Exosome-temperature-sensitive gel delivery system for in-situ administration of brain glioma postoperative resection as well as preparation method and application of exosome-temperature-sensitive gel delivery system

Abstract

The invention discloses an exosome-temperature-sensitive gel delivery system for brain glioma postoperative resection in-situ administration and a preparation method and application thereof.A drug-loaded exosome and an immunomodulator are jointly loaded in temperature-sensitive hydrogel, the drug-loaded exosome entraps mitoxantrone and siRNA, in the delivery system, the final concentration of mitoxantrone is 3-5 mg / kg, the final concentration of siRNA is 0.3-1.5 mg / kg, the final concentration of temperature-sensitive hydrogel is 0.3-1.5 mg / kg, and the final concentration of temperature-sensitive hydrogel is 0.3-1.5 mg / kg. The final concentration of the immunomodulator is 25-100 [mu] g / kg; the temperature-sensitive hydrogel is prepared from the following components in percentage by weight: 1 to 5 percent of chitosan, 5 to 15 percent of beta-sodium glycerophosphate, 0.01 to 2 percent of gelatin and the balance of water. The system can synchronously track and kill residual glioma starting cells and glioma cells in a targeted manner and remodel a tumor immune microenvironment, can bypass BBB through postoperative in-situ administration, is seamlessly connected with surgical treatment, and organically integrates chemotherapy, gene therapy and immunotherapy means.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an exosome-thermosensitive gel delivery system for in situ drug administration after brain glioma resection surgery, and a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is highly malignant, with a short median survival and an extremely low 5-year survival rate. The current standard clinical treatment for GBM is surgical resection supplemented with postoperative radiotherapy and chemotherapy. However, due to the invasive growth of GBM, it is difficult to achieve 100% resection of cancer cells during surgery. At the same time, the blood-brain barrier (BBB) ​​hinders the diffusion of drugs into the brain, resulting in GBM usually recurring rapidly in a short period of time. Recurrent tumors are often more malignant and resistant to radiotherapy and chemotherapy. Glioma initiating cells (GIC) are a special type of glioma with stem characteristics such as self-renewal and multidirectional differentiation capabilities. They play a key role in the recurrence of malignant gliomas and resistance to radiotherapy and chemotherapy, and are considered to be the key "seeds" of glioma recurrence. The complex tumor microenvironment of GBM, composed of immune cells and various factors secreted by them, astrocytes, extracellular matrix and blood vessels, is the "soil" on which the "seeds" grow. Although surgical treatment removes the main tumor of glioma and has largely removed the "soil" for tumor recurrence, the immunosuppressive microenvironment at the resection site has not been significantly improved by surgery. The recurrence of GBM is the result of the combined effects of the above factors. If you want to achieve the best effect of suppressing recurrence during postoperative in situ treatment, you must design a comprehensive treatment strategy that combines and regulates each key factor.

[0003] Current studies have found that postoperative in situ drug delivery can be seamlessly integrated with surgery, allowing the drug to bypass the BBB and act directly on the tumor resection site, thereby efficiently and low-toxicly removing residual glioma initiating cells and glioma cells. However, both the approved Glider implant and the postoperative in situ drug delivery system in existing preclinical studies have prolonged GBM survival to a certain extent, but have not yet achieved long-term survival, and their efficacy is very limited.

[0004] Hydrogels have good biocompatibility and safety, and can be used as effective drug carriers for local treatment of GBM after resection of the primary tumor. In particular, thermosensitive gels are solutions at low temperatures, which are very convenient for injection into the resection cavity after surgery. They can adhere to and fit the resection edge tissue in the resection cavity, and then quickly gel and form a gel structure at physiological temperature, improving the sustained release effect and achieving long-term sustained release of drugs in the resection cavity after surgery.

[0005] At present, a variety of methods are used to inhibit GBM recurrence, including surgical resection, chemotherapy, gene therapy, etc. In particular, the co-delivery system constructed for glioma initiating cells, intratumoral administration can kill glioma initiating cells and glioma cells, thereby inhibiting the growth of GBM. However, there are many causes of GBM recurrence, including glioma initiating cells, immunosuppressive microenvironment, drug resistance, etc. Only targeting one cause is not enough to completely inhibit the recurrence of GBM. Therefore, it is of great significance to construct a delivery system that simultaneously targets and tracks and kills residual glioma cells and glioma initiating cells and reshapes the tumor immune microenvironment to overcome the above shortcomings. Summary of the invention

[0006] In view of the problems existing in the prior art and the various complex causes of GBM postoperative recurrence and drug resistance, the present invention constructs an exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection and its preparation method and application, which can simultaneously target and track and kill residual glioma initiating cells and glioma cells and reshape the tumor immune microenvironment, thereby inhibiting the recurrence of brain glioma and prolonging survival.

[0007] The present invention is achieved in this way: an exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection after surgery, wherein drug-loaded exosomes and immunomodulators are co-loaded in a thermosensitive hydrogel, wherein the drug-loaded exosomes encapsulate mitoxantrone (MIT) and siRNA, wherein the final concentration of MIT is 3-5 mg / kg, the final concentration of siRNA is 0.3-1.5 mg / kg, and the final concentration of the immunomodulator is 25-100 μg / kg; the thermosensitive hydrogel is composed of chitosan, sodium β-glycerophosphate and gelatin, wherein by mass percentage, the chitosan is 1%-5%, the sodium β-glycerophosphate is 5%-15%, the gelatin is 0.01%-2%, and the rest is water.

[0008] The drug-loaded exosomes are prepared using the method of CN202310738539.6.

[0009] The exosomes are at least one of macrophage exosomes, HEK293T cell exosomes, bone marrow mesenchymal stem cell exosomes, umbilical cord mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

[0010] The immunomodulator is one of IL-12, TNF-α, and IFN-γ.

[0011] The preparation method of the exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection surgery comprises the following steps: (1) Preparation of drug-loaded exosomes: prepared using the method of CN202310738539.6; (2) Preparation of thermosensitive hydrogel: Weigh a certain amount of chitosan to prepare a chitosan solution with a concentration of 0.05-0.25 M and a pH of 2-5; Weigh a certain amount of sodium β-glycerophosphate to prepare a 1.5-5M sodium β-glycerophosphate solution with a pH of 8-10; Weigh a certain amount of gelatin to prepare a gelatin solution with a concentration of 0.01-0.5 mM and a pH of 1-4; After the chitosan solution and the gelatin solution are uniformly mixed, the sodium β-glycerophosphate solution is slowly added under stirring, and the pH is adjusted to 7.2-7.4 to obtain a thermosensitive hydrogel; (3) Preparation of exosome-thermosensitive gel delivery system: Add the immunomodulator and drug-loaded exosomes into the thermosensitive hydrogel at 4-15°C and mix them to obtain a liquid exosome-thermosensitive gel delivery system.

[0012] The application of the exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection obtained by the above preparation method in the preparation of anti-brain glioma drugs.

[0013] GBM was surgically resected and the injection was directly injected into the resection cavity at the end of the operation.

[0014] The advantages and technical effects of the present invention are as follows: The present invention can simultaneously target, track and kill residual glioma initiating cells and glioma cells and reshape the tumor immune microenvironment. It can bypass the BBB and seamlessly connect with surgical treatment through in situ administration after surgery. It can organically integrate chemotherapy, gene therapy and immunotherapy to simultaneously eliminate the "seeds" and "soil" of GBM recurrence, and effectively curb the postoperative recurrence of GBM through the synergistic effect of multiple mechanisms, reducing toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a mechanism diagram of the exosome-thermosensitive gel delivery system of the present invention for in situ drug delivery after brain glioma resection surgery.

[0016] Figure 2 Characterization diagram of the exosome-thermosensitive hydrogel delivery system ((A) Thermosensitivity of hydrogel and VEMR@Gel; (B) Changes in viscosity with increasing shear rate of hydrogel and VEMR@Gel; (C) Rheological analysis of hydrogel and VEMR@Gel; (D) Hemolysis of hydrogel; (E) Scanning electron microscopy results of hydrogel; (F) Scanning electron microscopy results of VEMR@Gel; (G) Analysis of degradation of hydrogel).

[0017] Figure 3Figure 3 is the in vivo pharmacodynamic evaluation of the exosome-thermosensitive hydrogel delivery system ((A) In vivo imaging of orthotopically resected mice on days 12, 20, 24, and 30 after different treatments; (B) Fluorescence quantification of (A); (C) HE staining results of brain tissue of mice in different treatment groups; (D) Survival analysis of mice in each group; (E) TUNEL staining results of brain tissue of mice in different treatment groups).

[0018] Figure 4 Figure 2 is the expression of CD133 and Notch1 in the brain tissue of mice in different treatment groups.

[0019] Figure 5 CD4 + T cells, CD8 + Expression of T cells and DC cells. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] The exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection of the present invention comprises drug-loaded exosomes and immunomodulators co-loaded in a thermosensitive hydrogel, wherein the drug-loaded exosomes encapsulate mitoxantrone (MIT) and siRNA, and wherein the final concentration of MIT is 3-5 mg / kg, the final concentration of siRNA is 0.3-1.5 mg / kg, and the final concentration of the immunomodulator is 25-100 μg / kg; the thermosensitive hydrogel is composed of chitosan, sodium β-glycerophosphate and gelatin, wherein the chitosan comprises 1%-5%, the sodium β-glycerophosphate comprises 5%-15%, the gelatin comprises 0.01%-2%, and the remainder is water.

[0022] The drug-loaded exosomes are prepared using the method of CN202310738539.6.

[0023] The exosomes are at least one of macrophage exosomes, HEK293T cell exosomes, bone marrow mesenchymal stem cell exosomes, umbilical cord mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

[0024] The immunomodulator is one of IL-12, TNF-α, and IFN-γ.

[0025] The preparation method of the exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection surgery comprises the following steps: (1) Preparation of drug-loaded exosomes: prepared using the method of CN202310738539.6; (2) Preparation of thermosensitive hydrogel: Weigh a certain amount of chitosan to prepare a chitosan solution with a concentration of 0.05-0.25 M and a pH of 2-5; Weigh a certain amount of sodium β-glycerophosphate to prepare a 1.5-5M sodium β-glycerophosphate solution with a pH of 8-10; Weigh a certain amount of gelatin to prepare a gelatin solution with a concentration of 0.01-0.5 mM and a pH of 1-4; After the chitosan solution and the gelatin solution are uniformly mixed, the sodium β-glycerophosphate solution is slowly added under stirring, and the pH is adjusted to 7.2-7.4 to obtain a thermosensitive hydrogel; (3) Preparation of exosome-thermosensitive gel delivery system: Add the immunomodulator and drug-loaded exosomes into the thermosensitive hydrogel at 4-15°C and mix them to obtain a liquid exosome-thermosensitive gel delivery system.

[0026] The application of the exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection obtained by the above preparation method in the preparation of anti-brain glioma drugs.

[0027] GBM was surgically resected and the injection was directly injected into the resection cavity at the end of the operation.

[0028] The present invention first performs surgical resection of GBM, and then directly administers the drug into the resection cavity at the end of the operation. The advantages are that the number of operations is reduced, the operation is seamlessly connected, and the treatment window period is filled; the drug crosses the blood-brain barrier and directly exerts an anti-tumor effect; and it plays a role in reducing toxicity and increasing efficacy.

[0029] Since GBM infiltrates into the brain parenchyma and grows, it is difficult to completely remove the tumor by surgery, and the residual glioma cells, especially the residual glioma initiating cells, will still cause GBM recurrence. For the residual glioma cells and glioma initiating cells, the present invention selects MIT as a chemotherapy drug to kill them; in addition, the present invention also uses siNotch1 to reduce the stemness of glioma initiating cells, thereby affecting the proliferation of glioma initiating cells.

[0030] GBM is generally considered a "cold" tumor because it lacks infiltration of anti-tumor immune cells. In view of the immunosuppressive microenvironment of GBM, the present invention selects MIT and IL-12, wherein MIT has an ICD effect and can activate the immune system to play a role; IL-12 is an immunomodulator that can promote the infiltration of immune cells. MIT and IL-12 together reverse the immunosuppressive microenvironment of GBM, turning the "cold" tumor into a "hot" tumor.

[0031] In order to solve the problem of GBM drug resistance, the present invention uses the chemotherapy drug MIT, because the research results show that GBM is very likely to develop drug resistance to the chemotherapy drug temozolomide (TMZ), and its killing effect on glioma initiating cells is not as good as MIT.

[0032] The drug of the present invention has good anti-tumor effect, good stability, and diverse functions. It can simultaneously target, track, and kill residual glioma initiating cells and glioma cells and reshape the tumor immune microenvironment, thereby inhibiting GBM recurrence.

[0033] Example 1 Construction and characterization of drug-loaded exosomes The construction and characterization results of drug-loaded exosomes were prepared using CN202310738539.6, a preparation and application of a siRNA co-delivery system that efficiently targets glioma initiating cells.

[0034] Example 2 Construction and characterization of exosome-thermosensitive gel drug delivery system S1. Prepare 2% chitosan (CS)-hydrochloric acid solution, 56% β-glycerophosphate (β-GP)-sodium bicarbonate (NaHCO 3 ) solution, 0.5% gelatin (GA)-hydrochloric acid solution. At 4°C and magnetic stirring, slowly add the gelatin solution to the chitosan solution and mix evenly, then slowly add the β-glycerophosphate sodium solution to the above solution while maintaining stirring, adjust the pH to 7.2-7.4 with sodium hydroxide (NaOH) to obtain a liquid thermosensitive hydrogel; then add the immunomodulator (IL-12, final concentration of 50μg / kg) and the exosome targeted delivery system (the final concentration of mitoxantrone is 3mg / kg, and the final concentration of siRNA is 0.375mg / kg) to the thermosensitive hydrogel and mix to obtain an exosome-gel drug delivery system. Place at 4°C and 37°C for 3-5 minutes respectively to observe the state of the hydrogel. Figure 2 A The results showed that the hydrogel was in liquid state at 4°C and in solid state at 37°C.

[0035] S2. The rheological properties of the hydrogel were measured on a rheometer. In order to detect the thixotropic properties of the hydrogel, a dynamic time scan was performed at a strain frequency of 1 rad / s. G′ (storage modulus) and G″ (loss modulus) were measured at a strain amplitude of 0.5% in the frequency range of 0.1-100 rad / s. The results are shown in Figure 2 As shown in B and 2C, the hydrogel can undergo shear thinning before and after drug loading ( Figure 2 B), both the storage modulus and loss modulus values ​​increased significantly with time, indicating gel formation ( Figure 2 C).

[0036] S3. Use physiological saline and ultrapure water as negative and positive controls respectively, incubate the gel or control group with diluted fresh mouse whole blood at 37°C for 60 minutes, centrifuge and take the supernatant, then use a UV detector to detect the absorbance of the supernatant at a wavelength of 545nm to calculate the hemolysis rate. The formula is as follows: Hemolysis rate (%) = (average absorbance of the experimental group - average absorbance of the negative control group) / (average absorbance of the positive control group - average absorbance of the negative control group). Figure 2 As shown in D, after calculation, the hemolysis rate of the hydrogel is 0.58%, which is less than 5% and meets the requirements of medical materials.

[0037] S4. The hydrogel and drug-loaded gel were freeze-dried and the microstructure of the freeze-dried hydrogel was observed using a scanning electron microscope. Figure 2 The E-2F results showed that the hydrogel exhibited multiple pore structures. After drug loading, the pore structures became fewer, indicating that the drugs were filled in the pores of the hydrogel.

[0038] S5. The in vitro degradation of the hydrogel was evaluated by adding 1 mL of hydrogel to the bottom of a 5-ml capped glass bottle and 2 ml of PBS or artificial cerebrospinal fluid. The capped glass bottle was then kept at 37 °C and the remaining mass of the hydrogel was recorded. Figure 2 G The results show that with the increase of time, the residual mass of the hydrogel in the artificial cerebrospinal fluid gradually decreases compared with PBS, indicating that the artificial cerebrospinal fluid accelerates the degradation of the hydrogel. In other words, under normal physiological conditions, the hydrogel can also be degraded well.

[0039] Example 3 Study on the in vivo pharmacodynamics and mechanism of action of exosome-thermosensitive gel drug delivery system S1. Select male C57BL / 6 mice weighing about 20 g. After anesthesia, remove the hair on the top of the head, make a midline incision on the scalp to expose the anterior fontanelle, clean the incision site with 75% alcohol, and carefully use an electric drill to cut a circular incision with a diameter of 3 mm 2 mm to the right of the midsagittal suture and 1 mm behind the coronal suture of the skull to expose the cerebral cortex. Use a microsyringe to slowly inject 5 μL of Luc-GL261 cells that stably express luciferase. The number of cells is 2×10 5 The injection depth was 2.5 mm, and the scalp was sutured to establish an intracranial GBM animal model. 12 days after Luc-GL261 was implanted into the mouse brain, the tumor growth was confirmed by in vivo imaging, and brain tumor resection surgery was performed under gas anesthesia to establish a GBM surgical resection mouse model.

[0040] S2. The mice with GBM surgical resection were randomly divided into 6 groups, with 10 mice in each group. After surgery, PBS, Gel, EM@Gel, EMR@Gel, VEMR@Gel, IL-12&VEMR@Gel were injected in situ into the tumor resection cavity (the dosage of MIT, siRNA and IL12 in each group remained the same). The scalp was sutured and the luminescence intensity of the tumor in each group of mice was detected by in vivo imaging technology to determine the recurrence and growth of the tumor. At the same time, the weight changes and survival time of the mice were recorded to investigate the differences in the therapeutic effects of inhibiting GBM recurrence among the groups. The mice were killed on the 30th day after cell inoculation, and the surrounding tissues of the surgical resection were collected. After fixation with paraformaldehyde and dehydration, HE staining was used to investigate the tissue pathology, and TUNEL staining was used to investigate the cell apoptosis. The results are shown in Figure 3 As shown in the figure, bioluminescence results showed that compared with the control group, mice in the EM@Gel, EMR@Gel, and VEMR@Gel groups showed significant tumor growth inhibition, and IL-12&VEMR@Gel treatment significantly inhibited the growth of glioma ( Figure 3 A-3B), the median survival time of mice was significantly prolonged to 150 days ( Figure 3 D). On the 30th day after tumor cell inoculation, the tumor-bearing mice were euthanized and brain tumor tissues were collected and stained with HE. The results showed that the tumor in the IL-12&VEMR@Gel group was the smallest ( Figure 3 C). In addition, TUNEL staining results showed that a large number of apoptotic cells were detected in the brain sections of tumor-bearing mice in the IL-12&VEMR@Gel group ( Figure 3 E). In summary, IL-12&VEMR@Gel significantly inhibited the recurrence of glioma.

[0041] S3. GBM mice were randomly divided into 6 groups, with 3 mice in each group. After the intracranial tumor was removed, each preparation group (PBS, Gel, EM@Gel, EMR@Gel, VEMR@Gel, IL-12&VEMR@Gel) was injected into the resection cavity in situ, and the scalp was sutured. The mice were killed on the 30th day after inoculation of GL261 cells, and the cut edge tissues were taken to prepare tissue sections. Immunofluorescence staining was used to analyze the expression of glioma initiating cell-related markers Notch1 and CD133 to explore the effects of different treatments on GICs of mouse brain tumors. Figure 4 As shown, CD133 and Notch1 were highly expressed in the control group and Gel group, while the relative expression levels in the EM@Gel, EMR@Gel, VEMR@Gel, and IL-12&VEMR@Gel groups were 0.83, 0.71, 0.49, and 0.36, respectively. In addition, compared with the control group, the CD133 and Notch1 fluorescence signals of IL-12&VEMR@Gel-treated mice were significantly weakened.

[0042] S4. GBM mice were randomly divided into 6 groups, 3 mice in each group. After intracranial tumor resection, each preparation group (PBS, Gel, EM@Gel, EMR@Gel, VEMR@Gel, IL-12&VEMR@Gel) was injected into the resection cavity in situ, and the scalp was sutured. The mice were killed on the 30th day after inoculation of GL261 cells, and the resection edge tissue was obtained and tissue sections were prepared. Immunofluorescence staining was used to analyze various immune cells (CD8 + T cells, CD4 + T cells, DC cells) to explore whether the exosome-hydrogel system can give mice strong immunity. It is worth noting that more CD4 + T cells, CD8 + T cells and DC cells ( Figure 5 ), indicating that intracavitary injection of IL-12&VEMR@Gel reversed the immunosuppressive microenvironment. In summary, intracavitary injection of exosome-hydrogel system in GBM mice after surgery can not only inhibit the growth of GICs, but also reverse the immunosuppressive microenvironment, thereby exerting effective anti-tumor activity and inhibiting GBM recurrence.

[0043] The exosomes in this system are mainly used as a drug delivery carrier for delivering mitoxantrone and siRNA. Therefore, the exosomes can be one of the exosomes of macrophages, exosomes of HEK293T cells, exosomes of bone marrow mesenchymal stem cells, exosomes of umbilical cord mesenchymal stem cells, and exosomes of adipose mesenchymal stem cells.

[0044] Immunomodulators include IL-12, TNF-α, IFN-γ, etc. Since one of the main functions of IL-12 is to promote the proliferation of activated T cells and NK cells and enhance the cytotoxic activity of T cells and NK cells, and IL-12 can also induce T cells and NK cells to produce interferon gamma (IFN-γ) and tumor necrosis factor beta (TNF-β), therefore, the immunomodulator IL-12 can also be replaced with TNF-α and IFN-γ.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection, characterized in that: The drug-loaded exosomes and the immunomodulator are co-loaded in a thermosensitive hydrogel, wherein the drug-loaded exosomes encapsulate mitoxantrone and siRNA. In the delivery system, the final concentration of mitoxantrone is 3-5 mg / kg, the final concentration of siRNA is 0.3-1.5 mg / kg, and the final concentration of the immunomodulator is 25-100 μg / kg. The thermosensitive hydrogel is composed of chitosan, sodium β-glycerophosphate and gelatin, wherein by mass percentage, the chitosan is 1%-5%, the sodium β-glycerophosphate is 5%-15%, the gelatin is 0.01%-2%, and the rest is water.

2. The exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection surgery according to claim 1, characterized in that: The exosomes are at least one of macrophage exosomes, HEK293T cell exosomes, bone marrow mesenchymal stem cell exosomes, umbilical cord mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

3. The exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection surgery according to claim 1, characterized in that: The immunomodulator is one of IL-12, TNF-α, and IFN-γ.

4. The method for preparing the exosome-thermosensitive gel delivery system for in situ drug delivery after brain glioma resection surgery according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of drug-loaded exosomes; (2) Preparation of thermosensitive hydrogel: Weigh a certain amount of chitosan to prepare a chitosan solution with a concentration of 0.05-0.25 M and a pH of 2-5; Weigh a certain amount of sodium β-glycerophosphate to prepare a 1.5-5M sodium β-glycerophosphate solution with a pH of 8-10; Weigh a certain amount of gelatin to prepare a gelatin solution with a concentration of 0.01-0.5 mM and a pH of 1-4; After the chitosan solution and the gelatin solution are uniformly mixed, the sodium β-glycerophosphate solution is slowly added under stirring, and the pH is adjusted to 7.2-7.4 to obtain a thermosensitive hydrogel; (3) Preparation of exosome-thermosensitive gel delivery system: Add the immunomodulator and drug-loaded exosomes into the thermosensitive hydrogel at 4-15°C and mix them to obtain a liquid exosome-thermosensitive gel delivery system.

5. Use of the exosome-thermosensitive gel delivery system for in situ administration after glioma resection surgery obtained by the preparation method as claimed in claim 4 in the preparation of anti-glioma drugs.

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