Preparation method of homologous tumor cell and American ginseng composite exosome

Abstract

The invention discloses a preparation method of a homologous tumor cell and American ginseng composite exosome, and belongs to the field of functional medical materials and nanotechnologies, and the preparation method comprises the following steps: S1, separating a U87 cell exosome from a U87 cell by using a continuous ultracentrifugation method, and preparing U87-Exos for later use; s2, separating American ginseng exosomes from American ginseng rhizomes by using a continuous ultracentrifugation method, and preparing AG-Exos for later use; s3, the U87-Exos obtained in the step S1 and the AG-Exos obtained in the step S2 are subjected to extrusion compounding, and U87 / AG-Exos is prepared; s4, temozolomide and resveratrol are dissolved in a solvent and mixed with the U87 / AG-Exos obtained in the step S3, and then the mixed solution is subjected to ultrasonic treatment. According to the exosome preparation method, a medicine delivery system based on homologous tumor cell composite Exos is prepared, TMZ and RES are jointly delivered and used for treating GBM, the medicine delivery system is based on the concept of traditional Chinese medicine combination therapy, the limitation of traditional chemotherapy is overcome, and a promising strategy is provided for treating GBM.

Description

Technical Field

[0001] The present invention relates to the fields of functional medical materials and nanotechnology, and in particular to a method for preparing homologous tumor cell and American ginseng composite exosomes. Background Art

[0002] Glioblastoma multiforme (GBM) is the most aggressive primary malignant tumor in the central nervous system (CNS), with a poor prognosis and high mortality, which seriously threatens human health. Currently, surgical resection is the main clinical practice for the treatment of GBM. However, due to the highly invasive growth of GBM, surgical eradication is impossible. In order to prevent local GBM recurrence, chemotherapy is usually performed after resection. However, due to the existence of the blood-brain barrier (BBB) ​​and insufficient tumor targeting, the effect of chemotherapy is hindered, and the use of chemotherapeutic drugs is often associated with severe systemic toxicity. So far, synthetic drug delivery vehicles have partially overcome these limitations, but their application is limited by inevitable defects, such as non-biodegradability and immunogenicity. Therefore, there is an urgent need to develop an advanced and safe drug delivery system that can quickly cross the BBB and accurately target the tumor site.

[0003] Exosomes (Exos) are natural nanoscale lipid vesicles secreted by almost all cell types, containing a large number of proteins, coding and noncoding RNAs, and lipids, which play an important role in intercellular communication. Recently, exosomes have attracted increasing attention as a promising alternative to nanopolymer-based drug delivery systems due to their good drug loading properties, nanoscale effects, biocompatibility, and circulation stability. Increasing evidence shows that tumor cell-derived Exos can specifically bind to homologous tumor cells with the same adhesion proteins, thus endowing them with inherent targeting capabilities. In view of these results, we hypothesized that American ginseng exosomes (AG-Exos) and U87 cell-derived Exos (U87-Exos) may have the ability to deliver therapeutic drugs to homologous GBM, including BBB penetration and tumor-specific targeting, while enhancing the activity of immune cells to play a synergistic role with American ginseng.

[0004] Temozolomide (TMZ) is now used as a first-line chemotherapeutic drug for GBM treatment. However, long-term use of TMZ inevitably leads to chemoresistance and adverse events. Therefore, there is an urgent need to develop a new therapeutic strategy to alleviate TMZ resistance and improve treatment efficacy. Combination therapy is a common clinical practice in cancer treatment, in which chemotherapeutic drugs are combined with safe and effective natural compounds to overcome chemoresistance, enhance the efficacy of chemotherapeutic drugs, and reduce drug-related adverse side effects. Resveratrol (RES) is a natural polyphenolic compound that exerts a powerful anticancer effect by regulating various proliferation signaling pathways and inhibiting the growth and survival of cancer cells, including GBM cells. Studies have shown that RES can increase the sensitivity of GBM cells to a variety of chemotherapeutic drugs. Therefore, we predict that the combination of TMZ and RES may provide promising synergistic therapeutic benefits in the treatment of GBM. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing homologous tumor cell and American ginseng composite exosomes, to prepare a drug delivery system based on homologous tumor cell composite Exos, to co-deliver TMZ and RES for the treatment of GBM, the drug delivery system is based on the concept of combined therapy of traditional Chinese medicine to overcome the limitations of traditional chemotherapy, and provides a promising strategy for the treatment of GBM.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] A method for preparing homologous tumor cell and American ginseng composite exosomes is provided, comprising the following steps:

[0008] S1: Preparation of U87-Exos: Separate U87 cell exosomes from U87 cells using continuous ultracentrifugation to prepare U87-Exos for use;

[0009] S2: Preparation of AG-Exos: Separate American ginseng exosomes from American ginseng rhizomes using continuous ultracentrifugation to prepare AG-Exos for use;

[0010] S3: Preparation of U87 / AG-Exos: The U87-Exos obtained in step S1 and the AG-Exos obtained in step S2 are extruded and compounded to prepare U87 / AG-Exos;

[0011] S4: Preparation of drug delivery system: Temozolomide and resveratrol are dissolved in a solvent and mixed with the U87 / AG-Exos obtained in step S3, and then the mixed solution is subjected to ultrasonic treatment.

[0012] Furthermore, in step S1, the centrifugation method of U87-Exos is: (1) centrifuging at 4°C and filtering through a 0.22 μm pore size; (2) further centrifuging the filtrate at 4°C, discarding the supernatant, and washing the precipitate.

[0013] Furthermore, in step (1), the cells are centrifuged at 300×g, 2000×g and 10000×g for 10 min, 10 min and 30 min, respectively, at 4°C;

[0014] In step (2), the filtrate is centrifuged at 4°C and 120,000×g for 70 min.

[0015] Furthermore, in step S2, the centrifugation method of AG-Exos is: at 4°C, centrifuge at 1000-2000×g, 1000-2000×g, 5000-10000×g, 100000-200000×g, 200000×g, and 100000×g for 15-25min, 30-40min, 1-3h, 2.5h, and 1h, respectively.

[0016] Furthermore, in step S3, the mass ratio of U87-Exos to AG-Exos is 1:1.

[0017] Furthermore, in step S4, the ratio of temozolomide to resveratrol is: 1.7 μg / mL: 1 μg / mL.

[0018] Furthermore, in step S4, the mass ratio of the U87 / AG-Exos to the mixed drug of temozolomide and resveratrol is: mU87 / AG-Exos: mdrug=5:1.

[0019] Furthermore, in step S4, the ultrasonic conditions are: amplitude 20%, on 4s, off 2s, and cycled 6 times;

[0020] Between each cycle, the sample was cooled on ice for 2 min, and the whole process was operated at low temperature.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The method for preparing composite exosomes of homologous tumor cells and American ginseng in the present invention, which is combined with synergistic temozolomide and resveratrol for the treatment of glioblastoma multiforme, effectively alleviates the problem of temozolomide resistance, thereby improving the therapeutic effect of glioblastoma multiforme;

[0023] 2. The method for preparing the composite exosomes of homologous tumor cells and American ginseng exemplified in the present invention uses the composite exosomes of homologous tumor cells and American ginseng as a drug delivery carrier to deliver TMZ / RES to treat GBM, thereby achieving targeted delivery of chemotherapeutic drugs, reducing drug-related toxic side effects, and synergistically exerting the therapeutic effect of American ginseng exosomes;

[0024] 3. The method for preparing exosomes from homologous tumor cells and American ginseng composites exemplified in the present invention verified the efficacy of U87 / AG-Exos@TMZ / RES in organoid models and in situ CDX and PDX models that are closer to the actual conditions of patient tumors, making the treatment results more clinically valuable. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0026] Figure 1 The extraction process of U87-Exos;

[0027] Figure 2 This is the heat map of the drug combination response of TMZ and RES;

[0028] Figure 3 Transmission electron microscopy characterization of U87-Exos, AG-Exos, and U87-Exos@TMZ / RES;

[0029] Figure 4 Particle size characterization of U87-Exos, AG-Exos, and U87-Exos@TMZ / RES;

[0030] Figure 5 Zeta potential characterization of U87-Exos, AG-Exos, and U87-Exos@TMZ / RES;

[0031] Figure 6 Characterization of surface marker proteins of U87-Exos, AG-Exos, and U87-Exos@TMZ / RES;

[0032] Figure 7 This is an investigation on the stability of U87 / AG-Exos@TMZ / RES. DETAILED DESCRIPTION

[0033] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0034] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0035] 1. Preparation method based on homologous tumor cells and American ginseng composite exosomes (U87 / AG-Exos@TMZ / RES).

[0036] 1. Extraction of U87 cell exosomes (U87-Exos)

[0037] Fetal bovine serum pretreatment: Fetal bovine serum was centrifuged at 4°C, 200,000 × g for 6 h to remove Exos in the serum and obtain Exos-free serum for subsequent cell culture;

[0038] Cell culture: After U87 cells were cultured to a density of about 80%, the complete medium was discarded, the cells were washed twice with PBS solution, and Exos-free serum medium was added for 24 h;

[0039] The cell culture medium was centrifuged at 4°C, 300×g, 2000×g and 10000×g for 10 min, 10 min and 30 min, respectively, to remove dead cells and cell debris, and then the supernatant was collected. The supernatant was filtered through a 0.22μM sterile filter to remove large extracellular vesicles. The filtrate was centrifuged at 4°C, 120,000×g for 70 min, the supernatant was discarded, and the obtained precipitate was resuspended in 5 mL of sterile pre-cooled PBS, and continued to be centrifuged at 4°C, 120,000×g for 70 min. The obtained U87-Exos precipitate was resuspended in 200μL of sterile pre-cooled PBS for use. The extraction process of U87-Exos is as follows. Figure 1 shown.

[0040] 2. Extraction of American ginseng exosomes (AG-Exos)

[0041] (1) Wash fresh American ginseng with deionized water, dry it, weigh it, cut it into small pieces, transfer it to a crusher, add PBS solution so that the liquid level covers the contents of the American ginseng, and crush it to obtain American ginseng juice; filter the American ginseng juice with gauze, remove the trapped filter residue, and obtain a crude American ginseng extract;

[0042] (2) centrifuging the crude extract of American ginseng for the first time, taking the supernatant after centrifugation and pouring it into a clean bottle, centrifuging it for the second time, and taking out the supernatant after centrifugation and retaining it;

[0043] (3) The supernatant is centrifuged for the third time, and the supernatant is discarded after centrifugation, and the crude American ginseng exosomes in the precipitate are retained and resuspended with a PBS solution to obtain the crude American ginseng exosomes diluted with a PBS solution;

[0044] (4) Prepare sucrose solutions of different concentrations, add them to the ultracentrifuge tube in order from high to low concentrations, and finally add the crude American ginseng exosomes diluted with the PBS solution, perform the fourth centrifugation, obtain the band containing American ginseng exosomes, remove the band, transfer it to a new centrifuge tube, add PBS solution, perform the fifth centrifugation, and after centrifugation, retain the precipitate for resuspending to obtain purified American ginseng exosomes.

[0045] The concentration of the PBS solution used in step (1) is 0.01 M, the mass ratio of the PBS solution to the fresh American ginseng is 5:(1-5), more preferably 5:(1-4), and the ratio used in this embodiment is 5:1.25; the crushing time is 1-3 min, more preferably 1.5-2.5 min, and 2 min in this embodiment. The gauze filter mesh number is 100-200 mesh, and 200 mesh is used in this embodiment.

[0046] In step (2), the centrifugal force of the first centrifugation is 1500×g; the centrifugation time is 20 min, and the centrifugation temperature is 4°C. The first centrifugation can remove large particle impurities in the American ginseng crude extract; the centrifugal force of the second centrifugation is 5000×g in this embodiment; the centrifugation time is 35 min, and the centrifugation temperature is 4°C. The second centrifugation can remove broken cell fragments in the American ginseng crude extract.

[0047] In step (3), the centrifugal force of the third centrifugation is 150000×g; the centrifugation time is 2h, the centrifugation temperature is 4°C, and the third centrifugation can remove macromolecular proteins and soluble impurities in the supernatant; the PBS solution concentration is 0.01M; the concentration of American ginseng exosomes after resuspension is 150-300mg / mL, preferably 180-250mg / mL, and 200mg / mL in this embodiment. The specific operation of resuspension is to absorb PBS with a 1000μL pipette, slowly inject the PBS in the pipette tip into the substance to be resuspended, and gently and repeatedly absorb several times until the solution is uniform and free of lumps.

[0048] In step (4), the concentration gradient of the sucrose solution is 5%, 10%, 30%, and 45% from low to high, and the added volume of the sucrose solution in the concentration gradient is 3 mL, 3 mL, 2 mL, and 2 mL, respectively; the centrifugal force of the fourth centrifugation is 200000×g, and the centrifugation time is 2.5 h. The fourth centrifugation can preliminarily remove some impurities in the American ginseng exosomes; the PBS solution concentration is 0.01 M; the centrifugal speed of the fifth centrifugation is 100000×g; the centrifugation time is 1 h; the fifth centrifugation can remove the residual sucrose solution in the strip.

[0049] 3. Preparation of exosome drug delivery system based on homologous tumor cells and American ginseng (U87 / AG-Exos@TMZ / RES)

[0050] The prepared U87-Exos and AG-Exos were uniformly mixed in a ratio of 1:1, 1:2, and 2:1, respectively, and extruded through a 0.22 μm filter membrane extruder to prepare U87 / AG-Exos. The particle size of U87 / AG-Exos prepared in different ratios was measured, and the results showed that U87 / AG-Exos prepared in a ratio of 1:1 had the smallest particle size and PDI.

[0051] The SynergyFinder online software was used to evaluate the synergistic effect of TMZ and RES when used in combination through the response surface model and the Zero Interaction Potential (ZIP) calculation method. A ZIP synergy score greater than 0 is considered a synergy, and a ZIP synergy score greater than 10 is considered a strong synergy. SynergyFinder was used to further evaluate the therapeutic significance of the combined use of TMZ and RES, and the drug concentrations at which the two drugs exerted the best synergy were obtained. The two drugs were mixed at different concentrations as shown in the table below. TMZ and RES were used in combination at different concentrations to further evaluate the synergy between the two drugs, and the combinations are shown in Table 1. The combined use of TMZ and RES showed a high degree of synergy in inhibiting tumor proliferation (ZIP synergy score>10). As Figure 2 As shown, when TMZ:RES = 400 μM:200 μM (i.e., TMZ:RES = 1.7 μg / mL:1 μg / mL), the ZIP score was the highest at 11.229, indicating that the combination of 400 μM TMZ and 200 μM RES was the optimal concentration for synergistic anti-proliferation.

[0052] Table 1 Different proportions of TMZ and RES added

[0053]

[0054]

[0055] Three batches of U87 / AG-Exos@TMZ / RES were prepared according to three different ratios of mU87 / AG-Exos:mdrug = 15:1, 10:1, and 5:1. As shown in Table 2, the loading efficiency of TMZ and RES was determined by UV-visible spectrophotometry. The drug loading efficiency of the mU87 / AG-Exos:mdrug = 5:1 group was the highest, so this ratio was determined as the optimal formulation.

[0056] Table 2 Results of UV-Vis spectrophotometry for the loading efficiency of TMZ and RES

[0057]

[0058] In the example, TMZ and RES were dissolved in 5% DMSO / PBS at a ratio of 1.7 μg / mL: 1 μg / mL. The collected U87 / AG-Exos solution (BCA was used to determine the protein content) was mixed with the drug dissolved in 5% DMSO / PBS at a ratio of mU87 / AG-Exos: mdrug = 5:1. Ultrasonic treatment was performed in an ultrasonic cell disruptor, and the setting conditions were: amplitude 20%, on 4s, off 2s, and 6 cycles. In the interval between each cycle, the sample was placed on ice to cool for 2 minutes, and the whole process was operated at low temperature. After the ultrasound is completed, the sample was incubated at 37°C for 1h to ensure that the integrity of the U87 / AG-Exos membrane was restored. Afterwards, the sample was centrifuged at 4°C and 120,000×g for 70 min, the supernatant was discarded, the precipitate was repeatedly washed with 5 mL of sterile pre-cooled PBS solution, and centrifuged again at 4°C and 120,000×g for 70 min, and the precipitate was resuspended with 200 μL of sterile pre-cooled PBS to obtain U87 / AG-Exos@TMZ / RES.

[0059] 2. Characterization of U87 / AG-Exos@TMZ / RES.

[0060] 1. Determination of drug encapsulation efficiency of U87 / AG-Exos@TMZ / RES

[0061] The amount of drug encapsulated in the prepared U87 / AG-Exos@TMZ / RES was determined by UV-visible spectrophotometry using NanoDrop One ultra-micro spectrophotometer, and the drug encapsulation efficiency was used as the evaluation index.

[0062] The loading efficiencies of TMZ and RES were measured to be 19.76% and 20.99%, respectively.

[0063] 2. TEM characterization of U87-Exos, AG-Exos and U87 / AG-Exos@TMZ / RES

[0064] The separated U87-Exos and AG-Exos were observed by TEM. Figure 3 It can be seen that U87-Exos and AG-Exos present a typical cup-shaped structure, and the TEM image of the prepared U87 / AG-Exos@TMZ / RES appears as a flat sphere with a depression inside.

[0065] 3. Particle size characterization of U87-Exos, AG-Exos and U87 / AG-Exos@TMZ / RES

[0066] The particle sizes of U87-Exos, AG-Exos, and U87-Exos@TMZ / RES were characterized by NTA. Figure 4 As shown in the figure, the peak particle size of U87-Exos is around 126.6nm, and the particle size of AG-Exos is around 60nm. After drug loading, the peak particle size of U87 / AG-Exos@TMZ / RES increases to around 133.2nm.

[0067] 4. Zeta potential characterization of U87-Exos, AG-Exos and U87 / AG-Exos@TMZ / RES

[0068] like Figure 5 As shown, the Zeta potential of U87-Exos was -47.00±0.43 mV, the Zeta potential of AG-Exos was -14.34±0.87 mV, and the Zeta potential of U87 / AG-Exos@TMZ / RES was -32.57±1.03 mV.

[0069] 5. Characterization of surface marker proteins of U87-Exos and U87 / AG-Exos@TMZ / RES

[0070] Extraction of surface marker proteins: Add protein lysis buffer to U87-Exos and U87 / AG-Exos@TMZ / RES solutions in equal proportions, mix thoroughly, and place on ice for 30 minutes, vortexing every 5 minutes for 30 seconds each time. After lysis, centrifuge at 13000rpm for 20 minutes at 4°C, take the supernatant and place it in a 1.5mL pre-cooled EP tube. Determine the protein concentration according to the BCA protein quantification kit. Add loading buffer to the protein solution in proportion, heat it in a metal bath for 10 minutes, and store it in a -80°C refrigerator for short-term storage.

[0071] Preparation of stacking gel and separation gel: Prepare 1.5 mm thick 10% gel according to the instructions of the Rapid SDS-PAGE Color Gel Kit.

[0072] Electrophoresis: 80V voltage electrophoresis, when bromophenol blue runs to the junction of the concentrated gel and the separation gel, adjust the voltage to 120V and continue electrophoresis. When bromophenol blue runs close to the bottom of the gel, stop electrophoresis. Take out the gel plate, pry it open slowly and gently, and carefully cut off the upper gel and empty holes.

[0073] Transfer and blocking: Cut the PVDF membrane and activate it by soaking it in 100% methanol for 1 minute. Add an appropriate amount of Tris-Glycine Transfer Buffer to a tray, place the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad in sequence with the black side of the transfer clip facing down. Align the black side of the transfer clip with the black electrode of the transfer tank, and transfer the membrane at a constant current of 220mA for 70 minutes at low temperature. After the transfer is completed, wash the membrane with the front side facing up in TBST buffer for 3 times, 10 minutes each time. After washing the membrane, block it with 5% BSA at room temperature for 90 minutes.

[0074] Incubation with primary antibody: The blocked membrane was washed three times in TBST buffer, each time for 10 min; then the primary antibody diluent (Alix) was added and incubated overnight at 4°C.

[0075] Incubation with secondary antibody: After the primary antibody incubation is completed, remove the membrane and wash it in TBST buffer for 3 times, 10 minutes each time; then add the secondary antibody dilution of the corresponding species and incubate at room temperature for 60 minutes; remove the membrane and wash it in TBST buffer for 3 times, 10 minutes each time.

[0076] ECL luminescence detection: Add the prepared ECL reaction solution onto the PVDF membrane, then use the chemiluminescence imaging system to expose and take pictures for preservation.

[0077] The results of WB are as follows Figure 6 As shown in the figure, U87-Exos is rich in Exos-related marker protein Alix. After drug loading, U87 / AG-Exos@TMZ / RES still retains the surface marker protein of U87-Exos well, indicating that the Exos membrane structure will not be destroyed after drug loading, which provides a basis for the subsequent use of U87 / AG-Exos as a drug delivery carrier to achieve targeted drug delivery.

[0078] 6. Stability investigation of U87 / AG-Exos@TMZ / RES

[0079] U87 / AG-Exos@TMZ / RES was stored at 4°C for 7 days, and the changes in particle size distribution were monitored using the NTA system to investigate the storage stability of U87 / AG-Exos@TMZ / RES.

[0080] like Figure 7 As shown, the particle size change of U87 / AG-Exos@TMZ / RES within 7 days. According to the NTA results, the particle size of U87 / AG-Exos@TMZ / RES was maintained in the range of 130-140 nm, and the particle size did not change much, indicating that U87 / AG-Exos@TMZ / RES had good stability within 7 days.

[0081] The homologous tumor cells and American ginseng composite exosomes of this embodiment co-deliver TMZ and RES for GBM treatment. TMZ and RES are co-encapsulated in U87 / AG-Exos by simple ultrasonic treatment to obtain composite U87 / AG-Exos@TMZ / RES, which can quickly migrate on the BBB and accurately accumulate at the GBM site. At the same time, TMZ and RES act in combination to enhance the therapeutic effect on GBM and significantly reduce drug-related side effects. The excellent therapeutic effect of U87 / AG-Exos@TMZ / RES has been well verified in GBM patient-derived organoids (PDO) and in situ GBM models. The U87 / AG-Exos@TMZ / RES prepared in this embodiment has multiple advantages, including the ability to cross the preferred blood-brain barrier with nanoscale size, intrinsic tumor-targeted accumulation with homologous effects, and enhanced anti-tumor activity with synergistic effects of TMZ and RES, while exerting the synergistic treatment of American ginseng to enhance immunity.

[0082] Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for preparing homologous tumor cell and American ginseng composite exosomes, characterized in that: The following steps are involved: S1: Preparation of U87-Exos: Separate U87 cell exosomes from U87 cells using continuous ultracentrifugation to prepare U87-Exos for use; S2: Preparation of AG-Exos: Separate American ginseng exosomes from American ginseng rhizomes using continuous ultracentrifugation to prepare AG-Exos for use; S3: Preparation of U87 / AG-Exos: The U87-Exos obtained in step S1 and the AG-Exos obtained in step S2 are extruded and compounded to prepare U87 / AG-Exos; S4: Preparation of drug delivery system: Temozolomide and resveratrol are dissolved in a solvent and mixed with the U87 / AG-Exos obtained in step S3, and then the mixed solution is subjected to ultrasonic treatment.

2. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S1, the centrifugation method of U87-Exos is as follows: (1) centrifugation at 4°C and filtering through a 0.22 μm pore size filter; (2) further centrifuging the filtrate at 4°C, discarding the supernatant, and washing the precipitate.

3. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 2, characterized in that: In step (1), the cells are centrifuged at 300×g, 2000×g and 10000×g for 10 min, 10 min and 30 min, respectively, at 4°C; In step (2), the filtrate is centrifuged at 4°C and 120,000×g for 70 min.

4. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S2, the centrifugation method of AG-Exos is: at 4°C, centrifuge at 1000-2000×g, 5000-10000×g, 100000-200000×g, 200000×g, and 100000×g for 15-25 min, 30-40 min, 1-3 h, 2.5 h, and 1 h, respectively.

5. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S3, the mass ratio of U87-Exos to AG-Exos is 1:

1.

6. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S4, the ratio of temozolomide to resveratrol is: 1.7 μg / mL: 1 μg / mL.

7. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S4, the mass ratio of the U87 / AG-Exos to the mixed drug of temozolomide and resveratrol is: mU87 / AG-Exos:mdrug=5:

1.

8. The method for preparing homologous tumor cell and American ginseng composite exosomes according to claim 1, characterized in that: In step S4, the ultrasonic conditions are: amplitude 20%, on 4s, off 2s, and cycled 6 times; Between each cycle, the sample was cooled on ice for 2 min, and the whole process was operated at low temperature.

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