CN116855439A

CN116855439A - Extraction method for improving stability of plant exosomes

Info

Publication number: CN116855439A
Application number: CN202311066023.8A
Authority: CN
Inventors: 王卫中; 张鹏飞; 连昕; 谭雅; 王子霆; 刘琳娜; 张晓松
Original assignee: Shenzhen Huajian Biotechnology Co ltd
Current assignee: Shenzhen Huajian Biotechnology Co ltd
Priority date: 2023-08-23
Filing date: 2023-08-23
Publication date: 2023-10-10
Anticipated expiration: 2043-08-23
Also published as: CN116855439B

Abstract

The invention provides an extraction method for improving stability of plant exosomes, and belongs to the technical field of plant exosomes. The extraction method of the invention comprises the following steps: (1) Preparing a crude solution of plant exosomes by adopting a polymer precipitation method; (2) Sequentially carrying out fatty acid modification and polyethylene glycol modification on the crude solution of the plant exosomes to obtain a plant exosome crosslinking system; (3) And adding a trehalose solution into the plant exosome crosslinking system, mixing, centrifuging and re-suspending to obtain the plant exosome with high stability. The invention combines a polymer precipitation method, a fatty acid modification method and a polyethylene glycol modification method, improves the yield of plant exosomes, increases the stability, the feasibility and the bioactivity of the plant exosomes, and reduces the degradation rate of the plant exosomes in the processes of extraction, preservation and the like.

Description

Extraction method for improving stability of plant exosomes

Technical Field

The invention relates to the technical field of plant exosomes, in particular to an extraction method for improving stability of plant exosomes.

Background

Exosomes are extracellular vesicles comprising a series of bioactive molecules, such as proteins, nucleic acids, lipids, etc., with a wide range of biological functions and application values. Plant exosome-like nanovesicles (PELNVs) are multi-vesicles derived from plant eukaryotic cells, and are released to the outside of the cells through fusion of the latter with plasma membranes, and are available for mass production because of their wide resources due to their plant origin. Meanwhile, PELNVs derived from medicinal plants are often rich in various bioactive lipid, protein, RNA and other components, are natural nano preparations, and have obvious regulation and control effects in aspects of tumor, immunoregulation, intestinal diseases, regenerative medicine and the like. In addition, PELNVs have the shape and the characteristics of nano-carriers, so the PELNVs can be used as low-toxicity nano-carriers to realize the delivery of exogenous drug molecules, and compared with mammal sources and artificially synthesized nano-vesicles, the drug delivery nano-platform based on the PELNVs has obvious advantages in the aspects of biocompatibility, stability, in-vivo distribution, half-life prolongation, cell internalization and the like. In addition, the PELNVs also have the advantages of small volume, strong tissue penetrability and the like, can maintain good physical and chemical stability under different pH values and temperatures, and all the characteristics enable the PELNVs to be ideal carrier selection in drug delivery such as transdermal delivery, targeted drug delivery, gene delivery and the like, and have wide application prospects.

However, plant exosomes are susceptible to environmental, physiological and chemical factors during collection, purification, storage and transport, resulting in their reduced stability, affecting their progress in research and use. How to improve the stability of the plant exosomes has important significance for improving the yield of the plant exosomes and developing the application of the plant exosomes.

Disclosure of Invention

The invention aims to provide an extraction method for improving stability of plant exosomes. The invention combines a polymer precipitation method, a fatty acid modification method and a polyethylene glycol modification method, improves the yield of plant exosomes, increases the stability, the feasibility and the bioactivity of the plant exosomes, and reduces the degradation rate of the plant exosomes in the processes of extraction, preservation and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an extraction method for improving stability of plant exosomes, comprising the following steps:

(1) Preparing a crude solution of plant exosomes by adopting a polymer precipitation method;

(2) Sequentially carrying out fatty acid modification and polyethylene glycol modification on the crude solution of the plant exosomes to obtain a plant exosome crosslinking system;

(3) And adding a trehalose solution into the plant exosome crosslinking system, mixing, centrifuging and re-suspending to obtain the plant exosome with high stability.

Preferably, the preparation method of the plant exosome crude solution in the step (1) comprises the following steps:

a. adding PBS solution into plant tissues, and crushing to obtain plant extract;

b. centrifuging the plant extract juice for 10-20 min at 800-1200 g, and taking supernatant;

c. centrifuging the supernatant obtained in the step b for 25-40 min at 4000-6000 g, and taking the supernatant;

d. c, mixing the supernatant obtained in the step c with polyethylene glycol-800 to ensure that the final concentration of the polyethylene glycol-800 is 5-15%, centrifuging, and taking a precipitate;

e. dissolving the precipitate in PBS solution, adding polyethylene glycol-800 to make the final concentration of the polyethylene glycol-800 be 2-5%, mixing uniformly, centrifuging, and re-suspending to obtain the plant exosome crude solution.

Preferably, in the step a, the volume ratio of the plant tissue to the PBS solution is (0.5 to 2): (0.5-2).

Preferably, in the step d, the original concentration of the polyethylene glycol-800 is 50-60%; the mixing temperature is 0-4 ℃, and the mixing time is 12-16 h.

Preferably, in the step e, the volume of the PBS solution is 2-8 mL; the original concentration of the polyethylene glycol-800 is 50-60%; the resuspended solvent is particle-free PBS buffer, and the volume of the particle-free PBS buffer is 50-500 mu L.

Preferably, in the step (2), the method for modifying fatty acid comprises mixing fatty acid carboxylic acid with the crude plant exosome solution and PBS solution to obtain fatty acid modified plant exosome;

the concentration of the fatty acid carboxylic acid is 0.1-10 mM; the volume ratio of the plant exosome crude solution to the fatty acid carboxylic acid is (8-12): 1, a step of; the addition amount of the PBS solution is 0.5-2 times of the total volume of the plant exosome crude solution and the fatty acid carboxylic acid; the temperature of the mixing is 20-25 ℃ and the time is 6-12 h.

Preferably, in the step (2), the polyethylene glycol modification method is to mix polyethylene glycol-800 with the plant exosome modified by fatty acid to make the final concentration of polyethylene glycol-800 be 1.3-2.5%, so as to obtain a plant exosome crosslinking system;

the original concentration of the polyethylene glycol-800 is 30-50%, the temperature of the mixing is 20-25 ℃ and the time is 1-3 h.

Preferably, in the step (4), the original concentration of the trehalose solution is 20-30%, and the final concentration of the trehalose solution is 10-15%; the temperature of the centrifugation is 2-6 ℃, the rotating speed is 120000 ～ 150000g, the time is 0.5-1.5 h, and the times are 1-3 times.

Preferably, the resuspended solvent is trehalose solution with the concentration of 20-30%, and the concentration of the plant exosomes with high stability is (0.5-1) mg/mL.

The invention provides an extraction method for improving stability of plant exosomes. The invention uses polymer precipitation method to extract crude solution of plant exosome, improves yield of plant exosome, utilizes fatty acid to modify plant exosome, increases membrane stability and bioactivity of plant exosome, utilizes polyethylene glycol to crosslink plant exosome, and further increases stability and feasibility of plant exosome. The method solves the problems of small extraction amount and poor preservation of the plant exosomes, improves the yield of the plant exosomes, and largely avoids the problem of easy degradation and inactivation in the processes of extraction, preservation and the like.

Drawings

FIG. 1 is a BCA protein standard curve of experimental example 1.

FIG. 2 is a graph showing the results of measurement of the total protein concentration of each group of exosomes measured in experimental example 1.

FIG. 3 is a BCA protein standard curve of experimental example 2.

FIG. 4 is a chart showing the WB development by gel electrophoresis of CD63 expression levels of exosomes of each group stored at 4℃in experimental example 2.

FIG. 5 is a chart showing the WB development by gel electrophoresis of the expression level of CD63 in each group of exosomes stored at 37℃in experimental example 2.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

In the embodiment, ginger is taken as a raw material, and a ginger exosome with high stability is prepared, and the specific preparation process is as follows:

(1) Cleaning fresh rhizoma Zingiberis recens with ultrapure water, drying, cutting into small pieces, squeezing with a food processor, adding equal volume of PBS solution, and squeezing for 3min in cold material processing mode to obtain rhizoma Zingiberis recens juice; filtering the squeezed rhizoma Zingiberis recens juice with a filter gauze and a funnel, and removing residue to obtain rhizoma Zingiberis recens extract;

balancing rhizoma Zingiberis recens extract, centrifuging at 1000g for 15min, removing large granule impurities, collecting supernatant, and discarding bottom precipitate;

balancing the supernatant, centrifuging at 5000g for 30min, removing broken cell fragments and large apoptotic bodies in the ginger extract, retaining the supernatant, and discarding the bottom precipitate;

mixing the supernatant of the previous step with a PEG-800 solution with an initial concentration of 55% to obtain a final concentration of PEG-800 of 10%, and incubating overnight at 4 ℃ after fully mixing; after overnight, centrifuge at 50000g for 1h at 4 ℃, pour out the conical tube after centrifugation is completed, and drain for five minutes, tap from time to remove excess PEG to obtain a precipitate;

diluting the precipitate in 5mL of PBS solution, and adding 55% of PEG-800 solution to reduce the final concentration of PEG-800 to 5%; the solution was centrifuged at 50000g at 4℃for 1 hour to obtain a precipitate, and the obtained precipitate was suspended in 200. Mu.L of a particle-free PBS buffer and shaken at room temperature for 20 minutes to obtain a crude solution of ginger exosomes.

(2) Mixing the solution obtained in the step 1 with fatty acid carboxylic acid (5 mM) according to a volume ratio of 10:1 to obtain a mixture; adding an equal volume of PBS solution as a buffer agent into the mixture, incubating for 10 hours at room temperature, modifying fatty acid on the surface of the ginger exosome, mixing with PEG-800 solution (with the concentration of 35%), and incubating for 2 hours at room temperature to obtain the ginger exosome crosslinking system.

(3) Adding a trehalose solution with the original concentration of 25% into the crosslinking system to ensure that the trehalose concentration in the system is 15%, thereby obtaining a ginger exosome ultracentrifugation system; the ginger exosome ultracentrifugation system was centrifuged at 150000g at 4℃for 1h, 2 times, the supernatant was discarded, and the precipitated fraction was the isolated ginger exosome. The mass of the obtained precipitate was weighed and resuspended with 25% trehalose solution to give a high stability ginger exosome at a concentration of 1 mg/mL.

Example 2

In the embodiment, the salvia miltiorrhiza bunge is taken as a raw material, and the salvia miltiorrhiza bunge exosome with high stability is prepared by the following specific preparation process:

(1) Cleaning fresh Saviae Miltiorrhizae radix with ultrapure water, drying, cutting into small pieces, squeezing with a processor, adding equal volume of PBS solution, and squeezing for 3min to obtain Saviae Miltiorrhizae radix juice; filtering the squeezed radix Salviae Miltiorrhizae juice with gauze and funnel, and removing residue to obtain radix Salviae Miltiorrhizae extract;

balancing Saviae Miltiorrhizae radix extract, centrifuging at 1000g for 15min, removing large granule impurities, collecting supernatant, and discarding bottom precipitate;

balancing the supernatant, centrifuging at 5000g for 30min, removing broken cell fragments and large apoptotic bodies in the Saviae Miltiorrhizae radix extract, retaining supernatant, and discarding bottom precipitate;

diluting the precipitate in 5mL of PBS solution, and adding 55% of PEG-800 solution to reduce the final concentration of PEG-800 to 5%; the mixture was centrifuged at 50000g at 4℃for 1 hour to obtain a precipitate, and the obtained precipitate was suspended in 200. Mu.L of a particle-free PBS buffer and shaken at room temperature for 20 minutes to obtain a crude extract solution of the Salviae Miltiorrhizae radix.

(2) Mixing the solution obtained in the step 1 with fatty acid carboxylic acid (5 mM) according to a volume ratio of 10:1 to obtain a mixture; adding PBS solution with equal volume as buffer agent into the mixture, incubating at room temperature for 10h, modifying fatty acid on the surface of the radix Salviae Miltiorrhizae exosome, mixing with PEG-800 solution (35%) to make the final concentration of PEG be 2%, and incubating at room temperature for 2h to obtain radix Salviae Miltiorrhizae exosome crosslinking system.

(3) Adding 25% trehalose solution into the crosslinking system to make the trehalose concentration in the system 15% to obtain radix Salviae Miltiorrhizae exosome ultracentrifugation system; centrifuging the ultra-centrifugation system of the red sage root exosomes at 150000g and 4 ℃ for 1h, centrifuging for 2 times, discarding the supernatant, and obtaining the precipitate as the separated red sage root exosomes. The mass of the obtained precipitate is weighed, and the suspension is re-suspended by adopting a trehalose solution with the concentration of 25 percent, so that the high-stability salvia miltiorrhiza exosome with the concentration of 1mg/mL is obtained.

Example 3

In the embodiment, the grape is taken as a raw material, and the grape exosome with high stability is prepared by the following specific preparation process:

(1) Cleaning fresh grape with ultrapure water, drying, cutting into small pieces, adding into a cooking machine, squeezing, adding equal volume of PBS solution, and squeezing for 3min in cold material treatment mode to obtain grape juice; filtering the squeezed grape juice with a filter gauze and a funnel, and removing filter residues to obtain grape extract;

balancing grape extract, centrifuging at 1000g for 15min, removing large granule impurities, collecting supernatant, and discarding bottom precipitate;

balancing the supernatant in the last step, centrifuging for 30min at 5000g, removing broken cell fragments and large apoptotic bodies in the grape extract, retaining the supernatant, and discarding the bottom sediment;

diluting the precipitate in 5mL of PBS solution, and adding 55% of PEG-800 solution to reduce the final concentration of PEG-800 to 5%; the precipitate was obtained by centrifugation at 50000g at 4℃for 1 hour, and the obtained precipitate was suspended in 200. Mu.L of a particle-free PBS buffer and shaken at room temperature for 20 minutes to obtain a crude solution of grape exosomes.

(2) Mixing the solution obtained in the step 1 with fatty acid carboxylic acid (5 mM) according to a volume ratio of 10:1 to obtain a mixture; adding an equal volume of PBS solution as a buffer agent into the mixture, incubating for 10 hours at room temperature, modifying fatty acid on the surface of the grape exosome, mixing the fatty acid with PEG-800 solution (with the concentration of 35%), and incubating for 2 hours at room temperature to obtain the grape exosome crosslinking system.

(3) Adding a trehalose solution with the original concentration of 25% into the crosslinking system to ensure that the trehalose concentration in the system is 15%, thereby obtaining a grape exosome ultracentrifugation system; and (3) centrifuging the grape exosome ultracentrifugation system for 1h at the temperature of 4 ℃ at 150000g, centrifuging for 2 times, discarding the supernatant, and obtaining a sediment part which is the separated grape exosome. The mass of the obtained precipitate was weighed and resuspended with a 25% trehalose solution to give a highly stable grape exosome at a concentration of 1 mg/mL.

Comparative example 1

This comparative example is different from example 1 in that only the treatment of step (1) was performed to obtain a crude ginger exosome solution.

Comparative example 2

This comparative example is different from example 2 in that only the treatment of step (1) was performed to obtain a crude extract solution of red sage root.

Comparative example 3

This comparative example is different from example 3 in that only the treatment of step (1) was performed to obtain a crude solution of grape exosomes.

Experimental example 1

In this experimental example, the BCA method was used to measure the total protein concentrations of exosomes in examples 1 to 3 and comparative examples 1 to 3, respectively. The specific process is as follows:

(1) The total protein concentration test was performed using a BCA protein concentration measurement kit (purchased from the company Hirscht Biotechnology, inc., lot:20201ES 76), kit components: KGPBCA (250 assay ELISA plate/50 assay 1ml cuvette), protein standard solution (0.5. Mu.g/. Mu.l) 5ml, BCA reagent A25ml×2, BCA reagent B1ml

(2) Drawing a standard curve: an ELISA plate was used to prepare a reaction system according to the types and contents of the reagents shown in Table 1.

TABLE 1 types and contents of reagents for each well

Hole number	0	1	2	3	4	5	6	7
									Protein standard solution (μl)	0	1	2	4	8	12	16	20
Deionized water (mul)	20	19	18	16	12	8	4	0
									Content of the corresponding protein (. Mu.g)	0	0.5	1	2	4	6	8	10

Adding BCA working solution required by a sample prepared by adding 1 volume of BCA reagent B into 50 volumes of BCA reagent A, fully and uniformly mixing, adding 200 μl of BCA working solution into each hole, placing an ELISA plate on a shaker for 30s, placing at 37 ℃ for 30 minutes, and then colorimetrically measuring at 562 nm. A standard curve is drawn with the protein content (. Mu.g) on the abscissa and the absorbance on the ordinate, as shown in FIG. 1.

(3) And (3) testing the total protein concentration of a sample to be tested: 10 μl of exosomes of examples 1 to 3 and comparative examples 1 to 3 were taken as samples to be measured, the control group was comparative examples 1 to 3, and the experimental group was examples 1 to 3. Diluting a sample to be detected, enabling the total volume of a sample diluent to be 20 mu l, adding 200 mu l of BCA working solution, fully and uniformly mixing, standing at 37 ℃ for 30 minutes, taking a standard curve number 0 tube as a reference, colorizing at 562nm wavelength, recording a light absorption value, obtaining the corresponding protein content (mu g) on the standard curve according to the light absorption value of the detected sample, dividing the protein content by the total volume of the sample diluent (20 mu l), multiplying the sample dilution by the sample actual concentration, and determining the total protein concentration of each group of exosomes as shown in figure 2.

(4) Analysis of results: as can be seen from fig. 2, the total protein concentration measured for the stably modified ginger, red sage and grape exosomes was higher than for the unmodified ginger, red sage and grape exosomes. That is, after the modification of the present invention is performed on the plant exosomes in the process of extracting the plant exosomes, the extraction amount of the plant exosomes is higher than that of the extraction method without modification of the plant exosomes.

Experimental example 2

In order to test the stability difference of the plant exosomes after modification and without modification, the CD63 protein is selected as a marker of the plant exosome immunoblotting, and the protein marker contents of the exosomes in examples 1 to 3 and comparative examples 1 to 3 are respectively detected by using a western blot immunoblotting experiment. The specific process is as follows:

(1) The exosomes of comparative examples 1 to 3 were used as control groups and the exosomes of examples 1 to 3 were used as experimental groups. 2ml of each of the three control groups, 1ml of which was stored at 4℃for 30 days and 1ml of which was stored at 37℃for 30 days; the experimental groups were each 2ml, 1ml of which was stored at 4℃for 30 days and 1ml was stored at 37℃for 30 days.

(2) Protein sample extraction preparation: taking 500 mu l of each of the exosomes of examples 1-3 and comparative examples 1-3 stored under different conditions, washing the exosomes with PBS buffer solution, removing pollutants on the surface of the exosomes, centrifuging for 30min with 100000g, discarding PBS, adding precooled RIPA lysate containing protease inhibitor, performing lysis on ice for 30min, blowing with a gun for many times until the exosomes are completely lysed, shaking for 30min at 4 ℃, centrifuging for 12000rpm for 20min at 4 ℃, gently sucking the supernatant, transferring to a newly precooled centrifuge tube, placing on ice to obtain a protein sample, and discarding precipitation.

(3) BCA protein quantification: the protein standard curve was measured according to the procedure shown in steps (1) and (2) in experimental example 1, as shown in FIG. 3. The total protein concentration of each group of samples was measured by continuing the procedure shown in step (3) in experimental example 1.

(4) Protein denaturation: diluting the sample stock solution to a uniform concentration of 5 mug/μl according to the sample concentration detected in the step (3), and diluting the sample stock solution according to the sample dilution: adding a 5 XSDS gel reduction type sample adding buffer solution into the sample adding buffer solution according to the volume ratio of 4:1, fully mixing, heating at 95 ℃ for 5min, placing the mixture on an ice box, cooling to 4 ℃ to obtain a sample, and placing the sample into the ice box for standby.

(5) SDS-PAGE gel preparation: the vertical electrophoresis plate is assembled, the two sides and the bottom of the two glass plates are sealed by agarose of 1%, the assembled glass electrophoresis plate is inclined to form an angle of 60 degrees, when gel is ready to be poured, after TEMED of 0.08% is added, the mixture is immediately mixed uniformly and slowly poured into a rubber bed between the two glass plates until liquid is close to overflow, a proper comb is immediately inserted, close attention is paid to preventing bubbles from being generated under comb teeth, the comb is clamped on one side of the glass plate by a clamp, then the glass plate is inclined to lean against an object to form an angle of 10 degrees, the chance of liquid leakage can be reduced, after polymerization for one hour at room temperature, the glass plate is inserted into an electrophoresis tank, and then is tightly screwed up, and 0.1 XTBE buffer is poured into the glass plate, and the comb is carefully taken out and sample is added.

(5) Electrophoresis and transfer: fixing a gel plate into an electrophoresis device, adding a proper amount of 1 XSDS electrophoresis buffer solution into an outer groove, adding the 1 XSDS electrophoresis buffer solution into an inner groove until the gel sample adding hole is just submerged, pulling out a comb in the gel plate, centrifuging 12000g of a sample in an ice box for 5min after electrophoresis is also full of the sample adding hole, loading the sample, carrying out electrophoresis for 30min under 80V until bromophenol blue dye concentrated gel enters a separation gel, carrying out voltage modulation for 120V for electrophoresis for 60min, taking PVDF membrane and activating in methanol for 5min, balancing the membrane and filter paper in the buffer solution for 15min, putting the membrane and the gel interlayer taken out after SDS-PAGE electrophoresis is completed into an experiment tray with transfer solution, taking out gel in the interlayer, rinsing the separation gel after removing the concentrated gel once by using a 1X membrane transfer buffer solution, transferring 180mA membrane in a membrane transfer groove for 2h in a mode of 'sponge → three layers of filter paper → separation gel + PVDF membrane +.three layers of filter paper +.sponges', placing the PVDF membrane after transferring the membrane in a TBST incubation box of 5% skimmed milk powder, shaking and sealing on a room temperature decolorization shaker for 1h, placing the protein surface of the membrane upwards in a primary antibody diluent with proper concentration after sealing, selecting an antibody corresponding to CD63 by the primary antibody, shaking and incubating on a decolorization shaker for overnight at 4 ℃, shaking and washing 3 times by TBST at room temperature for 10min each time, selecting anti-mouse IgG by the secondary antibody, and shaking and washing by the TBST for 3 times after incubating for 1.5h at room temperature each time for 10min.

(6) Color development: the film was placed in an imager and chemiluminescent reaction was performed with ECL luminescent solution added and photographed. The horizontal gel electrophoresis WB developed image of the CD63 expression of the exosome preserved at 4℃is shown in FIG. 4, and the horizontal gel electrophoresis WB developed image of the CD63 expression of the exosome preserved at 37℃is shown in FIG. 5.

(7) Analysis of results: from the WB visualizations of fig. 4 to 5, it can be seen that the content of the protein marker CD63 of the modified exosomes in the experimental group was higher than the content of CD63 of the unmodified exosomes in the control group after 30 days of exosomes preservation at 4 ℃. After the exosomes are stored for 30 days under the storage condition of 37 ℃, the content of the protein marker CD63 of the modified exosomes in the experimental group is higher than the content of the CD63 of the unmodified exosomes in the control group; when the exosomes are stored for 30 days, the content of the protein marker CD63 of the exosomes in the experimental group under the storage condition of 4 ℃ is higher than the content of the protein marker CD63 of the exosomes in the control group under the storage condition of 37 ℃. Therefore, the plant exosome modification method provided by the invention can improve the stability of the plant exosome.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An extraction method for improving stability of plant exosomes is characterized by comprising the following steps:

2. The method of extraction according to claim 1, wherein the method of preparing the crude solution of plant exosomes in step (1) comprises the steps of:

3. The extraction method according to claim 2, wherein in step a, the volume ratio of the plant tissue to the PBS solution is (0.5-2): (0.5-2).

4. The extraction method according to claim 3, wherein in step d, the original concentration of polyethylene glycol-800 is 50 to 60%; the mixing temperature is 0-4 ℃, and the mixing time is 12-16 h.

5. The method according to claim 4, wherein in step e, the volume of the PBS solution is 2-8 mL; the original concentration of the polyethylene glycol-800 is 50-60%; the resuspended solvent is particle-free PBS buffer, and the volume of the particle-free PBS buffer is 50-500 mu L.

6. The method according to claim 5, wherein in the step (2), the fatty acid modification method comprises mixing fatty acid carboxylic acid with the crude plant exosome solution and PBS solution to obtain fatty acid modified plant exosome;

7. The extraction method according to claim 6, wherein in the step (2), the polyethylene glycol-800 is mixed with the fatty acid modified plant exosome to obtain a plant exosome crosslinking system, wherein the final concentration of the polyethylene glycol-800 is 1.3-2.5%;

8. The method according to claim 7, wherein in the step (4), the initial concentration of the trehalose solution is 20 to 30%, and the final concentration of the trehalose solution is 10 to 15%; the temperature of the centrifugation is 2-6 ℃, the rotating speed is 120000 ～ 150000g, the time is 0.5-1.5 h, and the times are 1-3 times.

9. The method according to claim 8, wherein the resuspended solvent is a trehalose solution having a concentration of 20-30%, and the concentration of the highly stable plant exosomes is (0.5-1) mg/mL.