KR102183104B1 - A Method for Purifying Plant-Derived Extracellular Vesicles - Google Patents

Abstract

The present invention relates to a method for isolating extracellular vesicles from biological samples, particularly plant tissues. The present invention can be usefully used in an efficient separation and purification process capable of obtaining high purity and high quality while maintaining the inherent therapeutic activity and drug delivery activity of the extracellular vesicles.

Description

Method for Purifying Plant-Derived Extracellular Vesicles {A Method for Purifying Plant-Derived Extracellular Vesicles}

The present invention relates to a method for purifying plant-derived extracellular vesicles using size exclusion chromatography.

The extracellular endoplasmic reticulum has various sizes, and in particular, the smallest exosomes are small cell membrane-like blisters of 50-150 nm. Extracellular endoplasmic reticulum is secreted from human and animal as well as insect, plant, and microbial cells. In particular, they contain specific molecules such as proteins, nucleic acids, lipids, and carbohydrates possessed by cells, and are surrounded by a double layer of lipids to reliably protect these molecules and deliver them to other cells after secretion. Extracellular vesicles, found in high concentrations in body fluids such as blood, urine, and saliva in vivo, have potential as new biological materials due to this characteristic of.

Compared to humans, animals, and microorganisms, there have been few studies on plant-derived extracellular vesicles. However, in a recent study, in animal experiments using extracellular vesicles present in grapes, ginger, etc., it was confirmed that the orally administered extracellular vesicles are delivered to cells in the large intestine of mice, and in particular, it can be used to treat inflammatory diseases in the colon. There were reported results. In other words, it was found that plant-derived extracellular vesicles not only contain useful components of plants, but also can safely deliver these molecules to cells present in the large intestine after feeding, through which the representative diseases of the large intestine, such as colon cancer and colon There is a possibility that inflammatory diseases (such as Crohn's disease) can be treated.

Edible plant-derived extracellular endoplasmic reticulum 1) is a natural substance and has very few side effects; 2) Not only can it reach the large intestine through oral administration; 3) It can be applied to infants and children because it is more convenient than edible directly as food; 4) Mass production from plants is possible, leading a new paradigm of health functional food; 5) It has the advantage that it can be applied as an efficient drug delivery system that can be delivered to the large intestine by loading pharmacological ingredients.

However, despite these advantages, the biological functions and applications of plant-derived extracellular vesicles used as food, as well as their extraction and purification methods, have not been systematically studied. Plant-derived extracellular vesicles generally have a relatively high concentration of plant-derived impurities in the ultra-high-speed centrifugation method used to separate extracellular vesicles, and physical disruption of the extracellular vesicles may be caused by strong centrifugal force. Polymer sedimentation methods such as ExoQuick (SBI), which are commercially available and sold, have a very high concentration of impurities and have a contamination problem due to the use of PEG (polyethylene glycol). Therefore, for the industrial use of edible plant-derived extracellular vesicles, the development of a new technology capable of efficiently separating them in large quantities is required.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Patent Document 1. US Patent Publication No. 2010-0184046 Patent Document 2. US Patent Publication No. 2018-0369351

The present inventors have made intensive research efforts to develop an efficient method for obtaining extracellular vesicles derived from biological samples, specifically plant-derived extracellular vesicles. As a result, when separating and purifying using size exclusion chromatography, deviating from the conventionally used polymer sedimentation method or ultra-high-speed centrifugation method, the impurity content is greatly reduced, while there is no physical crushing applied to the endoplasmic reticulum. And by finding that it is possible to obtain a high-purity, high-quality extracellular endoplasmic reticulum that maintains the drug delivery activity as it is, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for separating extracellular vesicles from a biological sample containing extracellular vesicles.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for separating extracellular vesicles comprising the step of performing size exclusion chromatography on a biological sample including the extracellular vesicles.

The present inventors have made intensive research efforts to develop an efficient method for obtaining extracellular vesicles derived from biological samples, specifically plant-derived extracellular vesicles. As a result, when separating and purifying using size exclusion chromatography, deviating from the conventionally used polymer sedimentation method or ultra-high-speed centrifugation method, the impurity content is greatly reduced, while there is no physical crushing applied to the endoplasmic reticulum. And it was found that it is possible to obtain high purity, high quality extracellular vesicles maintaining the drug delivery activity as it is.

In the present specification, the term "extracellular vesicle" refers to a small vesicle of a membrane structure secreted from various cells, has a diameter in the range of approximately 30-1,000 nm, and the outer cell through the fusion of the polycyst and the plasma membrane. Released into the environment.

As used herein, the term “biological sample” refers to a sample that has been collected, isolated from, or derived from living organisms, and which has been processed or pretreated containing an effective amount of the component of interest. Biological samples include tissue sections from animals and plants, frozen or paraffin-embedded sections from animals and plants for pharmacological, diagnostic or food purposes, cells, cell cultures, primary cultures, and sections. It may include an explant, but is not limited thereto. In addition, the "biological sample" may be a material collected from an environment (eg, water, air, soil, etc.) suspected of the presence of a specific living organism.

Specifically, the biological sample of the present invention is at least one selected from the group consisting of an animal cell culture solution, an animal tissue, a plant cell culture solution, a plant tissue, a bacteria culture solution, and a yeast culture solution. More specifically, the biological sample is a plant cell culture medium or plant tissue, and most specifically, a plant tissue.

In the present specification, the term “size exclusion chromatography (SEC)” refers to a material with a size smaller than the diameter of the bead pores of the stationary phase among the materials constituting the heterogeneous mixture enters the pore and elutes relatively slowly. The large-sized material is rapidly eluted along the mobile phase without entering the pore, thereby separating the material based on the difference in moving speed according to the size of each material.

According to a specific embodiment of the present invention, in the size exclusion chromatography used in the present invention, the average pore diameter of the stationary phase is 30-75 nm.

According to a specific embodiment of the present invention, the method of the present invention further comprises the step of performing ultrafiltration on the biological sample including the extracellular vesicles in any order.

In the present specification, the term “ultrafiltration” refers to a membrane-based separation process in which each material constituting a heterogeneous mixed solution is separated along a semipermeable membrane by pressure or concentration gradient. The ultrafiltration membrane has a pore size with a constant cutoff value. According to a specific embodiment of the present invention, the ultrafiltration used in the present invention has a cut-off value of 3 to 100 kDa, more specifically, a cut-off value of 5 to 50 kDa, Most preferably, it has a cut-off value of 5 to 15 kDa.

When the step of performing ultrafiltration is additionally included in the method of the present invention, this may be performed before size exclusion chromatography or after completion of chromatography.

More specifically, the method of the present invention includes sequentially performing the ultrafiltration and the size exclusion chromatography. In the case of performing size-exclusion chromatography after ultrafiltration, the volume of the sample is reduced by concentration through ultrafiltration, and then size-exclusion chromatography is applied so that a more efficient separation process can be achieved.

According to a specific embodiment of the present invention, when the biological sample of the present invention is a real tissue, the plant tissue is obtained by a method comprising the following steps.

(a) crushing the plant and then extracting it to obtain an extract;

(b) filtering the extract to remove solids; And

(c) centrifuging the filtrate of step (b).

In the present specification, the term "juice" refers to a liquid fluid obtained by exhaling moisture contained in a plant tissue to the outside by applying a mechanical or physical force without applying heat or enzymes, for example, fruit juice, Vegetable juice and root juice are included, but are not limited thereto.

More specifically, the step (b) is filtered so that solids having a diameter of 1 mm or more are removed.

More specifically, the step (c) is carried out by performing the first centrifugation at 7,000-9,000 x g and second centrifugation of the supernatant at 19,000-21,000 x g. More specifically, the step (c) is performed by first centrifugation at 7,500-8,500 x g, and then second centrifugation of the supernatant at 19,500-20,500 x g.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for separating extracellular vesicles from biological samples, particularly plant tissues.

(b) The present invention can be usefully used in an efficient separation and purification process capable of obtaining it with high purity and high quality while maintaining the inherent therapeutic activity and drug delivery activity of the extracellular vesicles.

1 is a diagram showing a process of measuring weight, washing, juice, and filtering a strainer in order to separate plant-derived extracellular vesicles.
2 is a diagram showing a sample obtained by concentrating and purifying a juice containing impurities excluding plant-derived extracellular vesicles.
3 is a diagram showing the extracellular vesicles and impurity proteins from which the plant-derived juice was separated through size exclusion chromatography.
4 is a diagram showing the separation and purification of extracellular vesicles derived from cabbage (FIG. 4a ), red cabbage (FIG. 4b) and tomato (FIG. 4c ), respectively.
5 is a diagram showing the size distribution of cabbage-derived extracellular vesicles purified through size exclusion chromatography in each fraction.
6 is a diagram showing cabbage-derived extracellular vesicles separated by polymer sedimentation using PEG, ultrafast centrifugation, and size exclusion chromatography, respectively.
FIG. 7 is a diagram showing the size distribution of cabbage extracellular vesicles separated by polymer sedimentation using PEG, ultrafast centrifugation, and size exclusion chromatography, respectively.
FIG. 8 is a diagram showing the yield and purity of cabbage extracellular vesicles isolated by polymer sedimentation using PEG, ultrafast centrifugation, and size exclusion chromatography, respectively.
9 is a diagram showing the yield of extracellular vesicles versus the mass and price of edible plants using the present technology in the case of cabbage (FIG. 9a) and tomato (FIG. 9b ), respectively.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Juice for isolation of plant-derived extracellular vesicles

1) After measuring the weight to separate the extracellular vesicles from commonly consumed plants such as red cabbage, cabbage, tomatoes, cucumbers, and oranges, the pesticides of the plants were first removed with running tap water. Next, tap water and pesticides were secondarily removed using flowing ultrapure distilled water. After that, cut into pieces of less than 40 mm in width and 30 mm in length using a pan, and then extract the juice using a juicer, and then only lumps of 1 mm width and 1 mm length or less of the cotton bag were filtered to remove large substances from the juice. . Finally, centrifugation was performed at 8,000 x g for 1 hour and at 4°C to remove the protein and cell wall from the solution filtered with a cotton bag, and then only the supernatant was centrifuged again at 20,000 x g for 1 hour at 4°C. The supernatant obtained through this process was stored at -80°C until further experiments were conducted (FIG. 1).

Plant-derived extracellular vesicle isolation and purification method

1) In order to obtain high purity plant-derived extracellular vesicles, impurities including proteins contained in plants must be removed. Even after centrifugation of the juice, a large number of protein impurities other than extracellular vesicles are contained in the supernatant, and there is a limitation in removing these impurity proteins by ultra-high-speed centrifugation method and polymer-based sedimentation method. Therefore, in order to increase the purity after removing these substances other than the extracellular vesicles, the size exclusion chromatography method was used for the separation and purification of extracellular vesicles, and for efficient separation and purification, the separation efficiency and purity were accompanied by ultrafiltration. Tried to increase

2) Add 13 mL of the juice solution to the Amicon Ultra®-15 10kDa Centrifugal filter (Merck Millipore, Cat. No: UFC901024), centrifuge at 5,000 xg for 3 hours at 4°C, and add 0.5 mL of the resulting supernatant to 200 nm. It was added to a syringe filter and transferred to 1.5 mL of an Eppendorf tube (SPL, Cat. No: 50015) (Fig. 2).

3) After fixing qEVoriginal/70 nm (Izon science, Cat.No: SP1) on the experimental clamp stand, removing the PBS solution from the qEVoriginal column, and performing 10 mL equilibration by gravity with 1X PBS (pH7.4) solution. I did. Next, 0.5 mL of the juice solution filtered in the previous step was injected into the qEVoriginal column, and when all the solutions were transferred to the size exclusion chromatography beads, the PBS solution was added. At this time, PBS was continuously injected so as not to exceed 2 mL in order to keep the force of gravity subjected to the size exclusion chromatography constant. The resulting solution was added 0.5 mL each to 1.5 mL of one Eppendorf tube, resulting in a total of 33 tubes (FIG. 3).

Plant-derived extracellular endoplasmic reticulum size distribution, concentration and purity

1) To determine the concentration and purity of plant-derived extracellular vesicles, a nanoparticle tracking analyzer (NTA) and a BCA assay to quantify proteins were used. First, the tubes of the isolated extracellular vesicles were each diluted 1:100 with PBS, and then the concentration and size distribution of the extracellular vesicles were measured using NTA. Next, the protein concentration of each fraction was quantified through BCA assay in order to confirm the protein concentration of the solution subjected to size exclusion chromatography. The concentration dilution of the protein was measured in two ways. For the first time, the process of directly taking the solution without dilution and the sample diluted 1:10 with PBS were measured to confirm the concentration of the protein entering the BSA standard curve (Fig. 4 and Fig. 5).

Comparison of yield and purity according to the method of isolation and purification of plant-derived extracellular vesicles

1) Separation of extracellular vesicles is currently mainly carried out by ultra-high speed centrifugation and polymer sedimentation. However, in the case of the ultra-high-speed centrifugation method, a strong centrifugal force acts and crushing of the extracellular vesicles may occur due to a physical force, and the polymer sedimentation method is not preferable in terms of purity because it contains a large amount of impurities other than the extracellular vesicles.

2) Size exclusion chromatography, ultra-high-speed centrifugation, and polymer sedimentation were all using the same amount of 13 mL of cabbage juice to obtain a size distribution of plant-derived extracellular vesicles using a nanoparticle tracking analyzer, and BCA assay. By measuring the protein concentration, the yield, concentration, and purity were calculated as the amount of extracellular vesicles per 1 mg of protein, and compared. In the case of the polymer sedimentation method and the ultrafast centrifugation method, peaks of various sizes were observed, whereas the extracellular vesicles separated by size exclusion chromatography showed uniform peaks (FIG. 7). In the yield, all methods showed a similar level of yield without statistical significance (Fig. 8a), but in the case of purity, the case of separation using size exclusion chromatography was significantly higher than that of other methods (Fig. 8b).

Extracellular endoplasmic reticulum yield by plant weight

1) After measuring the ratio of 13 mL used for size exclusion chromatography to the total volume of the plant-derived juice used, multiplying the total weight of the plant used for the juice to determine the weight used for the separation of plant-derived extracellular vesicles. The plant-derived extracellular vesicles obtained per 1 g were measured. Next, the price of the plant per 1 g was checked and converted to obtain the price used from the obtained weight by concentration. The price of the plants used was calculated as the average price of the previous year (source: Agricultural Observation Headquarters, https://aglook.krei.re.kr/) (Fig. 9).

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

Performing ultrafiltration with a cut-off value of 5 to 15 kDa on the plant cell culture medium or plant tissue containing the extracellular vesicles; And
A method for separating extracellular vesicles comprising the step of performing size-exclusion chromatography having an average pore diameter of 30-75 nm of the stationary phase on the result of the ultrafiltration.
delete delete delete delete delete delete delete The method of claim 1, wherein the plant tissue is obtained by a method comprising the following steps:
(a) crushing the plant and then extracting it to obtain an extract;
(b) filtering the extract to remove solids; And
(c) centrifuging the filtrate of step (b).
The method of claim 9, wherein the step (b) is characterized in that filtering to remove solids having a diameter of 1 mm or more.
The method of claim 9, wherein the step (c) is carried out by performing the first centrifugation at 7,000-9,000 g and second centrifugation of the supernatant at 19,000-21,000 g.

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