High-yield nanovesicle preparation method based on ultrasonic disruption of walled cell protoplasts

Abstract

The application discloses a kind of high-yield nanovesicle preparation methods based on ultrasonic crushing of walled cell protoplast.The method comprises the following steps: ultrasonic crushing of walled cell protoplast, then standing to rearrange membrane structure to form nanovesicle; removing uncrushed bacteria and large fragments by low-speed centrifugation, further purifying to obtain narrow nanovesicle band by density gradient centrifugation, and finally obtaining uniform nanovesicle by ultracentrifugation.The application overcomes the shortcomings of low yield and long acquisition period of naturally secreted extracellular vesicles of walled cells, significantly improves the yield, and the yield of Escherichia coli as an example can reach about 600 times of naturally secreted vesicles;The process is simpler without extrusion, and the vesicles obtained by ultrasonic crushing and standing treatment can be used as efficient substitutes for natural extracellular vesicles, and applied in the fields of gene drug delivery and vaccine development.

Description

Technical Field

[0001] This invention relates to a method for preparing nanovesicles, and to the fields of biomedicine and nanoparticle technology, specifically to a high-yield method for preparing nanovesicles based on ultrasonic disruption of wall-walled cell protoplasts. Background Technology

[0002] Cells with cell walls, such as Escherichia coli and yeast, can secrete nanovesicles, each with its own unique advantages. Outer membrane vesicles (OMVs) secreted by E. coli possess natural immune adjuvant activity and can efficiently encapsulate biomolecules such as proteins and nucleic acids, making them classic carriers for vaccine development and tumor immunotherapy. Yeast nanovesicles exhibit high biocompatibility, low immunogenicity, and stable physicochemical properties, effectively avoiding the potential endotoxin-related risks associated with bacterial vesicles, making them suitable for in vivo drug delivery and functional molecule transport.

[0003] Despite the significant advantages of cell wall-derived nanovesicles, naturally secreted nanovesicles generally suffer from long acquisition cycles and low yields, failing to meet the demands of large-scale, industrial production. Existing methods often employ chemical induction or physical extrusion to increase nanovesicle yield. However, chemical induction methods carry the risk of toxic residues and offer limited yield increases, while physical extrusion methods suffer from high membrane costs, clogging issues, and cumbersome operations, resulting in poor economic viability for large-scale production. Therefore, developing a high-yield, chemical-residue-free, and uniformly sized cell wall-derived vesicle preparation method, particularly a universal method applicable to various cell wall-derived organisms such as E. coli and yeast, is of great value for promoting the large-scale application of nanovesicles. Summary of the Invention

[0004] To address the problems existing in the background art, this invention provides a high-yield nanovesicle preparation method based on ultrasonic disruption of wall-dwelling cell protoplasts. This method obtains a mixture containing nanovesicles through ultrasonic disruption and settling, followed by purification using density gradient centrifugation. The entire process eliminates the need for compression treatment, enabling efficient and large-scale preparation of wall-dwelling cell nanovesicles.

[0005] The technical solution adopted in this invention is: The present invention provides a method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts, comprising: Step 1) Resuspend protoplasts with wall cells in an ultrasonic buffer solution and perform ultrasonic disruption under ice bath conditions to obtain an ultrasonic disruption solution. Allow the ultrasonic disruption solution to stand to allow the membrane structure to rearrange and self-assemble, resulting in a mixed solution containing nanovesicles; specifically, resuspend each gram of protoplast precipitate in 10–30 mL of ultrasonic buffer solution.

[0006] Step 2) Centrifuge the mixed solution containing nanovesicles at low speed to precipitate impurities such as protoplast fragments and collect the supernatant.

[0007] Step 3) Spread the supernatant on a sucrose density gradient medium and perform density gradient centrifugation to separate the white nanovesicle zone.

[0008] Step 4) Collect the nanovesicle bands, perform ultracentrifugation, discard the supernatant, and the precipitate is the nanovesicle. Resuspend the precipitate in phosphate-buffered saline (PBS) for storage and subsequent experiments.

[0009] In step 1), the wall cells are either Escherichia coli or yeast.

[0010] In step 1), the ultrasound buffer contains a physiologically compatible pH buffer and an osmotic pressure stabilizer of 200–800 mmol / L, and the pH of the ultrasound buffer is 7.0–8.0; the physiologically compatible pH buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl), or phosphate buffer (PBS); the osmotic pressure stabilizer is sucrose or trehalose.

[0011] When the physiologically compatible pH buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES) or tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl), the concentration is 10–50 mmol / L; when the physiologically compatible pH buffer is phosphate-buffered saline (PBS), it contains 137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na₂HPO₄, and 1.8 mmol / L KH₂PO₄.

[0012] In step 1), the parameters for ultrasonic disruption are as follows: ultrasonic power is 100-300 W, ultrasonic treatment for 3-5 seconds and pause for 6-10 seconds constitutes one cycle, and the cycle is repeated so that the total ultrasonic disruption treatment time is 6-15 minutes, and the ice bath condition is 0℃; after ultrasonic disruption, the ultrasonic disruption fluid is left to stand at 4-25℃ for 30-90 minutes.

[0013] In step 2), the mixed solution containing nanovesicles is centrifuged at 3,000 × g for 10–30 minutes.

[0014] In step 3), the supernatant is spread on a 20% (w / v) to 60% (w / v) sucrose density gradient medium and centrifuged at 100,000 to 200,000 × g at 4°C for 5 to 10 hours.

[0015] In step 4), the nanovesicle zone is ultracentrifuged at 100,000–150,000 × g for 1–2 hours at 4°C.

[0016] The present invention relates to high-yield nanovesicles based on ultrasonic disruption of wall-walled cell protoplasts: the high-yield nanovesicles are prepared by the method described above.

[0017] Application of the high-yield nanovesicles of the present invention: The nanovesicles are used as delivery carriers in the preparation of gene drugs, vaccine drugs or anti-tumor drugs.

[0018] The beneficial effects of this invention are: 1) Extremely high yield: Taking Escherichia coli as an example, the purchased Escherichia coli BL21(DE3) derivative strain is used. Escherichia coli ClearColi BL21(DE3), when cultured in LB (Luria-Bertani) liquid medium to OD600 = 2.0, yielded a total of 10 nanovesicles per liter of culture. 12 The order of magnitude larger, while the production of naturally secreted OMVs under the same conditions is only about 10. 9 Order of magnitude. Calculations show that the yield of nanovesicles prepared by the method of this invention is approximately 600 times that of naturally secreted OMV, which can fully meet the needs of industrial production.

[0019] 2) High versatility: The method of this invention has good universality and can be widely applied to different types of wall-bound cells such as Escherichia coli and yeast. Its core preparation process is highly consistent: after obtaining protoplasts, ultrasonic disruption is used to induce membrane structure rearrangement and self-assembly, ultimately forming nanovesicles. The process obtains vesicles solely through ultrasonic disruption and static treatment, without the need for compression, making the process simpler. For different cell types, only simple adjustments to the ultrasonic parameters are required without changing the overall technical approach.

[0020] 3) No chemical toxic residues: The entire preparation process avoids the use of surfactants or organic solvents such as sodium dodecyl sulfate (SDS), and the obtained vesicles have no chemical toxic residues, have high biosafety, and are suitable for fields such as gene drug delivery, vaccine development and tumor immunotherapy.

[0021] 4) Highly concentrated and uniform particle size: The particle size of the prepared nanovesicles is comparable to that of naturally secreted extracellular vesicles. Taking Escherichia coli as an example, the particle size and number of particles were detected using a NanoCoulter single particle analyzer. The particle size of the Escherichia coli nanovesicles obtained in this invention is mainly concentrated in the range of 120–250 nm, with an average particle size of 202 nm, which is comparable to that of natural OMV and has good substitutability.

[0022] In summary, this invention overcomes the shortcomings of low yield and long acquisition cycle of naturally secreted extracellular vesicles from wall-dwelling cells, achieving a significant increase in yield. It provides a method for preparing wall-dwelling cell nanovesicles with high yield, uniform particle size, and no chemical toxicity residue. The obtained vesicles can serve as an effective substitute for naturally secreted vesicles and have broad industrial application prospects in fields such as gene drug delivery, vaccine development, and tumor immunotherapy. Attached Figure Description

[0023] Figure 1 This is a schematic flowchart of the preparation method of the present invention; Figure 2 The image shows the purification results of E. coli nanovesicles prepared in this invention under different ultrasonic buffer systems via sucrose density gradient centrifugation. Figure 3 Transmission electron microscope image of Escherichia coli nanovesicles prepared in this invention; Figure 4 Agarose gel electrophoresis analysis of the residual genomic DNA (gDNA) in Escherichia coli nanovesicles prepared in this invention; Figure 5 A comparison chart showing the yield of Escherichia coli nanovesicles prepared in this invention with that of natural outer membrane vesicles (OMVs); Figure 6 The particle size distribution diagram of the Escherichia coli nanovesicles prepared in this invention; Figure 7 The fluorescence detection results of the Escherichia coli nanovesicles prepared in this invention delivering pVAX1-mCherry to African green monkey kidney cells (Vero cells) are shown in the figure. Figure 8 Transmission electron microscope image of the yeast nanovesicles prepared in this invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Specific embodiments of the present invention are as follows: Example 1: In this embodiment, *E. coli* nanovesicles were prepared based on a PBS-sucrose ultrasonic buffer system, as detailed below: (1) Bacterial culture: The purchased Escherichia coli ClearColi BL21(DE3) strain was inoculated into 5 mL of LB liquid medium and cultured at 37°C and 200 rpm with shaking for 12–16 hours. The next day, it was transferred to 200 mL of LB liquid medium at a volume ratio of 1:100 and cultured under the same conditions until the bacterial culture reached OD600 = 2.0.

[0026] (2) Protoplast preparation: The cultured bacterial culture was centrifuged at 5000 × g for 15 minutes at 4°C, and the bacterial pellet was collected. The bacterial cells were washed twice with protoplastization buffer (containing 20 mmol / L HEPES, 500 mmol / L sucrose, 5 mmol / L EDTA, pH 7.4). Each gram of bacterial cells was resuspended in 10 mL of the above protoplastization buffer containing 2 mg / mL lysozyme, and incubated at 37°C and 100 rpm with shaking for 60 minutes. During the enzymatic digestion, samples were taken periodically and observed under an optical microscope. The enzymatic digestion was terminated when most cells in the field of view transformed into round, transparent protoplasts. After incubation, the pellet was centrifuged at 3000 × g for 10 minutes at 4°C, the supernatant was removed, and the pellet was resuspended in protoplastization buffer (without lysozyme), centrifuged at 3000 × g for 10 minutes, and washed three times to obtain the E. coli protoplast pellet.

[0027] (3) Ultrasonic fragmentation and membrane structure rearrangement: Each gram of protoplast precipitate was resuspended in 20 mL of sonication buffer (PBS containing 500 mmol / L sucrose, pH 7.4) and thoroughly mixed. The suspension was incubated on ice at 0°C and sonicated using an ultrasonic cell disruptor. The sonication parameters were: power 200 W, 3 seconds of sonication followed by a 6-second pause as one cycle, repeated for a total treatment time of 8 minutes; after every 2 minutes of sonication, the suspension was incubated on ice for 5 minutes to cool. After sonication, the disrupted solution was transferred to 4°C and allowed to stand for 90 minutes to allow for rearrangement and self-assembly of membrane fragments, yielding a mixed solution containing nanovesicles.

[0028] (4) Preliminary purification of nanovesicles: After centrifuging the mixed solution containing nanovesicles at 3,000 × g for 15 minutes at 4°C, the unbroken protoplasts and large fragments were precipitated, and the supernatant was collected.

[0029] (5) Density gradient centrifugation purification: Prepare a continuous sucrose density gradient of 20% (w / v) to 60% (w / v) (using PBS to prepare 20% (w / v) and 60% (w / v) sucrose solutions, and then using a gradient mixer to prepare the continuous gradient, pH 7.4). Carefully spread the supernatant on top of the sucrose density gradient medium. Centrifuge at 100,000 × g for 6 hours at 4°C. After centrifugation, distinct white vesicle bands were visible, as shown in the figure. Figure 2 As shown.

[0030] (6) Collection of nanovesicles: Take the white vesicle zone sample and dilute it with PBS (pH 7.4) to 2 / 3 to 3 / 4 of the total volume of the ultracentrifuge tube. Centrifuge at 100,000 × g for 1.5 hours at 4°C. Discard the supernatant and resuspend the precipitate in 50–500 μL of PBS (pH 7.4) to obtain E. coli nanovesicles, which are then characterized.

[0031] (7) Characterization of nanovesicles: Transmission electron microscopy (TEM) observation: The obtained nanovesicles were placed on a copper grid treated with glow discharge, negatively stained with phosphotungstic acid, dried, and observed under a TEM. Numerous nanovesicles with intact morphology and relatively uniform particle size were observed. The results are as follows: Figure 3 As shown.

[0032] Verification of the purification effect by density gradient centrifugation: Equal masses of *E. coli* cells, *E. coli* nanovesicles that were not purified by density gradient centrifugation, and *E. coli* nanovesicles purified by density gradient centrifugation (using the method of this invention) were taken, and gDNA was extracted from each. The results were then analyzed by agarose gel electrophoresis. The results are as follows: Figure 4 As shown in the figure. Lane M is the DNA Marker, with the largest band indicating 2000 bp; Lane 1 contains E. coli cell gDNA; Lane 2 contains unpurified nanovesicle gDNA; Lane 3 contains purified nanovesicle gDNA using a density gradient. The results show that no visible gDNA band was observed in Lane 3, indicating that density gradient centrifugation can effectively remove bacterial gDNA contamination.

[0033] Nanovesicle yield and particle size determination: 1 L of *E. coli* ClearColi BL21(DE3) culture was cultured to OD600 = 2.0. The culture was centrifuged at 5,000 × g for 15 minutes at 4°C, and the supernatant and bacterial precipitate were collected separately. The supernatant was filtered through a 0.45 µm filter and then ultracentrifuged at 100,000 × g for 1.5 hours to obtain natural OMVs. Simultaneously, *E. coli* nanovesicles were prepared from the bacterial precipitate according to the method in Example 1. The particle number and particle size distribution of the two types of vesicles were determined using a NanoCoulter single-particle analyzer (based on the principle of resistance pulse induction), with three biological replicates for each group. The results are as follows: Figure 5 As shown, starting with an equal volume of bacterial culture, the yield of natural OMVs is 10. 9 The yield is on the order of magnitude, while the yield of nanovesicles prepared in this invention is 10. 12 The magnitude is on the order of magnitude, with a yield increase of approximately 600 times. Particle size distribution results are as follows... Figure 6As shown, the nanovesicles prepared by the present invention have a particle size mainly concentrated in the range of 120–250 nm, with vesicles accounting for 82.86% within this range. The average particle size is 202 nm, and the particle size distribution width (Span) is 0.86, indicating that the nanovesicles prepared by the method of the present invention have a narrow particle size distribution and good uniformity.

[0034] (8) Applications of nanovesicles: The application of the *E. coli* nanovesicles prepared in this invention in synergistic enhancement of nucleic acid delivery with polyethyleneimine (PEI) is as follows: Preparation of transfection complex: PEI (molecular weight 40000) was mixed with pVAX1-mCherry plasmid and incubated for 15 minutes (1 μg pDNA and 2 μg PEI per well of a 24-well plate). Then, E. coli nanovesicles prepared in this invention were added (no nanovesicles were added to the control group, i.e., conventional PEI transfection). The mixture was mixed and incubated for another 30 minutes to obtain the transfection complex.

[0035] Cell seeding: Vero cells were seeded in 96-well plates and cultured overnight in DMEM containing 10% fetal bovine serum until the cell confluence reached 80%–90%.

[0036] Cell incubation with transfection complex: Before transfection, replace the culture medium with 120 μL serum-free DMEM, add 30 μL of transfection complex to the cells, and culture at 37℃ and 5% CO2 for 24 hours.

[0037] Fluorescence microscopy observation: Observe under a fluorescence microscope, photograph, and count the red fluorescent cells. Results are as follows: Figure 7 As shown, at nanovesicle concentration gradients of 0, 1, 5, 10, 20, and 50 μg / mL, compared to the control group, the experimental group with added nanovesicles prepared according to this invention exhibited a significantly enhanced red fluorescence signal, and this enhancement was concentration-dependent. This indicates that the *E. coli* nanovesicles prepared according to this invention can significantly enhance the transfection efficiency of PEI-mediated pVAX1-mCherry plasmid in Vero cells and can be used as a PEI delivery enhancer for nucleic acid delivery.

[0038] Example 2: In this embodiment, *E. coli* nanovesicles were prepared based on the HEPES-sucrose ultrasonic buffer system, as detailed below: The only difference between this embodiment and Example 1 is that the ultrasonic buffer in step (3) is replaced with a buffer containing 20 mmol / L HEPES and 500 mmol / L sucrose (pH 7.4). After ultrasonic treatment, the ultrasonically broken liquid is left to stand at 25°C for 30 minutes. The remaining steps and parameters are the same as in Example 1.

[0039] Example 3: In this embodiment, *E. coli* nanovesicles were prepared based on a Tris-HCl-sucrose ultrasonic buffer system, as detailed below: The only difference between this embodiment and Example 1 is that the ultrasonic buffer in step (3) is replaced with a buffer containing 50 mmol / L Tris-HCl and 500 mmol / L sucrose (pH 7.4). All other steps and parameters are the same as in Example 1.

[0040] Example 4: In this embodiment, *E. coli* nanovesicles were prepared based on the HEPES-sucrose ultrasonic buffer system, as detailed below: The only difference between this embodiment and Embodiment 1 is that the E. coli strain used is replaced with a commercially available one. Escherichia coli Nissle 1917 (EcN), the remaining steps and parameters are the same as in Example 1.

[0041] Example 5: In this embodiment, yeast nanovesicles were prepared based on the HEPES-sucrose ultrasonic buffer system, as detailed below: (1) Bacterial culture: The purchased Saccharomyces cerevisiae BY4741 strain was inoculated into 5 mL of yeast extract peptone glucose liquid medium (YPD) and shaken at 30℃ and 200 rpm for 12–16 hours. The next day, it was transferred to 200 mL of YPD liquid medium at a 1:100 volume ratio and cultured under the same conditions until OD600 = 1.5. The yeast cells were precipitated by centrifugation at 3,000 × g for 10 minutes.

[0042] (2) Protoplast preparation: Each gram of *Saccharomyces cerevisiae* cells was precipitated and resuspended in 10 mL of protoplastization buffer (containing 20 mmol / L Tris-HCl, 1 M sorbitol, 10 mmol / L EDTA, and 20 mg / mL snailase, pH 7.4) and incubated at 30°C for 60 minutes. Samples were taken periodically during the digestion and observed under an optical microscope. The digestion was terminated when most cells in the field of view transformed into round, translucent protoplasts. After digestion, the protoplasts were precipitated by centrifugation at 500 × g for 10 minutes and washed 2–3 times with protoplastization buffer (without snailase) to obtain purified *Saccharomyces cerevisiae* protoplasts.

[0043] (3) Ultrasonic fragmentation and membrane structure rearrangement: Each gram of protoplast precipitate was resuspended in 20 mL of sonication buffer (containing 20 mmol / L HEPES and 500 mmol / L sucrose, pH 7.4) and thoroughly mixed. The suspension was incubated at 0°C on an ice bath and sonicated using an ultrasonic cell disruptor. The sonication parameters were: power 260 W, 3 seconds of sonication followed by a 6-second pause as one cycle, repeated for a total treatment time of 14 minutes; after every 2 minutes of sonication, the suspension was incubated on an ice bath for 5 minutes to cool. After sonication, the disrupted solution was transferred to 4°C and allowed to stand for 60 minutes to allow for rearrangement and self-assembly of membrane structure fragments, yielding a mixed solution containing nanovesicles.

[0044] (4) Preliminary purification of nanovesicles: The mixed solution containing nanovesicles was centrifuged at 3,000 × g for 15 minutes at 4 °C to precipitate, and unbroken protoplasts and large fragments were removed. The supernatant was collected.

[0045] (5) Density gradient centrifugation purification: Prepare a continuous sucrose density gradient from 20% (w / v) to 60% (w / v) (using PBS to prepare 20% (w / v) and 60% (w / v) sucrose solutions, and then prepare the continuous gradient using a gradient mixer, pH 7.4). Carefully spread the supernatant on top of the sucrose density gradient medium. Centrifuge at 100,000 × g for 8 hours at 4°C. After centrifugation, distinct white vesicle bands are visible.

[0046] (6) Collection of nanovesicles: Take the white vesicle zone sample and dilute it with PBS (pH 7.4) to 2 / 3 to 3 / 4 of the total volume of the ultracentrifuge tube. Centrifuge at 100,000 × g for 1.5 hours at 4°C. Discard the supernatant and resuspend the precipitate in 50–500 μL of PBS (pH 7.4) to obtain Saccharomyces cerevisiae nanovesicles, which are then characterized.

[0047] (7) Characterization of nanovesicles: Transmission electron microscopy (TEM) observation: The obtained nanovesicles were placed on a copper grid treated with glow discharge, negatively stained with phosphotungstic acid, dried, and observed under a TEM. The *Saccharomyces cerevisiae* nanovesicles were then observed. The results are as follows: Figure 8 As shown.

[0048] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts, characterized in that, include: Step 1) The protoplasts with wall cells were resuspended in an ultrasonic buffer solution and subjected to ultrasonic disruption under ice bath conditions to obtain an ultrasonic disruption solution. The ultrasonic disruption solution was allowed to stand to allow the membrane structure to rearrange and self-assemble, resulting in a mixed solution containing nanovesicles. Step 2) Centrifuge the mixed solution containing nanovesicles at low speed and collect the supernatant; Step 3) Spread the supernatant on a sucrose density gradient medium and perform density gradient centrifugation to separate the nanovesicle zones; Step 4) Collect the nanovesicle zones, perform ultracentrifugation, discard the supernatant, and the precipitate is the nanovesicle.

2. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 1), the wall cells are either Escherichia coli or yeast.

3. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 1), the ultrasound buffer contains a physiologically compatible pH buffer and an osmotic pressure stabilizer of 200–800 mmol / L, and the pH of the ultrasound buffer is 7.0–8.0; the physiologically compatible pH buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl), or phosphate buffer (PBS); the osmotic pressure stabilizer is sucrose or trehalose.

4. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: When the physiologically compatible pH buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES) or tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl), the concentration is 10–50 mmol / L; when the physiologically compatible pH buffer is phosphate-buffered saline (PBS), it contains 137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na₂HPO₄, and 1.8 mmol / L KH₂PO₄.

5. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 1), the parameters for ultrasonic disruption are: ultrasonic power of 100-300 W, ultrasonic treatment for 3-5 seconds and pause for 6-10 seconds as one cycle, and the cycle is repeated so that the total ultrasonic disruption time is 6-15 minutes; after ultrasonic disruption, the ultrasonic disruption fluid is left to stand at 4-25℃ for 30-90 minutes.

6. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 2), the mixed solution containing nanovesicles is centrifuged at 3,000 × g for 10–30 minutes.

7. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 3), the supernatant is spread on a 20% (w / v) to 60% (w / v) sucrose density gradient medium and centrifuged at 100,000 to 200,000 × g at 4°C for 5 to 10 hours.

8. The method for preparing high-yield nanovesicles based on ultrasonic disruption of wall-dwelling protoplasts according to claim 1, characterized in that: In step 4), the nanovesicle zone is ultracentrifuged at 100,000–150,000 × g for 1–2 hours at 4°C.

9. A high-yield nanovesicle based on ultrasonic disruption of wall-dwelling protoplasts, characterized in that: The high-yield nanovesicles are prepared by the method described in any one of claims 1-8.

10. The application of the high-yield nanovesicles according to claim 9, characterized in that: The nanovesicles are used as delivery carriers in the preparation of gene drugs, vaccines, or anti-tumor drugs.

Images