Preparation method of plant exosome-like nano vesicles

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of plant exosome-like nano vesicles. The method comprises the steps of juicing, centrifugation, filtration, ultrafiltration and the like. According to the method, ultrafiltration tubes of different specifications are adopted for ultrafiltration and centrifugation, the plant exosome-like nano-vesicles can be rapidly and mildly separated, and the obtained vesicle membrane is complete in structure, obvious in double-layer membrane and good in active substance retention effect. The preparation method provided by the invention has the advantages of simple and easily available process and equipment, no need of additional addition of reagents, easiness in control, low cost and suitability for obtaining high-yield vesicles in a short time, can be used as a drug delivery carrier, and shows good application potential in the fields of skin care products, medical beauty products, functional foods and nutritional health-care products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing plant exosome-like nanovesicles. Background Technology

[0002] In recent years, plant exosome-like nanovesicles (PELNs) have emerged as a new type of cell-free nanomedicine, bringing new hope to disease treatment. Among them, plant-derived exosome-like nanovesicles, due to their good biocompatibility, potentially low immunogenicity, natural targeting, stability, and ability as drug carriers, have shown great application value in the biopharmaceutical field (especially in drug delivery, anti-inflammation, anti-tumor, and tissue repair), cosmetics, and functional foods. For example, ginger exosome-like nanovesicles have been shown to have therapeutic potential in treating rheumatoid arthritis, alleviating alcoholic liver damage, regulating gut microbiota metabolism, and periodontitis-induced tissue damage; white radish exosome-like nanovesicles have also been shown to have anti-inflammatory and immunomodulatory effects.

[0003] Plant exosome-like nanovesicles refer to nanoscale vesicles obtained by disrupting and physically extracting plant tissues. In existing methods for preparing plant exosome-like nanovesicles, the separation process mainly employs the following methods: (1) Tangential flow filtration (TFF) is a cross-flow filtration method in which a pump drives fluid through the surface of a filter membrane to flush away molecules trapped on it, thereby minimizing fouling on the membrane surface. Solutes and small molecules pass through the filter membrane through fluid shear forces parallel to the membrane surface, thus obtaining the desired vesicles. However, this method has high technical requirements, and parameters need to be monitored at all times during operation.

[0004] (2) Ultracentrifugation (UC) uses the centrifugal force generated by ultracentrifugation (100,000-200,000 × g) to separate exosomes. Alternating between low-speed and high-speed centrifugation can separate vesicle particles of similar size. However, ultracentrifugation also has disadvantages such as being time-consuming and labor-intensive, and the purity being affected by centrifugation parameters. In addition, the operation requires ultracentrifugation, which may damage the structure of exosomes, and the equipment requirements are high.

[0005] (3) Size exclusion chromatography (SEC) separates components based on the difference in migration rate of different particle sizes in the column, which is filled with porous gel particles. When the sample flows through the column, substances larger than the pore size of the gel cannot enter the pores and can only be eluted through the gaps between the particles; while smaller molecules can enter the channels inside the gel, thus achieving separation. However, this method has some limitations: on the one hand, the elution process dilutes the exosome sample, resulting in a low final exosome concentration; on the other hand, for large-volume samples, pre-concentration is usually required, making the entire operation time-consuming. Summary of the Invention

[0006] Therefore, the present invention provides a method for preparing plant exosome-like nanovesicles that can at least partially solve the above-mentioned problems.

[0007] For example, the preparation method provided by the present invention does not require expensive centrifugation and purification equipment, and the operation process is simple and fast; the plant exosome-like nanovesicle membranes prepared by the preparation method provided by the present invention have high structural integrity and high yield.

[0008] This disclosure provides the following technical solution: In a first aspect, the present invention provides a method for preparing plant exosome-like nanovesicles, the method comprising the following steps: S1. Clean the plant tissues, extract the juice, filter, remove the residue, and collect the filtrate; S2. Centrifuge the collected filtrate and collect the supernatant; S3. Filter the collected supernatant to remove impurities and collect the filtrate rich in exosomes; S4. The filtrate rich in exosomes was subjected to ultrafiltration and centrifugation to obtain plant exosome-like nanovesicles.

[0009] In one alternative embodiment, the plant tissue includes, but is not limited to, any one of ginger, white radish, and grape.

[0010] In one optional embodiment, the juicing conditions in step S1 are: low-speed spiral extrusion for 3-5 minutes at (23±2)℃.

[0011] In one alternative implementation, in step S2, centrifugation is differential centrifugation.

[0012] In one optional implementation, the differential centrifugation includes: Centrifuge at a centrifugal force of 3000~4000g for 30~60min and collect the supernatant. And / or, centrifuge at a centrifugal force of 10000~12000g and a centrifugation time of 60~90min, and collect the supernatant.

[0013] In one alternative implementation, the filtering includes: Use homogenizing bags or gauze for filtration; And / or, use a cell filter membrane with a thickness of 0.45~1.2μm for filtration.

[0014] In one optional implementation, step S4 includes two ultrafiltration centrifugation steps.

[0015] In one optional implementation, the conditions for the two ultrafiltration centrifugations are as follows: First ultrafiltration centrifugation: Use a 1000kDa ultrafiltration tube to centrifuge and concentrate the filtrate at 3~5℃, with a concentration ratio of 15~20:1; Second ultrafiltration centrifugation: Use a 300kDa ultrafiltration tube to centrifuge and concentrate the filtrate at 3~5℃, with a concentration ratio of 15~20:1.

[0016] Secondly, the present invention also provides a method for preparing plant exosome-like nanovesicles according to the above preparation method; In one optional embodiment, the plant exosome-like nanovesicles include any one of the following: ginger exosome-like nanovesicles, white radish exosome-like nanovesicles, or grape exosome-like nanovesicles.

[0017] Thirdly, the present invention also provides the application of plant exosome-like nanovesicles prepared according to the above-described method for preparing plant exosome-like nanovesicles as drug delivery carriers. In one alternative implementation, the application includes applications in skin care products, medical aesthetic products, functional foods, and nutritional supplements.

[0018] The technical solution of this invention has the following advantages: 1. This invention uses a combination of centrifugation and ultrafiltration to prepare plant nano-exosome vesicles. It eliminates the need for expensive centrifugation purification equipment, and the operation process is simple and fast, yielding plant nano-exosome vesicles with high membrane structure integrity and high yield.

[0019] 2. No additional reagents are added during the extraction process of this invention. A physical method is used for gentle crushing and extraction to reduce plant cell debris contamination. The resulting plant nano-like exosome vesicles have intact membrane structures, obvious double membrane structures, and high retention of active substances. They are aliquoted and frozen at -80°C to avoid repeated freeze-thaw cycles and maximize the retention of active substances.

[0020] 3. This invention uses ultrafiltration tubes of different specifications for ultrafiltration centrifugation, which can quickly separate plant nano-like exosome vesicles; centrifugation is performed twice, and the centrifugation time is short, which can prevent some large-diameter exosomes from clogging the ultrafiltration membrane, and can also prevent the membrane structure of some large-diameter exosomes from being damaged and ruptured due to shear force caused by excessive centrifugation time, thereby obtaining high yield of exosome-like nanovesicles with intact membrane structure.

[0021] 4. The preparation method employed in this invention is simple and rapid, suitable for obtaining high yields of plant-based nano-exosome vesicles in a short time. The exosome vesicles prepared by this method possess advantages such as extremely high biocompatibility, wide availability, and low cost. These exosome vesicles can serve as natural drug delivery carriers, exhibiting high biocompatibility and low immunogenicity, as well as anti-inflammatory, immunomodulatory, and tissue repair functions. In skin care and aesthetic applications, these exosome vesicles act as intercellular communication carriers, achieving deep repair through precise delivery of active ingredients in the skincare field. Their nanoscale size allows them to penetrate the stratum corneum, promoting collagen production and improving skin barrier function, demonstrating outstanding performance in anti-aging and repairing UV damage. In functional foods and nutritional supplements, relying on plant active ingredients, these exosome vesicles can enhance bioavailability, enabling the development of functional foods with antioxidant and immunomodulatory effects. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 These are transmission electron microscope (TEM) images of ginger exosome-like nanovesicles prepared in Example 1 of the present invention, viewed at 100 nm (A) and 500 nm (B). Figure 2 These are transmission electron microscope (TEM) images of the white radish exosome-like nanovesicles prepared in Example 2 of the present invention, viewed at 100 nm (A) and 500 nm (B). Figure 3 These are transmission electron microscope (TEM) images of grape exosome-like nanovesicles prepared in Example 3 of the present invention, viewed at 100 nm (A) and 500 nm (B). Figure 4 These are transmission electron microscope (TEM) images of ginger exosome-like nanovesicles prepared in Comparative Example 1 of this invention, viewed at 100 nm (A) and 500 nm (B). Figure 5This is a nanoparticle tracking analysis (NTA) diagram of ginger exosome-like nanovesicles prepared in Example 1 of this invention; Figure 6 This is a nanoparticle tracking analysis (NTA) diagram of the white radish exosome-like nanovesicles prepared in Example 2 of this invention; Figure 7 This is a nanoparticle tracking analysis (NTA) diagram of grape exosome-like nanovesicles prepared in Example 3 of the present invention; Figure 8 This is a nanoparticle tracking analysis (NTA) diagram of the ginger exosome-like nanovesicles prepared in Comparative Example 1 of this invention. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0025] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0026] Example 1 The preparation steps of a ginger exosome vesicle are as follows: S1. Wash the ginger, cut it into small pieces, and juice it at a low speed using a spiral press at 21℃ for 3 minutes; S2. Filter the solution through gauze to remove residue and collect the filtrate; Perform the first centrifugation at 3.4000g for 30min and collect the supernatant. Perform a second centrifugation at 4.12000g for 60min, and collect the supernatant. S5. Filter using a 0.8μm cell filter membrane to remove large-diameter impurities and collect the filtrate rich in exosomes; S6. Using a 1000kDa ultrafiltration tube (i.e., the pore size of the filter membrane allows molecules with a molecular weight ≤1000kDa to pass through), centrifuge at 3000g and 4℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 40min and the concentration ratio was 15. Collect ginger exosome-like nanovesicles. S7. Using a 300kDa ultrafiltration tube (i.e., the pore size of the filter membrane allows molecules with a molecular weight ≤300kDa to pass through), centrifuge at 3500g and 4℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 60min and the concentration ratio was 15. Collect ginger exosome-like nanovesicles and store them in a -80℃ freezer.

[0027] Example 2 The preparation steps of a white radish exosome vesicle are as follows: S1. Wash the white radish, cut it into small pieces, and juice it at a low speed using a spiral press at 25℃ for 4 minutes; S2. Filter the mixture through a homogenizing bag to remove residue and collect the filtrate; S3.3000g, 40min for the first centrifugation, and collect the supernatant; Perform a second centrifugation at 4.11000g for 80min, and collect the supernatant. S5. Filter using a 0.45μm cell filter membrane to remove large-diameter impurities and collect the filtrate rich in exosomes; S6. Using a 1000kDa ultrafiltration tube (i.e., the pore size of the filter membrane allows molecules with a molecular weight ≤1000kDa to pass through), centrifuge at 3200g and 5℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 45min and the concentration ratio was 20. Collect white radish exosome-like nanovesicles. S7. Using an ultrafiltration tube with a filter membrane specification of 300kDa (i.e., the filter membrane pore size allows molecules with a molecular weight ≤300 kDa to pass through), centrifuge at a centrifugal force of 3400g and 5℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 80min and the concentration ratio was 20. Collect the white radish exosome-like nanovesicles and store them in a -80℃ refrigerator.

[0028] Example 3 The preparation steps of a grape exosome vesicle are as follows: S1. Wash the grapes and juice them at 21°C using a low-speed spiral press for 4 minutes. S2. Filter the mixture through a homogenizing bag to remove residue and collect the filtrate; The sample was centrifuged for 3.3500g for 60min for the first time, and the supernatant was collected. S4. Centrifuge for 10000g for 90min and collect the supernatant. S5. Filter using a 1.2μm cell filter membrane to remove large-diameter impurities and collect the filtrate rich in exosomes; S6. Using a 1000kDa ultrafiltration tube (i.e., the pore size of the filter membrane allows molecules with a molecular weight ≤1000kDa to pass through), centrifuge at 3500g and 3℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 30min and the concentration ratio was 18. Collect grape exosome-like nanovesicles. S7. Using an ultrafiltration tube with a filter membrane specification of 300kDa (i.e., the filter membrane pore size allows molecules with a molecular weight ≤300 kDa to pass through), centrifuge at a centrifugal force of 3000g and 3℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 50min and the concentration ratio was 18. Collect grape exosome-like nanovesicles and store them in a -80℃ freezer.

[0029] Comparative Example 1 This comparative example provides a method for preparing ginger exosomes. The raw materials and preparation steps are the same as in Example 1, except that the 1000 kDa ultrafiltration tube in step S6 is omitted. The preparation operation steps are as follows: S1. Wash the ginger, cut it into small pieces, and juice it at a low speed using a spiral press at 21℃ for 3 minutes; S2. Filter the solution through gauze to remove residue and collect the filtrate; Perform the first centrifugation at 3.4000g for 30min and collect the supernatant. Perform a second centrifugation at 4.12000g for 60min, and collect the supernatant. S5. Filter using a 0.8μm cell filter membrane to remove large-diameter impurities and collect the filtrate rich in exosomes; S6. Using a 300kDa ultrafiltration tube (i.e., the pore size of the filter membrane allows molecules with a molecular weight ≤300kDa to pass through), centrifuge at 3500g and 4℃ to concentrate the filtrate. The specific centrifugation time depends on the concentration ratio. In this case, the centrifugation time was 150min and the concentration ratio was 15. Collect ginger exosome-like nanovesicles and store them in a -80℃ freezer.

[0030] Test Example 1: Transmission Electron Microscopy (TEM) Testing of Plant Exosome-like Nanovesicles The operating procedures for transmission electron microscopy (TEM) testing are as follows: S1. Adsorption: Take 10-20 μL of sample suspension, drop it onto the electron microscope grid, and let it stand for 10 minutes. Then use a pipette to remove the excess droplets and let it dry slightly.

[0031] S2. Staining: Take 20 μL of uranium acetate staining solution, drop it onto the electron microscope grid, and let it stand for 2-5 minutes.

[0032] S3. Drying: Use a pipette to remove excess dye and let it stand for 5-6 minutes to air dry.

[0033] S4. Image Acquisition: Observe and acquire images under a transmission electron microscope.

[0034] TEM image of ginger exosome-like nanovesicles prepared in Example 1 is shown below. Figure 1 As shown, under different fields of view, the intact nanovesicle membrane structure of ginger exosomes can be observed, and the double membrane structure is obvious.

[0035] TEM image of the white radish exosome-like nanovesicles prepared in Example 2 is shown below. Figure 2 As shown, radish exosome-like nanovesicles can be observed under different fields of view, with intact membrane structures and obvious bilayer membrane structures.

[0036] TEM image of the grape exosome-like nanovesicles prepared in Example 3 is shown below. Figure 3 As shown, grape exosome-like nanovesicles can be observed under different fields of view, with intact membrane structures and obvious bilayer membrane structures.

[0037] TEM image of ginger exosome-like nanovesicles prepared in Comparative Example 1 is shown below. Figure 4 As shown, ginger exosome-like nanovesicles can be observed under different fields of view. The membrane structure is relatively intact, showing a slightly wrinkled state, and the double membrane structure is obvious.

[0038] Test Example 2: Tracking Analysis (NTA) of Plant Exosome-like Nanovesicle Nanoparticles The procedure for nanoparticle tracking analysis (NTA) testing is as follows: Using a nanoparticle analyzer, load the diluted exosome solution onto a 1mL syringe and observe the particle image on the screen, adjusting the focus as needed. Adjust the dilution factor based on the loading results.

[0039] Specifically, the process includes: powering on → cleaning → loading samples → setting parameters → adjusting the field of view → setting the save path → detection → automatic analysis of results by the instrument → generating a report → saving, unloading samples, and powering off.

[0040] The NTA test results of ginger, white radish, grape, and ginger exosome-like nanovesicles prepared in Examples 1, 2, 3, and Comparative Example 1 are shown in Table 1. Figure 5 , Figure 6 , Figure 7 , Figure 8 As shown.

[0041] Table 1. NTA test results of exosome-like nanovesicles

[0042] According to the NTA test results, there are differences in the average particle size of exosome-like nanovesicles extracted from different plant species, and the particle concentration also varies depending on the plant species and the concentration ratio. Example 1 and Comparative Example 1 both use the same plant, but there are significant differences in average particle size and particle concentration. The preparation steps for Comparative Example 1 are the same as in Example 1, except that the 1000 kDa ultrafiltration tube in step S6 is omitted. Therefore, based on the experimental process and results, in order to achieve the same concentration ratio as in Example 1, the ultrafiltration centrifugation time in Comparative Example 1 was longer, causing some of the larger exosome-like nanovesicle membranes to rupture, resulting in a smaller average particle size and concentration of the harvested nanovesicles. The above data indicates that the single-pass ultrafiltration method used in Comparative Example 1 has significant limitations in terms of vesicle yield.

[0043] Test Example 3: Quantitative BCA Detection of Plant Exosome-like Nanovesicle Proteins The quantitative detection of plant exosome proteins using BCA was performed using the Micro BCA™ Protein Detection Kit (catalog number: 23235). For detailed operating procedures, please refer to the instruction manual.

[0044] The formula for calculating the particle-to-protein ratio is: Particle-to-protein ratio = Number of particles / Protein concentration.

[0045] The BCA protein detection results of ginger, white radish, grape and ginger exosome-like nanovesicles prepared in Examples 1, 2 and 3 and Comparative Example 1 are shown in Table 2.

[0046] Table 2. BCA protein detection results of exosome-like nanovesicles

[0047] The particle-to-protein ratio is an indicator used to evaluate the purity of nanoparticles such as exosomes. A higher ratio indicates that more particles are obtained with the same amount of protein, less contamination from impurity proteins or non-target particles, and higher purity. Example 1 and Comparative Example 1 both use the same plant, but the particle-to-protein ratio of Comparative Example 1 is much lower than that of Example 1. The preparation steps of Comparative Example 1 are the same as those of Example 1, except that the 1000 kDa ultrafiltration tube in step S6 is omitted. The above data shows that the single-pass ultrafiltration method used in Comparative Example 1 has significant limitations in terms of vesicle yield.

[0048] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing plant exosome-like nanovesicles, characterized in that, The method includes the following steps: S1. Clean the plant tissues, extract the juice, filter, remove the residue, and collect the filtrate; S2. Centrifuge the collected filtrate and collect the supernatant; S3. Filter the collected supernatant to remove impurities and collect the filtrate rich in exosomes; S4. The filtrate rich in exosomes was subjected to ultrafiltration and centrifugation to obtain plant exosome-like nanovesicles.

2. The method for preparing plant exosome-like nanovesicles according to claim 1, characterized in that, The plant tissues include, but are not limited to, any one of ginger, white radish, and grape.

3. The method for preparing plant exosome-like nanovesicles according to claim 1, characterized in that, In step S1, the juicing conditions are: low-speed spiral extrusion for 3-5 minutes at (23±2)℃.

4. The method for preparing plant exosome-like nanovesicles according to claim 1, characterized in that, In step S2, centrifugation is differential centrifugation.

5. The method for preparing plant exosome-like nanovesicles according to claim 4, characterized in that, The differential centrifugation includes: Centrifuge at a centrifugal force of 3000~4000g for 30~60min and collect the supernatant. And / or, centrifuge at a centrifugal force of 10000~12000g and a centrifugation time of 60~90min, and collect the supernatant.

6. The method for preparing plant exosome-like nanovesicles according to claim 1, characterized in that, The filtering includes: Use homogenizing bags or gauze for filtration; And / or, use a cell filter membrane with a thickness of 0.45~1.2μm for filtration.

7. The method for preparing plant exosome-like nanovesicles according to claim 1, characterized in that, In step S4, the ultrafiltration centrifugation step includes two ultrafiltration centrifugations.

8. The method for preparing plant exosome-like nanovesicles according to claim 7, characterized in that, The conditions for the two ultrafiltration centrifugations are as follows: First ultrafiltration centrifugation: Use a 1000kDa ultrafiltration tube to centrifuge and concentrate the filtrate at 3~5℃, with a concentration ratio of 15~20:1; Second ultrafiltration centrifugation: Use a 300kDa ultrafiltration tube to centrifuge and concentrate the filtrate at 3~5℃, with a concentration ratio of 15~20:

1.

9. Plant exosome-like nanovesicles are prepared by the preparation method according to any one of claims 1-8; Optionally, the plant exosome-like nanovesicles include any one of the following: ginger exosome-like nanovesicles, white radish exosome-like nanovesicles, or grape exosome-like nanovesicles.

10. The application of plant exosome-like nanovesicles prepared by the method of preparing plant exosome-like nanovesicles according to any one of claims 1-8 as drug delivery carriers; Optionally, the application includes applications in skin care products, medical aesthetic products, functional foods, and nutritional supplements.

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