Preparation method and application of EV-derived engineered nano-membrane vesicles

Abstract

The invention discloses a preparation method and application of an EV-derived engineered nano-membrane vesicle, and belongs to the technical field of engineered nano-membrane vesicles. According to the invention, the engineered nano-vesicle carrier is prepared from natural membrane components and different types of liposomes by means of membrane fusion, so that bioactive molecules RNA and DNA can be effectively loaded and delivered, and the spanning function of nucleic acid nano-drugs on cell barriers can be effectively exerted; the defects that traditional phospholipid lipidosome lacks biocompatibility and natural vesicle stability are overcome, and the method is easy to operate and large in sample treatment amount and has very good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of engineered nanovesicles, and particularly relates to the preparation of multi-component fusion nanovesicles by microbial extracellular vesicles and artificial liposomes and their application in loading exogenous nucleic acid molecules. Background Art

[0002] Nucleic acid therapeutics have become an important tool in modern biotechnology and precision medicine due to their unique role in gene regulation. Nucleic acid therapeutics can not only be used to treat diseases, but also show great potential in applications such as agriculture, bioengineering, and gene editing. Through different forms of nucleic acids such as siRNA, mRNA, and CRISPR-Cas systems, scientists can precisely regulate gene expression, achieve the improvement of target traits, the expression of functional proteins, and the inhibition of specific genes, bringing unprecedented possibilities for technological innovation in multiple disciplines. In addition, nucleic acid therapeutics can act synergistically with small molecule drugs, cooperate with each other, optimize pharmacokinetic properties, and expand the scope of application.

[0003] Despite the broad application prospects of nucleic acid therapeutics, their delivery remains the biggest challenge at present. Naked nucleic acid molecules are easily degraded in vivo and difficult to effectively cross cell barriers (cell wall, cell membrane) to enter cells. For this reason, various delivery systems have emerged to help nucleic acid molecules overcome multiple barriers in vivo. These delivery systems, including liposomes, polymer nanoparticles, exosomes, and novel engineered carriers, can protect nucleic acids while enhancing their stability, targeting, and cellular uptake efficiency, thus significantly improving the bioavailability of nucleic acid therapeutics.

[0004] Existing delivery systems have their own advantages and disadvantages in nucleic acid delivery. Synthetic carriers such as liposomes and nanoparticles have the advantages of designability and large-scale production, and can adapt to different application requirements; while exosome carriers of natural origin show excellent biocompatibility and are suitable for long-term delivery in the in vivo environment. However, delivery systems still have limitations. Traditional delivery carriers such as phospholipid liposomes, although they can protect these nucleic acid molecules to a certain extent, lack broad biocompatibility and have a single function; natural extracellular vesicles (EVs) are nanoparticles secreted by microorganisms and plants, have a natural membrane structure and good biocompatibility, and show great potential as nucleic acid delivery carriers. These vesicles can not only effectively protect RNA and DNA molecules from degradation by the external environment, but also achieve effective targeted delivery through their complex membrane structure. However, the low yield and lack of homogeneity of natural extracellular vesicles limit their wide application. For this reason, developing a new generation of delivery systems and improving their performance has become a key breakthrough direction for the wider application of nucleic acid therapeutics. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for preparing and applying EV-derived engineered nanovesicles. The present invention prepares highly loaded and highly biocompatible engineered nanovesicles by means of membrane fusion from natural membrane components and different types of liposomes, which can effectively load and deliver small molecule compounds, bioactive molecule RNAs, and DNAs, effectively exert the function of nucleic acid nanodrugs to cross cell barriers, and are applicable to the biological precision control of field agricultural diseases, solving the drawbacks of traditional phospholipid liposomes lacking biocompatibility and the stability of natural vesicles.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for increasing the yield of microbial extracellular vesicles. By changing the ethanol concentration, sugar content and pH value in the culture medium and disrupting cell homeostasis to form protoplasts, and using microfiltration, the yield of extracellular vesicles (EVs) can be significantly increased.

[0008] For the above technical solution, preferably, the method includes the following steps: inoculating the microorganism in a culture medium containing ethanol, culturing at 28 - 32 °C and 150 - 250 rpm for 20 - 24 hours, centrifuging to collect the cells, inoculating into a fresh culture medium, adding 1% - 10% of snailase, culturing for 10 - 20 h, centrifuging at 6000 - 9000 g and 1 - 5 °C for 8 - 12 min to remove cell precipitates, centrifuging at 16000 - 18000 g and 1 - 5 °C for 8 - 12 min to remove cell debris, filtering the obtained supernatant through a 0.22 μm hydrophilic filter membrane to remove impurities, collecting EVs through a 0.02 μm inorganic membrane, washing 1 - 4 times with phosphate buffer, and collecting the EVs enriched on the membrane into a clean centrifuge tube with phosphate buffer.

[0009] For the above technical solution, preferably, the culture medium is yeast extract - peptone - glucose medium (YPD) and nutrient broth peptone medium (LB), the ethanol concentration in the culture medium is 3% - 15%; the glucose concentration is 3% - 6%; the pH value of the culture medium is 6 - 8; the concentration of snailase is 1% - 3%.

[0010] For the above technical solution, preferably, the microorganisms include Saccharomyces cerevisiae, Lactococcus lactis, and Escherichia coli.

[0011] The second object of the present invention is to provide the microbial extracellular vesicles and cell membrane components prepared by the above method.

[0012] The third objective of the present invention is to provide a method for preparing EV-derived engineered nanovesicles. By means of membrane fusion, the above-mentioned microbial extracellular vesicles, cell membrane components, and liposomes are used to prepare engineered nanovesicles with high loading rates and high biocompatibility.

[0013] For the above-mentioned technical solution, preferably, the liposomes include phospholipid-based neutral liposomes (LNP) and non-phospholipid cationic liposomes (STE).

[0014] For the above-mentioned technical solution, preferably, the preparation method includes a mixed incubation method and a freeze-thaw extrusion method:

[0015] Mixed incubation method: Mix the prepared microbial EV solution and the non-phospholipid cationic liposome (STE) or phospholipid-based neutral liposome (LNP) solution evenly, and incubate with shaking at 35-39 °C for 10-60 min to obtain engineered nanovesicles;

[0016] Freeze-thaw extrusion method: Mix the prepared microbial EV solution and the non-phospholipid cationic liposome (STE) or phospholipid-based neutral liposome (LNP) solution evenly, immediately freeze in liquid nitrogen for 0.5-1.5 min, then completely melt in a water bath at 35-39 °C, incubate for 3-10 min, repeat the freezing and melting process 2-5 times, and then use a manual liposome extruder to pass through filters with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm in sequence to equalize the particle size and obtain engineered nanovesicles.

[0017] For the above-mentioned technical solution, preferably, the concentration of the microbial EV solution and the non-phospholipid cationic liposome (STE) or phospholipid-based neutral liposome (LNP) solution is 10 8 -10 11 particles / mL, and the volume ratio of the two is 1:5-5:1.

[0018] For the above-mentioned technical solution, preferably, the preparation process of the non-phospholipid cationic liposome (STE) is as follows: Dissolve equimolar amounts of octadecylamine and cholesterol in a benzene / methanol solution with a volume ratio of 85:15-95:5, freeze-dry to remove the organic solvent to obtain a powder, dissolve the obtained powder in a Tris-Mes buffer solution with pH = 5, complete self-assembly by means of the repeated freeze-thaw method, and finally obtain the non-phospholipid cationic liposome after ultrasonication.

[0019] For the technical solutions described above, preferably, in the preparation process of non-phospholipid cationic liposomes (STE), the final concentrations of octadecylamine and cholesterol powder in Tris-Mes buffer are 0.5 - 5 mg / mL; the specific process of the repeated freeze-thaw method is to freeze in liquid nitrogen for 0.5 - 1.5 min, water bath at 70 °C for 5 - 15 min, and alternate and cycle 2 - 5 times, with vortex oscillation for 20 - 40 s during each cycle.

[0020] For the technical solutions described above, preferably, the preparation process of phospholipid-based neutral liposomes (LNP) is as follows: Dissolve distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and DMG-PEG2000 with a molar ratio of 4:13:1 in a citric acid buffer with pH = 4, incubate at room temperature for 5 - 20 min, then assemble into lipid nanoparticles through a 0.22 μm filter, and dialyze in a 10 kDa dialysis bag at 1 - 5 °C to obtain phospholipid-based neutral liposomes.

[0021] For the technical solutions described above, preferably, in the preparation process of phospholipid-based neutral liposomes, the concentration of distearoylphosphatidylcholine (DSPC) is 1 - 10 mM, and the dialysis solution is a phosphate buffer with pH = 7.2.

[0022] The fourth object of the present invention is to provide the engineered nanomembrane vesicles prepared by the above preparation method.

[0023] The fifth object of the present invention is to provide the application of the above engineered nanomembrane vesicles in the efficient loading and delivery of small molecule drugs, dsRNA, and plasmid DNA.

[0024] For the technical solutions described above, preferably, when loading small molecule drugs, dsRNA, or plasmid DNA, first mix the small molecule drugs, dsRNA, or plasmid DNA with liposomes to form lipid nanoparticles, and then add microbial EVs for mixing incubation or freeze-thaw extrusion.

[0025] For the technical solutions described above, preferably, the mass ratio of dsRNA or plasmid DNA to liposomes is 1 - 10:20.

[0026] The beneficial effects of the present invention compared with the prior art are as follows:

[0027] The present invention prepares an engineered nanovesicle carrier by means of membrane fusion with natural membrane components and different types of liposomes, which can effectively load and deliver small molecule drugs, bioactive molecule RNAs, and DNAs, effectively exert the function of nucleic acid nanodrugs to cross cell barriers, solve the drawbacks of traditional phospholipid-based liposomes lacking biocompatibility and the stability of natural vesicles, and the method is simple to operate, can handle a large amount of samples, and has very good application prospects. Description of the Drawings

[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings related to the embodiments will be briefly introduced below.

[0029] Figure 1 It is the particle characterization (A) and transmission electron micrograph (B) of Saccharomyces cerevisiae EV in Example 1.

[0030] Figure 2 It is the particle characterization (A) and transmission electron micrograph (B) of Lactococcus lactis EV in Example 1.

[0031] Figure 3 It is the particle characterization (A) and transmission electron micrograph (B) of EV-LNP particles prepared by the mixed incubation method in Example 2.

[0032] Figure 4 It is the particle characterization (A) and transmission electron micrograph (B) of EV-STE particles prepared by the freeze-thaw extrusion method in Example 3.

[0033] Figure 5 It is the experimental result of the efficiency of loading dsRNA into Saccharomyces cerevisiae EV by electroporation in Comparative Example 1. Among them, A is the gel electrophoresis pattern of loading dsRNA into Saccharomyces cerevisiae EV by different methods, and B is the statistical data of the efficiency of loading dsRNA into Saccharomyces cerevisiae EV. In the figure, 1: incubation method, 2: freeze-thaw method, 3: CaCl 2 treatment method, 4: electroporation method.

[0034] Figure 6 It is the result graph of the efficiency of loading dsRNA into different membrane fusion vesicles in Example 4. Among them, A is the gel electrophoresis pattern of loading dsRNA, and B is the statistical data of the efficiency of loading dsRNA. In the figure, 1: dsRNA@STE, 2: dsRNA@EV-STE, 3: dsRNA@LNP, 4: dsRNA@EV-LNP, 5: dsRNA@Saccharomyces cerevisiae EV).

[0035] Figure 7 It is the particle characterization (A) and transmission electron micrograph (B) of the fusion vesicles in Example 5.

[0036] Figure 8 It is the safety evaluation graph of Saccharomyces cerevisiae EV-LNP and Saccharomyces cerevisiae EV-STE on tomato seedlings in Examples 2 and 3.

[0037] Figure 9 It is the agglomeration (A) and breakage (B) graphs of cationic liposome STE on the cells of the pathogen (P. infestans) in Example 6.

[0038] Figure 10Experimental result graphs showing that dsRNA@EV-LNP and dsRNA@EV-STE in Example 6 successfully delivered through multiple barriers of cell wall and cell membrane into oomycete cells.

[0039] Figure 11 Particle characterization (A) and transmission electron microscopy image (B) of engineered Lpp-OmpA-eGFP Escherichia coli EVs in Example 7.

[0040] Figure 12 Particle characterization (A) and transmission electron microscopy image (B) of Escherichia coli EV-STE fusion vesicles in Example 7.

[0041] Figure 13 Experimental result of the efficiency of plasmid DNA loading into Escherichia coli EV-STE fusion vesicles in Example 7, where A is plasmid DNA@Escherichia coli EV-STE, B is plasmid DNA@Escherichia coli EV, C is plasmid DNA@STE, and D is plasmid DNA. Detailed implementation manners

[0042] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be purchased from chemical companies, and the strains used are all purchased from conventional preservation institutions.

[0043] Example 1

[0044] High-yield preparation of extracellular vesicles (EVs) from Saccharomyces cerevisiae: Saccharomyces cerevisiae was cultured in yeast extract-peptone-dextrose (YPD) medium. The composition of the medium was 1% yeast extract, 2% peptone, 4% dextrose, 5% ethanol, and the balance was water, with the pH adjusted to 7.5. The shaking culture conditions were: 30 °C, 200 rpm. The Saccharomyces cerevisiae liquid was taken from the bacterial preservation and inoculated into the YPD medium at a ratio of 1:1000 for culture to resuscitate and activate the bacterial liquid, and the activation time was 18 h. After activation, it was transferred to fresh YPD medium at a ratio of 1:100 and placed on a shaker for further scale-up culture for 22 h. Then, it was centrifuged at 8000 rpm for 10 min to obtain the Saccharomyces cerevisiae cell precipitate. The precipitate was continuously transferred to an equal volume of fresh YPD medium for continued culture, and 2% snailase (purchased from Solarbio S8280) was added for continued culture for 12 h. The bacterial liquid was centrifuged at 8000 g at 4 °C for 10 minutes to remove the cell precipitate, and then centrifuged at 17000 g at 4 °C for 10 min to remove cell debris. The supernatant was filtered through a 0.22 μm polyethersulfone hydrophilic filter membrane to remove impurities, and then EVs were collected through a 0.02 μm Anodisc inorganic membrane under a suction filtration pressure of 60 kPa. Finally, after repeated washing twice with phosphate buffer, the EVs enriched on the membrane were collected into a clean centrifuge tube with 1 mL of PBS buffer, aliquoted, and stored at -80 °C. The results are as Figure 1 shown. The characterization of Saccharomyces cerevisiae EVs showed a particle size of 75.40 ± 3.20 nm, a concentration of 1.83×10 10 particles / mL, a Zeta potential of -8.54 ± 0.90 mV, a PDI of 0.25 ± 0.09, and a typical teacup shape of EVs was presented under transmission electron microscopy.

[0045] Using the same extraction method as above, extracellular vesicles (EVs) of Lactococcus lactis were isolated and obtained with high yield. The characterization of Lactococcus lactis EVs showed that the results are as Figure 2 shown. The measured particle size was 89.60 ± 15.60 nm, the concentration was 3.95×10 10 particles / mL, the Zeta potential was -13.42 ± 1.63 mV, the PDI was 0.34 ± 0.03, and a typical teacup shape of EVs was presented under transmission electron microscopy.

[0046] Example 2

[0047] Preparation of EV-LNP by the mixed incubation method. The preparation process includes the following steps:

[0048] (1) Preparation of Saccharomyces cerevisiae EVs. Take 40 μL with a particle concentration of 4×10 9 particles / mL;

[0049] (2) Preparation of LNP liposome solution: Dissolve distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and DMG-PEG2000 at a molar ratio of 4:13:1 in 10 mM citric acid buffer with pH = 4 (the concentration of distearoylphosphatidylcholine DSPC is 5.5 mM). After incubating at room temperature for 10 minutes, form lipid nanoparticles by assembling through a 0.22 μm filter, place them in a 10 kDa dialysis bag and dialyze overnight at 4°C. The dialysis solution is PBS (pH = 7.2) phosphate buffer. Finally, obtain LNP. The concentration of the LNP liposome solution is 2.26×10 11 particles / mL;

[0050] (3) Dilute the Saccharomyces cerevisiae EV in step (1) and the LNP liposome solution obtained in step (2) to 4×10 9 particles / mL. Take 40 μL each (volume ratio 1:1) and mix well, then incubate with shaking at 37°C for 30 min to obtain the fused vesicles EV-LNP.

[0051] The detection results are as Figure 3 shown. The particle size of EV-LNP is 95.90 ± 0.2 nm, and the transmission electron microscopy detection results show that the formed hybrid carrier has a spherical-like structure.

[0052] Example 3

[0053] Preparation of EV-STE by freeze-thaw extrusion method: The preparation process includes the following steps:

[0054] (1) Prepare Saccharomyces cerevisiae EV, take 40 μL with a particle concentration of 4×10 9 particles / mL;

[0055] (2) Preparation of STE liposome solution: Dissolve octadecylamine and cholesterol at a molar ratio of 1:1 in a benzene / methanol (V / V = 90 / 10) solution, freeze-dry for 16 h to remove the organic solvent to obtain a powder. Dissolve the powder in Tris-Mes buffer with pH = 5, with a final concentration of 1 mg / mL. Complete the self-assembly process using the freeze-thaw method. The reaction conditions are freezing in liquid nitrogen for 1 minute, water bath at 70°C for 10 minutes, alternating and cycling 3 times, vortexing for 30 s during each cycle, and finally placing it in an ultrasonic crusher for 20 minutes, with a cycle of turning on for 20 s and turning off for 5 s during the process, and an amplitude of 40%. Finally, obtain STE. The concentration of the STE liposome solution is 7.10×10 11 particles / mL;

[0056] (3) The Saccharomyces cerevisiae EV of step (1) and the STE liposome solution obtained in step (2) were fully mixed at a concentration ratio of 1:4, immediately placed in liquid nitrogen for freezing for 1 min, and then completely thawed in a 37°C water bath and incubated for about 5 min. The operation was repeated 3 times and then the particle size was homogenized by passing through filter membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm using a manual extruder (Genizer, USA) to obtain fusogenic vesicles EV-STE.

[0057] Test results such as Figure 4 As shown, the particle size of EV-STE is 98.60±3.30nm, and the transmission electron microscopy results show that the formed hybrid carrier has a spherical structure.

[0058] Comparative Example 1

[0059] Using electroporation to load dsRNA, 1 μg of dsRNA (SEQ ID NO: 1) was mixed with 40 μL of particles at a concentration of 2×10 9 The Saccharomyces cerevisiae EVs were mixed with 2000 μg / mL of Saccharomyces cerevisiae particles / mL, and a pulse time of 15 ms, a pulse voltage of 400 V, and 50 μF were used. The pulses were cycled twice, and the samples were finally visualized by agarose gel electrophoresis.

[0060] The results are as follows Figure 5 As shown in the figure, electroporation allows dsRNA to be partially loaded into EVs and retained in the agarose gel pores. Under electroporation, the concentration is 2×10 9 The efficiency of loading 1 μg of dsRNA into EVs with a particle size of 1.54 particles / mL was approximately 23.1%.

[0061] Incubation method: 1 μg of dsRNA was mixed with 40 μL of particles at a concentration of 2 × 10 9 Saccharomyces cerevisiae EVs with a mixing and shaking condition of 50 particles / mL were incubated at 37°C for 30 min, and agarose gel electrophoresis results showed that almost no dsRNA was actively loaded into EVs.

[0062] Freeze-thaw method: Mix the dsRNA and Saccharomyces cerevisiae EVs under the same conditions as above, immediately freeze them in liquid nitrogen for 1 min, and then completely thaw them in a 37°C water bath. Incubate for about 5 min, and repeat the freeze-thaw operation 3 times. The results of agarose gel electrophoresis showed that only a small amount of dsRNA could be actively loaded into EVs, with a loading rate of about 3%.

[0063] CaCl 2 Treatment method: Add 0.1M CaCl to the above solvent 2 Then perform the freeze-thaw operation.

[0064] Example 4

[0065] Fusion membrane vesicle EV-STE loaded with dsRNA (dsRNA@EV-STE): 10 μL of dsRNA with a concentration of 1 μg / μL (the nucleotide sequence is the same as that of the dsRNA in Comparative Example 1) was mixed and incubated with the STE liposome solution prepared in Example 3 (10 μL, 1.6×10 10 particles / mL) at 37 °C for 30 min with shaking. Then, Saccharomyces cerevisiae EV (10 μL, 4×10 9 particles / mL) was added, and dsRNA loading was achieved after repeated freezing and thawing and extrusion. The specific process was as follows: immediately frozen in liquid nitrogen for 1 min, then completely melted in a 37 °C water bath, incubated for about 5 min, and after repeating the operation 3 times, the particle size was homogenized using a manual extruder (Genizer, USA) through filters with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm to obtain the fusion membrane vesicle EV-STE loaded with dsRNA. Finally, the loading efficiency of the hybrid vector was investigated by agarose gel electrophoresis, and the gray scale of the band was measured using image J analysis software, with unencapsulated dsRNA as the control.

[0066] Fusion membrane vesicle EV-LNP loaded with dsRNA (dsRNA@EV-LNP): dsRNA was first loaded into the LNP prepared in Example 2 at a nitrogen-to-phosphorus ratio of 3:1, that is, 10 μL of dsRNA with a concentration of 1 μg / μL (the nucleotide sequence is the same as that of the dsRNA in Comparative Example 1) was added to a mixture of 55 nmol of distearoylphosphatidylcholine DSPC, 178.75 nmol of cholesterol Chol, and 13.75 nmol of DMG-PEG2000. After incubation at room temperature for 10 minutes, lipid nanoparticles were formed by assembling through a 0.22 μm filter, placed in a 10 kDa dialysis bag, and dialyzed overnight at 4 °C. The dialysis solution was PBS (pH = 7.2) phosphate buffer. Then, Saccharomyces cerevisiae EV (10 μL, 4×10 9 particles / mL) was added, and dsRNA loading was achieved after mixing and incubating for 30 min. Finally, the loading efficiency of the hybrid vector was investigated by agarose gel electrophoresis, and the gray scale of the band was measured using image J analysis software, with unencapsulated dsRNA as the control.

[0067] Loading dsRNA into LNP (dsRNA@LNP): 10 μL of dsRNA with a concentration of 1 μg / μL (the nucleotide sequence is the same as that of the dsRNA in Comparative Example 1) was added to a mixed powder of 55 nmol of distearoylphosphatidylcholine (DSPC), 178.75 nmol of cholesterol (Chol), and 13.75 nmol of DMG-PEG2000. After incubation at room temperature for 10 minutes, lipid nanoparticles were assembled through a 0.22-μm filter and placed in a 10-kDa dialysis bag for overnight dialysis at 4°C. The dialysis solution was PBS (pH = 7.2) phosphate buffer.

[0068] Loading dsRNA into STE (dsRNA@STE): 10 μL of dsRNA with a concentration of 1 μg / μL (the nucleotide sequence is the same as that of the dsRNA in Comparative Example 1) was mixed and incubated with the STE liposome solution (10 μL, 1.6×10 10 particles / mL) prepared in Example 3 at 37°C for 30 min with shaking.

[0069] Loading dsRNA into Saccharomyces cerevisiae EV (dsRNA@EV): The same as Comparative Example 1.

[0070] The experimental results are as Figure 6 shown. The dsRNA loading efficiency of EV-STE for 1 μg of dsRNA was 94.65 ± 1.09%, and the dsRNA loading efficiency of EV-LNP for 1 μg of dsRNA was approximately 80.81 ± 12.34%. Both were higher than the dsRNA loading by electroporation of EVs alone and also higher than the loading capacity of LNP alone.

[0071] Based on the above results, it shows that the fusion membrane vesicles EV-STE and EV-LNP maintain the performance of high loading rate for dsRNA, and have a higher dsRNA loading capacity compared to microbial EVs; compared to artificial liposomes LNP and STE, the lipid composition of the fusion membrane vesicles can be closer to the structure of biological membranes, thereby reducing the risk of triggering immune responses or other adverse reactions in vivo.

[0072] Example 5

[0073] Loading the small molecule drug sodium butyrate into the cell membrane component-STE fusion membrane vesicle:

[0074] (1) Obtaining cell membrane components: Lactococcus lactis was cultured with reference to Example 1. 100 mL of the overnight culture was centrifuged at 8000 g for 10 min at 4 °C to obtain a Lactococcus lactis precipitate. After resuspension with PBS buffer, it was centrifuged and washed again at 8000 g for 10 min at 4 °C, and this washing process was repeated twice. The obtained precipitate was placed in 20 mL of a highly alkaline solution (sodium carbonate, pH = 11) and sonicated for 10 - 15 min. During the sonication treatment, it was cycled with 9 s on and 1 s off, and the amplitude was 40%. After fragmentation, it was centrifuged at 8000 g and 17000 g for 10 min at 4 °C in a gradient manner to remove the unbroken cell precipitate and impurities, and then passed through a 0.22 μm filter membrane. Then the supernatant was ultracentrifuged at 170000 g for 15 min to obtain a rich transparent gel precipitate at the bottom, which was the membrane sheet. It was resuspended in 1 mL of buffer (Tris 50 mM, MES 50 mM, NaCl 130 mM, EDTA 0.5 mM, pH = 5.0). Finally, the freshly obtained cell membrane was frozen in liquid nitrogen for 30 s, heated in a 40 °C water bath for 5 min, vortexed for 10 s, and this cycle was repeated 3 times. Then it was diluted 10 times with buffer (Tris 50 mM, MES 50 mM, NaCl 130 mM, EDTA 0.5 mM, pH = 5.0) for standby.

[0075] (2) Preparation of sodium butyrate@STE: Referring to the preparation method of STE in Example 3, 50 mg of the freeze-dried powder was weighed and dissolved in 10 mL of buffer (Tris 50 mM, MES 50 mM, NaCl 130 mM, EDTA 0.5 mM, pH = 5.0). After dissolution and stirring evenly, 12 mg of sodium butyrate was added, frozen in liquid nitrogen for 1 min, then heated in a 70 °C water bath for 10 min, and vortexed for 30 s. To ensure good hydration, this cycle was repeated 5 times. Finally, it was placed in an ultrasonic crusher and treated for 20 min. During the treatment, it was cycled with 9 s on and 3 s off, and the amplitude was 40%. Then, free sodium butyrate was removed by centrifugation at 3500 g for 25 min through an ultrafiltration centrifugal tube (10 kD). Finally, sodium butyrate@STE was collected.

[0076] (3) Preparation and characterization of sodium butyrate@cell membrane component-STE: 2 mL of the diluted cell membrane solution was mixed with 2 mL of sodium butyrate@STE and placed in an ultrasonic crusher for treatment for 5 min. During the treatment, it was cycled with 9 s on and 3 s off, and the amplitude was 40%; thus, the fusion vesicles of Lactococcus lactis cell membrane components and STE loaded with the small molecule drug sodium butyrate were achieved.

[0077] The results are as Figure 7 shown. The prepared fusion vesicles had a particle size of 70.8 nm and a concentration of 1.27×10 12particles / mL, spherical with a multi-layer membrane structure under transmission electron microscopy. The encapsulation efficiency for sodium butyrate is 31.08%, and the drug loading rate is 6.47%.

[0078] Example 6

[0079] Plant safety evaluation of the fusion membrane vesicles: For tomato seeds, the paper towel germination method was used. When white dots of germination appeared in 2 - 3 days, they were transplanted into commercial potted soil and cultured for 5 - 6 weeks in an environment with 16 h of daytime, relative humidity of 40%, light intensity of 33%, and temperature of 22°C; 8 h of night, relative humidity of 80%, no light, and temperature of 18°C. After cultivation, plants with the same height were selected for the next experiment. Control group: 1 mL of deionized water was evenly sprayed on tomato plants; Experimental group: 1 mL of EV-STE (7×10 10 particles / mL) and EVs-LNP (2×10 10 particles / mL) from Examples 2 and 3 were respectively and evenly sprayed on tomato plants. After seven days, the growth status of the plants was observed and evaluated.

[0080] The results are as Figure 8 shown. Whether it is the tomato plants sprayed with water or the fusion vesicles, on the seventh day, the leaves grew well and no symptoms such as curling, yellowing, or withering appeared. This indicates that neither of the two EV-STE and EV-LNP had an adverse effect on the plants. Therefore, the fusion membrane vesicles have advantages in improving drug loading performance and biocompatibility.

[0081] In the field of plant protection, the delivery process of nucleic acid drugs is more challenging. RNA and DNA molecules are extremely easily degraded in the open natural environment; at the same time, due to the existence of multiple barriers such as cell walls and cell membranes in most pests or pathogens, penetrating these barriers has become the core challenge. Both EV-LNP and EV-STE can effectively penetrate the cell barriers of pathogens and ensure the integrity and viability of cells, realizing the function of delivering nucleic acid drugs to the target. Taking the penetration ability of pathogens (P. infestans) as an example:

[0082] The pathogen (P. infestans) mycelial blocks were inoculated on rye solid medium (80 g of rye was crushed, 20 g of sucrose, 15 g of agar powder, 1 g of calcium carbonate, dissolved in 1 L of water, sterilized at 120°C for 20 min and then poured into plates), and cultured in the dark at 18°C for 7 - 9 days until the surface of the medium was covered with white mycelia. Sterile water was added to the petri dish to wash and collect the sporangia of the pathogen, and impurities and large particle debris were removed by filtering through two layers of degreased cotton gauze, obtaining a buffer solution rich in sporangia. 1 μL of the sporangia-enriched buffer solution was aspirated under an ordinary microscope, and the sporangia particle concentration was calculated. Then the sporangia buffer solution was diluted to approximately 10 5-10 6 in the range of particles / mL, placed at 4°C. Take 5 μL of the STE solution (7×10 10 particles / mL) in Example 3 and mix it with 15 μL of the sporangium suspension;

[0083] Label the dsRNA with fluorescein-UTP (the nucleotide sequence is the same as that of the dsRNA in Comparative Example 1, using the commercial labeling kit Fluorescein RNA Labeling Mix Kit, 11685619910, sigma). Prepare dsRNA@EV-STE and dsRNA@EV-LNP according to the method in Example 4. Take 5 μL of each and mix it with 15 μL of the sporangium suspension, then incubate overnight at 18°C in the dark. Subsequently, observe the uptake of dsRNA by oomycetes under a fluorescence microscope.

[0084] Compared with the cell aggregation and damage caused by treating the pathogen with STE alone ( Figure 9 ), the fusion membrane vesicles exhibit biosafety and nucleic acid delivery ability. As Figure 10 shown, the uptake of fluorescently labeled dsRNA by oomycetes was visualized by a Zeiss fluorescence confocal microscope. At the initial stage of sporangia, germination, and hyphal formation stages, exogenous naked dsRNA was hardly transferred into oomycetes. However, both dsRNA@EV-STE and dsRNA@EV-LNP, the two engineered fusion membrane vesicles, could well transfer dsRNA into oomycetes. This indicates that they can better target organisms and help exogenous dsRNA cross multiple barriers such as cell walls and cell membranes during sporangium germination.

[0085] Example 7

[0086] Preparation of engineered bacterial extracellular vesicles and preparation of bacterial EV-STE by freeze-thaw extrusion method:

[0087] (1) Preparation of a laboratory-engineered Escherichia coli strain and extraction and characterization of Escherichia coli EV: A laboratory-engineered Escherichia coli was prepared, and an Lpp-OmpA-eGFP fusion plasmid (SEQ ID NO; 2) was designed and transformed into the Escherichia coli BL21 strain to prepare an Lpp-OmpA-eGFP engineered bacterium. Cultivate in LB medium for 16 h, and the shaking culture conditions are: 38°C, 200 rpm. Then transfer it into fresh 100 mL LB medium at a ratio of 1:100, and place it on a shaker to continue the enlarged culture until OD 600The value was 0.6. IPTG with a final concentration of 0.1 mM was added, and induction expression was carried out at 30 °C and 200 rpm for 12 h. Then, centrifugation was performed at 8000 rpm for 10 min to obtain the culture medium supernatant. Centrifugation was carried out at 8000 g and 4 °C for 10 min to remove the cell precipitate, and then centrifugation was carried out at 17000 g and 4 °C for 10 min to remove cell debris. The supernatant was filtered through a 0.22-μm polyethersulfone hydrophilic filter membrane to remove impurities, and then EVs were collected through a 0.02-μm Anodisc inorganic membrane under a suction filtration pressure of 60 kPa. Finally, after repeated washing twice with phosphate buffer solution, the EVs enriched on the membrane were collected into a clean centrifuge tube with 1 mL of PBS buffer solution, aliquoted, and stored at -80 °C. The results are as Figure 11 shown. The characterization of engineered Escherichia coli EVs showed a particle size of 68 ± 28 nm and a concentration of 1.15×10 10 particles / mL. The fluorescence of eGFP on the vesicles was detected, and the labeling efficiency reached 100%. Under transmission electron microscopy, it presented the typical teacup shape of EVs.

[0088] (2) Bacterial EV-STE was prepared by referring to the method of Example 3. The detection results are as Figure 12 shown. The particle size of bacterial EV-STE was 82.2 ± 35.8 nm and the concentration was 1.09×10 11 particles / mL. The detection results of transmission electron microscopy showed that the formed hybrid carrier presented a spherical-like structure.

[0089] Plasmid DNA was loaded using bacterial EV-STE: 10 μL of plasmid DNA (SEQ ID NO; 3) with a concentration of 1 μg / μL was mixed and oscillated with the diluted bacterial EV-STE solution (10 μL, 1.6×10 10 particles / mL) at 37 °C for 30 min to achieve the loading of plasmid DNA. As Figure 13 shown, the gel retardation assay showed that the fusion vesicles could effectively load plasmid DNA.

Claims

1. A method for increasing the yield of extracellular vesicles of microorganisms, characterized in that: By changing the ethanol concentration, sugar content and pH value in the culture medium and breaking the cell homeostasis to form protoplasts, the yield of extracellular vesicles (EVs) was significantly increased using micromembrane filtration.

2. The method according to claim 1, characterized in that The method comprises the following steps: inoculating the microorganism in a culture medium containing ethanol, culturing at 28-32°C and 150-250rpm for 20-24 hours, collecting the bacteria by centrifugation, inoculating into a fresh culture medium, adding 1%-10% snail enzyme, culturing for 10-20 hours, centrifuging at 6000-9000g and 1-5°C for 8-12 minutes to remove the cell precipitate, centrifuging at 16000-18000g and 1-5°C for 8-12 minutes to remove the cell debris, filtering the obtained supernatant through a 0.22μm hydrophilic filter membrane to remove impurities, collecting EVs through a 0.02μm inorganic membrane, adding phosphate buffer for washing 1-4 times, and collecting the EVs enriched on the membrane into a clean centrifuge tube with phosphate buffer; The culture medium is a yeast extract-peptone-glucose medium (YPD) and a meat extract peptone medium (LB), wherein the ethanol concentration in the culture medium is 3% to 15%; the glucose concentration is 3% to 6%; the pH value of the culture medium is 6 to 8; and the concentration of snail enzyme is 1% to 3%; The microorganisms include saccharomyces cerevisiae, Lactococcus lactis and Escherichia coli.

3. The microbial extracellular vesicle or cell membrane component prepared by the method according to claim 1 or 2.

4. A method for preparing EV-derived engineered nanomembrane vesicles, characterized in that: The microbial extracellular vesicles or cell membrane components described in claim 3 and liposomes are used for membrane fusion to prepare engineered nano-membrane vesicles with high loading rate and high biocompatibility; The liposomes include phospholipid neutral liposomes (LNP) and non-phospholipid cationic liposomes (STE).

5. The preparation method according to claim 4, characterized in that: The preparation method includes a mixed incubation method and a freeze-thaw extrusion method: Mixed incubation method: The prepared microbial EV solution and non-phospholipid cationic liposome (STE) or phospholipid neutral liposome (LNP) solution are mixed evenly, and incubated at 35-39°C for 10-60 minutes to obtain engineered nanomembrane vesicles; Freeze-thaw extrusion method: The prepared microbial EV solution and non-phospholipid cationic liposome (STE) or phospholipid neutral liposome (LNP) solution were mixed evenly, immediately placed in liquid nitrogen for freezing for 0.5-1.5 min, and then completely thawed in a water bath at 35-39°C, incubated for 3-10 min, and the freezing and thawing process was repeated 2-5 times. The particle size was homogenized by passing through filter membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm in sequence using a manual liposome extruder to obtain engineered nanomembrane vesicles.

6. The preparation method according to claim 5, characterized in that: 10% of microbial EV solution and non-phospholipid cationic liposome (STE) or phospholipid neutral liposome (LNP) solution 8 ~10 11 particles / mL, and the volume ratio of the two is 1:5 to 5:

1.

7. The preparation method according to claim 5, characterized in that: The preparation process of non-phospholipid cationic liposomes (STE) is as follows: dissolving equimolar octadecylamine and cholesterol in a benzene / methanol solution with a volume ratio of 85:15 to 95:5, freeze-drying, removing the organic solvent to obtain a powder, dissolving the obtained powder in a Tris-Mes buffer at pH=5, completing self-assembly by repeated freeze-thaw method, and finally obtaining non-phospholipid cationic liposomes after ultrasonication; The preparation process of phospholipid neutral liposomes (LNPs) is as follows: distearoylphosphatidylcholine DSPC, cholesterol Chol and DMG-PEG2000 with a molar ratio of 4:13:1 are dissolved in a citric acid buffer at pH = 4, incubated at room temperature for 5 to 20 minutes, and then assembled through a 0.22 μm filter to form lipid nanoparticles, which are placed in a 10 kDa dialysis bag and dialyzed at 1 to 5°C to obtain phospholipid neutral liposomes.

8. The engineered nanomembrane vesicles prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the engineered nanomembrane vesicles according to claim 8 in the efficient loading and delivery of small molecule drugs, dsRNA, and plasmid DNA.

10. The use according to claim 9, characterized in that: When loading small molecule drugs, dsRNA or plasmid DNA, the small molecule drugs, dsRNA or plasmid DNA are first mixed with liposomes to form lipid nanoparticles, and then microbial EVs are added for mixed incubation or freeze-thaw extrusion.

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