figure 10As shown, ICG and α-M solutions were prepared in DMSO, respectively, and mixed at a molar ratio of 1:8. The mixed solution was slowly added dropwise to the aqueous phase under magnetic stirring. Stirring was continued for 30 minutes after the addition, followed by centrifugation and the supernatant discarded. The nanoparticles were resuspended in triple-distilled water and centrifuged again twice to remove unencapsulated free ICG. Finally, the resuspended nanoparticles were added and filtered through a 0.22 μm microporous filter to remove unencapsulated α-M, thereby producing IM. GL261-MUT cells were harvested and digested, washed three times with PBS, and resuspended in 10 mM hypotonic TM buffer (containing protease inhibitors) and incubated at 4°C overnight. The cells were then freeze-thawed five times, transferred to a Dounce homogenizer, and repeatedly ground on ice. The homogenate was transferred to a centrifuge tube and centrifuged at 1000 g and 10,000 g, respectively. Separate the supernatant and centrifuge at 100,000 g. Discard the supernatant to obtain the GL261-MUT cell membrane pellet. Extrude the cell membrane suspension through a 400 nm polycarbonate membrane 15 times. Measure the protein concentration of the cell membrane suspension using a test kit. Mix the IM and cell membranes and extrude them through a 200 nm polycarbonate membrane 15 times. Centrifuge at 12,000 rpm for 15 minutes. Discard the supernatant to obtain the free cell membranes not encapsulated on the IM. Resuspend the pellet in PBS to obtain the 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, namely the biomimetic self-assembly delivery system GIMM.

[0059] The preparation method of PIMM is as follows: insert methoxy-terminated polyethylene glycol on the surface of IMM 2000 The nanoparticles were formed after the addition of α-M and ICG. The particle size and potential of GIMM were characterized by dynamic light scattering, and the morphology of the nanoparticles was observed by transmission electron microscopy, and the encapsulation efficiency of α-M and ICG was determined.

[0060] GL261-MUT cells, GL261-MUT cell membranes, IMMs, and GIMMs were collected and lysed with RIPA lysis buffer on ice. The cells were centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was collected and SDS-PAGE protein loading buffer was added to adjust the protein concentration. The samples were mixed thoroughly, heated at 95°C for 10 minutes, and stored at -80°C until ready to use. According to the instructions of the SDS-PAGE gel preparation kit, the corresponding stacking gel and separating gel were prepared and added to the mold. After gelation, the samples were loaded into the lanes and electrophoresed at 60V for 1 hour in constant voltage mode, then switched to 120V and continued for another hour. After electrophoresis, the gel was placed in Coomassie Brilliant Blue staining solution and stained for 1 hour at room temperature with slow shaking. The gel was removed and rinsed three times with triple-distilled water. Destaining solution was added and rinsed repeatedly until the gel background was removed. The gel was then placed in a gel imager for imaging. Western blotting: After electrophoresis, transfer, and blocking, add primary antibodies such as Anti-E-Cadherin and Anti-N-Cadherin, and the rest of the steps remain 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 PDIs were 0.118±0.051, 0.151±0.052, 0.156±0.033 and 0.448±0.035, respectively. The Zeta potentials 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, approaching that 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 well. 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 5Cells were seeded at a density of 100 cells / well in 6-well plates and allowed to adhere for 24 hours. The supernatant was discarded and replaced with ICG-S (ICG solution), IM, IMM, PIMM, and GIMM. The cells were then incubated for 2 hours. The cells were harvested and analyzed by flow cytometry. GL261-MUT cells and RAW264.7 cells were seeded in 12-well plates and incubated for 2 hours with ICG-S, IM, IMM, PIMM, and GIMM. The cells were fixed with 4% paraformaldehyde, stained with Hoechst, and observed under a fluorescence microscope.

[0064] In vitro trans-BBB assay: bEnd.3 cells in logarithmic growth phase were seeded in 24-well Transwell chambers at a density of 20,000 cells 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 instead of Glucose-PEG 2000 -DSPE) and GIMM, incubated for 4 and 8 hours, respectively. After incubation, the culture medium was discarded, and the GL261-MUT cells were harvested and analyzed by flow cytometry. At the same time, after incubation, the culture medium in the well plate was discarded, and the well plate was rinsed three times with PBS, followed by nuclear staining with 4% paraformaldehyde solution and Hoechst, followed by observation under a fluorescence microscope.

[0065] Pharmacokinetic experiment: 15 SD rats were randomly divided into 5 groups, with 3 rats in each group. 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 anticoagulant 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 =810 nm). The drug concentration in plasma was calculated using 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 via tail vein at a dose of 1.2 mg / kg ICG. Eight hours after injection, the mice were anesthetized and perfused with 0.9% NaCl solution and 4% paraformaldehyde solution sequentially. The brains were dissected and isolated, and images were captured using a small animal in vivo imager for semi-quantitative analysis.

[0067] An orthotopic mouse glioma model was established. Ten days after tumor cell inoculation, ICG solution (ICG-S), IM, IMM, PIMM, and GIMM were injected into the tail vein at a dose of 1.2 mg / kg. Images were captured using a small animal imaging device at 1, 2, 4, 8, and 12 hours after injection. Twelve hours after the imaging session, the mice were anesthetized and perfused transcardially with 0.9% NaCl solution and then 4% PFA. The heart, liver, spleen, lungs, kidneys, brain, thymus, and lymph nodes of the tumor-bearing mice were dissected and analyzed for ex vivo imaging.

[0068] Flow cytometry was used to assess intratumoral uptake of the agents. An orthotopic GL261-MUT-RFP mouse glioma model was established. Ten days after tumor cell inoculation, ICG-S, IM, IMM, PIMM, and GIMM were injected into the tail vein at a dose of 1.2 mg / kg. Eight hours after injection, mice were anesthetized and perfused with 0.9% NaCl solution. Brain tumor tissue was dissected and single-cell suspensions were obtained by passing through a 40 μm cell sieve. Cells were collected by centrifugation at 500 g for 5 minutes. Red blood cell lysis buffer was added, and the suspension was lysed at room temperature for 2 minutes. Cells were then centrifuged at 500 g for 5 minutes. After collection, cells were washed three times with PBS and stained with antibodies according to the manufacturer's instructions. After incubation at 4°C for 40 minutes, the cells were washed three 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 of GL261-MUT cells in the IMM group was significantly increased; compared with the IMM group, the ICG fluorescence intensity of GL261-MUT cells in the GIMM group was increased by 2.72 times and 2.26 times, respectively. Figure 4 As shown in EH, the GIMM group had the highest ICG fluorescence intensity in the tumor cells in the Transwell chamber. The above results indicate 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-term 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. Flow cytometric analysis of tumor cell ICG fluorescence intensity and distribution cells 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 100 cells / well in a 6-well plate and cultured for 24 hours. 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 hours, the cells were rinsed with PBS three times and irradiated with a laser at 808 nm (0.8 W / cm 2 , 1 min), then added DCFH-DA staining solution and incubated for 30 min. Cells were harvested and DCF fluorescence intensity analyzed by flow cytometry. Additionally, GL261-MUT cells were seeded in a confocal dish and treated according to the above protocol. They were then 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 collected and plated at 5×10 3 The cells were seeded at a density of 1000 cells / 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, respectively, and the corresponding α-M concentrations were 840, 1680, 3360, and 4200 ng / mL, respectively. The cells were incubated for 4 h, rinsed once with PBS, and replaced with DMEM culture medium. The cells were irradiated with a laser at 808 nm (0.8 W / cm 2 , 1 min), where laser irradiation is indicated by "L." Incubate for 20 h. Add 5 mg / mL MTT solution and incubate in an incubator for 4 h. Discard the supernatant, add 150 μL DMSO, and shake at room temperature for 20 min to dissolve the crystallized formazan. Measure the OD value at 490 nm using a microplate reader.

[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 hours. 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 hours and irradiated with a laser at 808 nm (0.8 W / cm 2 The cells were cultured for 20 h, the culture medium was discarded, the cells were rinsed three times with PBS, and the cells were observed and photographed under a laser confocal microscope according to the instructions of the Calcein / PI Cell Viability and Cytotoxicity Assay Kit.

[0074] Apoptosis detection: GL261-MUT cells in the logarithmic growth phase were collected and 1×10 5The cells were seeded at a density of 100 cells / well in a 24-well plate and cultured for 24 hours. 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 hours and irradiated with a laser at 808 nm (0.8 W / cm 2 , 1 min) and culture for 20 h. Collect the supernatant. Combine the collected cells and the supernatant, centrifuge at 500 g for 5 min, and discard the supernatant. Adjust the cell concentration to 10 using 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 minutes. Finally, 400 μL of binding buffer was added, the cell suspension was mixed, and the cell suspension was analyzed by flow cytometry.

[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 Figures CF, GIMM+L exhibited the highest cytotoxic effect and induced the greatest apoptosis in GL261-MUT cells.

[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 cells / well in a 6-well plate and cultured for 24 hours. 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 hours, the supernatant was discarded, the cells were rinsed 3 times with PBS, and DMEM complete culture medium was added. The cells were irradiated with a laser at 808 nm (0.8 W / cm 2 , 1 min) and incubate for 20 h. Discard the supernatant, collect the cells, add FITC anti-mouse CRT antibody according to the manufacturer's instructions, incubate at 4°C in the dark, wash three times with PBS, and analyze 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 1000 cells / dish in a confocal culture dish and cultured for 24 hours. 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 hours, the supernatant was discarded, DMEM culture medium was added, and the cells were rinsed with PBS three times. The cells were irradiated with a laser at 808 nm (0.8 W / cm 2 , 1 min) and incubate for 20 h. Discard the supernatant, rinse three times with PBS, and after fixation and blocking, add FITC anti-mouse CRT antibody and AF647 anti-mouse HMGB1 antibody according to the manufacturer's instructions. Incubate overnight at 4°C in the dark. After incubation, rinse three times with PBS, stain nuclei with Hoechst, rinse three times with PBS, and observe and photograph under a laser confocal microscope.

[0079] ELISA analysis: After incubation as described above, remove the supernatant and centrifuge at 2000 rpm for 5 minutes to separate the supernatant. Perform subsequent procedures according to the ELISA kit instructions to determine the HMGB1 concentration.

[0080] Result analysis: As shown in the attached Figure 6 As shown in Figures AE, compared with the PBS control group, the CRT exposure on the surface of GL261 cells in the GIMM+L group was significantly increased, the expression of HMGB1 in the cell nucleus was reduced, and the levels of HMGB1 and ATP in the cell culture medium were increased, indicating that GIMM combined with PDT can significantly promote the 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), GIMM+L-treated tumor cells significantly promoted the expression of CD80 and CD86 in DCs, indicating that GIMM combined with PDT enhanced the immunogenicity of tumor cells and effectively promoted DC activation.

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

[0082] An orthotopic GL261-MUT model was established in mice, and the mice were randomly divided into PBS, PBS+L, ICG-S+L, α-M, IM, IM+L, IMM+L, and GIMM+L groups. After administration, mice were weighed and sacrificed by cervical dislocation after anesthesia. The thymus and spleen were dissected, photographed, and weighed. The immune organ index was calculated using 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 fixed with 4% paraformaldehyde for 48 hours. The cells were then dehydrated and precipitated using 15% and then 30% sucrose solutions, embedded in OCT, and cryosectioned for immunofluorescence staining. After administration, the spleen, thymus, peripheral blood, and brain tumor were isolated and passed through a 70μm sieve to obtain single-cell suspensions. The suspensions were washed three times with PBS and stained with flow cytometry antibodies. The suspensions were fixed with 4% paraformaldehyde and analyzed by flow cytometry. Cytokine levels in tumors and peripheral blood were measured using ELISA.

[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 compared with the PBS+L control group, and the number of spleen cells and thymocytes increased significantly. 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, increase 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. 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 gliomas

[0087] The GL261-MUT-luc model was constructed and randomly divided into 8 groups, namely 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: Figure 9As shown in Figure A. On days 6, 12, and 18, tumor-bearing mice were intraperitoneally injected with D-luciferin potassium solution (150 mg / kg). Ten minutes later, the mice were anesthetized with isoflurane. Images were captured using a small animal in vivo imaging device, and intracranial tumor progression was monitored using bioluminescence technology and semi-quantitatively analyzed. After the end of dosing, the survival of the tumor-bearing mice was observed, and changes in body weight were recorded. When the body weight loss exceeded 20% or significant neurological dysfunction occurred, the mice were anesthetized and sacrificed by cervical dislocation.

[0088] The results show that: Figure 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 ( Figure 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 Figure 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. Figure 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 the recovery of immune function.

Claims

1. A biomimetic self-assembly delivery system for remodeling systemic and local immune functions, characterized in that: The biomimetic self-assembly delivery system includes a core containing two drugs, the two drugs can self-assemble to form nanoparticles, one drug is a small molecule drug α-mangostin that regulates systemic immune function, and the other drug is a photosensitizer indocyanine green that regulates local tumor immune function. The surface of the nanoparticles is coated with a cell membrane, the cell membrane is a tumor cell membrane, and the cell membrane is modified with a brain-targeting molecule selected from glucose-polyethylene glycol. 2000 -Distearoylphosphatidylethanolamine.

2. The method for preparing a biomimetic self-assembly delivery system for remodeling system and local immune function according to claim 1, characterized in that The steps include: 1) A step for preparing nanoparticles (IMs) formed by self-assembly of α-mangostin, a small molecule drug that regulates systemic immune function, and indocyanine green, a photosensitizer that regulates local tumor immunity; 2) a step to prepare tumor cell membranes; 3) a step of coating the tumor cell membrane with IM to form nanoparticles IMM; 4) A glucose-polyethylene glycol is inserted into the surface of the nanoparticle IMM 2000 - Steps for forming the biomimetic self-assembly delivery system GIMM after distearoylphosphatidylethanolamine.

3. The method for preparing a biomimetic self-assembly delivery system for remodeling system and local immune function according to claim 2, 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 separately using organic solvents, and the mixture is uniformly mixed at a molar ratio of indocyanine green to α-mangostin of 1:2 to 1:

12. The mixed solution is added dropwise to the aqueous phase under magnetic stirring. After the addition is complete, stirring is continued, and the supernatant is discarded by centrifugation. The nanoparticles are resuspended in triple-distilled water to remove unencapsulated free indocyanine green. The nanoparticles are filtered through a 0.22 μm microporous membrane to remove unencapsulated α-mangostin, thereby preparing nanoparticles IM formed by self-assembly of α-mangostin and indocyanine green. 2) In step 2), digest and collect GL261-MUT cells expressing IDH1-R132H, wash with PBS, and then resuspend in TM buffer containing protease inhibitors. Incubate the cells in a hypotonic atmosphere at 4°C overnight, freeze-thaw repeatedly 3-8 times, transfer the cells to a Dounce homogenizer, and grind repeatedly on ice. Transfer the homogenate to a centrifuge tube, centrifuge, and separate the supernatant. Centrifuge again and discard the supernatant to obtain a tumor cell membrane pellet. Extrude the cell membrane suspension through a 400 nm polycarbonate membrane 10-20 times to obtain tumor cell membranes. 3) In step 3), the nanoparticles IM self-assembled from α-mangostin and indocyanine green are mixed with the cell membranes and extruded through a 200 nm polycarbonate membrane 10-20 times. The mixture is centrifuged and the free cell membranes not encapsulated on the IM are discarded. The precipitate is resuspended in PBS to obtain the nanoparticles IMM formed by the tumor cell membranes 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 tumor cell membrane coating IM. The mixture was incubated at 37°C for 1-2 h and the unencapsulated glucose-polyethylene glycol was removed by centrifugation. 2000 -Distearoylphosphatidylethanolamine, thus obtaining the biomimetic self-assembly delivery system GIMM.

4. Use of the biomimetic self-assembly delivery system for remodeling system and local immune function according to claim 1 in the preparation of a drug for treating glioma.

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