A method of extraction and engineering of fecal-derived extracellular vesicles

Abstract

The application provides a method for extracting and engineering fecal-derived extracellular vesicles, which comprises the following steps: obtaining pure fecal-derived extracellular vesicles by using ultracentrifugation and density gradient centrifugation; and successfully loading selenium nanoparticles on the surface of the fecal-derived extracellular vesicles in situ by pre-mixing the fecal-derived extracellular vesicles with sodium selenite for incubation and then adding ascorbic acid for in-situ reaction, so as to form engineered SeNPs-FEVs, which can not only retain the cell affinity of the fecal-derived extracellular vesicles, but also endow the fecal-derived extracellular vesicles with excellent antioxidant properties, thereby effectively improving the application effect of the fecal-derived extracellular vesicles in intestinal inflammatory diseases.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a method for the extraction and engineering of fecal extracellular vesicles. Background Technology

[0002] Inflammatory bowel diseases such as inflammatory bowel disease (IBD) are chronic and recurrent diseases with unclear causes. Precision treatment of these diseases faces two core challenges: first, the lack of non-invasive diagnostic tools that can reflect real-time changes in the gut microbiota; and second, the low targeted delivery efficiency and significant systemic toxicity of existing anti-inflammatory drugs. Extracellular vesicles (EVs), as natural nanocarriers, have become an innovative platform for disease diagnosis and treatment in recent years due to their biocompatibility, ability to cross biological barriers, and endogenous signal transduction functions. Fecal vesicles (FEVs), because they originate directly from the gut microenvironment and carry unique molecular markers of host cells and microbiota (such as miRNAs, proteins, and metabolites), provide a breakthrough window for the non-invasive diagnosis of intestinal diseases. However, FEVs have relatively limited functions; when faced with severe oxidative damage, their endogenous antioxidant capacity is insufficient to reverse the pathological process, thus seriously affecting their effectiveness. Summary of the Invention

[0003] This application addresses the technical problem that existing fecal extracellular vesicles (FEVs) have relatively limited functions and insufficient endogenous antioxidant capacity to reverse pathological processes when faced with severe oxidative damage, thus seriously affecting their efficacy. It proposes a method for extracting and engineering fecal extracellular vesicles. Pure FEVs are obtained through ultracentrifugation and density gradient centrifugation. Simultaneously, selenium nanoparticles are loaded in situ onto the surface of FEVs to form engineered SeNPs-FEVs. This method retains the cell affinity of FEVs while endowing them with excellent antioxidant properties, thereby effectively improving their application in inflammatory bowel diseases.

[0004] To achieve the above objectives, this application adopts the following technical solution: A method for extracting and engineering extracellular vesicles derived from feces, comprising the following steps: Extraction of fecal extracellular vesicles Obtain a fecal sample and centrifuge it once to obtain the supernatant; The supernatant was subjected to ultracentrifugation, and the precipitate was resuspended in buffer solution to obtain a first resuspension. After density gradient centrifugation of the primary resuspension, the precipitate is resuspended in buffer solution to obtain a secondary resuspension. After filtering the secondary resuspension, fecal-derived extracellular vesicles were obtained; Engineering of fecal extracellular vesicles The fecal-derived cell vesicles were premixed with sodium selenite solution, and then ascorbic acid was added to carry out an in-situ reduction reaction to obtain a reaction mixture. The reaction mixture was subjected to a second centrifugation to obtain fecal-derived extracellular vesicles loaded with selenium nanoparticles.

[0005] Furthermore, the centrifugation process is as follows: the fecal sample is added to a buffer solution and mixed evenly, then centrifuged at 500×g for 3-7 minutes at 4°C to remove food residue and fine particles that have settled to the bottom; then centrifuged at 2000×g for 8-12 minutes to remove cell debris and intestinal bacteria that have settled to the bottom; finally, centrifuged at 14000×g for 25-35 minutes to remove large extracellular vesicles that have settled to the bottom.

[0006] Furthermore, the ultracentrifugation process is carried out at a temperature of 4°C, a centrifugal force of 120,000 × g, and a time of 1.5 to 3 hours.

[0007] Furthermore, the density gradient centrifugation process is as follows: prepare sucrose solutions with concentrations of 10%, 20%, 40%, and 50%, and add the four concentrations of sucrose solutions to centrifuge tubes in ascending order of concentration. Then, add the obtained resuspension to the top layer of the corresponding centrifuge tube. Centrifuge at 4°C and 120,000 × g for 6.5 to 7.5 hours to obtain a centrifuged solution. Divide the centrifuged solution into 10 equal parts by volume from top to bottom. Take the 6th middle part of the solution, add buffer solution, and continue centrifuging at 4°C and 120,000 × g for 1.5 to 2.5 hours. Then, collect the precipitate.

[0008] Furthermore, the filtration process uses a filter pore size of 0.22 μm.

[0009] Furthermore, the concentration of the sodium selenite solution is 0.3 mM.

[0010] Furthermore, the premixing process involves placing the mixture of fecal-derived cell vesicles and the sodium selenite solution in a constant-temperature shaker and incubating it for 1.5 to 3 hours. The temperature of the constant-temperature shaker is 35 to 40°C, and the rotation speed is 160 to 200 rpm.

[0011] Furthermore, the concentration of the ascorbic acid is 12 mM.

[0012] Furthermore, the in-situ reduction reaction process involves adding the ascorbic acid and then placing it at 35-40°C in the dark for 1.5-3 hours.

[0013] Furthermore, the secondary centrifugation process involves centrifuging at 120,000 × g for 1.5 to 3 hours at 4°C.

[0014] The beneficial effects of this application are: In the centrifugation process of this application, centrifugation is carried out by gradually increasing the centrifugal force, which can remove the settled food residue, cells, intestinal bacteria and large-diameter extracellular vesicles step by step. At the same time, density gradient centrifugation is used in the subsequent process to ensure that pure fecal extracellular vesicles are obtained, which lays a good foundation for the subsequent engineering of fecal extracellular vesicles.

[0015] This application involves in-situ loading of selenium nanoparticles onto the surface of extracellular vesicles derived from fecal microbiota to form engineered SeNPs-FEVs. This process retains the cell affinity of fecal microbiota-derived extracellular vesicles while endowing them with excellent antioxidant properties, thereby effectively improving their application efficacy in intestinal inflammatory diseases. Specifically: The surface of extracellular vesicles derived from fecal microorganisms extracted from fecal samples is not smooth lipid, but rather rich in transmembrane proteins and polysaccharides, carrying numerous pockets of locally positively charged amino residues. In this application, sodium selenite is first added to the system. Selenite anions are adsorbed and anchored to the protein macromolecular network on the surface of the fecal microorganism-derived extracellular vesicles through strong electrostatic attraction. Then, ascorbic acid is added, causing tetravalent selenium ions to be reduced in situ to elemental zero-valent selenium on the vesicle surface. The amino and thiol groups on the surface of the fecal microorganism-derived extracellular vesicles contain abundant lone pairs of electrons, which immediately undergo coordination complexation reactions with the newly formed highly active nano-selenium surface, forming strong coordination bonds. This process not only overcomes the tendency of selenium nanoparticles to aggregate during mixing but also effectively enhances the "chemical antioxidant and biological tissue repair" effects of fecal microorganism-derived extracellular vesicles in the treatment of colitis. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Transmission electron micrographs of fecal extracellular vesicles obtained in Example 1 and Comparative Example 1 of this application; Figure 2 This is a tracking analysis diagram of fecal extracellular vesicles obtained in Example 1 and Comparative Example 1 of this application; Figure 3Transmission electron microscopy image of fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 of this application; Figure 4 This is a comparison diagram of the particle size and zeta potential of fecal-derived extracellular vesicles and fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 of this application. Figure 5 This is an elemental mapping analysis diagram of fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 of this application; Figure 6 X-ray photoelectron spectroscopy of fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 of this application; Figure 7 This figure shows the results of an in vivo mouse experiment on fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 of this application. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In the description of this application, the experimental methods, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, are all commercially available.

[0019] The following disclosure provides many different implementations or examples for carrying out this application. To simplify the disclosure of this application, specific examples of components and arrangements are described below. Of course, these are merely examples and are not intended to limit the scope of this application. Those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0020] Example 1

[0021] This application provides a method for extracting and engineering extracellular vesicles from feces, comprising the following steps: Extraction of fecal extracellular vesicles Weigh the collected fresh wild-type C57BL / 6 mouse feces, add 40g of mouse feces to 400ml of 1×PBS solution, dispense into 6 50mL centrifuge tubes, place the centrifuge tubes on a shaker and shake well. The temperature during the shaking process is 4℃, the speed is 200rpm, and the time is 2h.

[0022] After shaking, centrifuge each of the six centrifuge tubes at 4°C and 500×g for 5 minutes. Then, collect the supernatant and discard the precipitate at the bottom of the centrifuge tube to remove food residue and fine particles. Next, centrifuge at 2000×g for 10 minutes and collect the supernatant, discarding the precipitate at the bottom of the centrifuge tube to remove cell debris and intestinal bacteria. Finally, centrifuge at 14000×g for 30 minutes and collect the supernatant, discarding the precipitate at the bottom of the centrifuge tube to remove large extracellular vesicles.

[0023] The six supernatants obtained after centrifugation were added one-to-one to six 40mL ultracentrifuge tubes. The volume of each ultracentrifuge tube was brought up to 40mL with 1×PBS solution. The ultracentrifuge tubes were weighed on an analytical balance, balanced, and placed in the rotor of an ultracentrifuge. Centrifuged at 120,000×g for 12 minutes at 4°C. The supernatant was discarded, but the bottom precipitate was retained. The ultracentrifuge tubes were then inverted on absorbent paper for 5 minutes to allow the liquid to drain completely.

[0024] The precipitate obtained after ultracentrifugation was resuspended in 18 mL of 1×PBS solution and divided equally into six 40 mL ultracentrifuge tubes. The volume of each tube was brought up to 40 mL with 1×PBS solution. The tubes were weighed on an analytical balance, balanced, and placed in the rotor of an ultracentrifuge. Centrifuged at 120,000 × g for 12 minutes at 4°C. The supernatant was discarded, and the bottom precipitate was retained. The tubes were then inverted on absorbent paper for 5 minutes to allow the liquid to drain completely. The precipitate obtained after the second ultracentrifugation was resuspended in 18 mL of 1×PBS solution for later use.

[0025] Prepare 20 mL each of 10%, 20%, 40%, and 50% sucrose solutions. Add 3 mL of each of these solutions sequentially to six 15 mL ultracentrifuge tubes. Then, add 3 mL of the solution resuspended after two ultracentrifugations to the top layer of each tube. Weigh all six tubes on an analytical balance, balance them, and place them in the rotor of an ultracentrifuge. Centrifuge at 120,000 × g for 7 hours at 4°C. Divide each tube into 10 equal portions (1.5 mL each). Carefully pipette each portion using a 1 mL pipette, reserving the sixth portion.

[0026] The solution from the sixth portion of each of the six obtained ultracentrifuge tubes was added to six corresponding 40mL ultracentrifuge tubes. Then, 1×PBS solution was added to each ultracentrifuge tube to bring the total volume to 40mL. The ultracentrifuge tubes were weighed on an analytical balance, balanced, and placed in the rotor of an ultracentrifuge. Centrifuged at 120,000×g for 2 hours at 4°C. The supernatant was discarded, and the bottom precipitate was retained. The ultracentrifuge tubes were inverted on absorbent paper for 5 minutes to allow the liquid to flow out completely and obtain the precipitate. The precipitate was resuspended in 1mL of 1×PBS solution. The resuspended solution was filtered through a 0.22μm filter in a clean bench to obtain fecal extracellular vesicles, which were then transferred to 1.5mL sterile centrifuge tubes.

[0027] Engineering of fecal extracellular vesicles The extracted fecal extracellular vesicles were mixed with a 0.3 mM sodium selenite solution and incubated for 2 hours in a constant-temperature shaker at 37°C and 180 rpm. After incubation, ascorbic acid to a final concentration of 12 mM was added, and the mixture was allowed to stand at 37°C in the dark for 2 hours to carry out an in-situ reduction reaction to obtain a reaction mixture. The reaction mixture was centrifuged at 120,000 × g for 2 hours at 4°C. The supernatant was discarded, and the bottom precipitate was collected to obtain fecal extracellular vesicles loaded with selenium nanoparticles.

[0028] Comparative Example 1 Extraction of extracellular vesicles from feces, comprising the following steps: Weigh the collected fresh wild-type C57BL / 6 mouse feces, add 40g of mouse feces to 400ml of 1×PBS solution, dispense into 6 50mL centrifuge tubes, place the centrifuge tubes on a shaker and shake well. The temperature during the shaking process is 4℃, the speed is 200rpm, and the time is 2h.

[0029] After shaking, centrifuge each of the six centrifuge tubes at 4°C and 500×g for 5 minutes. Then, collect the supernatant and discard the precipitate at the bottom of the centrifuge tube to remove food residue and fine particles. Next, centrifuge at 2000×g for 10 minutes and collect the supernatant, discarding the precipitate at the bottom of the centrifuge tube to remove cell debris and intestinal bacteria. Finally, centrifuge at 14000×g for 30 minutes and collect the supernatant, discarding the precipitate at the bottom of the centrifuge tube to remove large extracellular vesicles.

[0030] The six supernatants obtained after centrifugation were added one-to-one to six 40mL ultracentrifuge tubes. The volume of each ultracentrifuge tube was brought up to 40mL with 1×PBS solution. The ultracentrifuge tubes were weighed on an analytical balance, balanced, and placed in the rotor of an ultracentrifuge. Centrifuged at 120,000×g for 12 minutes at 4°C. The supernatant was discarded, but the bottom precipitate was retained. The ultracentrifuge tubes were then inverted on absorbent paper for 5 minutes to allow the liquid to drain completely.

[0031] The precipitate obtained after ultracentrifugation was resuspended in 18 mL of 1×PBS solution and then evenly divided into six 40 mL ultracentrifuge tubes. The volume of each tube was brought up to 40 mL with 1×PBS solution. The tubes were weighed on an analytical balance, balanced, and placed in the rotor of an ultracentrifuge. Centrifuged at 120,000 × g for 12 minutes at 4°C. The supernatant was discarded, and the bottom precipitate was retained. The tubes were then inverted on absorbent paper for 5 minutes to allow the liquid to drain completely and obtain the precipitate. The precipitate was resuspended in 1 mL of 1×PBS solution and filtered through a 0.22 μm filter in a clean bench to obtain fecal extracellular vesicles, which were then transferred to 1.5 mL sterile centrifuge tubes.

[0032] Performance testing test 1 The protein concentration of fecal extracellular vesicles obtained in Example 1 and Comparative Example 1 was detected using the biuret method. Simultaneously, the morphology of the fecal extracellular vesicles in both groups was examined using transmission electron microscopy, and their particle size distribution was determined using nanoparticle tracking analysis. The protein concentration results are shown in Table 1, and the electron microscopy results are shown in... Figure 1 As shown, the particle size distribution is as follows: Figure 2 As shown.

[0033] Table 1. Results of protein concentration detection in fecal extracellular vesicles obtained in Example 1 and Comparative Example 1.

[0034] As can be seen from the test results in Table 1, the protein content of the fecal extracellular vesicles obtained in Comparative Example 1 is about 6 times that of Example 1. However, this does not mean that the method in Comparative Example 1 extracts more fecal extracellular vesicles, because the protein in the fecal extracellular vesicles extracted in Comparative Example 1 may come from lipoproteins and other protein-containing impurities.

[0035] exist Figure 1 The two images on the left in the middle are the detection results of Example 1, and the two images on the right are the detection results of Comparative Example 1. Figure 1The test results clearly show that the extracellular vesicles derived from feces in Comparative Example 1 contain a large number of lipoprotein particles and fine fibrous impurities in the background under transmission electron microscopy, while the extracellular vesicles derived from feces in Example 1 have a cleaner background under transmission electron microscopy, and the above-mentioned impurities have been basically removed.

[0036] exist Figure 2 The left side of the image shows the detection results of Example 1, and the right side shows the detection results of Comparative Example 1. Figure 2 It can be seen that the peak particle size distribution of both Example 1 and Comparative Example 1 is around 120 nm. Although the method steps in Example 1 are more numerous, they do not affect the particle size of the obtained fecal extracellular vesicles. Therefore, the extraction method of the present invention can obtain purer fecal extracellular vesicles.

[0037] Performance testing test 2 The morphology of fecal extracellular vesicles loaded with selenium nanoparticles obtained in Example 1 was examined using transmission electron microscopy. The results are as follows: Figure 3 As shown, by Figure 3 It can be seen that it has typical "coffee saucer-like" extracellular vesicles, and its surface is loaded with dense black granules.

[0038] Zeta potentials were measured on fecal extracellular vesicles extracted in Example 1 and fecal extracellular vesicles loaded with selenium nanoparticles. The results are shown below. Figure 4 As shown, by Figure 4 It can be seen that after selenium nanoparticles were synthesized in situ from extracellular vesicles derived from feces, the particle size increased from 118.5 nm to 165.4 nm, and the zeta potential increased from -31.97 mV to -36.48 mV.

[0039] Elemental mapping analysis was performed on the fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1. The results are as follows: Figure 5 As shown in the figure, selenium nanoparticles are uniformly bound to the membrane of extracellular vesicles derived from feces.

[0040] X-ray photoelectron spectroscopy analysis was performed on the fecal-derived extracellular vesicles loaded with selenium nanoparticles obtained in Example 1. The detection results are as follows: Figure 6 As shown in the figure, selenium nanoparticles are in a zero-valent state on the membrane surface of fecal extracellular vesicles, and the proportion of selenium nanoparticles in all elements on the surface of fecal extracellular vesicles is about 0.74%.

[0041] From the above Figures 3 to 6 The test results clearly show that the engineered method of this application can successfully load selenium nanoparticles in situ onto the surface of fecal extracellular vesicles to construct engineered SeNPs-FEVs.

[0042] Performance testing test 3 The fecal-derived extracellular vesicles obtained in Example 1 and the fecal-derived extracellular vesicles loaded with selenium nanoparticles were used in a DSS-induced acute mouse colitis model to evaluate the in vivo intervention effects of FEVs and SeNPs-FEVs. The experimental results are as follows: Figure 7 As shown. In Figure 7 In the figure, Part A shows the timeline of animal modeling and drug administration. Part B shows the results of general physical examination of mice. This part shows that the DSS group mice experienced a continuous and significant decrease in body weight from the onset of the model, and these mice also experienced severe diarrhea and bloody stools during the experiment. Part C shows the Disease Activity Index (DAI) results of the mice. This part shows that after the mice were given DSS in their drinking water, the DAI score continuously increased, reaching a peak on days 8-9. After stopping DSS on day 7 and administering drug intervention, the symptoms of all groups of mice were controlled to varying degrees within 1-2 days. Among them, the SeNPs-FEVs group showed the most rapid recovery and the most significant decrease in DAI score, approaching healthy levels by day 14. Figure 7 Part D shows the gross morphology and measurement results of mice in each group. In the DSS group, the colon length of mice atrophied (shortened to approximately 5.5 cm); while the colon length of mice in the SeNPs-FEVs group was largely salvaged (compared to the DSS group, P < 0.01), exceeding 6.5 cm, almost returning to the control group level. Figure 7 In the middle section (E), there are HE pathological staining images of colon tissue from each group of mice. The DSS group showed severe damage to the colonic mucosa, with extensive destruction or even disappearance of crypt structures, accompanied by a large number of inflammatory cells infiltrating the mucosa and submucosa. In contrast, the SeNPs-FEVs group maintained the integrity of its intestinal crypt structures, significantly restored the number of goblet cells, and showed a significant reduction in inflammatory cell infiltration. These results collectively confirm that SeNPs-FEVs effectively alleviates acute colitis in mice compared to FEVs.

[0043] The above provides a detailed description of a method for extracting and engineering extracellular vesicles from feces, as provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the technical solutions and core ideas of this application. Those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for extracting and engineering extracellular vesicles from feces, characterized in that, Includes the following steps: Extraction of fecal extracellular vesicles Obtain a fecal sample and centrifuge it once to obtain the supernatant; The supernatant was subjected to ultracentrifugation, and the precipitate was resuspended in buffer solution to obtain a first resuspension. After density gradient centrifugation of the primary resuspension, the precipitate is resuspended in buffer solution to obtain a secondary resuspension. After filtering the secondary resuspension, fecal-derived extracellular vesicles were obtained; Engineering of fecal extracellular vesicles The fecal-derived cell vesicles were premixed with sodium selenite solution, and then ascorbic acid was added to carry out an in-situ reduction reaction to obtain a reaction mixture. The reaction mixture was subjected to a second centrifugation to obtain fecal-derived extracellular vesicles loaded with selenium nanoparticles.

2. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The centrifugation process is as follows: the fecal sample is added to a buffer solution and mixed evenly, and then centrifuged at 500×g for 3-7 minutes at 4°C to remove food residue and fine particles that have settled to the bottom; Centrifuge at 2000×g for 8-12 minutes to remove cell debris and intestinal bacteria that have settled to the bottom; finally, centrifuge at 14000×g for 25-35 minutes to remove large extracellular vesicles that have settled to the bottom.

3. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The ultracentrifugation process is carried out at a temperature of 4°C, a centrifugal force of 120,000 × g, and a time of 1.5 to 3 hours.

4. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The density gradient centrifugation process is as follows: Prepare sucrose solutions with concentrations of 10%, 20%, 40%, and 50%, and add the four concentrations of sucrose solutions to centrifuge tubes in descending or ascending order of concentration. Then, add the obtained resuspension to the top layer of the corresponding centrifuge tube. Centrifuge at 4°C and 120,000 × g for 6.5 to 7.5 hours to obtain a centrifuged solution. Divide the centrifuged solution into 10 equal parts by volume from top to bottom. Take the 6th middle part of the solution, add buffer solution, and continue centrifuging at 4°C and 120,000 × g for 1.5 to 2.5 hours. Then, collect the precipitate.

5. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The filtration process uses a filter pore size of 0.22 μm.

6. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The concentration of the sodium selenite solution is 0.3 mM.

7. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The premixing process involves placing the mixture of fecal-derived cell vesicles and the sodium selenite solution in a constant-temperature shaker and incubating it for 1.5 to 3 hours. The temperature of the constant-temperature shaker is 35 to 40°C, and the rotation speed is 160 to 200 rpm.

8. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The concentration of ascorbic acid is 12 mM.

9. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The in-situ reduction reaction process involves adding the ascorbic acid and then placing it at 35-40°C in the dark for 1.5-3 hours.

10. The method for extracting and engineering extracellular vesicles from feces as described in claim 1, characterized in that: The secondary centrifugation process involves centrifuging at 120,000 × g for 1.5 to 3 hours at 4°C.

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