A nanomaterial, a preparation method and application thereof

Abstract

The application discloses a kind of nanomaterial and its preparation method and application, the nanomaterial includes exosome and liposome;The exosome expresses immune checkpoint, the exosome is derived from brain glioma cell;The liposome is loaded with I type photosensitizer.The application is by PDT, immune checkpoint and brain glioma cell exosome antigen three tubes simultaneously to remodel tumor local immune microenvironment, greatly improve the efficiency of cancer immunotherapy, can activate strong anti brain glioma immune response and overcome immune escape.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a nanomaterial, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common primary intracranial tumors of the central nervous system. Currently, the treatment strategy for gliomas is primarily surgery, supplemented by radiotherapy and chemotherapy, but the treatment outcomes are not optimistic. Gliomas often occur in the motor, language, auditory, and visual centers. The boundaries between the lesion and normal brain tissue are indistinct, making complete surgical removal difficult and potentially leading to significant neurological deficits and severe sequelae. Radiotherapy and chemotherapy are used as adjunctive treatments after glioma surgery; however, chemotherapy can easily damage normal tissues and lead to drug resistance. Furthermore, the blood-brain barrier restricts the penetration of chemotherapy drugs into the tumor, resulting in unsatisfactory clinical efficacy for glioma patients undergoing radiotherapy and chemotherapy.

[0003] Tumor immunotherapy utilizes the body's innate immune system to specifically attack tumor cells, boasting advantages such as high efficacy and few side effects, and has become a new standard clinical treatment. However, in practical applications, immunotherapy for gliomas has shown poor efficacy. Research analysis indicates that the main reasons are limitations imposed by the complex immunosuppressive microenvironment of tumors and tumor heterogeneity. One of the reasons for the severe immunosuppressive microenvironment in gliomas is the presence of a large number of tumor-associated macrophages (TAMs) and immune checkpoints (ICs). TAMs are classified into M2 and M1 phenotypes. M2 macrophages can promote tumor angiogenesis and metastasis by secreting pro-tumor cytokines and inhibit the activation of immune cells (such as dendritic cells (DCs) and T cells). M1 macrophages secrete pro-inflammatory cytokines and phagocytose microorganisms, contributing to bactericidal and anti-tumor immunity. ICs, as regulators of the immune system, are crucial for maintaining autoimmune tolerance and regulating the duration and extent of peripheral tissue immune responses. However, these pathways can be "hijacked" and continuously activated by tumors, suppressing anti-tumor immunity and promoting tumorigenesis. Therefore, ICs proteins have always been a major target for cancer immunotherapy. Currently, FDA-approved checkpoint inhibitors can block CTLA4, PD-1, and PD-L1. Notably, tumor-associated macrophages typically possess a large number of immune checkpoint molecules on their surface, including SIRPa, Fcr, and Siglec-10. Furthermore, glioma tumor cells highly express these immune checkpoint ligands (e.g., CD47, CD20, CD24), releasing "don't eat me" signals to avoid macrophage phagocytosis and clearance. For example, the binding of tumor Siglec-10 to CD24 leads to macrophage exhaustion.

[0004] Furthermore, gliomas exhibit significant tumor heterogeneity, making them highly susceptible to immune escape by tumor cells. This is because glioma cells are prone to mutation, and each tumor cell carries unique tumor-associated antigens. Antitumor immune responses activated by a single or a few antigens cannot completely kill all tumor cells. Current methods for generating tumor antigens include: direct extraction of tumor-associated antigen proteins, genetic engineering expression of antigen proteins, chemical lysis of tumor cells to generate antigens, and physical methods to directly lyse tumor cells to generate tumor-associated antigens, among others. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a nanomaterial, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a nanomaterial comprising exosomes and liposomes; wherein the exosomes express immune checkpoints and are derived from glioma cells; and wherein the liposomes are loaded with a type I photosensitizer.

[0008] Exosomes (Exo) are small vesicles secreted by cells, ranging in diameter from 30 nm to 150 nm. Their lipid bilayer contains proteins such as CD63, CD81, and CD9, as well as endosomal membrane proteins flotillin and ALIX. Exosomes are secreted by various cell types in biological fluids such as blood, cerebrospinal fluid, and urine. Exosomes contain functional biomolecules (proteins, lipids, RNA, and DNA), can be transported to recipient cells, act as intercellular communication agents, and play important roles in various physiological and pathological processes, including tumor-related processes. Exosomes are also excellent drug delivery systems, possessing advantages such as deep tissue penetration, long circulation periods, high bioavailability, and the ability to cross biological barriers such as the blood-brain barrier (BBB). In this invention, exosomes are secreted by glioma cells, enabling them to carry all tumor-associated antigens of glioma cells. This activates the production of various tumor-specific T cells, killing different subtypes of glioma cells, avoiding the inability of single-antigen anti-tumor immunity to kill tumor cells with high tumor heterogeneity, and reducing or even preventing tumor cell immune escape. Exosomes expressing macrophage immune checkpoints can block the binding of tumor cell immune checkpoints to macrophage immune checkpoint receptors, relieving macrophage exhaustion and reshaping the immunosuppressive microenvironment of gliomas. Glioma cell exosomes, acting as carriers of type I photosensitizers, can cross the blood-brain barrier and deliver them to the brain tumor. The type I photosensitizers in the nanomaterials, through ROS generated by photodynamic therapy (PDT), convert M2 macrophages into M1 macrophages, thereby improving the tumor immunosuppressive microenvironment and blocking immune checkpoint reversal of macrophage exhaustion, thus enhancing anti-tumor capabilities. By employing a three-pronged approach—PDT, immune checkpoints, and glioma cell exosome antigens—to reshape the local tumor immune microenvironment, the efficiency of cancer immunotherapy is significantly improved, activating a powerful anti-glioma immune response and overcoming immune escape.

[0009] In some embodiments of the present invention, the exosomes are fused with liposomes to form nanoparticles; preferably, the exosomes are modified on the surface of the liposomes; preferably, the average hydrated particle size of the nanoparticles is 195 nm to 205 nm.

[0010] In some embodiments of the present invention, the mass ratio of exosomes to liposomes is 1:0.1 to 10.

[0011] In some embodiments of the present invention, the proportion of immune checkpoint positive exosomes in the exosomes is 90% to 100%.

[0012] In some embodiments of the present invention, the liposomes comprise a polymer-encapsulated type I photosensitizer; the polymer and the photosensitizer are non-covalently bonded.

[0013] In some embodiments of the present invention, the mass ratio of the polymer to the photosensitizer in the liposomes is 2 to 4:1.

[0014] In some embodiments of the present invention, the polymer includes at least one of polyethylene glycol, polylactic acid-glycolic acid copolymer, polylactic acid or polycaprolactone, and PLGA polymer.

[0015] In some embodiments of the present invention, the immune checkpoints include at least one of Siglec-10, SIRPα, LILRB2, and Tcr.

[0016] In some embodiments of the present invention, the type I photosensitizer includes at least one of Fs, Fo, Fc, and Ce6.

[0017] A second aspect of the present invention provides a method for preparing the aforementioned nanomaterial, comprising the following steps:

[0018] (1) A lentiviral vector carrying an immune checkpoint gene was transferred into glioma cells to obtain glioma cells expressing immune checkpoints. After expansion culture, the cell supernatant was collected and exosomes of glioma cells expressing immune checkpoints were isolated.

[0019] (2) The nanomaterial is prepared by mixing exosomes and liposomes and then freezing and melting.

[0020] In some embodiments of the present invention, in step (1), the lentiviral vector is transferred into glioma cells with the electrostatic assistance of a cationic polymer; preferably, the cationic polymer includes at least one of polybrene and lipofectamine 2000.

[0021] In some embodiments of the present invention, in step (1), exosomes are obtained by multiple centrifugations, specifically: the cell supernatant is centrifuged sequentially at a centrifugal force of 10000g to 15000g for 25min to 45min, the supernatant is collected, the supernatant is filtered, and then centrifuged at a centrifugal force of 100000g to 150000g for 80 to 100min to remove the supernatant, and then centrifuged at a centrifugal force of 100000g to 150000g for 80 to 100min to collect the precipitate, which is the exosome; preferably, the multiple centrifugations are all performed at 2℃ to 6℃.

[0022] In some embodiments of the present invention, the method for preparing the liposomes includes the following steps: dissolving the polymer and type I photosensitizer in an organic solvent, mixing by blowing, and then removing the organic solvent by solvent evaporation to obtain the liposomes.

[0023] In some embodiments of the present invention, the organic solvent includes at least one of dichloromethane and chloroform.

[0024] In some embodiments of the present invention, in step (2), during the freeze-melt process, the freezing temperature is -100°C to -60°C and the time is 10 min to 20 min; the melting temperature is 35°C to 38°C and the time is 15 min to 30 min; preferably, the freeze-melt process is repeated 2 to 8 times.

[0025] A third aspect of the invention provides a pharmaceutical composition comprising the aforementioned nanomaterial and pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutical molecules loaded in the nanomaterial.

[0027] In some embodiments of the present invention, the drug molecule includes at least one of TMZ chemotherapy drug, photosensitizer, photothermal agent, and immunomodulator.

[0028] In some embodiments of the present invention, the administration methods of the pharmaceutical composition include: oral, intravenous, intramuscular, subcutaneous, intratumoral, or intracavitary administration, preferably oral, intravenous, or intratumoral administration.

[0029] A fourth aspect of the invention provides for the use of the nanomaterial and / or the pharmaceutical composition in the preparation of medicaments for photodynamic and / or photothermal therapy and / or chemotherapy and / or radiotherapy for the treatment of glioma.

[0030] The beneficial effects of this invention are:

[0031] This invention successfully expresses macrophage immune checkpoints on glioma cell exosomes, fully utilizing the advantages of engineered exosomes as delivery carriers to achieve the safe in vivo delivery of type I photosensitizers across the blood-brain barrier to target glioma sites. This invention successfully combines photodynamic therapy and immune checkpoint blockade therapy, solving the problems of insufficient targeting and inadequate immune microenvironment regulation in current conventional antibody therapies and photodynamic therapies.

[0032] Exosomes derived from glioma cells fuse with the tumor cell membrane during their production and are secreted extracellularly. All tumor antigens on the tumor cell membrane are transferred to the exosome membrane, which can be called tumor cell exosome antigens. These tumor cell exosome antigens maintain their natural immunogenicity, activating the body's immune system to produce different types of tumor-specific T cells. This avoids the inability of a single antigen to kill the highly heterogeneous tumor cells, thus preventing tumor cell immune escape. In this way, exosomes derived from glioma cells carry all antigens on the surface of glioma cells and can also serve as a high-quality collection of tumor-associated antigens. This can generate different tumor-specific T cells to kill different types of glioma cells, maximally inhibiting tumor metastasis and recurrence, and preventing tumor immune escape. Ultimately, this improves the efficacy of glioma immunotherapy. Attached Figure Description

[0033] Figure 1 This is the expression result of Siglec10 on the surface of GL261 cells in Experiment Example 1 of this invention.

[0034] Figure 2 This is the expression result of Siglec10 on the surface of exosomes in Experiment Example 1 of this invention.

[0035] Figure 3 This is a transmission electron microscope image of eExo@P-NPs in Experimental Example 2 of the present invention.

[0036] Figure 4 This refers to the hydrated particle size of eExo@P-NPs in Experimental Example 2 of this invention.

[0037] Figure 5 The variations in hydrated particle size (A) and PDI value (B) of eExo@P-NPs in Experimental Example 2 of this invention are shown.

[0038] Figure 6 This refers to the change in hemolysis rate of eExo@P-NPs red blood cells in Experiment Example 2 of this invention.

[0039] Figure 7 This is the effect of eExo@P-NPs on the phagocytic capacity of macrophages in Experimental Example 3 of this invention.

[0040] Figure 8 The results of the phenotypic transformation performance test of eExo@P-NPs on macrophages (A) and M2 macrophages (B) in Experiment Example 4 of this invention are shown.

[0041] Figure 9 This is the result of antigen identification for eExo@P-NPs in Experiment Example 5 of this invention. Detailed Implementation

[0042] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0043] Example 1

[0044] This embodiment prepares a nanomaterial, and the specific process is as follows:

[0045] (1) Construction of GL261 glioma cell line stably expressing Siglec10 protein and extraction of exosomes

[0046] A lentiviral vector carrying the murine Siglec10 gene was efficiently transfected into the glioma cell line GL261 with the electrostatic assistance of polybrene. GL261 cell lines stably expressing the Siglec10 protein were selected using puromycin. The selected Siglec10-EMT-6 cells were seeded into 15cm cell culture dishes for expansion culture. After 24 hours of culture, the cells were washed 1-2 times with PBS, replaced with serum-free medium, and cultured for another 48 hours. The supernatant was collected for exosome extraction. Centrifuge the above culture medium at 2000g for 15 min and collect the supernatant. Then centrifuge the sample at 10000g for 30 min at 4℃ in a high-speed refrigerated centrifuge and collect the supernatant. Filter the supernatant of the previously centrifuged sample using a 0.22μm syringe filter moistened with PBS. Transfer the filtered sample to a Himac ultracentrifuge tube and centrifuge at 120000g for 90 min at 4℃. Remove the supernatant, resuspend the precipitate with PBS, and centrifuge again at 120000g for 90 min at 4℃. Collect the precipitate, which is the engineered exosome (eExo). The exosome protein content of engineered glioma cells was determined using a protein concentration assay kit via the BCA method, controlling the exosome protein concentration to be 0.5–0.8 mg / mL. After aliquoting, store at -80℃.

[0047] (2) Preparation of P-NPs nanocarriers

[0048] 10 mg of DSPE-PEG-NH2 and 4 mg of photosensitizer Fc were dissolved together in a 50 mL centrifuge container containing 5 mL of chloroform. After thorough mixing, 10 mL of deionized water was slowly added, resulting in a two-layer solution. Nitrogen gas was then bubbled through the mixture until no obvious layering occurred and the chloroform completely evaporated. The resulting solution contained P-NPs. This solution was then transferred to a 7000 kDa dialysis bag and dialyzed in distilled water for 48 hours, with the solution changed every 6 hours to remove free small molecules. The resulting PNPs solution was further concentrated with PEG, and the concentration was controlled at 0.2 mg / mL using a UV-Vis spectrophotometer. The solution was then stored at 4°C.

[0049] (3) Preparation of engineered exosome-modified nanoparticles eExo@P-NPs

[0050] Engineered exosomes expressing Siglec10 protein were mixed with P-NPs nanoparticle solution at a mass ratio of 1:1 and subjected to a three-cycle freeze-thaw cycle. The freezing temperature was -80℃ and the freezing time was 15 min. The thawing temperature was 37℃ and the thawing time was 15 min under water bath conditions. The mixture was repeatedly blown and stirred to mix, and the process was repeated three times to obtain the engineered exosome-modified nanocarrier eExo@PNPs.

[0051] Example 2

[0052] This embodiment prepares a nanomaterial, and the specific process is as follows:

[0053] Referring to the preparation method of Example 1, in step (3), the engineered exosomes expressing Siglec10 protein and P-NPs nanoparticle liposomes were mixed evenly at a mass ratio of 1:N (N = 0.1~10), and the mixture was subjected to a freeze-thaw cycle 3~6 times. The freezing temperature was -80℃ and the freezing time was 15min. The thawing temperature was 37℃ and the thawing time was 15min under water bath conditions. The mixture was repeatedly blown and mixed, and the process was repeated 3~6 times to obtain the engineered exosome-modified nanocarrier eExo@PNPs.

[0054] Example 3

[0055] This embodiment prepares a nanomaterial, and the specific process is as follows:

[0056] Following the preparation method of Example 1, in step (2), 10 mg of DSPE-PEG / PEG-PLGA and 4 mg of photosensitizer Fo were dissolved together in a 50 mL centrifuge containing 5 mL of chloroform. After mixing thoroughly, 10 mL of deionized water was slowly added, resulting in a two-layer solution. Nitrogen gas was then introduced into the mixture until no obvious layering occurred and the chloroform completely evaporated. The resulting solution was the P-NPs-containing solution. The solution was then transferred to a 7000 kDa dialysis bag and dialyzed in distilled water for 48 hours, with the solution replaced every 6 hours to remove free small molecules. The resulting PNPs solution was further concentrated with polyethylene glycol, and the concentration was controlled to be 0.2 mg / mL using a UV-Vis spectrophotometer. The solution was then stored at 4°C.

[0057] Comparative Example

[0058] This comparative example prepared a nanomaterial, and the specific process is as follows:

[0059] The preparation method is the same as in Example 1, except that glioma cells are replaced with microglia and breast cancer cells to obtain microglia exosomes and breast cancer cell exosomes.

[0060] Experimental Example 1

[0061] This experiment measured the expression of Siglec10 in the cell membrane and exosomes of the glioma cell line GL261. The specific procedure was as follows:

[0062] Siglec10-expressing mouse glioma cells GL261 were divided into 8×10 groups. 3 Cells were seeded at a density of [number] cells / well in 8-well confocal microscopes and cultured overnight. The supernatant was removed, and the cells were washed 1-2 times with pre-chilled PBS. Immobilization with 4% paraformaldehyde solution was performed for 10 min. The fixative was removed, and the cells were washed 2-3 times with pre-chilled PBS. Primary antibody anti-Siglec10 solution was then added, and the cells were incubated at room temperature for 4 h. The primary antibody was removed, and secondary antibody tagged with Alexa Fluor 488 was added. The cells were incubated at 37°C for 30 min. The secondary antibody was removed, and the cells were washed 1-2 times with PBS. Cell membrane dye DIR was added, and the cells were incubated for 10 min. The dye was removed, and the cells were washed 1-2 times with PBS. PBS solution was added again, and the cells were observed under a confocal microscope. Results are as follows: Figure 1 As shown, Siglec10 was successfully expressed on the GL261 cell membrane. Figure 2 As shown, Western blotting laboratory results confirmed the expression of Siglec10 on exosomes.

[0063] Experimental Example 2

[0064] This experimental example characterizes the eExo@P-NPs prepared in Example 1. The specific process is as follows:

[0065] The particle size variation of engineered glioma cell exosome-modified mouse nanocarriers was determined using a Zetasizer Nano ZS nanoparticle size analyzer. Results are as follows: Figure 3 and 4 As shown, the hydrated particle sizes of P-NPs and eExo@P-NPs are 190±5nm and 200±5nm, respectively, indicating that exosomes were successfully modified onto the surface of P-NPs, and eExo@P-NPs were successfully prepared.

[0066] The changes in hydrated particle size (A) and PDI value (B) of P-NPs and eExo@P-NPs in DMEM solution were measured, and the results are as follows: Figure 5 As shown, this indicates that eExo@P-NPs have good stability.

[0067] Mouse blood was separated and centrifuged to obtain red blood cells. P-NPs and eExo@P-NPs nanoparticles were then added to the cells, respectively. Red blood cell lysis was measured after 24 hours and 48 hours, and the hemolysis rate was calculated. The results are as follows: Figure 6 As shown, the hemolysis rate of both was less than 3% after 24 hours and less than 9% after 48 hours, indicating that eExo@P-NPs has superior biocompatibility and safety.

[0068] Experimental Example 3

[0069] This experiment tested the effect of eExo@P-NPs on the phagocytic capacity of macrophages. The specific procedure was as follows:

[0070] Mouse macrophages (Raw 264.7) in logarithmic growth phase were collected in 15 mL centrifuge tubes. The prepared fluorescent probe CSFE was added to a final concentration of 10 μg / mL. After incubation at 37°C for 30 min, the dye was removed by centrifugation, and the cells were washed three times with PBS. The cell count was then calculated, and the cells were stored at 4°C for later use. Mouse glioma cells (GL261) were collected in 15 mL centrifuge tubes. The prepared fluorescent probe Alex647 was added to a final concentration of 10 μg / mL. After incubation at 37°C for 30 min, the dye was removed by centrifugation, and the cells were washed three times with PBS. The cell count was then calculated, and the cells were stored at 4°C for later use. The treated Raw 264.7 and GL261 cells were seeded in 6-well plates at a cell ratio of 3:1 (total cell count: 5 × 10⁶ cells / well). 4 Cells were added to 6-well plates with PNPs (10 μg / mL photosensitizer) and eExo@P-NPs (10 μg / mL photosensitizer) respectively. After treatment for 24 h, the treated cells were collected, and the number of cells labeled with the dual probes was determined by flow cytometry. The results are shown below. Figure 7As shown, the eExo@P-NPs treatment group had the highest proportion of dual-fluorescent cells, indicating that the phagocytic capacity of macrophages was significantly enhanced.

[0071] Test Example 4

[0072] This experiment tested the ability of eExo@P-NPs to regulate macrophage phenotypic transition. The specific process was as follows:

[0073] Mouse macrophages Raw 264.7 were divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured overnight. Then, P-NPs (10 μg / mL photosensitizer) and eExo@P-NPs (10 μg / mL photosensitizer) were added to each well. After 1 hour of treatment, the medium was replaced with fresh medium, and the cells were irradiated with a 660 nm laser for 15 minutes. Following treatment, the cells were incubated in a CO2 incubator for 24 hours. After washing with PBS three times, the cells were collected, and anti-CD86-FITC was added. The change in the proportion of M1 macrophages was measured by flow cytometry. The results are as follows: Figure 8 As shown in Figure A, after treatment with a 660nm laser, the proportion of M1 macrophages in the eExo@P-NPs group increased by approximately 10-fold compared to the control group, indicating that the ROS induced by eExo@P-NPs under laser treatment can promote macrophage polarization towards the M1 type. Mouse macrophages (Raw 264.7) were classified as M2 macrophages. Following the above procedure, the treated cells were collected, and anti-CD206-APC was added. Flow cytometry was used to measure the change in the proportion of M2 macrophages. The results are shown in Figure A. Figure 8 As shown in Figure B, the proportion of M2 macrophages in the eExo@P-NPs group was significantly reduced by about 4 times compared with the control group after treatment with 660nm laser, indicating that the ROS induced by eExo@P-NPs under laser treatment can reduce the proportion of M2 macrophages.

[0074] Experimental Example 5

[0075] This experiment identified antigens carried by exosomes. The specific process was as follows:

[0076] The expression of antigen proteins in cells was detected using Western blotting, and the results are as follows: Figure 9 As shown.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nanomaterial, characterized in that: The formulation includes exosomes and liposomes; the exosomes express immune checkpoints and are derived from glioma cells; the liposomes are loaded with a type I photosensitizer; the exosomes and liposomes are fused to form nanoparticles and the exosomes are modified on the surface of the liposomes; the liposomes include a polymer encapsulating a type I photosensitizer; the polymer and the photosensitizer are bound in a non-covalent manner; the immune checkpoint includes Siglec-10; the type I photosensitizer includes Fc.

2. The nanomaterial according to claim 1, characterized in that: The mass ratio of exosomes to liposomes is 1:0.1~10.

3. The nanomaterial according to claim 1, characterized in that: The proportion of exosomes that are positive for immune checkpoints is 90% to 100%.

4. The nanomaterial according to claim 1, characterized in that: In the liposomes, the mass ratio of the polymer to the photosensitizer is 2-4:

1.

5. The nanomaterial according to claim 1, characterized in that: The polymer includes polyethylene glycol and polylactic acid. At least one of glycolic acid copolymer, polylactic acid or polycaprolactone, or PLGA polymer.

6. The nanomaterial according to claim 1, characterized in that: The average hydrated particle size of the nanoparticles is 195 nm to 205 nm.

7. A method for preparing the nanomaterial according to any one of claims 1 to 6, comprising the following steps: (1) A lentiviral vector carrying an immune checkpoint gene was transferred into glioma cells to obtain glioma cells expressing immune checkpoints. After expansion culture, the cell supernatant was collected and exosomes of glioma cells expressing immune checkpoints were isolated. (2) The nanomaterial is prepared by mixing exosomes and liposomes and then freezing and melting.

8. A pharmaceutical composition comprising the nanomaterial as described in any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

9. The use of a nanomaterial as described in any one of claims 1 to 6 or a pharmaceutical composition as described in claim 8 in the preparation of a medicament for photodynamic and / or photothermal therapy and / or chemotherapy and / or radiotherapy for the treatment of glioma.

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