Method for efficiently separating and purifying plant exosome and application

Abstract

The invention discloses a method for efficiently separating and purifying plant exosomes and application, and belongs to the technical field of biological nanotechnology. The method comprises the following key steps: (1) performing sterile cleaning on plant raw materials, and then performing peeling or tissue slitting pretreatment; (2) carrying out mechanical crushing treatment by adopting a phosphate buffer solution (PBS), and filtering through a 200-mesh screen to obtain a primary extracting solution; (3) performing gradient centrifugal purification on the extracting solution, collecting supernate, and filtering the supernate through a 0.45 mu m microporous filter membrane; (4) adding 8-12% of polyethylene glycol for low-temperature phase separation and precipitation; (5) purifying by using an electrodialysis technology; and (6) carrying out final concentration by adopting a 100kDa ultrafiltration membrane to obtain the high-purity PELNs preparation. According to the method, the process, equipment and reagents are simple and easy to obtain, the final yield is high, the purity is high, particle size distribution is uniform, and an innovative solution is provided for large-scale industrial production of natural-source nano-drug carriers.

Description

Technical Field

[0001] The present invention belongs to the intersection of bionanotechnology and pharmaceutical preparations, and specifically relates to a method and application for efficiently separating and purifying plant exosomes, and in particular to a method for efficiently separating and purifying plant-derived exosome-like nanoparticles (PELNs). Background Art

[0002] Plant-derived exosome-like nanoparticles are lipid bilayer membrane vesicles secreted by plant cells with a particle size distribution of 50-200 nm (verified by transmission electron microscopy). They carry plant-specific transmembrane proteins (such as annexin ANNEXIN and heat shock protein HSP) on their surface and encapsulate functional microRNAs (such as miR166 and miR398), metabolites (phenolic acids, flavonoids) and enzyme active substances. Studies have shown that after PELNs enter mammalian cells through endocytosis, they can regulate signaling pathways such as NF-κB and Nrf2, thereby exerting antioxidant (SOD activity increased by 42% when the protein concentration was 16 μg / mL) (Pan Q, Bao Z, Wang Y, et al. RETRACTED: Nrf2 pathway activation with natural plant-derived exosome-like nanovesicle / hydrogel preparations for oxidative stress modulation in inflammation related diseases[J]. Chemical Engineering Journal, 2024, 480: 1482822024.) and anti-inflammatory (effectively inhibiting the expression of proinflammatory cytokines (TNF-α, IL-6, IL-12 and IL-1β) and myeloperoxidase (MPO), and increasing the level of anti-inflammatory cytokine (IL-10)) (Zhu M, Xu H, Liang Y, et al. Edible exosome-like nanoparticles from portulaca oleracea L mitigate DSS-induced colitis via facilitating double-positive CD4+CD8+T cells expansion[J].Journal of nanobiotechnology,2023,21(1):309.) and promoting tissue repair (0.5×10 9particle / mL closure rate increased significantly to 82.38%, significantly higher than the control group)( Y, OK, Bozkurt B T, et al. Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing [J]. Food & function, 2021, 12 (11): 5144-5156.) and other activities. Compared with animal exosomes, PELNs have significant advantages: no risk of pathogen contamination, low immunogenicity (the IgE level in mice is only 1 / 5 of that in liposomes, p < 0.01); the structure remains intact after incubation in simulated gastric fluid (pH 2.0) for 2 hours (dynamic light scattering verification); the raw material cost is reduced by more than 90% compared with mesenchymal stem cell culture. Current mainstream PELN separation technologies face the following bottlenecks: differential ultracentrifugation requires 100,000 × g for more than 4 hours and requires expensive equipment, resulting in low yields (only 2.1 ± 0.5 mg / kg of tomato fruit raw material), vesicle rupture rates >30% (flow cytometric nanoparticle tracking analysis), and strong equipment dependence (requiring an ultracentrifuge and a single processing volume <200 mL). Size exclusion chromatography (SEC) offers higher purity (protein impurities <2.5 μg / mg), but yields less than 15%, and scalability is expensive (the packing life is only 50 cycles). Polymer precipitation is simple to operate, but the co-precipitated protein content is as high as 8.7 μg / mg (BCA assay), requiring secondary purification (such as ultrafiltration), which extends the total purification time to 18 hours. To address these shortcomings, the present invention proposes a three-stage combined technique of "gradient centrifugation-polymer precipitation-electrodialysis." Summary of the Invention

[0003] The present invention aims to provide a method for isolating and purifying plant exosome-like nanoparticles and its specific application examples, offering new insights and technical solutions to address the aforementioned challenges. This method can be applied to extract exosome-like nanoparticles from various plants, resulting in high yields and high purity of PELNs. It can also be prepared on a large scale and applied to a wide range of plants, laying the foundation for the application of PELNs.

[0004] The object of the present invention is achieved through the following technical solution: a method for efficiently separating and purifying plant exosomes, comprising the following steps:

[0005] (1) Pretreatment of plant raw materials: fresh plant tissues were washed with sterile deionized water, then peeled and cut;

[0006] (2) Mechanical disruption and primary filtration: The plant tissue treated in step (1) was mixed with phosphate buffered saline (PBS) at a mass ratio of 1:4-4:1, disrupted by a homogenizer, and filtered through an 80-200 mesh sieve to collect the primary filtrate;

[0007] (3) Gradient centrifugation and microfiltration: The primary filtrate is subjected to three-stage gradient centrifugation in sequence;

[0008] The supernatant after centrifugation was filtered through a microporous membrane with a pore size of 0.22-0.45 μm to obtain a clarified extract;

[0009] (4) Polyethylene glycol (PEG) phase separation: PEG-6000 was added to the clarified extract at a final concentration of 8-15% (w / v, g / mL). The extract was allowed to stand at 2-8°C for 12-16 hours. The extract was then centrifuged at 8000-8500 × g for 20-30 minutes at 4°C to collect the enriched precipitate.

[0010] (5) Electrodialysis purification: The precipitate was resuspended in PBS buffer at pH 7.2 and placed in a dialysis bag with a molecular weight cutoff of 100 kDa. A constant current of 300 ± 50 mA was applied in a glycine-Tris buffer with a molar ratio of glycine to Tris of 4-5:0.5-1.5. The dialysis cycle was repeated 4-6 times, each time for 30-40 minutes.

[0011] (6) Ultrafiltration concentration: The dialysate was concentrated by 100 kDa ultrafiltration tube, and the resulting precipitate was resuspended in PBS buffer to obtain plant exosome-like nanoparticles (PELNs) with a particle size of 50-150 nm and a PDI < 0.35.

[0012] Furthermore, the plant raw material is selected from the group consisting of at least one of Morus alba L., Paris polyphylla, Cucumis sativus, Citrus aurantium, Lonicera japonica, Pelargonium graveolens, Luffa cylindrica, Rubus idaeus, Crocus sativus, Perilla frutescens, and Houttuynia cordata.

[0013] Furthermore, the three-stage gradient centrifugation parameters are:

[0014] First stage centrifugation: Centrifuge at 1000-2000 × g for 10-15 minutes at 4°C to remove cell debris;

[0015] Second stage centrifugation: Centrifuge at 4°C, 5000-6000 × g for 20-30 minutes to remove large molecular impurities;

[0016] Third stage centrifugation: Centrifuge at 4°C, 10,000-11,000 × g for 30-40 minutes and collect the supernatant.

[0017] Furthermore, the final concentration of PEG-6000 in step (4) is 10% (w / v), the standing temperature is 4° C., and the centrifugation conditions are 4° C., 8000×g, and 25 minutes.

[0018] Furthermore, the electrodialysis parameters in step (5) are:

[0019] The dialysate had a molar ratio of glycine:Tris=4-5:0.5-1.5, a constant current of 300 mA, and dialysis was performed 5 times, each time for 35 minutes.

[0020] Furthermore, the ultrafiltration tube in step (6) is a regenerated cellulose membrane with a molecular weight cutoff of 100 kDa, and the ultrafiltration pressure is 0.1-0.3 MPa.

[0021] In a second aspect, the present invention provides a plant exosome-like nanoparticle, which is prepared by the method according to any one of claims 1 to 5 and meets the following characteristics:

[0022] (a) Particle size distribution PDI < 0.35;

[0023] (b) Protein content detected by BCA method was ≤3 μg / mg.

[0024] In a third aspect, the present invention provides an application of plant exosome-like nanoparticles in the preparation of antioxidant preparations.

[0025] In a fourth aspect, the present invention provides a use of plant exosome-like nanoparticles as a drug delivery carrier.

[0026] Furthermore, the antioxidant preparation includes cosmetics, functional foods or medical dressings, and its dosage form is hydrogel, nanoemulsion or freeze-dried powder injection.

[0027] Beneficial effects of the present invention:

[0028] This method innovatively integrates triple purification technologies of gradient centrifugation, polymer precipitation, and electrodialysis, increasing the yield by 30% compared to traditional differential centrifugation (Kong T, Zhang K, Wang Y, et al. Cucumber-Derived Extracellular Vesicle-Functionalized Metal-Organic Frameworks for Enhanced Photodynamic Therapy of Hypertrophic Scars[J]. Advanced Functional Materials, 2024, 34(29): 2400379.), and reducing the protein impurity content to 1.2 μg / mg (BCA assay). This method is generally applicable to plants, and its standardized preparation system is applicable to 36 medicinal plants from 12 families, including Solanaceae and Cruciferae. The process, equipment, and reagents of the present invention are simple and easy to obtain, and the final product has a high yield, high purity, and uniform particle size distribution, providing an innovative solution for the large-scale industrial production of natural-source nanoparticle drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 TEM images and NTA results of some plant exosomes prepared by the method of the present invention, among which A,a is the TEM image and NTA of exosomes from Paris polyphylla root; B,b is the TEM image and NTA of exosomes from Luffa cylindrica; C,c is the TEM image and NTA of exosomes from cucumber.

[0031] Figure 2 Comparison of the NTA results of cucumber exosomes prepared by the method of the present invention and ultrafiltration combined with size exclusion chromatography, wherein A is the NTA graph of cucumber exosomes prepared by PEG precipitation combined with electrophoresis dialysis (ELD) and ultrafiltration combined with size exclusion chromatography (UF+SEC); B is the DLS particle size distribution of cucumber exosomes by PEG precipitation combined with electrophoresis dialysis (ELD).

[0032] Figure 3 This is the change in protein concentration during the purification of cucumber exosomes by the method of the present invention.

[0033] Figure 4 This is the antioxidant effect of cucumber exosomes prepared by the method of the present invention. DETAILED DESCRIPTION

[0034] The following examples are used to further illustrate the technical solutions of the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the experimental methods described are all implemented in accordance with national or international standards, and the reagents and equipment described are all conventional commercial products in the field.

[0035] The separation and purification method provided by the present invention can be used to prepare exosome-like nanoparticles from plants such as cucumber and loofah, comprising the following steps:

[0036] Step 1: Pre-process the plants (cucumber, loofah), wash, peel and cut;

[0037] Step 2: Mix the treated plants with PBS solution to break the wall, pass through a sieve, and obtain the filtrate;

[0038] Step 3: After the filtrate is centrifuged at low speed, the supernatant is taken and filtered through a microporous filter membrane to obtain a filtrate;

[0039] Step 4: Add PEG to the filtrate, let it stand, centrifuge at low speed, and collect the precipitate;

[0040] Step 5: Resuspend the collected precipitate in PBS solution and perform electrophoresis and dialysis;

[0041] Step 6: Collect the dialyzed solution, ultrafilter, and resuspend the precipitate with PBS solution to obtain plant exosome-like nanoparticles.

[0042] Example

[0043] The method for isolating and purifying cucumber plant exosome-like nanoparticles provided in this embodiment specifically comprises the following steps:

[0044] Example 1: Preparation and performance verification of cucumber-derived PELNs

[0045] Step 1: Raw material pretreatment

[0046] 1000 g of fresh cucumber (Cucumis sativus L.) was washed three times with tap water, sterile deionized water, and pre-cooled PBS buffer (pH 7.2, 0.01 M), peeled, and cut into 2 cm × 1 cm tissue blocks.

[0047] Step 2: Mechanical crushing and primary filtration

[0048] The treated cucumber tissue was mixed with pre-cooled PBS buffer at a mass ratio of 4:1 and homogenized using a BARBOSA (30 s). The resulting homogenate was filtered through a 200-mesh nylon sieve (pore size 75 μm) to collect the primary filtrate.

[0049] Step 3: Gradient centrifugation and microfiltration

[0050] First stage centrifugation: centrifuge at 4°C, 1500 × g for 15 min and discard the pellet (to remove cell debris >1 μm);

[0051] Second stage centrifugation: centrifuge at 4°C, 6000 × g for 25 min and discard the pellet (to remove subcellular organelles (mitochondria / chloroplasts));

[0052] The third stage of centrifugation: centrifuge at 4°C, 10,000 × g for 35 minutes and collect the supernatant;

[0053] Microfiltration: Filter through 0.45 μm and 0.22 μm polyethersulfone (PES) membranes in sequence to obtain a clarified extract.

[0054] Step 4: PEG phase separation and enrichment

[0055] PEG-6000 was added to the clarified extract to a final concentration of 10% (w / v). After standing at 4°C for 16 hours, the extract was centrifuged at 8000×g for 25 minutes and the precipitate was collected.

[0056] Step 5: Electrodialysis purification

[0057] The pellet was resuspended in PBS buffer (pH 7.2), transferred to a 300 kDa molecular weight cutoff regenerated cellulose dialysis bag, and placed in a glycine-Tris electrodialysis buffer (mass ratio 4.8:1). A constant current of 300 mA was applied, and the dialysis cycle was repeated five times (35 minutes each, with buffer changes at intervals). Based on the zeta potential of the vesicle surface (-10 mV to -30 mV), the vesicles migrated in a glycine-Tris buffer system, resulting in specific removal of free proteins (SDS-PAGE showed a 90% reduction in unwanted bands).

[0058] Step 6: Ultrafiltration Concentration

[0059] The dialysate was concentrated by 100 kDa ultrafiltration tube (regenerated cellulose membrane, 0.2 MPa), and the precipitate was resuspended in PBS buffer to obtain cucumber-derived PELNs.

[0060] Performance characterization:

[0061] Yield: 130 ± 1.2 mg / kg (fresh weight);

[0062] Particle size distribution: dynamic light scattering (DLS) detection is 149 ± 2.3 nm, PDI = 0.236; (such as Figure 2 B)

[0063] Purity: BCA assay detected protein impurity content of 1.1±0.3μg / mg;

[0064] Functional activity: ABTS free radical scavenging rate 82±3% (1 mg / mL).

[0065] Example 2

[0066] Example 2: Preparation of PELNs from Luffa (PEG concentration optimization)

[0067] Steps 1-3: Same as Example 1, except that the raw material is replaced with fresh loofah (Luffa cylindrica).

[0068] Step 4: The final concentration of PEG-6000 was adjusted to 8% (w / v), and the rest was the same as in Example 1.

[0069] Performance characterization:

[0070] Yield: 98 ± 0.4 mg / kg;

[0071] Particle size distribution: 159.1 ± 12 nm, PDI = 0.28;

[0072] Protein impurities: 1.4±0.2μg / mg;

[0073] Example 3: Preparation of PELNs from Paris polyphylla roots

[0074] Step 1: Take 200 g of Paris polyphylla roots, cut into 5 cm segments, and clean them as in Example 1.

[0075] Step 2: Adjust the mass ratio of PBS buffer to raw material to 1:1, and the disruption parameter to 30s×2 times.

[0076] Step 6 modification: The final product is PELNs derived from Paris polyphylla roots.

[0077] Performance characterization:

[0078] Yield: 16.2 ± 0.6 mg / kg (due to high lignin content);

[0079] Particle size distribution: 186.0 ± 3.2 nm, PDI = 0.3;

[0080] Protein impurities: 1.8±0.3μg / mg;

[0081] Example 4: Large-scale preparation of mulberry leaf-derived PELNs

[0082] Step 1: Take 1 kg of fresh mulberry leaves (Morus alba L.) and cut them into 5 cm pieces.

[0083] Step 2: The mass ratio of PBS buffer to raw material was adjusted to 3:1, and the disruption parameters were optimized to 30 s × 3 times.

[0084] Step 6 modification: The final product is mulberry leaf-derived PELNs.

[0085] Performance characterization:

[0086] Yield: 32 ± 0.3 mg / kg;

[0087] Particle size distribution: 155.2 ± 3.2 nm, PDI = 0.32;

[0088] Protein impurities: 2.4±0.6μg / mg;

[0089] This experimental example uses the BCA method to determine the total protein concentration of the plant exosome-like nanoparticles in the above example. The specific process is as follows:

[0090] The total protein concentration was determined using the BCA protein concentration assay kit (enhanced) (Biyuntian). The kit's protein standard curve was obtained according to the instructions. The prepared plant exosome-like nanoparticles were diluted a certain multiple, and 20 μL was added to a 96-well plate according to the instructions. Then, 200 μL of the working solution was added. The plate was wrapped in tin foil to protect from light and incubated at 37°C for 30 minutes. The absorbance was measured at a wavelength of 562 nm. The total protein concentration of the sample was then calculated based on the standard curve and the dilution multiple.

[0091] Test example:

[0092] The four plant exosome-like nanoparticles obtained in the above examples were taken out in appropriate amounts and washed with PBS buffer.

[0093] Dilute to an appropriate concentration. In this example, the performance of three samples of exosome-like nanoparticles obtained in Example 3 above, namely, loofah / cucumber / Paridis root hair, was tested and analyzed.

[0094] 1. NTA test of exosome-like nanoparticles

[0095] Use NanoSight NS300 to test the particle size and concentration of exosomes. Use PBS to clean the sample pool through a syringe, turn on the device switch and software for self-test, draw 1ul of standard sample into 2ml of PBS and mix well to test whether the particle size meets the requirements. Draw an appropriate volume of exosomes and dilute it with PBS to 2ml. Use a 1ml syringe to inject it into the sample pool and start the test. Figure 1As shown in the figure, the particle sizes of exosomes from Paris polyphylla root, loofah, and cucumber are 186.0 nm, 159.1 nm, and 151.2 nm, respectively, and the particle numbers are 9.81E+10 (Particles / ml), 6.2E+10 (Particles / ml), and 5.21E+10 (Particles / ml), respectively. At the same time, NTA tests were performed on cucumber exosomes prepared by PEG precipitation combined with electrophoresis dialysis (ELD) and ultrafiltration combined with size exclusion chromatography (UF+SEC), and the results were compared, as shown in the figure. Figure 2 As shown in the figure, the exosomes obtained by size exclusion chromatography have a relatively uniform particle size and high purity. The NTA results show that the exosomes obtained by this method have a particle size distribution similar to that of size exclusion chromatography, with a particle size of about 150nm. At the same time, the protein concentration during the electric field dialysis purification process was tracked and tested, such as Figure 3 As shown, the protein concentration showed a downward trend, proving that during the purification process, electrophoresis can remove a large amount of impurities and further purify extracellular vesicles.

[0096] 2.TEM test

[0097] Use a pipette to draw 10ul of exosome suspension onto the disposable sealing film.

[0098] Invert and allow to adsorb naturally for 15 minutes; use a pipette to draw 10ul of 2wt% paraformaldehyde fixative solution and drop it on the front of the copper mesh, and let it sit for 20 minutes; use a pipette to draw 10ul of 2wt% phosphotungstic acid solution and drop it on the front of the copper mesh, and let it sit for 90 seconds; use a filter paper strip to absorb excess droplets, and dry it in the dark at room temperature for half an hour; observe under a transmission electron microscope.

[0099] like Figure 1 As shown, TEM showed that the morphology of exosomes was a typical saucer-shaped structure, and the size was consistent with the exosome particle size range.

[0100] 3. Antioxidant test

[0101] The antioxidant capacity of cucumber exosomes extracted by this patented method was tested using the ABTS method.

[0102] Experimental methods:

[0103] Preparation of ABTS test solution: ABTS stock solution (7.4 mmol / L, 0.4 mL): 0.0045 g ABTS, 1.1025 mL distilled water (MW = 548.7); K2S2O8 stock solution (2.6 mmol / L, 1.43 mL): 0.0025 g K2S2O8, 3.575 mL distilled water (MW = 270.32), mixed, incubated in the dark at room temperature for 12 hours, and then diluted with PBS to an absorbance of approximately 0.7 (A734 nm). A0 value determination: 0.2 mL of this ABTS solution was thoroughly mixed with 50 μL of PBS solution, and the absorbance was measured at 734 nm. A value determination: 50 mL of ABTS test solution was thoroughly mixed with 50 μL of exosomes at a gradient concentration, and the absorbance was measured at 734 nm. The experiment was repeated three times.

[0104] like Figure 4 As shown, cucumber exosomes were tested at different concentrations against a positive control. Cucumber exosomes exhibited a concentration-dependent clearance of ABTS at low concentrations, but remained stable at high concentrations. Based on protein concentration, exosomes at a concentration of 0.25 mg / mL were comparable to 0.6 mg / mL EGCG and VC, with an average clearance of 85.1%.

[0105] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for efficiently separating and purifying plant exosomes, characterized in that: The following steps are involved: (1) Pretreatment of plant raw materials: fresh plant tissues were washed with sterile deionized water, then peeled and cut; (2) Mechanical disruption and primary filtration: The plant tissue treated in step (1) was mixed with phosphate buffered saline (PBS) at a mass ratio of 1:4-4:1, disrupted by a homogenizer, and filtered through an 80-200 mesh sieve to collect the primary filtrate; (3) Gradient centrifugation and microfiltration: The primary filtrate is subjected to three-stage gradient centrifugation in sequence; The supernatant after centrifugation was filtered through a microporous membrane with a pore size of 0.22-0.45 μm to obtain a clarified extract; (4) Polyethylene glycol (PEG) phase separation: PEG-6000 was added to the clarified extract at a final concentration of 8-15% (w / v, g / mL). The extract was allowed to stand at 2-8°C for 12-16 hours. The extract was then centrifuged at 8000-8500 × g for 20-30 minutes at 4°C to collect the enriched precipitate. (5) Electrodialysis purification: The precipitate was resuspended in PBS buffer at pH 7.2 and placed in a dialysis bag with a molecular weight cutoff of 100 kDa. A constant current of 300 ± 50 mA was applied in a glycine-Tris buffer with a molar ratio of glycine to Tris of 4-5:0.5-1.

5. The dialysis cycle was repeated 4-6 times, each time for 30-40 minutes. (6) Ultrafiltration concentration: The dialysate was concentrated by 100 kDa ultrafiltration tube, and the resulting precipitate was resuspended in PBS buffer to obtain plant exosome-like nanoparticles (PELNs) with a particle size of 50-150 nm and a PDI < 0.

35.

2. The method according to claim 1, characterized in that The plant raw material is selected from the group consisting of at least one of Morus alba L., Paris polyphylla, Cucumis sativus, Citrus aurantium, Lonicera japonica, Pelargonium graveolens, Luffacylindrica, Rubus idaeus, Crocus sativus, Perilla frutescens, and Houttuynia cordata.

3. The method according to claim 1, characterized in that The parameters for the three-stage gradient centrifugation are: First stage centrifugation: Centrifuge at 1000-2000 × g for 10-15 minutes at 4°C to remove cell debris; Second stage centrifugation: Centrifuge at 4°C, 5000-6000 × g for 20-30 minutes to remove large molecular impurities; Third stage centrifugation: Centrifuge at 4°C, 10,000-11,000 × g for 30-40 minutes and collect the supernatant.

4. The method according to claim 1, wherein The final concentration of PEG-6000 in step (4) is 10% (w / v), the standing temperature is 4°C, and the centrifugation conditions are 4°C, 8000×g for 25 minutes.

5. The method according to claim 1, wherein The electrodialysis parameters in step (5) are: The dialysate had a molar ratio of glycine:Tris=4-5:0.5-1.5, a constant current of 300 mA, and dialysis was performed 5 times, each time for 35 minutes.

6. The method according to claim 1, characterized in that The ultrafiltration tube in step (6) is a regenerated cellulose membrane with a molecular weight cut-off of 100 kDa, and the ultrafiltration pressure is 0.1-0.3 MPa.

7. A plant exosome-like nanoparticle, characterized in that: It is prepared by the method according to any one of claims 1 to 6 and meets the following characteristics: (a) Particle size distribution PDI < 0.35; (b) Protein content detected by BCA method was ≤3 μg / mg.

8. Use of the plant exosome-like nanoparticles according to claim 7 in the preparation of an antioxidant preparation.

9. Use of the plant exosome-like nanoparticles according to claim 7 as a drug delivery carrier.

10. The use according to claim 8 or 9, characterized in that: The antioxidant preparation includes cosmetics, functional foods or medical dressings, and its dosage form is hydrogel, nanoemulsion or freeze-dried powder injection.

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