Universal nano biological vesicle preparation method

Abstract

The invention relates to a universal nano biological vesicle preparation method. The preparation method comprises the following steps: carrying out crude extraction on cells from bacteria, fungi, plants or animals to obtain a crude extracting solution; the crude extracting solution is placed in a low-temperature and high-pressure homogenizer to be subjected to low-temperature and high-pressure homogenizing treatment, then the nano vesicles are obtained, the low-temperature and high-pressure homogenizer comprises a high-pressure cavity, a homogenizing valve, a working cavity, a conveying system and a temperature control system, a slit is formed in the homogenizing valve, and the crude extracting solution located in the high-pressure cavity enters the working cavity through the slit of the homogenizing valve, the pressure intensity of the high-pressure cavity is 300-3000 bar, and the working temperature of the high-pressure homogenizer is 0-10 DEG C. The activation energy is reduced by controlling high-pressure and low-temperature environments, so that the original cell membrane structure is broken, the cell wall structure is destroyed, and phospholipid bimolecules are driven to aggregate and self-assemble to form the vesicles. For biological membranes derived from bacteria, fungi, plants and animals, the vesicles can be prepared by the preparation method disclosed by the invention, and the method has universality.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a universal method for preparing nano biovesicles. Background Art

[0002] Extracellular vesicles (EVs) are cell-derived submicron-sized vesicles that can transfer substances and information between cells. Based on this feature, EVs have been used as effective therapeutic carriers for drug delivery. Compared with synthetic drug nanocarriers, natural membrane proteins, lipids and polysaccharides enable EVs to escape phagocytosis, exhibit excellent biocompatibility and inherent stability, and protect the encapsulated therapeutic drugs. However, the yield of EVs obtained by cell secretion is extremely low and the cost is extremely high, making it impossible to achieve industrial-scale production. Extracting natural nanobiovesicles through biotechnology or engineering methods is a complex technical process involving a variety of methods and technologies, which mainly depends on the source of the vesicles and application requirements. The common methods currently include (1) high-energy ball milling: grinding the raw material powder into nano-sized vesicles through the action of mechanical force by steel balls in a ball mill. However, this method of forming nanovesicles by physically squeezing cells will cause biological genetic material to be squeezed from the cell nucleus into the vesicles. After the genetic DNA fragments are taken up by the target cells, they will activate the strong cellular innate immune response mediated by the cGAS-STING signaling pathway, causing biosafety risks. (2) Ultrasonic-assisted method: The raw material powder is broken by the mechanical effect of sound waves in the liquid phase to produce uniformly dispersed nanovesicles. However, this ultrasonic-assisted method may cause structural changes or inactivation of the material, and the operation and maintenance costs of the equipment are high. (3) Enzymatic hydrolysis: The enzymatic hydrolysis method effectively decomposes the plant cell wall through enzymatic reaction to release plant exosomes. However, the enzymatic hydrolysis method is more suitable for cells with cell walls such as plants and bacteria. It has high yield and low cost, and is not applicable to animal cells without cell walls. In addition, the enzymes commonly used in the enzymatic hydrolysis method include lysozyme, mutalysin, polymerase, lysozyme and protease. These enzymes are used in combination with tissue digestion enzymes (such as collagenase, chondroitinase, hyaluronidase) to dissolve or destroy structures such as cell walls and outer membranes. However, the above enzymes will affect the proteins and other active ingredients on the vesicles, thereby affecting the function of the nanovesicles. In addition, the enzymatic hydrolysis method is highly dependent on the type of enzyme and its activity under specific conditions. The operation technology is complex and requires the control of precise parameters such as temperature, pH value, reaction time, etc., which is not universal. Therefore, there is an urgent need for a method for preparing nanovesicles that is applicable to microorganisms, plants, and animal cells. Summary of the invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the problem of lack of a universal method for preparing nano-biovesicles in the prior art.

[0004] To solve the above technical problems, the present invention provides a general method for preparing nano biovesicles. The present invention completes the self-assembly of biovesicles by controlling the temperature and pressure during the preparation process, and finally obtains nanovesicles with uniform morphology and multiple specific proteins, polysaccharides and lipids. Vesicle self-assembly is the aggregation behavior of amphiphilic molecules in water. These molecules have a hydrophilic head and a hydrophobic tail. When they are dispersed in water, the hydrophilic head faces outward to contact water, and the hydrophobic tail faces inward to form a double-layer structure. This structure enables the vesicle to exist stably in water and has a closed double-layer membrane structure. There are two main formation mechanisms of vesicles: micelle cavitation mechanism and membrane curling closure mechanism. However, in these two mechanisms, the vesicle process occurs randomly, and its formation dynamics is uncontrollable, resulting in polydispersity of vesicle size, and for vesicles from different sources, due to the different components on the cell membranes of different sources, it is difficult to form vesicles with uniform size and stable morphology through a preparation method. Especially for cells derived from bacteria, fungi and plants, due to the presence of their cell walls, enzymes or external forces are often required to destroy the cell walls to form vesicles. These methods will inevitably affect the active ingredients of the cell membrane, thereby affecting the composition of the vesicles. In the preparation process, the present invention reduces the activation energy by controlling the high pressure and low temperature environment, thereby breaking the original cell membrane structure and destroying the cell wall structure, and driving the phospholipid bimolecules to aggregate and self-assemble to form vesicles. The present invention is a nanovesicle formed by the self-assembly of a biological membrane, which has good biocompatibility, can also retain the active ingredients of the original cell membrane, and for biological membranes from bacteria, fungi, plants, and animals, it can be prepared by the preparation method of the present invention, and has universality.

[0005] The first object of the present invention is to provide a method for preparing nanovesicles, comprising the following steps:

[0006] S1. crudely extracting cells from bacteria, fungi, plants or animals to obtain a crude extract;

[0007] S2, placing the crude extract in a low-temperature and high-pressure homogenizer for low-temperature and high-pressure treatment to obtain the nanovesicles, wherein the low-temperature and high-pressure homogenizer includes a high-pressure chamber, a homogenizing valve, a working chamber, a transmission system and a temperature control system, a slit is formed in the homogenizing valve, and the crude extract in the high-pressure chamber enters the working chamber through the slit of the homogenizing valve, the transmission system is used to transmit the liquid in the working chamber into the high-pressure chamber, and the temperature control system is used to control the working temperature of the high-pressure homogenizer;

[0008] The pressure of the high-pressure chamber is 300-3000 bar, and the working temperature of the low-temperature and high-pressure homogenizer is 0-10°C.

[0009] Furthermore, the crude extract is placed in the high-pressure pump of the homogenizer, and the crude extract with high pressure is passed through the slit of the homogenizing valve to form a high-speed jet flow. In this process, the cells in the crude extract are impacted by high pressure and broken; at the same time, the high-speed flowing crude extract generates high-frequency vibration at the slit, forming a cavitation effect, further promoting the dispersion and homogenization of the biofilm; in the high-pressure jet flow, the cells in the crude extract collide with the homogenizing valve and other components at high speed, and this impact force further breaks the cells and further promotes the formation of nanovesicles. The crude extract that enters the working chamber through the homogenizing valve enters the high-pressure chamber again through the transmission system and undergoes low-temperature and high-pressure homogenization again.

[0010] Furthermore, when the pressure of the high-pressure chamber is 300-3000 bar, the crude extract is subjected to stronger shear force, impact force and cavitation effect when passing through the homogenizing valve, making the cells more easily broken and dispersed, and the formed nanovesicles are more uniform in morphology. However, too high a pressure can also lead to increased wear of the equipment, increased energy consumption, excessive damage to the biofilm, resulting in too small nanovesicles, and even affect the activity of the nanovesicles.

[0011] Furthermore, the operating temperature of the low-temperature and high-pressure homogenizer is 0-10°C, that is, the temperature of the crude extract in the high-pressure chamber, the homogenizing valve, the working chamber and the conveying system is 0-10°C. The temperatures of the crude extract in the high-pressure chamber, the homogenizing valve, the working chamber and the conveying system can be the same or different, as long as they are within the range of 0-10°C.

[0012] Furthermore, the resuspension solution in step S1 includes phosphate buffer and water.

[0013] Furthermore, the pH of the crude extract in step S1 is 6-8.

[0014] Furthermore, in step S2, the flow rate of the crude extract through the slit is 100-150 m / s.

[0015] Furthermore, the crude extract in step S2 passes through the slit 2-15 times, that is, the crude extract in the high-pressure chamber enters the working chamber through the slit of the homogenization valve, and enters the high-pressure chamber again through the transmission system for one cycle, and the cycle is repeated 2-15 times, and the nanovesicles obtained at this time have a uniform morphology. Too many low-temperature and high-pressure homogenization times may cause excessive cell fragmentation, and the resulting nanovesicles are too small, thereby affecting the stability of the nanovesicles and even affecting the biological activity of the nanovesicles. Too few homogenization times may cause uneven distribution of the particle size of the nanovesicles, affecting the final quality and stability of the nanovesicles.

[0016] Furthermore, in step S2, the crude extract is freeze-dried after low-temperature and high-pressure treatment to obtain the nanovesicles.

[0017] Furthermore, the freeze-drying temperature is -60°C to -100°C.

[0018] The second object of the present invention is to provide a nanovesicle prepared by the above preparation method.

[0019] Furthermore, the particle size of the nanovesicle is 50-500 nm, and the zeta potential of the nanovesicle is -20 mV-50 mV.

[0020] Beneficial effects of the present invention:

[0021] The present invention creates conditions for the self-assembly of vesicles by changing the environment during the vesicle preparation process, thereby obtaining nanovesicles with good stability. By controlling the temperature and pressure, the original cell membrane and the cell wall structure are broken, and the biofilm is driven to self-assemble to form nanovesicles. The obtained nanovesicles have uniform morphology and can well retain the active ingredients on the biofilm. In addition, the preparation method of the present invention is universal and is applicable to cells derived from bacteria, fungi, plants or animals, and the preparation method is simple and efficient. In addition, the nanovesicles prepared by the present invention still have good stability after freeze-drying and re-dissolution, and can be stably stored for more than one week at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0023] Figure 1 is a transmission electron micrograph of nanovesicles derived from yeast (a kind of fungus) in Example 1 (I);

[0024] Figure 2 This is a graph showing the particle size change of yeast-derived nanovesicles at room temperature in Example 1 (I);

[0025] Figure 3 This is the potential distribution diagram of the yeast-derived nanovesicles in Example 1 (I);

[0026] Figure 4 This is a graph showing the single preparation yield of yeast-derived nanovesicle powder in Example 1 (I);

[0027] Figure 5 This is a diagram of the composition analysis of yeast-derived nanovesicles in Example 1 (I);

[0028] Figure 6 The protein concentration comparison diagram and SDS-PAGE gel electrophoresis diagram of yeast-derived nanovesicles in Example 1 (I);

[0029] Figure 7This is a graph showing the single preparation yield of nanovesicle powder derived from Grifola frondosa (an edible fungus) in Example 1 (II);

[0030] Figure 8 This is a transmission electron micrograph of the nanovesicles derived from Grifola frondosa in Example 1 (ii);

[0031] Fig. 9 The particle size and potential distribution diagram of the nanovesicles derived from Grifola frondosa in Example 1 (ii);

[0032] Fig.10 This is a monosaccharide component analysis diagram of nanovesicles derived from Grifola frondosa in Example 1 (II);

[0033] Fig.11 This is a transmission electron micrograph of nanovesicles derived from Phellinus linteus (a medicinal fungus) in Example 1 (iii);

[0034] Fig.12 The particle size and potential distribution diagram of the nanovesicles derived from Phellinus igniarius in Example 1 (iii);

[0035] Fig.13 This is a transmission electron micrograph of nanovesicles derived from Coriolus versicolor (a medicinal fungus) in Example 1 (iv);

[0036] Fig.14 The particle size and potential distribution diagram of the nanovesicles derived from Coriolus versicolor in Example 1 (IV);

[0037] Fig.15 This is a graph showing the single preparation yield of nanovesicles derived from garlic (a plant) in Example 2 (I);

[0038] Fig.16 This is a transmission electron micrograph of garlic-derived nanovesicles in Example 2 (I);

[0039] Fig.17 The particle size distribution diagram of garlic-derived nanovesicles in Example 2 (I);

[0040] Fig.18 This is a graph showing the particle size change of garlic-derived nanovesicles at -20°C in Example 2 (I);

[0041] Fig.19 The protein composition analysis diagram and SDS-PAGE gel electrophoresis diagram of garlic-derived nanovesicles in Example 2 (I);

[0042] Fig. 20 This is a lipid composition analysis diagram of garlic-derived nanovesicles in Example 2 (I);

[0043] Fig.21 This is a transmission electron micrograph of the onion (a plant)-derived nanovesicles in Example 2 (ii);

[0044] Fig. 22 This is the particle size distribution diagram of onion-derived nanovesicles in Example 2 (ii);

[0045] Fig.23 This is a transmission electron micrograph of nanovesicles derived from ginger (a plant) in Example 2 (iii);

[0046] Fig.24 This is the particle size distribution diagram of ginger-derived nanovesicles in Example 2 (iii);

[0047] Fig.25 This is a transmission electron micrograph of the nanovesicles derived from mouse mesenchymal stem cells (a type of animal cell) in Example 3 (I);

[0048] Fig.26 This is the particle size distribution diagram of the mouse mesenchymal stem cell-derived nanovesicles in Example 3 (I);

[0049] Fig. 27 This is a transmission electron micrograph of the nanovesicles derived from mouse dendritic cells (a type of animal cell) in Example 3 (ii);

[0050] Fig.28 The particle size and potential distribution diagram of the mouse dendritic cell-derived nanovesicles in Example 3 (ii);

[0051] Fig.29 This is a transmission electron micrograph of the mouse dendritic cell-derived nanovesicles after freeze-drying and reconstitution in Example 3 (ii);

[0052] Fig.30 The particle size and potential distribution diagram of mouse dendritic cell-derived nanovesicles after freeze-drying and re-dissolving in Example 3 (ii);

[0053] Fig.31 This is the SDS-PAGE gel electrophoresis image of the mouse dendritic cell-derived nanovesicles in Example 3 (ii);

[0054] Fig.32 The particle size and potential distribution diagram of mouse dendritic cell-derived nanovesicles obtained under different preparation conditions in Example 3 (ii);

[0055] Fig.33 is the protein concentration of mouse dendritic cell-derived nanovesicles obtained under different preparation conditions in Example 3 (ii);

[0056] Fig.34 This is a transmission electron micrograph of nanovesicles derived from Clostridium butyricum (a type of bacteria) in Example 4 (I);

[0057] Fig.35 This is the particle size distribution diagram of the nanovesicles derived from Clostridium butyricum in Example 4 (I). DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0059] Example 1: Preparation and characterization of microbial derived nanovesicles

[0060] (I) Preparation and characterization of yeast-derived nanovesicles

[0061] The food-grade dry yeast was broken, the contents were removed and washed thoroughly, and then stored at room temperature after drying. 1g of yeast cell powder was weighed and dispersed in 1L 0.01M phosphate buffer with a pH of 7.2 and resuspended. A low-temperature high-pressure nano homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) was used for crushing. The working temperature was 4°C, the working pressure was 1700bar, and the number of operations was 10 times. The obtained product was filtered through a 0.8μm pore size filter membrane to obtain yeast-derived nanovesicles of uniform size. After freezing in a -80°C refrigerator, vacuum freeze drying was performed to obtain yeast-derived nanovesicle powder. The yield of yeast-derived nanovesicle powder was 16.2% (wt / wt).

[0062] The morphology of yeast-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Figure 1 and Figure 2 As shown in Figure 1, the particle size is about 300 nm, with a spherical morphology and uniform distribution. Zeta potential instrument (model: NanoZS90, manufacturer MALVERN) was used to evaluate the stability of the reconstituted nanovesicles under room temperature storage conditions. The results are shown in Figure 1. Figure 3 As shown in Figure 2, it can be stably stored at room temperature for at least one week, and the surface charge is about -12mV, which has good stability. The protein concentration of yeast-derived vesicles of different sizes was analyzed by BCA protein concentration detection kit, and the results are shown in Figure 2. Figure 5 As shown in Figure 2, there is basically no difference in protein concentration. SDS-PAGE gel electrophoresis shows that the proteins in the nano-sized vesicles are consistent with those in the large-sized vesicles and have not changed during the preparation process, such as Figure 6 shown.

[0063] (II) Preparation and characterization of nanovesicles derived from Grifola frondosa

[0064] Wash the maitake mushroom, weigh 3g and mix it with 500mL ultrapure water, and then squeeze it with a wall breaking machine to obtain micron-sized maitake mushroom vesicles. Use 300 mesh gauze to filter and remove the oversized vesicles, and then use a low-temperature high-pressure homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) to crush the obtained suspension. The working temperature is 4°C, the working pressure is 1800bar, and the working number is 10 times. The obtained product is filtered through a filter membrane with a pore size of 0.8μm to make the particle size of the nanovesicle reach the ideal range. After freezing in a -80°C refrigerator and vacuum freeze drying, the maitake mushroom-derived nanovesicle powder can be obtained, wherein the yield of the nanovesicles derived from maitake mushrooms is 19.7% (wt / wt).

[0065] The morphology of nanovesicles derived from Grifola frondosa was characterized using a transmission electron microscope (model: FEI TF20). Figure 8 and Fig. 9 As shown in the figure, the particle size is about 70nm, spherical and uniformly distributed. Zeta potential meter (model: NanoZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size and surface charge of nanovesicles derived from Grifola frondosa. The size is about 70nm, and the surface charge is about -20mV. Fig.10 As shown, the surface of nanovesicles derived from Grifola frondosa is also rich in various monosaccharide components.

[0066] (III) Preparation and characterization of nanovesicles derived from Phellinus igniarius

[0067] Wash Phellinus igniarius, weigh 20g and mix with 500mL ultrapure water, and then squeeze the juice with a wall-breaking machine to obtain micron-sized Phellinus igniarius vesicles. Use 300-mesh gauze to filter out oversized vesicles and then use a low-temperature high-pressure homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) to crush the resulting suspension. The working temperature is 4°C, the working pressure is 1800bar, and the number of operations is 10. The obtained product is filtered through a filter membrane with a pore size of 0.8μm to make the particle size of the nanovesicles reach the ideal range. After freezing in a -80°C refrigerator, vacuum freeze drying is performed to obtain the Phellinus igniarius-derived nanovesicle powder.

[0068] The morphology of the nanovesicles derived from Phellinus igniarius was characterized using a transmission electron microscope (model: FEI TF20). Fig.11 As shown in Figure 1, the morphology is spherical and the distribution is uniform. Zeta potential instrument (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size and surface charge of the nanovesicles derived from Phellinus igniarius. The results are shown in Figure 1. Fig.12 As shown, the size is about 250 nm and the surface charge is about -17 mV.

[0069] (IV) Preparation and characterization of nanovesicles derived from Coriolus versicolor

[0070] Wash the Versicolor, weigh 20g and mix with 500mL ultrapure water, and then squeeze the juice with a wall-breaking machine to obtain micron-sized Versicolor vesicles. Use 300-mesh gauze to filter out oversized vesicles, and then use a low-temperature high-pressure homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) to crush the resulting suspension. The working temperature is 4°C, the working pressure is 1800bar, and the working number is 10 times. The obtained product is filtered through a filter membrane with a pore size of 0.8μm to make the particle size of the nanovesicles reach the ideal range. After freezing in a -80°C refrigerator, vacuum freeze-drying is performed to obtain the Versicolor-derived nanovesicle powder.

[0071] The morphology of the nanovesicles from Coriolus versicolor was characterized using a transmission electron microscope (model: FEI TF20). Fig.13 As shown in Figure 1, the particle size is about 100 nm, with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size and surface charge of the nanovesicles derived from Versicolor. The results are shown in Figure 1. Fig.14 As shown, the size is about 100 nm and the surface charge is about -10 mV.

[0072] Example 2: Preparation and characterization of plant-derived nanovesicles

[0073] (I) Preparation and characterization of garlic-derived nanovesicles

[0074] The garlic purchased from the market was peeled, 15 g of garlic was weighed and mixed with 500 mL of 0.01 M phosphate buffer solution with a pH of 7.2, and the garlic particles were squeezed by a wall-breaking machine to obtain micron-sized garlic particles. After filtering with 300 mesh gauze to remove oversized particles, the resulting suspension was crushed by a low-temperature high-pressure nano-homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.), with an operating temperature of 4 ° C, a working pressure of 1500 bar, and a working number of 10 times. The resulting product is uniformly sized garlic-derived nanovesicles, which are frozen in a -80 ° C refrigerator and then vacuum-freeze-dried to obtain garlic-derived nanovesicle powder, and a single preparation can reach the gram level, such as Fig.15 As shown, the yield of garlic-derived nanovesicles was 10% (wt / wt).

[0075] The morphology of garlic-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Fig.16As shown in Figure 2, the particle size is about 120 nm (scale bar: 200 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size of garlic-derived nanovesicles. The results are shown in Figure 2. Fig.17 The stability of the reconstituted nanovesicles stored at -20°C was evaluated. Fig.18 As shown in Figure 2, it can be stably stored at -20°C for at least four weeks, indicating that it has good stability. Protein and lipid analysis of the prepared garlic nanovesicles was performed, as shown in Figure 2. Fig.19 and Fig. 20 As shown, the results showed that the protein and lipid compositions of garlic nanovesicles did not change before and after freeze-drying.

[0076] (II) Preparation and characterization of onion-derived nanovesicles

[0077] The onions purchased from the market were peeled, 500g of onions were weighed and mixed with 500mL of 0.01M phosphate buffer solution with a pH of 7.2, and the onion particles were squeezed by a wall breaking machine to obtain micron-sized onion particles. After filtering with 300-mesh gauze to remove oversized particles, the suspension was crushed by a low-temperature high-pressure nano homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.), with an operating temperature of 4°C, a working pressure of 1500 bar, and a working number of 10 times. The obtained product is an onion-derived nanovesicle of uniform size, which is frozen in a -80°C refrigerator and then vacuum-freeze-dried to obtain an onion-derived nanovesicle powder.

[0078] The morphology of onion-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Fig.21 As shown in Figure 1, the particle size is about 110 nm (scale bar: 200 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size of onion-derived nanovesicles. The results are shown in Figure 1. Fig. 22 As shown, it is consistent with the transmission electron microscopy image.

[0079] (III) Preparation and characterization of ginger-derived nanovesicles

[0080] The ginger purchased from the market was peeled, 500g of ginger was weighed and mixed with 500mL of 0.01M phosphate buffer solution with a pH of 7.2, and the micron-sized ginger particles were obtained after being squeezed by a wall-breaking machine. After filtering with 300-mesh gauze to remove oversized particles, the suspension was crushed using a low-temperature and high-pressure nano-homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.), with an operating temperature of 4°C, a working pressure of 1500bar, and a working number of 10 times. The resulting product is a uniform-sized ginger-derived nanovesicle, which is frozen in a -80°C refrigerator and then vacuum-freeze-dried to obtain a ginger-derived nanovesicle powder.

[0081] The morphology of ginger-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Fig.23 As shown in Figure 2, the particle size is about 180 nm (scale bar: 200 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size of ginger-derived nanovesicles. The results are shown in Figure 2. Fig.24 As shown, it is consistent with the transmission electron microscopy image.

[0082] Example 3: Preparation and characterization of animal cell-derived nanovesicles

[0083] (I) Preparation and characterization of mouse mesenchymal stem cell-derived nanovesicles

[0084] After mouse mesenchymal stem cells were cultured and collected, they were mixed with pure water, fully broken under ultrasound, and the cell membrane material was collected by ultracentrifugation, resuspended in 0.01M phosphate buffer with a pH of 7.2, and treated with a low-temperature and high-pressure nano-homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) at a working temperature of 4°C, a working pressure of 1500 bar, and a working number of 10 times. The resulting product is a uniform-sized nanovesicle derived from mouse mesenchymal stem cells, which is frozen at -80°C and then vacuum-freeze-dried to obtain a nanovesicle powder derived from mouse mesenchymal stem cells.

[0085] The morphology of mouse mesenchymal stem cell-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Fig.25 As shown in Figure 1, the particle size is about 120 nm (scale bar: 200 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer: MALVERN) was used to evaluate the hydrodynamic size of mouse mesenchymal stem cell-derived nanovesicles. The results are shown in Figure 1. Fig.26 As shown, it is consistent with the transmission electron microscopy image.

[0086] (II) Preparation and characterization of mouse dendritic cell-derived nanovesicles

[0087] After mouse dendritic cells were cultured and collected, they were washed with phosphate buffer and resuspended in homogenization buffer (0.25M sucrose, 1mM ethylenediaminetetraacetic acid (EDTA), 20mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), protease inhibition). The suspension was treated with a low-temperature high-pressure nano-homogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.) at a working temperature of 4°C, a working pressure of 500 bar, and a working number of 2 times. The obtained product was centrifuged at 3000rpm for 5min to remove large particle fragments, and the obtained product was a mouse dendritic cell-derived nanovesicle of uniform size. After being frozen in a -80°C refrigerator and vacuum freeze-dried, the mouse dendritic cell-derived nanovesicle powder was obtained.

[0088] The morphology of mouse dendritic cell-derived nanovesicles was characterized using a transmission electron microscope (model: FEI TF20). Fig. 27 As shown in Figure 2, the particle size is about 120 nm (scale bar: 200 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer: MALVERN) was used to evaluate the hydrodynamic size of mouse dendritic cell-derived nanovesicles. The results are shown in Figure 2. Fig.28 As shown, it is consistent with the transmission electron microscope image, and the surface charge is about -8mV. Fig.29 and Fig.30 As shown in Figure 2, the nanovesicle powder can be stably stored at room temperature for at least one month and has good stability. SDS-PAGE gel electrophoresis showed that the proteins of dendritic cell-derived nanovesicles were highly similar to those of dendritic cells and did not change during freeze-dried storage. Fig.31 shown.

[0089] The working pressure and working temperature were changed to prepare mouse dendritic cell-derived nanovesicles. The vesicle hydrodynamic size and location were evaluated by Zeta potential instrument. The protein concentration of vesicles obtained by different preparation methods was analyzed by BCA protein concentration detection kit. The results are as follows: Fig.32 and Fig.33 As shown, the vesicle size, potential and protein concentration obtained at working pressure of 300-500 bar and temperature of 3-4°C had no significant difference. However, the vesicle size obtained at 2000 bar and 4°C was similar, but the protein concentration dropped significantly. However, the vesicle size obtained at 2000 bar and 10°C increased significantly, and the protein concentration dropped significantly, indicating that this condition is not suitable for preparing animal cell-derived nanovesicles.

[0090] Example 4: Preparation and characterization of bacterial-derived nanovesicles

[0091] (I) Preparation and characterization of Clostridium butyricum-derived nanovesicles

[0092] After the C. butyricum culture was collected, it was mixed with pure water, fully broken under ultrasound, and resuspended in 0.01M phosphate buffer with a pH of 7.2. The resulting suspension was processed using a low-temperature high-pressure nanohomogenizer (model: JN-Mini Pro; manufacturer: Guangzhou Juneng Nano Biotechnology Co., Ltd.), with a working temperature of 4°C, a working pressure of 1500 bar, and a working number of 10 times. The resulting product is a uniform-sized C. butyricum-derived nanovesicle, which is frozen in a -80°C refrigerator and then vacuum-freeze-dried to obtain a C. butyricum-derived nanovesicle powder.

[0093] The morphology of the nanovesicles derived from Clostridium butyricum was characterized using a transmission electron microscope (model: FEI TF20). Fig.34 As shown in Figure 1, the particle size is about 190 nm (scale bar: 500 nm), with a spherical morphology and uniform distribution. Zeta potential instrument (model: Nano ZS90, manufacturer: MALVERN) was used to evaluate the hydrodynamic size of Clostridium butyricum-derived nanovesicles. The results are shown in Figure 1. Fig.35 As shown, it is consistent with the transmission electron microscopy image.

[0094] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing nano biovesicles, characterized in that: The following steps are involved: S1. crudely extracting cells from bacteria, fungi, plants or animals to obtain a crude extract; S2, placing the crude extract in a low-temperature and high-pressure homogenizer for low-temperature and high-pressure homogenization to obtain the nanovesicles, wherein the low-temperature and high-pressure homogenizer includes a high-pressure chamber, a homogenization valve, a working chamber, a transmission system and a temperature control system, wherein a slit is formed in the homogenization valve, and the crude extract in the high-pressure chamber enters the working chamber through the slit of the homogenization valve, the transmission system is used to transmit the crude extract in the working chamber into the high-pressure chamber, and the temperature control system is used to control the working temperature of the low-temperature and high-pressure homogenizer; The pressure of the high-pressure chamber is 300-3000 bar, and the working temperature of the low-temperature and high-pressure homogenizer is 0-10°C.

2. The preparation method according to claim 1, characterized in that: The crude extraction in step S1 is to crush cells derived from bacteria, fungi, plants or animals, and add a resuspension solution to obtain the crude extract.

3. The preparation method according to claim 2, characterized in that: The resuspension solution in step S1 includes phosphate buffer and water.

4. The preparation method according to claim 1, characterized in that: The pH of the crude extract in step S1 is 6-8.

5. The preparation method according to claim 1, characterized in that: In step S2, the flow rate of the crude extract through the slit is 100-150 m / s.

6. The preparation method according to claim 1, characterized in that: In step S2, the crude extract passes through the slit 2-15 times.

7. The preparation method according to claim 1, characterized in that: In step S2, the crude extract is subjected to low-temperature and high-pressure homogenization and then freeze-dried to obtain the nanovesicles.

8. The preparation method according to claim 7, characterized in that: The temperature of the freeze-drying treatment is -60°C to -100°C.

9. A nanovesicle prepared by the preparation method according to any one of claims 1 to 8.

10. The nanovesicle according to claim 9, characterized in that The particle size of the nanovesicle is 50-500nm, and the zeta potential of the nanovesicle is -20mV-50mV.

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