Method for large-scale preparation of plant exovesicles

Abstract

The invention relates to the technical field of preparation of extracellular vesicles, and particularly discloses a method for preparing plant extracellular vesicles on a large scale, which comprises the following steps: (1) carrying out gradient centrifugation on a filtrate obtained by juicing and filtering plants to obtain a crude extract; (2) filtering the crude extract by using a filter membrane, removing small-particle insoluble substances in filtrate, and collecting the filtrate; (3) fully mixing the collected filtrate and the aqueous two-phase solution, performing centrifugal separation, discarding an upper phase, and collecting a lower phase; and further concentrating to obtain a plant extracellular vesicle solution. Strong denaturation chemical reagents and violent reaction are not introduced in the whole process, damage to the outer vesicles is small, an ultra-speed centrifugal machine or immunomagnetic beads are not needed, the method is suitable for industrial production, and the outer vesicles from various sources can be treated.

Description

Technical Field

[0001] The invention belongs to the technical field of extracellular vesicle preparation, and particularly relates to a method for large-scale preparation of plant extracellular vesicles. Background Art

[0002] Extracellular vesicles are secreted by cells and wrapped by lipid bilayers. They are a heterogeneous group of membrane structures, including exosomes and microvesicles, which originate from the endosomal system or fall off from the plasma membrane. The membrane structure of extracellular vesicles is similar to that of cell membranes, wrapped by lipid bilayers, and contains various biomolecules, including proteins, lipids, RNA (such as mRNA and miRNA) and DNA fragments.

[0003] Extracellular vesicles play an important role in intercellular communication. They can transfer their contents to other cells by directly fusing with target cells or being taken up by target cells, thereby affecting the function of target cells. They have shown important biological functions and potential clinical application value in the fields of immune response, tumor microenvironment regulation, neurodegenerative diseases, cardiovascular diseases, and regenerative medicine. In recent years, the study of extracellular vesicles as potential biomarkers and drug carriers has attracted much attention. Due to their ability to carry and protect their contents from degradation, as well as their stable presence in body fluids such as blood and urine, extracellular vesicles are believed to have broad application prospects in disease diagnosis, prognosis assessment, and personalized treatment.

[0004] Plant extracellular vesicles are a type of tiny membrane bubble-like structures secreted by plant cells, with diameters usually ranging from 30 to 150 nanometers. They have a double lipid membrane structure and carry a variety of bioactive molecules, including proteins, lipids, RNA molecules, and secondary metabolites. They play an important role in intercellular communication, immune regulation, and resistance to pests and diseases. In addition, due to their natural origin and biocompatibility, plant extracellular vesicles also show potential medicinal value and have attracted widespread attention in the fields of drug delivery systems and cancer treatment.

[0005] There are many technologies for extracting extracellular vesicles at present, the most commonly used method is ultracentrifugation. The main disadvantage of this method is that although the operation process is simple, it is easy to be contaminated by riboproteins and aggregated proteins during the extraction process. The isolated exosomes have low purity, low total protein concentration, and low yield. They cannot meet the needs of large-scale production and the purity does not meet clinical requirements.

[0006] The kit method is not convenient for isolating EVs from a large volume, and unknown contaminants may be introduced into the sample.

[0007] Sucrose density gradient centrifugation: Although this method achieves better results in terms of EV purity and EV protein and RNA content, it can lead to considerable EV loss, and the operation is complex, laborious, time-consuming, and requires expensive equipment. Therefore, it is not suitable for clinical use.

[0008] None of the above methods can meet the requirements of large-scale extraction and purification, which limits the application scope of the industrialization of extracellular vesicles. Summary of the invention

[0009] In view of the above problems, the present invention aims to provide a method for large-scale preparation of plant exosomes, thereby providing a new approach for large-scale preparation of plant exosomes.

[0010] To achieve the above purpose, Figure 1 As shown, the plant extracellular vesicle scale extraction method adopted by the present invention comprises the following steps: (1) Wash the plants, squeeze the juice and filter it. Centrifuge the filtrate at 1000-2000g for 10-30 minutes at 4°C to collect the supernatant; centrifuge the supernatant at 3000-6000g for 20-40 minutes at 4°C to collect the supernatant; centrifuge the supernatant at 8000-10000g for 40-60 minutes at 4°C to collect the supernatant, thus completing the gradient low-speed centrifugation operation.

[0011] (2) Filter the filtrate collected in step (1) through a filter membrane with a pore size of 0.22-0.45 um and collect the filtrate.

[0012] (3) The filtrate collected in step (2) and the aqueous biphasic solution are fully mixed and centrifuged to discard the upper phase and collect the lower phase; the step is repeated 1 to 2 times for the lower phase to further concentrate the extracellular vesicle solution.

[0013] Furthermore, the two-phase aqueous solution is prepared by dissolving polyethylene glycol and dextran in distilled water or PBS buffer.

[0014] Furthermore, the concentration of polyethylene glycol in the aqueous two-phase solution is 20-80 g / L, and the concentration of dextran is 14-60 g / L.

[0015] Furthermore, the volume ratio of the filtrate to the aqueous two-phase solution is preferably 1:0.3-3.

[0016] The molecular weight of the polyethylene glycol is 5kDa to 35kDa, and the molecular weight of the dextran is 70kDa to 650kDa.

[0017] The centrifugal force of the centrifugal separation is 1000-3000g, and the centrifugal time is 10-40 minutes.

[0018] The present invention mixes the plant filtrate with a two-phase aqueous solution composed of polyethylene glycol and dextran, and then centrifuges the mixture. Through concentration and preliminary purification, the exosomes are concentrated to the dextran phase, and the upper phase is removed. After repeating this process 1-2 times, the concentration of the exosomes reaches a concentration effect of 6-15 times that of the initial filtrate.

[0019] The present invention is simple to operate, avoids the limitation of ultracentrifugation on the extraction scale, and is suitable for industrial extraction and concentration. The whole experimental process does not introduce strong denaturing chemical reagents and violent reactions, causes less damage to the exocysts, and does not require ultracentrifuges or immunomagnetic beads, is suitable for industrial production, and can process plant exocysts from various sources.

[0020] Attached photos Figure 1 Schematic diagram of the process flow for large-scale preparation of plant extracellular vesicles.

[0021] Figure 2 This is the nanoparticle tracking analysis result of the ginger extracellular vesicles obtained in Example 1.

[0022] Figure 3 This is a TEM image of the ginger extracellular vesicles obtained in Example 1.

[0023] Figure 4 This is the detection result of nanoparticle tracking analysis of pasteurized ginger extracellular vesicles obtained in Example 1.

[0024] Figure 5 This is a TEM image of the pasteurized ginger extracellular vesicles obtained in Example 1.

[0025] Figure 6 This is the zeta potential of the ginger extracellular vesicles in Example 1 and the ginger extracellular vesicles after pasteurization.

[0026] Figure 7 This is the detection result of nanoparticle tracking analysis of the turmeric extracellular vesicles obtained in Example 2.

[0027] Figure 8 This is a TEM image of the turmeric extracellular vesicles obtained in Example 2.

[0028] Fig. 9 is the zeta potential of the turmeric extracellular vesicles obtained in Example 2. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples. It should be correctly understood that the examples or drawings of the present invention are only used to illustrate the present invention, rather than to limit the present invention.

[0030] First, the present invention provides a method for preparing plant extracellular vesicles.

[0031] In some embodiments, the plant extracellular vesicle has a particle size of 23-400 nm and has a double-layer phospholipid layer vesicle structure.

[0032] The following is an exemplary description of the preparation method of plant extracellular vesicles provided by the present invention, which mainly comprises the following steps: (1) Wash the plants, squeeze the juice and filter the juice. Centrifuge the filtrate at 1000-2000g for 10-30 minutes at 4°C to collect the supernatant. Centrifuge the supernatant at 3000-6000g for 20-40 minutes at 4°C to collect the supernatant. Centrifuge the supernatant at 8000-10000g for 40-60 minutes at 4°C to collect the supernatant. The gradient low-speed centrifugation operation is completed. The filtered ginger juice filtrate is subjected to gradient low-speed centrifugation. The sedimentation coefficients of different components in the plant cells are used to separate various components under the action of different centrifugal forces to remove components that may interfere with the extraction of plant extracellular vesicles.

[0033] (2) Filter the supernatant after gradient low-speed centrifugation with a 0.22-0.45 μm filter membrane to remove small insoluble particles in the filtrate and collect the filtrate; (3) The collected filtrate and the aqueous two-phase solution are fully mixed and then centrifuged to discard the upper phase and collect the lower phase; this step is repeated 1 to 2 times for the lower phase to further concentrate the extracellular vesicle solution.

[0034] The plant extracellular vesicle extraction method described in the present invention can be widely applied to various plants.

[0035] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples in a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0036] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0037] Example 1 Preparation of ginger extracellular vesicles.

[0038] The ginger juice was squeezed with a juicer, and the obtained ginger juice filtrate was filtered using a Buchner funnel and centrifuged at 1000 g for 10 minutes at 4°C to collect the supernatant; The supernatant was centrifuged at 4000 g for 20 minutes at 4°C to collect the supernatant; The supernatant was centrifuged at 10000 g for 40 minutes at 4°C to collect the supernatant, completing the gradient low-speed centrifugation operation; The supernatant was filtered through a 0.22 μm filter membrane to remove small insoluble particles in the filtrate, and the filtrate was collected and stored in a refrigerator at 4° C. Formulated with 7.7% PEG 35k A PBS solution with 3.3% dextran-T500 was used as the aqueous two-phase solution.

[0039] The collected filtrate and the aqueous two-phase solution were fully mixed in a 1:1 ratio and then centrifuged at 1000 g for 40 minutes, the upper phase was discarded, and the lower phase was collected; this step was repeated 1 to 2 times for the lower phase to further purify the extracellular vesicle solution.

[0040] The obtained exosome solution was diluted 10 times and then pasteurized for long-term storage. The particle size of the pasteurized exosome solution did not change much, but the potential increased.

[0041] Figure 2 The nanoparticle tracking analysis results of ginger extracellular vesicles obtained in Example 1 are as follows: Figure 3 is a TEM image of ginger extracellular vesicles obtained in Example 1, Figure 4 The nanoparticle tracking analysis results of the extracellular vesicles of pasteurized ginger obtained in Example 1 are as follows: Figure 5 This is a TEM image of the pasteurized ginger extracellular vesicles obtained in Example 1. Figure 6 is the zeta potential of the ginger extracellular vesicles and the pasteurized ginger extracellular vesicles in Example 1; wherein, Figure 2 The horizontal axis is the particle size, in nm; the vertical axis is the concentration, in ×10 9 particles / mL. Figure 4 The horizontal axis is the particle size in nm, and the vertical axis is the concentration in 10 8 particles / mL. . From Figure 2 It can be seen that the size of ginger extracellular vesicles is about 23~400nm, and the concentration is 1.48×10 11 particles / mL. Figure 3 It can be seen that the size of the pasteurized ginger extracellular vesicles is about 51~500nm, and the concentration is 6.73×10 10 particles / mL. Figure 6 It can be seen that the Zeta potential of ginger extracellular vesicles before and after pasteurization is -20 to -40 mV.

[0042] Example 2 Preparation of turmeric extracellular vesicles.

[0043] After squeezing the turmeric with a juicer, the ginger juice filtrate was filtered using a sand core funnel and centrifuged at 2000 g for 30 minutes at 4°C to collect the supernatant; The supernatant was centrifuged at 4000 g for 40 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 10000 g for 60 minutes at 4°C to obtain the supernatant, thereby completing the gradient low-speed centrifugation operation; The supernatant was filtered through a 0.45 um filter membrane to remove small insoluble particles in the filtrate, and the filtrate was collected and stored in a refrigerator at 4°C; Formulated with 7.7% PEG 35k A PBS solution with 3.3% dextran-T500 was used as the aqueous two-phase solution.

[0044] The collected filtrate and the aqueous biphase solution were fully mixed in a 1:1 ratio and then centrifuged at 1000 g for 20 minutes, the upper phase was discarded, and the lower phase was collected; this step was repeated 1 to 2 times for the lower phase to further purify the extracellular vesicle solution.

[0045] Figure 7 The nanoparticle tracking analysis results of the turmeric extracellular vesicles obtained in Example 2 are as follows: Figure 8 is a TEM image of the turmeric extracellular vesicles obtained in Example 2, Fig. 9 is the zeta potential of turmeric extracellular vesicles; Figure 7 The horizontal axis is the particle size in nm, and the vertical axis is the concentration in 10 8 particles / mL. Figure 7 and Fig. 9 It can be seen that the size of turmeric extracellular vesicles is around 23~400nm, and the concentration is 1.47×10 10 particles / mL, and the Zeta potential is -15~-20mV.

[0046] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for large-scale preparation of plant extracellular vesicles, characterized in that: The following steps are involved: 1) Centrifugal extraction: wash the plant, squeeze the juice and filter it, centrifuge the filtrate at 1000-2000g for 10-30 minutes at 4°C, and collect the supernatant; centrifuge the supernatant at 3000-6000g for 20-40 minutes at 4°C to collect the supernatant; centrifuge the supernatant at 8000-10000g for 40-60 minutes at 4°C to collect the supernatant, and complete the gradient low-speed centrifugation operation; 2) Filtration: Filter through a filter membrane with a pore size of 0.22-0.45um and collect the filtrate; 3) Two-phase separation: the filtrate collected in step 2) and the two-phase aqueous solution are fully mixed and centrifuged to discard the upper phase and collect the lower phase; The lower phase was repeated 1-2 times for further concentration to obtain an extracellular vesicle solution.

2. The method for preparing plant extracellular vesicles according to claim 1, characterized in that: The two-phase aqueous solution is prepared by dissolving polyethylene glycol and dextran in distilled water or PBS buffer.

3. The method for preparing plant extracellular vesicles according to claim 1 or 2, characterized in that: The concentration of polyethylene glycol in the two-phase aqueous solution is 20-80 g / L, and the concentration of dextran is 14-60 g / L.

4. The method for preparing plant extracellular vesicles according to claim 1 or 2, characterized in that: The volume ratio of the filtrate to the aqueous two-phase solution is preferably 1:0.3-3.

5. The method for preparing plant extracellular vesicles according to claim 1 or 2, characterized in that: The molecular weight of the polyethylene glycol is 5kDa to 35kDa, and the molecular weight of the dextran is 70kDa to 650kDa.

6. The method for preparing plant extracellular vesicles according to claim 1 or 2, characterized in that: The centrifugal force of the centrifugal separation is 1000-3000g, and the centrifugal time is 10-40 minutes.

7. The method for preparing plant extracellular vesicles according to claim 1 or 2, characterized in that: The plant extracellular vesicle has a particle size ranging from 23 to 400 nm, has a double-layer phospholipid layer vesicle structure, and a Zeta potential ranging from -15 to -40 mV.

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