Use of chitooligosaccharides in inhibiting formation of extracellular vesicle plasma protein corona

Abstract

The application discloses application of chitooligosaccharide in inhibition of extracellular vesicle plasma protein corona formation, and belongs to the technical field of medicines. Specifically, chitooligosaccharide is modified to the surface of extracellular vesicles through co-incubation, and the formation of plasma protein corona is effectively inhibited. While reducing the uptake of the mononuclear phagocyte system (such as liver and spleen), the uptake of organs such as brain, heart and kidney is increased, so that the bioavailability of extracellular vesicles to organs outside the liver is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of chitosan oligosaccharide in inhibiting the formation of extracellular vesicle plasma protein crowns. Background Technology

[0002] In recent years, extracellular vesicles (EVs) have attracted widespread attention as an emerging therapeutic approach. EVs are nanoscale membrane vesicles released from cells into the external environment, including exosomes, microvesicles, and apoptotic bodies. EVs possess natural biocompatibility and low immunogenicity, showing promising potential as drug delivery carriers. Compared to traditional synthetic nanoparticles, EVs offer unique advantages in drug delivery. Furthermore, certain EVs derived from specific sources, such as mesenchymal stem cells (MSC-EVs), have demonstrated significant therapeutic effects. These EVs participate in antigen presentation, immune regulation, and tissue repair through their carried genetic material and proteins, improving cell proliferation and reducing inflammation and fibrosis.

[0003] However, EVs face the challenge of forming a protein corona (PC) on the surface of the drug delivery vehicle after cyclic administration. A protein corona refers to one or more protein structures adsorbed on the surface of nanoparticles released into the biological environment (such as blood, plasma, and cytoplasm). The presence of the protein corona affects the size, aggregation state, and interfacial properties of nanomaterials, endowing them with new biological characteristics and consequently influencing phagocytosis, biodistribution, and immune responses. For example, after intravenous injection, when nanoparticles adsorb opsonin proteins (such as complement proteins) from plasma, they promote phagocytosis by macrophages, leading to rapid clearance of the nanoparticles by the mononuclear phagocytic system. Therefore, there is an urgent need in this field to develop a method to inhibit the formation of a protein corona on the surface of drug delivery vehicles, thereby overcoming the adverse effects of the protein corona on drug delivery vehicles. 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 that inhibit the formation of extracellular vesicle plasma protein crowns.

[0005] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of chitosan oligosaccharide in the preparation of drugs that inhibit the formation of extracellular vesicle plasma protein crowns.

[0006] The chitosan oligosaccharide is a chitosan hydrolysis product obtained through chemical or biological synthesis. It has a degree of polymerization of 2 to 20, a molecular weight of ≤3200 Da, and positively charged polysaccharide properties.

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

[0008] The extracellular vesicles include exosomes, microvesicles, migratory bodies, apoptotic bodies, detached vesicles, or microparticles.

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

[0010] The method for modifying extracellular vesicles with chitosan oligosaccharide involves adding an extracellular vesicle solution dropwise into a chitosan oligosaccharide solution and incubating it on a rotary mixer; the resulting solution is then purified by centrifugation using an ultrafiltration tube to obtain the extracellular vesicles modified with chitosan oligosaccharide.

[0011] The concentration of the chitosan oligosaccharide solution is 50–150 μg / ml.

[0012] The drug is either a single-ingredient preparation or a compound preparation.

[0013] The dosage forms of the drug include liquid preparations, dry suspensions, spray preparations, tablets, capsules, or ointments.

[0014] The method for modifying EVs with chitosan oligosaccharides according to the present invention specifically includes the following steps:

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

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

[0017] Furthermore, this invention modifies EVs derived from human umbilical cord mesenchymal stem cells (MSCs) and human embryonic kidney cell line HEK293F with chitosan oligosaccharide (COS), thereby demonstrating the ability of chitosan oligosaccharide to inhibit the formation of plasma protein corona 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 extrahepatic organs to extracellular vesicles.

[0019] The average particle size of the MSC-EVs modified with chitosan oligosaccharide in this invention is about 170.6 ± 2.4 nm, and the average particle size of the 293F-EVs is about 179.3 ± 5.1 nm. The absolute value of the potential of both EVs is reduced after modification.

[0020] This invention demonstrates through experiments that the chitosan oligosaccharide-modified EVs (EV-COS) adsorb less protein after incubation with EV-removed plasma in vitro compared to unmodified EVs.

[0021] Furthermore, this invention demonstrates that chitosan oligosaccharide modification of EVs reduces macrophage uptake of EVs and decreases uptake 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 kidney) in septic mice.

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

[0024] Beneficial effects: This invention modifies extracellular vesicles with chitosan oligosaccharides, which can effectively inhibit the formation of protein crowns, reduce the uptake of extracellular vesicles by mononuclear phagocytic systems (such as liver and spleen), and increase the uptake by organs such as brain, heart and kidney, thereby improving the bioavailability of extracellular vesicles by extrahepatic organs. Attached Figure Description

[0025] Figure 1 Identification diagrams for chitosan oligosaccharide-modified EVs: (A) Particle size distribution diagram, left is the diagram after MSC-EV modification, right is the diagram after 293F-EV modification; (B) Potential diagram; where MSC-EV is extracellular vesicle secreted by MSC cells, MSC-EV-COS is extracellular vesicle secreted by chitosan oligosaccharide-modified MSC cells, 293F-EV is extracellular vesicle secreted by 293F cells, and 293F-EV-COS is extracellular vesicle secreted by chitosan oligosaccharide-modified 293F cells;

[0026] Figure 2 The following diagrams illustrate the effect of chitosan oligosaccharide-modified EVs on inhibiting plasma corona formation: (A) Particle size distribution; (B) Protein content determined by BCA method; (C) Evaluation of the corona inhibition effect of MSC-PC-COS by silver staining; (D) Evaluation of the corona inhibition effect of EV-COS by Western blot. MSC-EV-PC refers to extracellular vesicles secreted by MSC cells with corona, MSC-PC-COS refers to extracellular vesicles secreted by chitosan oligosaccharide-modified MSC cells with corona, 293F-EV refers to extracellular vesicles secreted by 293F cells, 293F-EV-PC refers to extracellular vesicles secreted by 293F cells with corona, and 293F-PC-COS refers to extracellular vesicles secreted by chitosan oligosaccharide-modified 293F cells with corona.

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

[0028] Figure 4 Biodistribution maps of EV-COS in normal mice and septic mice: (A) Biodistribution map and average fluorescence intensity statistics of MSC-EV-PC and MSC-PC-COS in normal mice; (B) Biodistribution map and average fluorescence intensity statistics of MSC-EV-PC and MSC-PC-COS in septic mice; (C) Biodistribution map and average fluorescence intensity statistics of 293F-EV-PC and 293F-PC-COS in normal mice; (D) Biodistribution map and average fluorescence intensity statistics of 293F-EV-PC and 293F-PC-COS in septic mice; Among them, the Sham group was the sham-operated control group, and the CLP group was the cecal ligation and perforation (septic model) group;

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

[0030] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0031] In this invention, statistical data are presented as mean ± standard error. Data were processed using SPSS 26 statistical software. One-way ANOVA was used for comparisons between groups, and t-tests were used for comparisons between two groups. p < 0.05 was considered statistically significant. All experimental results were repeated at least three times.

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

[0033] 1. Preparation of EV-COS

[0034] (1) Extract EVs:

[0035] a. Serum-free culture medium (Beijing Huakan Biotechnology Co., Ltd., RMZ112) was used to culture 3×10⁻⁶ umbilical cord mesenchymal stem cells (umbilical cord provided by Zhongda Hospital Affiliated to Southeast University, ethics approval number: 2023ZDSYLL118-P01). 6 (cells) cultured to passage P3, then added to cells with 3D... The microcarrier (Beijing Huakan Biotechnology Co., Ltd., catalog number W01-200) was placed in a 125ml roller bottle and placed in a 3D The cells were cultured in a miniSPIN bioreactor. When the glucose level in the roller flask dropped to 10 mmol / L, the medium was replaced with fresh medium. The supernatant of the old medium was collected. Once all the cells on the microcarriers had aggregated, the collection of the supernatant was stopped.

[0036] b. Suspend Expi293F cells (Thermo Fisher, A14527, 4.5 × 10⁻⁶) 5 (100 cells) were transferred to a 250ml roller bottle (Wuxi Nice Life Science Co., Ltd., 782011) and cultured in a shaker at 37℃ and 120rpm. After 2-3 days, the cell culture medium (Shanghai Aopumai Biotechnology Co., Ltd., OPM-293CD05) was changed and the supernatant was collected at the same time.

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

[0038] d. Next, size exclusion chromatography (Beijing Enzekangtai Biotechnology Co., Ltd., ES914) 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) Co-incubation of EVs and COS: MSC-EVs and 293F-EVs (5×10⁻⁶) were incubated with COS respectively. 10 200 μl of each particle was added to a 75 μg / ml COS solution (TICIE (Shanghai) Chemical Industry Development Co., Ltd., C2849, 300 μl), and incubated on a shaker at 37°C for 1 h. The solution was then purified using a 0.5 ml 100 kDa ultrafiltration tube (Millipore, UFC5100) and centrifuged at 3000 g for 2 min at 4°C.

[0040] 2. Identification of EV-COS

[0041] Particle size and potential were measured using the German PMX Zetaview nanoparticle tracking and analysis instrument. Results are as follows: Figure 1As shown, the average particle size of COS-modified MSC-EVs is approximately 170.6 ± 2.4 nm, while that of COS-modified 293F-EVs is approximately 179.3 ± 5.1 nm. Simultaneously, the absolute potential values ​​of both EVs decreased after COS modification, with the modified MSC-EVs having a potential of -12.3 ± 2.2 mV and the 293F-EVs having a potential of -12.6 ± 1.0 mV. This demonstrates that the positively charged COS was successfully modified onto the negatively charged EVs.

[0042] The results of this embodiment show that the average particle size of the COS-modified MSC-EVs prepared by this 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 EV potential decreased after modification, proving that COS was successfully modified on the EV surface.

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

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

[0045] Plasma separation: Whole blood from healthy individuals (collected from Zhongda Hospital Affiliated to Southeast University, aged 20-30 years, ethics approval number: 2024ZDSYLL249-P01) was centrifuged at 2500g for 15 min at 4℃; the supernatant was transferred to another centrifuge tube and centrifuged at 2500g for 15 min at 4℃, the supernatant being plasma; the plasma sample was centrifuged at 10000g for 40 min at 4℃ to remove large vesicles; the supernatant after centrifugation was filtered through a 0.22μm filter membrane and centrifuged at 167000g for 4 h at 4℃; EVs were further removed from the supernatant after ultrafiltration using a 100kD ultrafiltration tube, and the filtrate was obtained by centrifugation at 10000g for 30 min at 4℃; the filtrate was concentrated by 3kD: centrifuged at 10000g for 10 min at 4℃, thus obtaining EVs-free plasma (EVDP).

[0046] The MSC-EVs and 293F-EVs prepared in Example 1 were incubated with EVDP in a constant flow pump to simulate blood circulation for 30 min, with a volume ratio of MSC-EVs or 293F-EVs to EVDP of 1:1. The mixture was centrifuged at 167000g for 4 h at 4°C, purified by size exclusion chromatography, collected, and concentrated using a 3kDa ultrafiltration tube to obtain EVs with protein crowns, namely MSC-EV-PC and 293F-EV-PC, respectively, thus simulating the formation of plasma protein crowns in vitro. Using the same method as in Example 1, COS was modified onto MSC-EV-PC and 293F-EV-PC to obtain MSC-PC-COS and 293F-PC-COS, respectively.

[0047] 2. The effect of chitosan oligosaccharide-modified EVs on inhibiting protein crown formation

[0048] The inhibitory effect of EV-COS on the protein crown was evaluated using a nanoparticle tracking analyzer, BCA protein concentration assay, silver staining method, and Western blot method.

[0049] (1) Particle size detection: All samples were analyzed using the German PMX Zetaview nanoparticle tracking and analysis instrument. The results are as follows: Figure 2 As shown in Figure A, the particle size of COS-modified MSC-EV-PC and 293F-EV-PC is smaller than that of EVs that form protein crowns.

[0050] (2) Using the BCA protein concentration assay, silver staining method, and Western blot method: A protein standard curve was prepared according to the instructions of the BCA protein concentration assay kit (Jiangsu Kaiji Biotechnology, KGPBCA), and the test samples were prepared. The absorbance of the test samples at 562 nm was then detected using a microplate reader. The protein concentration and total protein amount were obtained from the standard curve, such as... Figure 2 As shown in Figure B, the EV protein content increased after MSC-EV-PC and 293F-EV-PC were incubated with plasma, while COS modification of MSC-EV-PC and 293F-EV-PC significantly reduced protein adsorption. Protein expression was detected using a silver staining kit (Beyotime Biotechnology, P0017S). The results showed that MSC-EV-PC and 293F-EV-PC exhibited increased protein bands after the formation of a protein crown, and the expression profile after the formation of the protein crown was similar to that of plasma without EVs. COS modification of EVs reduced protein adsorption, and Western blot analysis demonstrated that it significantly reduced the adsorption of proteins such as Alpha-2-macroglobulin, fibrinogen α, complement protein C3, and immunoglobulin IGHG1. Figure 2 As shown in C and 2D.

[0051] The results of this embodiment demonstrate that COS-modified EVs can inhibit protein crown formation and reduce the expression of plasma proteins.

[0052] Example 3: Chitosan oligosaccharide-modified EVs reduce macrophage uptake of them.

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

[0054] (2) Obtain DiD-MSC-PC-COS and DiD-293F-PC-COS: Take DiD-MSC-PC and DiD-293F-PC (1×10 10 (100 particles) were added to 75 μg / ml COS solution and incubated on a shaker at 37℃ for 1 h; purified using a 4 ml 100 kDa ultrafiltration tube and centrifuged at 4℃ and 3000g for 2 min to obtain DiD-MSC-PC-COS and DiD-293F-PC-COS.

[0055] (3) Flow cytometry analysis of uptake: RAW264.7 cells were seeded into 12-well plates. When the RAW264.7 cells reached 50-60% confluence, they were washed twice with 1×PBS (pH=7.4) and then cultured in serum-free medium for 12 h for synchronization. After synchronization, DiD-labeled DiD-MSC-Ev, DiD-293F-EV, DiD-MSC-PC, DiD-293F-PC, DiD-MSC-PC-COS, and DiD-293F-PC-COS were added and incubated for 12 h. The cells were resuspended in 1 ml PBS, washed twice with PBS, and then resuspended in PBS. The fluorescence intensity of intracellular DiD was measured by flow cytometry. Figure 3 As shown in Figure A, MSC-EV-PC and 293F-EV-PC increase macrophage uptake of EVs, while COS modification significantly reduces their average fluorescence intensity.

[0056] (4) Fluorescence microscopy imaging of macrophage uptake of EVs: RAW264.7 cells were seeded in confocal dishes. When the cells reached 50-60% confluence, they were washed twice with PBS and cultured in serum-free medium for 12 hours for synchronization. After synchronization, DiD-labeled DiD-MSC-EV, DiD-293F-EV, DiD-MSC-PC, DiD-293F-PC, MSC-PC-COS, and 293F-PC-COS were added and incubated for 12 hours. The cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes. 0.25% Triton X-100 was added to cover the bottom of the plate, and the membrane was ruptured at room temperature for 5 minutes. Nucleus staining: DAPI was added to stain the cell nuclei for 5 minutes. The cells were washed three times with PBS for 5 minutes each time. Mounting: Glycerol was added to cover the bottom of the plate. The uptake was observed under a fluorescence microscope. Figure 3As shown in B, macrophages do not take up EVs at all, but those with protein crowns take up more EVs, while COS modification reduces their uptake.

[0057] The results of this embodiment demonstrate that chitosan oligosaccharide-modified EVs reduce macrophage uptake of them.

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

[0059] (1) Preparation of DiD-labeled EVs and EV-COS: MSC-EVs and 293F-EVs were DiD-labeled using the method described in Example 3. The DiD-labeled EVs or EV-COS were injected into normal mice (Sham) and septic mice (CLP) via tail vein injection, with each mouse receiving 1×10⁻⁶ EVs. 10 Each particle was injected into a control mouse with only 1×PBS (pH=7.4). The mice were divided into groups: Sham+PBS, Sham+EV, Sham+EV-COS, CLP+PBS, CLP+EV, and CLP+EV-COS.

[0060] (2) Sepsis model in mice: mice were anesthetized by intraperitoneal injection. 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. A No. 5 needle was used to puncture between the ligation point and the tip of the cecum along the ligation direction. After removing the needle, a small amount of feces was squeezed out from the two punctures. After ensuring patency, 1 ml of physiological saline was injected subcutaneously. The cecum of the sham-operated group mice was not ligated or perforated.

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

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

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

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

[0065] (1) Intervention of macrophages with MSC-EV, MSC-EV-PC, MSC-PC-COS, 293F-EV, 293F-EV-PC, and 293F-PC-COS: RAW264.7 cells were seeded into 12-well plates. When the RAW264.7 cells in the plates reached 70-80% confluence, they were washed twice with PBS and then cultured in serum-free medium for 6 hours for synchronization. After synchronization, MSC-EV, MSC-EV-PC, MSC-PC-COS, 293F-EV, 293F-EV-PC, and 293F-PC-COS (1×10⁻⁶) were added respectively. 8 (one particle), intervention for 12 hours;

[0066] (2) Extracting cellular RNA and detecting inflammatory factor expression by RT-qPCR: Discard the cell culture medium, wash once with 1×PBS, and discard the waste liquid; add 500 μl of FreeZol Reagent (Nanjing Novizan Biotechnology, R711) to each well to fully cover the cell surface, and then repeatedly pipette the cells to detach them; transfer the lysis buffer to a 1.5 ml centrifuge tube, add 150 μl of Dilution Buffer to the above lysis buffer; centrifuge at 11200 rpm and 4℃ for 15 min; remove the EP tube, and aspirate 500 μl of the supernatant to a new EP tube; add an equal volume of isopropanol to the obtained supernatant, mix thoroughly by inverting, and let stand at 4℃ for 30 min; centrifuge at 11200 rpm and 4℃ for 10 min, after centrifugation, a white gel-like precipitate can be seen on the side and bottom of the tube, discard the supernatant; add 1 ml Centrifuge at 9100 rpm for 3 min at 4°C with 75% ethanol (prepared with DEPC water), discard the supernatant; air dry on ice, add 10 μl of DEPC water to dissolve the precipitate; detect RNA concentration using Nanodrop 2000: after calibrating the instrument with DEPC water, drop 1 μl of the sample onto the probe for detection, and record the RNA concentration and A260 / A280 ratio. For reverse transcription and real-time fluorescence PCR, refer to the kit instructions (Nanjing Novizan, R323-01, Q331-02) to obtain the corresponding expression values. Figure 5 As shown, after the formation of the protein crown, the levels of inflammatory factors IL-1B, IL-6, TNF-A, CCL2, and CCL5 in both types of EVs were significantly increased, while intervention with MSC-PC-COS and 293F-PC-COS could significantly reduce the protein crown-mediated inflammatory response.

[0067] The results of this embodiment demonstrate that chitosan oligosaccharide-modified EVs can significantly reduce the protein crown-mediated inflammatory response.

Claims

1. The application of chitosan oligosaccharide in the preparation of drugs that inhibit the formation of extracellular vesicle plasma protein coronas, characterized in that, The chitosan oligosaccharide is a chitosan hydrolysis product obtained through chemical or biological synthesis. It has a degree of polymerization of 2 to 20, a molecular weight of ≤3200 Da, and positively charged polysaccharide properties. The drug is prepared by modifying extracellular vesicles with chitosan oligosaccharide.

2. The application according to claim 1, characterized in that, The extracellular vesicles include vesicles with lipid membrane structures secreted by cells of humans, animals, plants, or microorganisms.

3. The application according to claim 1 or 2, characterized in that, The extracellular vesicles include exosomes, microvesicles, migratory bodies, apoptotic bodies, detached vesicles, or microparticles.

4. The application according to claim 1, characterized in that, The method for modifying extracellular vesicles with chitosan oligosaccharide involves adding an extracellular vesicle solution dropwise into a chitosan oligosaccharide solution and incubating it on a rotary mixer; the resulting solution is then purified by centrifugation using an ultrafiltration tube to obtain the extracellular vesicles modified with chitosan oligosaccharide.

5. The application according to claim 4, characterized in that, The concentration of the chitosan oligosaccharide solution is 50~150 μg / ml.

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

7. The application according to any one of claims 1 to 6, characterized in that, The drug is a single-ingredient preparation or a compound preparation.

8. The application according to any one of claims 1 to 6, characterized in that, The dosage forms of the drug include liquid preparations, dry suspensions, spray preparations, tablets, capsules, or ointments.

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