Application of chitosan oligosaccharide in inhibition of plasma protein crown formation of extracellular vesicles

Abstract

The invention discloses application of chitosan oligosaccharide in inhibiting plasma protein crown formation of extracellular vesicles, belongs to the technical field of medicines, and particularly relates to application of chitosan oligosaccharide in inhibiting plasma protein crown formation of extracellular vesicles by modifying chitosan oligosaccharide to the surfaces of extracellular vesicles through co-incubation, effectively inhibiting plasma protein crown formation, reducing uptake of mononuclear phagocytic systems (such as liver and spleen), and improving the activity of the extracellular vesicles. The uptake of organs such as brain, heart and kidney is increased, so that the bioavailability of the extracellular vesicles by the extracellular organs is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of chitosan oligosaccharide in inhibiting the formation of extracellular vesicle plasma protein corona. Background Art

[0002] In recent years, extracellular vesicles (EVs) have attracted widespread attention as an emerging therapeutic method. EVs are nanoscale membrane vesicles released by cells into the external environment, including exosomes, microvesicles, and apoptotic bodies. EVs have natural biocompatibility and low immunogenicity, and have shown new application prospects as drug delivery carriers. Compared with traditional synthetic nanoparticles, EVs have unique advantages in drug delivery. In addition, some specific EVs, such as mesenchymal stem cell-derived EVs (MSC-EVs), have significant therapeutic effects. These EVs participate in antigen presentation, immune regulation, and tissue repair through the genetic material and proteins they carry, and can improve cell proliferation, reduce inflammation, and reduce fibrosis.

[0003] However, EVs face the challenge of forming a plasma protein corona (PC) after circulatory administration. The protein corona refers to one or more layers of protein structure adsorbed on the surface of nanoparticles when they are released into the biological environment (such as blood, plasma, and cytoplasm). The presence of the protein corona affects the size, aggregation state, and interface properties of nanomaterials, giving them new biological characteristics, which in turn affects the phagocytosis, biodistribution, and immune response of nanoparticles. For example, after intravenous injection, when nanoparticles adsorb opsonin proteins (such as complement proteins) in plasma, they promote phagocytosis of macrophages, resulting in rapid clearance of nanoparticles by the mononuclear phagocytic system. Therefore, there is an urgent need in the art to develop a method for inhibiting the formation of a plasma protein corona on the surface of drug delivery carriers, thereby overcoming the adverse effects of the plasma protein corona on drug delivery carriers. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of chitosan oligosaccharide in the preparation of drugs for inhibiting the formation of extracellular vesicle plasma protein corona.

[0005] Technical solution: In order to solve the above technical problems, the present invention provides the use of chitosan oligosaccharide in the preparation of drugs for inhibiting the formation of extracellular vesicle plasma protein corona.

[0006] The chitosan oligosaccharide is a chitosan hydrolyzate obtained by chemical synthesis or biosynthesis, has a polymerization degree of 2 to 20, a molecular weight of ≤3200 Da, and has the characteristics of a positively charged polysaccharide.

[0007] The extracellular vesicles include vesicles with a lipid membrane structure secreted by cells of humans, animals, plants or microorganisms.

[0008] Wherein, the extracellular vesicles include exosomes, microvesicles, migrasomes, apoptotic bodies, shedding vesicles or microparticles.

[0009] The drug is prepared by modifying extracellular vesicles with chitosan oligosaccharide.

[0010] The method for modifying extracellular vesicles with chitosan oligosaccharide is to drop an extracellular vesicle solution into a chitosan oligosaccharide solution and incubate the solution on a rotary mixer; the obtained solution is purified by centrifugation using an ultrafiltration tube to obtain the extracellular vesicles modified with chitosan oligosaccharide.

[0011] Wherein, the concentration of the chitosan oligosaccharide solution is 50-150 μg / ml.

[0012] Wherein, the medicine is a single preparation or a compound preparation.

[0013] The dosage form of the drug includes liquid preparation, dry suspension, spray preparation, tablet, capsule or ointment.

[0014] The method for modifying EVs with chitosan oligosaccharides of the present invention specifically comprises the following steps:

[0015] (1) Add EVs solution to chitosan oligosaccharide solution, place on a rotary mixer, and incubate at 37°C for 1 h;

[0016] (2) The resulting solution was centrifuged at 4°C and 3000 g using a 0.5 mL 100 kDa ultrafiltration tube to obtain the chitosan oligosaccharide-modified EVs.

[0017] Furthermore, the present invention modified EVs derived from human umbilical cord mesenchymal stem cells (MSC) and human embryonic kidney cell line HEK293F with chitosan oligosaccharides (COS), thereby demonstrating the ability of chitosan oligosaccharides to inhibit the formation of plasma protein coronas of all types of extracellular vesicles.

[0018] The chitosan oligosaccharide-modified EVs can effectively inhibit the formation of plasma protein corona, reduce the uptake of EVs by the mononuclear phagocytic system (such as liver and spleen), and increase the uptake by organs such as brain, heart, and kidney, thereby improving the bioavailability of extracellular vesicles to extrahepatic organs.

[0019] The average particle size of MSC-EVs modified with chitosan oligosaccharides of the present invention is about 170.6±2.4 nm, and the average particle size of 293F-EVs is about 179.3±5.1 nm. The absolute values ​​of the potentials of the two EVs are reduced after modification.

[0020] The present invention proves through experiments that the chitosan oligosaccharide-modified EVs (EV-COS) and unmodified EVs adsorb less protein after incubation with EVs-removed plasma in vitro.

[0021] Furthermore, the present invention demonstrates that chitosan oligosaccharide modification of EVs reduces the uptake of EVs by macrophages and reduces the uptake of EVs by the mononuclear phagocytic system.

[0022] Furthermore, chitosan oligosaccharide-modified EVs can increase the uptake of EVs by damaged organs (such as brain, heart, and kidneys) in septic mice.

[0023] Furthermore, chitosan oligosaccharide-modified EVs can attenuate the macrophage inflammatory response mediated by protein corona.

[0024] Beneficial effects: The present invention modifies EVs with chitosan oligosaccharides, which can effectively inhibit the formation of protein corona, reduce the uptake of EVs by the mononuclear phagocytic system (such as liver and spleen), and increase the uptake by organs such as the brain, heart, and kidneys, thereby improving the bioavailability of extracellular vesicles to extrahepatic organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The identification diagram of chitosan oligosaccharide-modified EVs: (A) Particle size distribution diagram, the left is the diagram after MSC-EVs modification, and the right is the diagram after 293F-EVs modification; (B) Potential diagram; Among them, MSC-EV is the extracellular vesicle secreted by MSC cells, MSC-EV-COS is the extracellular vesicle secreted by MSC cells modified with chitosan oligosaccharide, 293F-EV is the extracellular vesicle secreted by 293F cells, and 293F-EV-COS is the extracellular vesicle secreted by 293F cells modified with chitosan oligosaccharide;

[0026] Figure 2 The effect diagram of chitosan oligosaccharide-modified EVs in inhibiting the formation of plasma protein corona: (A) particle size distribution diagram; (B) protein content determination diagram by BCA method; (C) silver staining method was used to evaluate the protein corona inhibitory effect of MSC-PC-COS; (D) Western blot method was used to evaluate the protein corona inhibitory effect of EV-COS; wherein, MSC-EV-PC is the extracellular vesicle secreted by MSC cells with protein corona, MSC-PC-COS is the extracellular vesicle secreted by MSC cells with protein corona modified by chitosan oligosaccharide, 293F-EV is the extracellular vesicle secreted by 293F cells, 293F-EV-PC is the extracellular vesicle secreted by 293F cells with protein corona, 293F-PC-COS is the extracellular vesicle secreted by 293F cells with protein corona modified by chitosan oligosaccharide;

[0027] Figure 3The analysis diagram of EV-COS uptake by mouse macrophages RAW264.7: (A) Average fluorescence intensity of mouse macrophages RAW264.7 after EV-COS uptake; (B) Fluorescence microscopy observation of EV-COS uptake by mouse macrophages RAW264.7;

[0028] Figure 4 The biodistribution diagrams of EV-COS in normal mice and septic mice: (A) Biodistribution diagrams and mean fluorescence intensity statistics of MSC-EV-PC and MSC-PC-COS in normal mice; (B) Biodistribution diagrams and mean fluorescence intensity statistics of MSC-EV-PC and MSC-PC-COS in septic mice; (C) Biodistribution diagrams and mean fluorescence intensity statistics of 293F-EV-PC and 293F-PC-COS in normal mice; (D) Biodistribution diagrams and mean fluorescence intensity statistics of 293F-EV-PC and 293F-PC-COS in septic mice; Among them, the Sham group is the sham operation control group, and the CLP group is the cecal ligation and puncture (sepsis model) group;

[0029] Figure 5 Schematic diagram of COS modification inhibiting the inflammatory response caused by EV plasma protein corona: (A) COS modification inhibits the inflammatory response caused by MSC-EV plasma protein corona; (B) COS modification inhibits the inflammatory response caused by 293F-EV plasma protein corona. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] The statistical data in the embodiments of the present invention are given in the form of mean ± standard error, SPSS26 statistical software is used to process the data, one-way analysis of variance is used for inter-group comparison, and t-test is used for two-group comparison, and p < 0.05 is considered to be significantly different. The experimental results are repeated more than 3 times.

[0032] Example 1 Identification of chitosan oligosaccharide-modified EVs

[0033] 1. Preparation of EV-COS

[0034] (1) Extraction of EVs:

[0035] a. Serum-free culture medium (Beijing Huakan Biotechnology Co., Ltd., RMZ112) was used to culture umbilical cord mesenchymal stem cells (umbilical cord was provided by Zhongda Hospital Affiliated to Southeast University, ethics approval number: 2023ZDSYLL118-P01, 3×10 6 Cells) were cultured to P3 and added to 3D Microcarriers (Beijing Huakan Biotechnology Co., Ltd., catalog number w01-200) were placed in a 125 ml spinner bottle on a 3D Cultivate in miniSPIN bioreactor, replace medium with new medium when glucose level in the spinner bottle drops to 10mmol / L, collect supernatant of old medium, and stop collecting supernatant when all cells on microcarriers aggregate;

[0036] b. Expi293F suspension cells (Thermo Fisher, A14527, 4.5×10 5 The cells were transferred into a 250 ml spinner bottle (Wuxi Nice Life Science Co., Ltd., 782011), and cultured in a shaker at 37°C and 120 rpm. After 2-3 days, the cell culture medium (Shanghai Aopuma Biotech Co., Ltd., OPM-293CD05) was replaced and the supernatant was collected.

[0037] c. Extracellular vesicles of umbilical cord mesenchymal stem cells and Expi293F were extracted by ultracentrifugation: the supernatant was first centrifuged at 4°C and 2000g for 20 minutes to remove dead cells; the supernatant was then transferred to a new centrifuge tube and centrifuged at 4°C and 10000g for 30 minutes to remove small cell fragments; the supernatant was then transferred to an ultracentrifuge tube and centrifuged at 4°C and 100000g for 2 hours to obtain MSC-EVs and 293F-EVs, respectively;

[0038] d. Then, size exclusion chromatography (ES914, Beijing Enze Kangtai Biotechnology Co., Ltd.) was used to purify MSC-EVs and 293F-EVs respectively: the 4th to 7th fractions collected were the purified MSC-EVs and 293F-EVs.

[0039] (2) EV and COS co-incubation: MSC-EVs and 293F-EVs (5×10 10 The resulting product was added with 100 μg / ml COS solution (C2849, 300 μl, TCI (Shanghai) Chemical Industry Development Co., Ltd.) and incubated on a shaker at 37°C for 1 h. The product was purified using a 0.5 ml 100 kDa ultrafiltration tube (Millipore, UFC5100) and centrifuged at 4°C and 3000 g for 2 min.

[0040] 2. Identification of EV-COS

[0041] Particle size and potential detection: both were performed using the German PMX nanoparticle tracking analyzer Zetaview. The results are as follows: Figure 1As shown, after COS modified MSC-EVs, its average particle size was about 170.6±2.4nm, and after modified 293F-EVs, its average particle size was about 179.3±5.1nm; at the same time, the absolute values ​​of the potentials of the two EVs decreased after being modified by COS. After modification, the potential of MSC-EVs was -12.3±2.2mV, and that of 293F-EVs was -12.6±1.0mV, proving that the positively charged COS was successfully modified on the negatively charged EVs.

[0042] The results of this example show that the average particle size of COS-modified MSC-EVs prepared by the present invention is about 170.6±2.4 nm, and the average particle size of 293F-EVs is about 179.3±5.1 nm. The absolute value of the EVs potential decreases after modification, proving that COS is successfully modified on the surface of EVs.

[0043] Example 2 Effect of chitosan oligosaccharide-modified EVs on inhibiting protein corona formation

[0044] 1. In vitro simulation of protein corona formation process

[0045] Plasma separation: healthy whole blood (healthy blood was collected from Zhongda Hospital affiliated to Southeast University, the age range of healthy people was 20-30 years old, ethics approval number: 2024ZDSYLL249-P01) was centrifuged at 4°C, 2500g for 15 minutes; the supernatant was taken to another centrifuge tube and centrifuged at 4°C, 2500g for 15 minutes, and the supernatant was plasma; the plasma sample was centrifuged at 10000g for 40 minutes at 4°C to remove large vesicles; the supernatant after centrifugation was filtered with a 0.22μm filter membrane and centrifuged at 4°C, 167,000g for 4 hours; EVs were further removed from the supernatant after ultrafiltration using a 100kD ultrafiltration tube, and the filtrate was obtained by centrifugation at 4°C, 10,000g for 30 minutes; the filtrate was concentrated by 3kD: centrifuged at 4°C, 10,000g for 10 minutes to obtain EVs-free plasma (EVDP).

[0046] The MSC-EVs and 293F-EVs prepared in Example 1 were placed in a constant flow pump with EVDP to simulate blood circulation and incubated for 30 minutes, with a volume ratio of 1:1 between MSC-EVs or 293F-EVs and EVDP; centrifuged at 4°C and 16 7000g for 4 hours, purified by size exclusion chromatography, collected and concentrated with a 3kDa ultrafiltration tube, and the obtained EVs with protein coronas were obtained, namely MSC-EV-PC and 293F-EV-PC, respectively, simulating the process of plasma protein corona formation in vitro. COS was modified on MSC-EV-PC and 293F-EV-PC using the same method as in Example 1 to obtain MSC-PC-COS and 293F-PC-COS, respectively.

[0047] 2. Effect of chitosan oligosaccharide-modified EVs on the inhibition of protein corona formation

[0048] Nanoparticle tracking analyzer, BCA protein concentration assay, silver staining and Western blot were used to evaluate the protein corona inhibitory effect of EV-COS.

[0049] (1) Particle size detection: The German PMX nanoparticle tracking analyzer Zetaview was used. The results are as follows Figure 2 As shown in A, the particle size of MSC-EV-PC and 293F-EV-PC modified by COS is smaller than that of EVs forming a protein corona.

[0050] (2) Using BCA protein concentration determination method, silver staining method and Western blot method: Prepare protein standard curve and test samples according to the instructions of BCA protein concentration determination kit (Jiangsu Keygene Biotechnology, KGPBCA), and then use a microplate reader to detect the absorbance of the test samples at 562nm. According to the standard curve, the protein concentration and total protein amount are obtained, such as Figure 2 As shown in B, after MSC-EV-PC and 293F-EV-PC were incubated with plasma, the protein content of EVs increased, and COS modification of MSC-EV-PC and 293F-EV-PC significantly reduced the amount of protein adsorption. According to the silver staining kit (Biyuntian Biotechnology, P0017S) to detect protein expression, the results showed that the protein bands of MSC-EV-PC and 293F-EV-PC increased after the formation of the protein corona, and the expression spectrum after the formation of the protein corona was similar to the bands of EV-free plasma, while COS modification of EVs can reduce protein adsorption, and Western blot proved that it can significantly reduce the adsorption of proteins such as Alpha-2-macroglobulin, fibrinogen α, complement protein C3, and immunoglobulin IGHG1, as shown in Figure 2. Figure 2 C, as shown in 2D.

[0051] The results of this example show that COS-modified EVs can inhibit protein corona formation and reduce the expression of plasma proteins.

[0052] Example 3: Chitosan oligosaccharide modification of EVs reduces their uptake by macrophages

[0053] (1) DiD-labeled EVs: 1×10 11 MSC-EV, 1×10 11 293F-EV, 1×10 11 MSC-PC and 1×10 111 mM DiD dye (Invitrogen, V22887) was added to each 293F-PC, and the mixture was incubated in a decolorizing shaker at 90 rpm in the dark for 2 h; free dye was removed by size exclusion chromatography, and the mixture was concentrated using a 3 kDa ultrafiltration tube to obtain DiD-MSC-Ev, DiD-293F-EV, DiD-MSC-PC, and DiD-293F-PC.

[0054] (2) Obtaining DiD-MSC-PC-COS and DiD-293F-PC-COS: DiD-MSC-PC and DiD-293F-PC (1×10 10 The MSCs (particles) were added to 75 μg / ml COS solution, placed on a shaker at 37°C and incubated for 1 h. DiD-MSC-PC-COS and DiD-293F-PC-COS were obtained by purification using a 4 ml 100 kDa ultrafiltration tube and centrifugation at 4°C and 3000 g for 2 min.

[0055] (3) Flow cytometry to detect uptake: RAW264.7 cells were seeded into a 12-well plate. When the RAW264.7 cells in the plate grew to 50-60%, they were washed twice with 1×PBS (pH=7.4) and synchronized by adding serum-free medium for 12 h. After synchronization, DiD-MSC-Ev, DiD-293F-EV, DiD-MSC-PC, DiD-293F-PC, DiD-MSC-PC-COS and DiD-293F-PC-COS labeled with DiD were added and incubated for 12 h. The cells were resuspended in 1 ml PBS, washed twice with PBS and resuspended in PBS. The fluorescence intensity of DiD in the cells was measured by flow cytometry, as shown in Figure 2. Figure 3 As shown in A, MSC-EV-PC and 293F-EV-PC increased the uptake of EVs by macrophages, while their mean fluorescence intensity decreased significantly after COS modification.

[0056] (4) Fluorescence microscopy to capture macrophage uptake of EVs: RAW264.7 was seeded into a confocal dish. When the cells in the dish grew to 50-60%, they were washed twice with PBS and synchronized with serum-free culture medium for 12 h. After synchronization, DiD-MSC-Ev, DiD-293F-EV, DiD-MSC-PC, DiD-293F-PC, MSC-PC-COS, and 293F-PC-COS labeled with DiD were added and incubated for 12 h. 4% paraformaldehyde was used for fixation at room temperature for 20 min. 0.25% TritonX-100 was added to cover the bottom of the plate and the membrane was broken at room temperature for 5 min. Nuclear staining: DAPI was added to stain the cell nucleus for 5 min. PBS was rinsed three times, 5 min each time. Seal the slides: glycerol was added to cover the bottom of the plate. The uptake was observed under a fluorescence microscope, as shown in Figure 4. Figure 3As shown in B, macrophages basically do not take up EVs, the uptake of EVs with protein corona increases, and after COS modification, their uptake decreases.

[0057] The results of this example show that chitosan oligosaccharide modification of EVs reduces their uptake by macrophages.

[0058] Example 4: Chitosan oligosaccharide-modified EVs can reduce the uptake of EVs by the mononuclear phagocytic system (such as liver and spleen) and increase the uptake of EVs by damaged organs (such as brain, heart, kidney, etc.) in septic mice

[0059] (1) Preparation of DiD-labeled EVs and EV-COS: MSC-EVs and 293F-EVs were labeled with DiD using the method of Example 3. DiD-labeled EVs or EV-COS were injected into normal mice (Sham) and septic mice (CLP) via the tail vein, with 1×10 10 particles, the control mice were only injected with 1×PBS (pH=7.4), and the groups were Sham+PBS, Sham+EV, Sham+EV-COS, CLP+PBS, CLP+EV, and CLP+EV-COS.

[0060] (2) Sepsis mouse model: Mice were anesthetized by intraperitoneal injection, and a small incision was made at the longitudinal midline of the cecum to expose the cecum. The cecum was then ligated 1 cm from the end, and a No. 5 needle was used to puncture the cecum between the ligation site and the tip of the cecum along the direction of the cecal ligation. After the needle was removed, a small amount of feces was squeezed out from the two punctures. After ensuring that the cecum was unobstructed, 1 ml of normal saline was injected subcutaneously. The cecum of mice in the sham operation group was not ligated or perforated.

[0061] (3) Observation of organ uptake using IVIS Spectrum: 10 hours after injection of DiD-labeled EV or EV-COS, the mice were anesthetized, the abdominal cavity was opened, and the main organs (brain, heart, lung, liver, spleen, and kidney) were collected for in vitro fluorescence imaging. The fluorescence intensity (total radiant efficiency) of each organ was obtained using IVIS Spectrum.

[0062] like Figure 4 As shown, the uptake of COS-modified EVs in the liver and spleen was significantly reduced compared with simple EVs in normal mice, and there was no significant difference in accumulation in other organs; in septic mice, in addition to the significant decrease in liver and spleen uptake, the uptake in the brain, heart, and kidneys was significantly increased.

[0063] The results of this example show that chitosan oligosaccharide-modified EVs reduce the uptake by the mononuclear phagocytic system (such as liver and spleen), while increasing the uptake by organs such as the brain, heart, and kidneys, thereby improving the bioavailability of extracellular vesicles to extrahepatic organs.

[0064] Example 5: Effects of chitosan oligosaccharide-modified EVs on protein corona-mediated macrophage inflammatory response

[0065] (1) MSC-EV, MSC-EV-PC, MSC-PC-COS, 293F-EV, 293F-EV-PC, and 293F-PC-COS intervention of macrophages: RAW264.7 cells were seeded into 12-well plates. When the RAW264.7 cells in the plate grew to 70-80%, they were washed twice with PBS and synchronized with serum-free medium for 6 h. After synchronization, MSC-EV, MSC-EV-PC, MSC-PC-COS, 293F-EV, 293F-EV-PC, and 293F-PC-COS (1×10 8 particles), intervention for 12 h;

[0066] (2) Extracting cell RNA and detecting the expression of inflammatory factors by RT-qPCR: discard the cell culture medium, wash once with 1× PBS, and discard the waste liquid; add 500μl FreeZol Reagent (Nanjing Novezan Biotechnology, R711) to each well to fully cover the cell surface, and then use a pipette to repeatedly blow the cells to make them fall off; transfer the lysate to a 1.5ml centrifuge tube, add 150μl Dilution Buffer to the above lysate; centrifuge at 11200rpm and 4℃ for 15min; remove the EP tube and pipette 500μl supernatant solution into a new EP tube; add an equal volume of isopropanol to the obtained supernatant solution, mix thoroughly by turning it upside down, and let it stand in a refrigerator at 4℃ for 30min; centrifuge at 11,200rpm and 4℃ for 10min. After centrifugation, white gel-like precipitates can be seen on the sides and bottom of the tube, and the supernatant is discarded; add 1ml 75% ethanol (prepared with DEPC water); centrifuge at 9100rpm and 4℃ for 3min, discard the supernatant; place on ice to dry, add 10μl DEPC water to dissolve the precipitate; use Nanodrop2000 to detect RNA concentration: after calibrating the instrument with DEPC water, take 1μl of the sample to be tested and drop it on the probe for detection, record the RNA concentration and A260 / A280 ratio. For the reverse transcription and real-time fluorescence PCR steps, refer to the instructions of the kit (Nanjing Novezan, R323-01, Q331-02) to obtain the corresponding expression values. Figure 5 As shown in the data, the inflammatory factors IL-1B, IL-6, TNF-A, CCL2, and CCL5 were significantly increased in the two EVs after the formation of the protein corona, while intervention with MSC-PC-COS and 293F-PC-COS could significantly reduce the inflammatory response mediated by the protein corona.

[0067] The results of this example show that chitosan oligosaccharide-modified EVs can significantly reduce the inflammatory response mediated by protein corona.

Claims

1. Application of chitosan oligosaccharides in the preparation of drugs that inhibit the formation of plasma protein corona in extracellular vesicles.

2. The use according to claim 1, characterized in that: The chitosan oligosaccharide is a chitosan hydrolyzate obtained by chemical synthesis or biosynthesis, has a polymerization degree of 2-20, a molecular weight of 3200 Da, and has the characteristics of a positively charged polysaccharide.

3. The use according to claim 1, characterized in that: The extracellular vesicles include vesicles with a lipid membrane structure secreted by cells of humans, animals, plants or microorganisms.

4. The use according to claim 1 or 3, characterized in that: The extracellular vesicles include exosomes, microvesicles, migratory bodies, apoptotic bodies, shedding vesicles or microparticles.

5. The use according to claim 1, characterized in that: The medicine is prepared by modifying extracellular vesicles with chitosan oligosaccharide.

6. The use according to claim 5, characterized in that: The method for modifying extracellular vesicles with chitosan oligosaccharide comprises the following steps: dripping an extracellular vesicle solution into a chitosan oligosaccharide solution, and incubating the solution on a rotary mixer; the obtained solution is purified by centrifugation using an ultrafiltration tube to obtain the extracellular vesicles modified with chitosan oligosaccharide.

7. The use according to claim 6, characterized in that: The concentration of the chitosan oligosaccharide solution is 50-150 μg / ml.

8. The use according to claim 1 or 5, characterized in that: The extracellular vesicles are derived from human umbilical cord mesenchymal stem cells or human embryonic kidney cell lines.

9. The use according to any one of claims 1 to 8, characterized in that: The medicine is a single preparation or a compound preparation.

10. The use according to any one of claims 1 to 8, characterized in that: The dosage form of the drug includes liquid preparation, dry suspension, spray preparation, tablet, capsule or ointment.

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