Purification method of plasma extracellular vesicles

Abstract

The invention discloses a method for purifying plasma extracellular vesicles, which is a collagen affinity capture method and is used for purifying the plasma extracellular vesicles by using telopeptide-removed soluble collagen. Compared with an affinity capture method using an antibody, the purification method disclosed by the invention does not need to be coupled to a solid-phase matrix, so that the time is shortened, and the cost is relatively low. The collagen affinity capture method is high in affinity, the obtained extracellular vesicles are high in purity and yield, meanwhile, collagenase is used for digestion and decomposition of collagen, the extracellular vesicles are easily and thoroughly released, and loss is extremely low. The purification method is simple to operate, does not need expensive instruments and consumables, and is easy to popularize and use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for purifying plasma extracellular vesicles. BACKGROUND

[0002] The basis and key of extracellular vesicles (EV) research lies in obtaining high-purity EV products, especially in researches that need to quantitatively detect the number of extracellular vesicles, the contents or the role of extracellular vesicles in the occurrence of diseases. Many damaged and diseased tissues secrete specific extracellular vesicles into the blood, such as tumors. However, it is a great challenge to separate pure EVs from body fluids such as plasma. The components in plasma are extremely complex, and many components have similar physicochemical properties such as size, density, isoelectric point, charge distribution, and antigenic epitopes to extracellular vesicles. In addition, a large amount of plasma proteins such as albumin change the viscosity of plasma and increase weak interactions, so it is very difficult to separate high-purity EVs from plasma.

[0003] The commonly used methods at present include ultracentrifugation, density gradient centrifugation, polymer precipitation, size exclusion chromatography (SEC), affinity capture, ultrafiltration, etc., or a combination of the above methods, but there is no standardized and universal method.

[0004] The most commonly used method at present is ultracentrifugation, and the evaluation of many new methods is compared with this method. The advantage of ultracentrifugation is simple operation and no additional reagents are needed, but the yield and purity of this method are very low, and the ultracentrifuge is expensive and has low popularization rate. As for the affinity capture method, most of them use the principle of specific binding of antigen-antibody, such as fixing anti-CD63 monoclonal antibody on magnetic beads or agarose microspheres to capture extracellular vesicles expressing CD63 molecules, but due to the high cost of antibodies, this method is not suitable for mass production of EVs.

[0005] In order to solve the above problems, the present application adopts affinity capture method as the basic technical route, and provides a low-cost purification method which far exceeds the yield and purity of ultracentrifugation and does not depend on expensive instruments, i.e. collagen affinity capture method. SUMMARY

[0006] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application aims to provide a method for purifying plasma extracellular vesicles, collagen co-precipitation (CCP). Since the outermost membrane of the extracellular vesicle is derived from the plasma membrane of the parent cell, its membrane protein component is similar to that of the parent cell plasma membrane, and also expresses integrin molecules (membrane receptors of extracellular matrix proteins). By utilizing this feature, the present application uses collagen molecules as ligands to capture EVs expressing integrin molecules. Since collagen is a linear molecule, it is easy to precipitate after forming a cross-linking product with EVs, thereby separating EVs from other soluble components in the plasma. Then, collagenase I is used to degrade collagen to release EVs, followed by polyethylene glycol concentration of EVs, and finally pure EVs are obtained.

[0007] To achieve the above-mentioned object, the technical solution adopted by the present application is: In a first aspect of the present application, a method for purifying plasma extracellular vesicles is provided, the method comprising the following steps: adding collagen to the plasma sample and mixing, centrifuging, removing the supernatant, adding extracellular vesicle separation solution and mixing, centrifuging, and separating the supernatant to obtain the plasma extracellular vesicles; the extracellular vesicle separation solution comprises at least one of collagen digestion solution, acid eluent and alkali eluent.

[0008] In some embodiments of the present application, the mixing time of the added collagen is 10-60 minutes.

[0009] In some embodiments of the present application, the mixing time of the added collagen is 20-40 minutes.

[0010] In some embodiments of the present application, the extracellular vesicle separation solution is mixed for 1-5 hours.

[0011] In some embodiments of the present application, the extracellular vesicle separation solution is mixed for 2-4 hours.

[0012] In some embodiments of the present application, the protease comprises at least one of collagenase, metalloproteinase and elastase. In some embodiments of the present application, the serine protease inhibitor comprises at least one of benzylsulfonyl fluoride, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 4-amino-2-methylbenzenesulfonyl fluoride.

[0013] In some embodiments of the present application, the serine protease inhibitor is used to inhibit the proteolytic enzyme activity of non-collagenase in collagenase.

[0014] In some embodiments of the present invention, the enzyme promoter comprises a reagent containing at least one of calcium ions and zinc ions.

[0015] In some embodiments of the present invention, the reagent containing calcium ions includes at least one of calcium chloride, calcium sulfate, and calcium nitrate.

[0016] In some embodiments of the present invention, the zinc ion-containing reagent includes at least one of zinc acetate and zinc sulfate.

[0017] In some embodiments of the present invention, the acid elution solution includes at least one of hydrochloric acid solution and acetic acid solution.

[0018] In some embodiments of the present invention, the alkaline eluent includes at least one of ammonia, sodium hydroxide solution, and potassium hydroxide solution.

[0019] In some embodiments of the present invention, the collagenase includes at least one of collagenase type I, II, III, IV and V.

[0020] In some embodiments of the present invention, the collagenase is collagenase type I.

[0021] In some embodiments of the present invention, the amount of collagenase used is 1-5 mg of collagenase per milligram of plasma sample.

[0022] In some embodiments of the present invention, the amount of collagenase used is 2-4 mg of collagenase per milliliter of plasma sample.

[0023] In some embodiments of the present invention, the mass ratio of collagenase to collagen is 0.5-1:1.

[0024] In some embodiments of the present invention, the mass ratio of collagenase to collagen is 0.6-0.9:1.

[0025] In some embodiments of the present invention, the collagen includes soluble collagen and insoluble collagen.

[0026] In some embodiments of the present invention, the soluble collagen includes determinated collagen.

[0027] In some embodiments of the present invention, the terminated collagen refers to collagen monomers with a triple helix structure after being digested by pepsin; it is neither denatured nor polymerized collagen.

[0028] In some embodiments of the present invention, the determinate collagen contains only the GFOGER motif.

[0029] In some embodiments of the present invention, the amount of collagen used is 1-5 mg of collagen added per milliliter of plasma sample.

[0030] In some embodiments of the present invention, the amount of collagen used is 2-5 mg of collagen added per milliliter of plasma sample.

[0031] In some embodiments of the present invention, the step includes adding at least one of a cross-linking promoter and an activator to the plasma sample and mixing it before adding collagen.

[0032] In some embodiments of the present invention, the step includes adding a cross-linking promoter and mixing the plasma sample before adding collagen.

[0033] In some embodiments of the present invention, the crosslinking accelerator includes covalent crosslinking accelerators and non-covalent crosslinking accelerators.

[0034] In some embodiments of the present invention, the covalent crosslinking promoter is used to promote the formation of covalent chemical bonds between molecules.

[0035] In some embodiments of the present invention, the non-covalent crosslinking promoter is used to promote the connection between molecules through non-covalent forces such as ionic bonds, hydrogen bonds, or van der Waals forces.

[0036] In some embodiments of the present invention, the covalent crosslinking promoter includes at least one of glutaraldehyde, carbodiimide, and genipin.

[0037] In some embodiments of the present invention, the non-covalent crosslinking promoter includes a reagent containing at least one of magnesium ions and calcium ions.

[0038] In some embodiments of the present invention, the crosslinking promoter is a reagent containing magnesium ions.

[0039] In some embodiments of the present invention, the step includes adding a reagent containing magnesium ions and mixing it before adding collagen to the plasma sample.

[0040] In some embodiments of the present invention, the magnesium ion-containing reagent is used to activate and promote the cross-linking of collagen.

[0041] In some embodiments of the present invention, the magnesium-containing reagent includes at least one of magnesium chloride, magnesium sulfate, and magnesium carbonate.

[0042] In some embodiments of the present invention, the activator includes a reagent containing calcium ions, copper ions, magnesium ions, or iron ions.

[0043] In some embodiments of the present invention, the step further includes concentrating the collagen lysis buffer after lysis.

[0044] In some embodiments of the present invention, the concentration includes the use of at least one of precipitation, centrifugation and membrane separation.

[0045] In some embodiments of the present invention, the centrifugation method includes centrifugation after adding a PEG solution.

[0046] In some embodiments of the present invention, the PEG solution includes at least one of PEG400, PEG1500, PEG4000 and PEG8000.

[0047] In some embodiments of the present invention, the method for purifying plasma extracellular vesicles includes removing platelets from the plasma sample.

[0048] In some embodiments of the present invention, the removal of platelets includes removal using centrifugation or filtration.

[0049] In some embodiments of the invention, the filtration includes filtering plasma using a filter membrane.

[0050] In some embodiments of the present invention, the pore size of the filter membrane is 0.2-1 micrometer.

[0051] In some embodiments of the present invention, the pore size of the filter membrane is 0.5-1 micrometer.

[0052] In some embodiments of the present invention, the purification method of the plasma extracellular vesicles includes the following steps: adding determinate collagen to a plasma sample, mixing well, centrifuging, removing the supernatant, adding collagenase, mixing well, centrifuging, separating the supernatant, and obtaining the plasma extracellular vesicles.

[0053] In some embodiments of the present invention, the purification method of the plasma extracellular vesicles includes the following steps: filtering the plasma sample to remove platelets, adding magnesium chloride solution and then adding determinate collagen, mixing for 10-60 minutes, centrifuging, removing the supernatant, adding collagenase and calcium chloride solution, mixing for 1-5 hours, centrifuging, separating the supernatant, adding PEG8000 solution, mixing, centrifuging, removing the supernatant, and thus obtaining the plasma extracellular vesicles.

[0054] A second aspect of the present invention provides the use of collagen in the purification of extracellular vesicles from plasma, wherein the collagen is telopeptide-free collagen.

[0055] The beneficial effects of this invention are: This invention provides a method for purifying extracellular vesicles from plasma: collagen co-precipitation (CCP). This invention selects telopeptide-free collagen, which is collagen that has been cleaved by pepsin, leaving only the triple helix structure. This means that this collagen contains only the GFOGER motif and not the RGD motif, and therefore cannot gel into a hydrogel. Collagen containing the RGD motif, as well as fibrinogen and other substances in plasma, competes with extracellular vesicles for binding, reducing the capture rate. While immobilization modifications to collagen, such as coupling collagen to agarose microspheres or polystyrene plates and using activation methods like carbodiimide and glutaraldehyde, can activate carboxyl or amino groups, they also activate the hydroxyl groups on the hydroxyproline residues in the GFOGER motif, leading to GFOGER structure destruction and preventing the capture of extracellular vesicles.

[0056] The collagen affinity capture method of this invention uses collagen to directly purify plasma samples. Compared with affinity capture methods using antibodies, it is lower in cost, which is conducive to large-scale use. Furthermore, it eliminates the need for coupling to a solid matrix, further reducing time and economic costs. The collagen affinity capture method of this invention exhibits strong affinity, yielding high purity extracellular vesicles and completely capturing extracellular vesicles in plasma with a high yield. This invention uses collagenase I to enzymatically decompose collagen, facilitating the complete release of extracellular vesicles with extremely low loss. Simultaneously, the technical solution of this invention is simple to operate, requires no expensive instruments or consumables, and is easy to promote and use. Attached Figure Description

[0057] Figure 1 The figure shows the experimental results of adding different proportions of plasma and collagen in the purification method of this invention.

[0058] Figure 2 This image shows the morphological observation results of the extracellular vesicles extracted in this invention using a transmission electron microscope.

[0059] Figure 3 This is a magnified image showing the morphological observation results of the extracellular vesicles extracted in this invention using a transmission electron microscope.

[0060] Figure 4 This is a graph showing the particle size distribution and concentration analysis of extracellular vesicles extracted using the purification method of this invention.

[0061] Figure 5 The image shows the results of protein immunoblotting of extracellular vesicles extracted from the same volume of sample using the purification method of this invention and ultracentrifugation.

[0062] Figure 6 This image shows the protein immunoblotting results of the purification method of this invention and the ultracentrifugation method using the same total protein loading. Detailed Implementation

[0063] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0064] Example 1 This embodiment provides a method for purifying extracellular vesicles in plasma according to the present invention: collagen co-precipitation (CCP), the specific steps of which are as follows: 1. Collagen processing: (1) Weigh 100mg of lyophilized atelocollagen (acid-soluble atelocollagen, lyophilized, Guangdong Shengchi Biotechnology Co., Ltd.), add 20mL of 0.05mol / L acetic acid solution, and stir at room temperature for 2 hours or at 4℃ overnight using a magnetic stirrer. (2) After stirring in (1), remove the magnetic rod with tweezers, rinse the magnetic rod with acetic acid solution, and make up the volume of the solution to 25 mL. The final concentration of collagen is 4 mg / mL. Store in a refrigerator at 4℃ for later use and use within 1 month.

[0065] 2. Blood sample processing: (1) Take 2 mL of peripheral blood containing EDTA anticoagulant, centrifuge at 2500×g for 15 minutes at room temperature using a benchtop centrifuge to remove blood cells and separate plasma; (2) Centrifuge again at 2500×g for 15 minutes, separate the supernatant, remove cell debris and some platelet precipitate, transfer the obtained supernatant to a 1.5mL centrifuge tube, and store it in a -80°C refrigerator for later use.

[0066] 3. Capture of extracellular vesicles (1) After 1 mL of plasma is taken out of the refrigerator, it is thawed at room temperature; (2) Take another 0.8-micron needle filter (PES membrane, Guangzhou Jete Biotechnology Co., Ltd.), place it flat on a 15mL collection tube, connect a 1mL medical syringe to the top, remove the push rod, transfer the plasma into the syringe, let the plasma filter into the collection tube by gravity, and then rinse the filter membrane with PBS solution containing 1mmol / L MgCl2 to make the volume of the filtered liquid reach 2mL. This step can further remove platelets. (3) Dispense 2 mL of the filtrate into two 2 mL centrifuge tubes, add 2.5 mg of the above collagen solution to each tube, and then place them on a rotary mixer and rotate at room temperature for 30 minutes. (4) After mixing in step (3), remove the centrifuge tube, put it into a small benchtop centrifuge, centrifuge at 2000×g for 5 minutes, and then remove the supernatant; (5) Gently wash the precipitate once with 1 mL of PBS, centrifuge again at 2000×g for 5 minutes, and discard the supernatant; 4. Release of extracellular vesicles (1) Prepare collagenase I (Biosharp® Collagenase I, Lanjieke Technology Co., Ltd.) with physiological saline to prepare 4mg / mL, dispense into 800μL vials, and freeze at -30℃ for later use; (2) Take one collagenase I, thaw it, add a certain amount of protease inhibitor benzyl sulfonyl fluoride (PMSF) to a final concentration of 5 mmol / L, mix well, and add it equally to the two centrifuge tubes obtained in step 3. Then add a certain amount of CaCl2 solution to the centrifuge tubes to a final concentration of 2 mmol / L. (3) Place the centrifuge tube back onto the rotary mixer and rotate it at room temperature for 2 hours until the precipitate disappears; (4) Centrifuge at 2000×g for 5 minutes at room temperature, separate the supernatant, and add the supernatant from the two centrifuge tubes into a clean 1.5mL centrifuge tube with a volume of about 1mL.

[0067] 5. Concentrated EV (1) Weigh 25g of PEG 8000 (Shanghai Sangon Biotech Co., Ltd.) into a 50mL centrifuge tube, add ultrapure water to make up to 50mL, shake on a shaker at 4℃ overnight to prepare a 50% (w / v) solution, and store in a refrigerator at 4℃. (2) Add 250 μL of PEG 8000 solution to the supernatant obtained in step 4 until the final concentration of PEG 8000 is 10% (w / v). (3) Place the centrifuge tube on ice and incubate for 1 hour. After incubation, transfer it to a benchtop centrifuge and centrifuge at 4°C and 10,000×g for 15 minutes. Remove the supernatant as much as possible. Finally, a gray precipitate the size of rice grains will be visible. (4) The precipitate is suspended in PBS to obtain the extracellular vesicle solution.

[0068] Example 2 This embodiment provides an optimization of the ratio of plasma to collagen using the method of Example 1. The specific experimental steps are as follows: EDTA-anticoagulated peripheral blood was collected from five different individuals (healthy, Han Chinese women aged 20-35 years). Plasma was separated according to the method in Example 1, mixed, and divided into six volumes containing 100, 200, 300, 400, 500, and 600 μL of plasma, respectively, with one replicate for each volume.

[0069] Extracellular vesicles were extracted from samples of different volumes according to the method in Example 1, and then 2 mg of collagen was accurately added to each sample.

[0070] Extracellular vesicles were lysed with an equal volume of radioimmunoprecipitation lysis buffer (RIPA buffer, Beyotime Biotechnology Co., Ltd.), followed by the addition of 1 / 4 volume of 5× reducing Laemmli buffer. The mixture was denatured at 70°C for 10 minutes, cooled, and centrifuged. The supernatant was used for SDS-PAGE electrophoresis analysis, and subsequently electroporated onto a 0.22 μm PVDF membrane for Western blot detection of CD63 protein. The specific steps are as follows: Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk, and then incubated with a specific antibody. The antibody used was anti-CD63 rabbit monoclonal antibody (MCE), incubated overnight at 4°C. The next day, the membrane was incubated with a secondary antibody (Proteintech, HRP-conjugated Goat anti-Rabbit IgG (H+L)) at room temperature for 1-2 hours. After incubation, the membrane was washed three times with TBST buffer, blotted dry with filter paper, and then placed in a prepared ECL chemiluminescence reagent (Shenzhen Maikes Biotechnology Co., Ltd.) for appropriate reaction. The PVDF membrane was then placed in a developing plate with tweezers, and finally exposed and detected using a functional imaging analysis system. The results were analyzed based on the obtained bands.

[0071] Experimental results are as follows Figure 1 As shown, the grayness of the protein band gradually deepens with the increase of plasma volume, indicating that 2 mg of collagen is excessive for 100-500 μL of plasma. Therefore, considering individual differences, 5 mg per milliliter of plasma is chosen to ensure complete capture of plasma EV and save collagenase I and digestion time.

[0072] Example 3 This embodiment provides morphological observation and particle size analysis of extracellular vesicles obtained using the collagen affinity capture method of the present invention. The specific steps are as follows.

[0073] Six EDTA-anticoagulated peripheral blood samples were collected from different individuals (healthy Han Chinese women aged 20-35 years). Extracellular vesicles (EVs) were extracted using the collagen affinity capture method described in Example 1. The EVs were then suspended in 100 μL of PBS, and their particle size distribution and concentration were detected using the ZetaView platform (an NTA platform). Simultaneously, the morphology of the extracellular vesicles extracted in this invention was observed using transmission electron microscopy.

[0074] Figure 2 and Figure 3 The observation results of transmission electron microscopy are images of different fields of view and magnification. Since the processing steps of each sample are the same, this qualitative experiment can prove that the extracellular vesicles obtained by the collagen affinity capture method of the present invention are typical vesicle-like structures.

[0075] Figure 4 The image shows the particle size distribution of one example. The extracellular vesicles obtained by the collagen affinity trapping method of this invention mainly have a particle size between 70-200 nm (the particle size measured by NTA is usually larger than that measured by TEM). The concentrations of the six samples are: 2.7 × 10⁻⁶. 10 1.7×10 10 1.3×10 10 1.6×10 10 3.1×10 10 2.6×10 10 Particle count / mL.

[0076] Example 4 This embodiment provides a comparison between the purification method of the present invention and the extraction of extracellular vesicles by ultracentrifugation, as detailed below.

[0077] Peripheral venous blood samples were collected from healthy adult women (20-30 years of age) with added EDTA anticoagulant. In the assay for marker proteins, samples from different individuals were first mixed to form homogeneous plasma, which was then divided into two equal portions. One portion was extracted using ultracentrifugation (UC), and the other portion was extracted using the method described in Example 1 (collagen co-precipitation, CCP).

[0078] The steps of ultracentrifugation are as follows: (1) Take a plasma sample, centrifuge at 2500×g for 15 minutes at room temperature to remove residual cells and cell debris, and collect the supernatant; (2) The supernatant from step (1) was filtered using a 0.8-micron needle filter (Jinteng, 5mm×0.8μm) to remove platelets and obtain filtrate; (3) Dilute the filtrate from step (2) with PBS solution to a total volume of 10 mL, transfer it to a Beckman ultracentrifuge tube, and then place it in the SW 41 Ti rotor of a Beckman Optima XPN-100 ultracentrifuge. Centrifuge at 120000×g for 2 hours. (4) After centrifugation in step (3), remove the centrifuge tube, gently pour out the supernatant, invert the centrifuge tube onto filter paper, absorb the excess water, and suspend the precipitate in PBS to obtain extracellular vesicles.

[0079] Subsequently, the extracellular vesicles obtained from peripheral blood samples via ultracentrifugation and the collagen affinity capture method of this invention were lysed using the same volume of radioimmunoprecipitation lysis buffer (RIPA solution, Beyotime Biotechnology Co., Ltd.). Then, Western blot was used to detect CD63, CD9, and TSG101 proteins, as detailed below: Protein concentration was measured using the BCA method on equal volumes of lysed extracellular vesicles. Western blot experiments were performed using the same total protein amount. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, and then incubated with specific antibodies. Antibodies used included anti-CD9 mouse monoclonal antibody (MCE), anti-CD63 rabbit monoclonal antibody (MCE), and anti-TSG101 rabbit monoclonal antibody (MCE), incubated overnight at 4°C. The next day, secondary antibody (Proteintech, HRP-conjugated Goat anti-Rabbit IgG (H+L)) was incubated at room temperature for 1-2 hours. After incubation, the membranes were rinsed three times with TBST reagent. The liquid on the PVDF membranes was blotted dry with filter paper, and the membranes were placed in prepared ECL chemiluminescence reagent (Shenzhen Maikes Biotechnology Co., Ltd.) for appropriate reaction. The PVDF membranes were then placed in a developing plate with tweezers, and finally, exposure and detection were performed using a functional imaging analysis system. The results were analyzed based on the obtained bands.

[0080] Western blot results are as follows Figure 5 and Figure 6 As shown, Figure 5 The results, using the same loading volume for both ultracentrifugation (UC) and the collagen affinity capture method (CCP) of this invention, show that the collagen affinity capture method of this invention yields significantly more extracellular vesicles than the ultracentrifugation method. Figure 6 Results of loading the same total protein amount using ultracentrifugation (UC) and the collagen affinity capture method (CCP) of this invention show that the purity of the extracellular vesicles obtained by this invention is higher than that obtained by ultracentrifugation.

[0081] In summary, the extracellular vesicles extracted by the collagen affinity capture method of this invention can simultaneously detect CD63, CD9, and TSG101 proteins. Morphological observation shows that they have typical vesicle-like structures, and their particle size is within the typical range of extracellular vesicle particle size. This conforms to the identification of extracellular vesicles in the MISEV 2023 (Minimal Information for Studies of Extracellular Vesicles 2023) guidelines published by the International Society for Extracellular Vesicles (ISEV), proving that the collagen affinity capture method of this invention can be used for extracellular vesicle extraction.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for purifying extracellular vesicles in plasma, characterized in that, The method includes the following steps: adding collagen to a plasma sample, mixing well, centrifuging, removing the supernatant, adding extracellular vesicle separation solution, mixing well, centrifuging, separating the supernatant, and thus obtaining the plasma extracellular vesicles; the extracellular vesicle separation solution includes at least one of protease solution, acid elution solution, and alkaline elution solution.

2. The method for purifying extracellular vesicles from plasma according to claim 1, characterized in that, The protease includes at least one of collagenase, metalloproteinase and elastase.

3. The method for purifying extracellular vesicles from plasma according to claim 1, characterized in that, The step includes adding an enzyme promoter and a serine protease inhibitor to an extracellular vesicle separation medium containing proteases; the enzyme promoter includes a reagent containing at least one of calcium ions and zinc ions.

4. The method for purifying extracellular vesicles from plasma according to claim 3, characterized in that, The serine protease inhibitors include at least one of benzyl sulfonyl fluoride, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and 4-amino-2-methylbenzenesulfonyl fluoride.

5. The method for purifying extracellular vesicles from plasma according to claim 2, characterized in that, The collagenase includes at least one of collagenase type I, II, III, IV and V.

6. The method for purifying extracellular vesicles of plasma according to claims 1-5, characterized in that, The amount of collagen used is 1-5 mg of collagen per milliliter of plasma sample.

7. The method for purifying extracellular vesicles from plasma according to claim 1, characterized in that, The collagen includes soluble collagen and insoluble collagen; the soluble collagen includes determinated collagen.

8. The method for purifying extracellular vesicles from plasma according to claim 1, characterized in that, The step includes adding at least one of a cross-linking promoter and an activator to the plasma sample and mixing it before adding collagen.

9. The method for purifying extracellular vesicles of plasma according to claim 8, wherein the cross-linking promoter comprises a covalent cross-linking promoter and a non-covalent cross-linking promoter; preferably, the covalent cross-linking promoter comprises at least one of glutaraldehyde, carbodiimide and genipin; preferably, the non-covalent cross-linking promoter comprises a reagent containing at least one of magnesium ions and calcium ions.

10. The application of collagen in the purification of extracellular vesicles from plasma, characterized in that, The collagen is determinated collagen.

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