Method for producing extracellular vesicles

Abstract

This invention provides a purification method that enables the separation and purification of extracellular vesicles derived from plants with high recovery rates and low cost. [Solution] The present invention relates to a method for producing extracellular vesicles, comprising the steps of: (a) obtaining a liquid fraction containing extracellular vesicles derived from plants; (b) adjusting the electrical conductivity of the liquid fraction to 4 mS / cm or more and 12 mS / cm or less; (c) contacting the adjusted liquid fraction with a positively charged carrier; and (d) contacting the carrier with a buffer solution containing a metal salt to recover the extracellular vesicles.

Description

[Technical Field]

[0001] This invention relates to a method for producing extracellular vesicles contained in plants by purifying them with a high recovery rate. [Background technology]

[0002] Extracellular vesicles are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from almost all living cells, and are broadly classified into exosomes, microvesicles, and apoptotic bodies based on differences in their production mechanisms.

[0003] Exosomes are formed by inward budding of the late endosomal membrane, and then fuse with the cell membrane to become complete particles, which are secreted extracellularly by exocytosis. Exosomes are extracellular vesicles with a diameter of approximately 30-150 nm released by eukaryotic cells in animals, fungi, etc., and are known to play an important role in intercellular communication, such as encapsulating proteins, mRNA, and miRNA in body fluids. In recent years, it has been suggested that exosomes are involved in the enhancement of cancer metastasis and cancer progression, and diagnostic markers focusing on exosomes are being developed, such as biomarkers that use miRNA expressed from cancer-derived exosomes as tumor markers, and biomarkers that predict cancer progression, metastasis, and prognosis.

[0004] Furthermore, by isolating and purifying exosomes from living organisms, they are expected to be applied to drug delivery systems (DDS) as biologically derived transporters that encapsulate nucleic acids, proteins, and other substances within cells. Therefore, there is a need for technology to isolate and purify exosomes with high purity.

[0005] Microvesicles are generated when the cell membrane buddings outward and separates. Microvesicles vary considerably in size, ranging from 100 to 1,000 nm in diameter. Apoptotic bodies are produced when cells undergo apoptosis, and their size also varies widely, from 50 to 5,000 nm in diameter. Similar applications to exosomes are expected for microvesicles and apoptotic bodies.

[0006] Methods for separating and purifying extracellular vesicles include ultracentrifugation methods such as centrifugal fractionation and density gradient centrifugation, precipitation methods using polymers such as polyethylene glycol, and chromatographic methods such as size exclusion chromatography and affinity chromatography. Ultracentrifugation is a common method, and high-purity extracellular vesicles can sometimes be obtained by density gradient centrifugation. For example, Patent Document 1 discloses a method for producing exosomes by ultrafiltration of a biological sample followed by anion exchange column chromatography.

[0007] In recent years, it has become clear that extracellular vesicle particles are also found in foods such as plants, fruits, and vegetables, and DDS development using extracellular vesicles derived from milk and grapefruit has been progressing (Non-Patent Literature 1). Plant-derived extracellular vesicles are active ingredients derived from natural substances and are in demand for DDS and pharmaceutical applications due to their safety and other factors. For example, Patent Literature 2 discloses an antioxidant that uses extracellular vesicles derived from dragon fruit to improve symptoms in living organisms such as the skin. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2020 / 027185 [Patent Document 2] International Publication No. 2024 / 048161 [Non-Patent Document 1] Yusuke Yoshioka, Takahiro Ochitani, Development of a Novel DDS Carrier Using Exosomes, Drug Delivery System, 35-1 (2020) [Overview of the project] [Problems that the invention aims to solve]

[0009] Ultracentrifugation has problems such as structural changes and destruction of extracellular vesicles during the centrifugation process, resulting in reduced quality, contamination by impurities, and low recovery rates. Furthermore, methods using anion exchange column chromatography have drawbacks, such as insufficient adsorption of extracellular vesicles per unit volume.

[0010] The present invention aims to provide a purification method that can separate and purify extracellular vesicles with high recovery rate and low cost, and a method for producing extracellular vesicles by said purification method. [Means for solving the problem]

[0011] The inventors of this invention have diligently studied methods for preparing extracellular vesicles derived from plants and have found that by optimizing a series of processes from the liquid fraction obtained from plants, removal of impurities, separation of extracellular vesicles, and recovery, it is possible to recover the target extracellular vesicles with high purity and high recovery rate.

[0012] In other words, we discovered that the above problems can be solved by the means described below, and thus arrived at the present invention. [1] A method for producing extracellular vesicles of plant origin, Step (a) Step of obtaining a liquid fraction containing extracellular vesicles from the plant. Step (b) A step of adjusting the electrical conductivity of the liquid fraction to 4 mS / cm or more and 12 mS / cm or less. Step (c) A step of bringing the prepared liquid fraction into contact with a positively charged carrier to adsorb the extracellular vesicles onto the carrier. A method characterized by comprising step (d) contacting the carrier with a buffer containing a metal salt to recover extracellular vesicles. [2] The method according to [1], characterized in that between step (a) and step (b), there is a step of removing at least a portion of impurities from the liquid fraction obtained from the plant. [3] The method according to [1] or [2], characterized in that the step of removing at least a portion of the impurities is one or more selected from the group consisting of centrifugal separation, filtration, microfiltration, ultrafiltration, and dialysis. [4] In the step (c), the method according to any one of [1] to [3] above, characterized in that the carrier is a separation membrane having an anion exchange capacity. [5] In the step (c), the method according to any one of [1] to [4] above, characterized in that the carrier has a tertiary amine and / or a quaternary ammonium group. [6] The method according to any one of [1] to [5] above, characterized by including a step of contacting a buffer solution with the carrier to remove contaminants between the step (c) and the step (d). [7] In the step (d), the method according to any one of [1] to [6] above, characterized in that the buffer solution containing the metal salt is any one of a Tris-hydrochloric acid buffer solution, a phosphate buffered saline, and a Tris buffered saline. [8] In the step (d), the method according to any one of [1] to [7] above, characterized in that the metal salt is NaCl. [Effect of the Invention]

[0013] The present invention can maximize the adsorption amount of the target extracellular vesicles to the carrier by applying a step of adjusting the electrical conductivity of the liquid fraction obtained from a plant before contacting the liquid fraction obtained from the plant with a carrier having a positive charge in a method for purifying plant-derived extracellular vesicles. [Brief Description of the Drawings]

[0014] [Figure 1] It is a graph (calibration curve) showing the relationship between salt concentration and electrical conductivity. <00000�0> [Embodiments for Carrying Out the Invention]

[0015] The present invention relates to a new method for producing extracellular vesicles from plants. Hereinafter, the present invention will be described, but the present invention is not limited to the following specific examples. In the present disclosure, numerical ranges of "x to y" shall include x and y.

[0016] Extracellular vesicles are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from almost all living cells. Extracellular vesicles are broadly classified into three types based on differences in their intracellular production mechanisms: exosomes, microvesicles, and apoptotic bodies. Exosomes are 30-150 nm in diameter and are formed by inward budding of the late endosomal membrane. They then fuse with the cell membrane to form complete particles and are secreted extracellularly by exocytosis. Microvesicles, on the other hand, are produced by outward budding and separation of the cell membrane. They are 100-1,000 nm in diameter. Apoptotic bodies are produced when cells undergo systematic cell death (apoptosis). They are 50-5,000 nm in diameter.

[0017] 1. Preparation process for liquid fractions obtained from plants In this step, a liquid fraction containing extracellular vesicles derived from plants, which are the target product, is prepared.

[0018] The plant-derived material is not particularly limited as long as it contains extracellular vesicles that are the target of purification and production, but the whole plant may be used or any part of the plant may be used. Examples of such parts include fruit, fruit peel, pulp, seeds, pulp containing seeds, leaves, stems, and roots. Only one type of such part may be used, or a mixture of any two types of parts may be used. Preferably, the part used is fruit or pulp. The fruit may be fully ripe or unripe, but fully ripe is preferred.

[0019] In this invention, "extracellular vesicles" may be any extracellular vesicles contained in a plant-derived sample, and there are no particular restrictions on the size or morphology of the extracellular vesicles themselves. Furthermore, they may be natural or unnatural (artificial) extracellular vesicles.

[0020] The term "plant" is not particularly limited to any specific type of plant, but refers to organisms that perform photosynthesis, and includes divisions such as Gymnosperms, Angiosperms, Ferns, Mosses, Chlorophytes, and Euglena. For example, within the Angiosperms division, it includes plants such as the Dicotyledonous and Monocotyledonous classes, and within the Dicotyledonous class, it includes plants such as the Magnolia subclass, Hamamelis subclass, Caryophyll subclass, Loquat subclass, Rosa subclass, and Chrysanthemum subclass. Considering availability and other factors, it is preferable to use the Solanum genus of the Solanaceae family, order Solanales, subclass Asteridae, and it is preferable to use eggplant, potato, and tomato. Examples of tomatoes include Aiko, Yellow Plum, Evergreen, Cocktail Tomato, Green Zebra, Sicilian Rouge, Zucca, Delicious Kanemitsu Tomato, Tomato Berry, Piccola Canaria, Piccola Rouge, First, Fruit Ruby EX, Frutica, Pomorosso, Buongiorno, Mountain Gold, Plum Tomato, Red Pear, Lemon Tomato, Orange Chika, Toscana Violet, Hula Girl, San Marzano, Renaissance, Momotaro, and Reika.

[0021] The extracellular vesicles derived from the plant can be prepared from a liquid fraction. The liquid fraction may be a sap obtained by juicing the plant, or a plant extract. The juicing method is not particularly limited, but examples include pressing, rotary pressing, and rotary pressing. In order to prevent the destruction of the extracellular vesicles, it is desirable to extract them using a method that does not involve excessive heating or pressure. Specifically, it is preferable to use the fruit, the pulp, or the pulp containing the seeds, and to use the sap obtained by juicing it. The sap is preferably the pulp sap, the pulp sap containing the seeds, and more preferably the sap from which the seeds have been removed. Seed removal can be performed by centrifuging the pulp sap containing the seeds, or by using a nonwoven fabric or membrane filter. The extraction method is not particularly limited, but an example is to add a solvent to the crushed plant material and recover the liquid fraction as an extract. The solvent may be an organic solvent, a buffer such as Tris-HCl, a salt solution such as an aqueous sodium chloride solution, or an aqueous solvent such as water. The liquid fraction may be the juice, the extract, a concentrate of the extract, or a mixture of the juice, the extract, and the concentrate of the extract. The crushed plant material may also be in paste form.

[0022] The plant parts and crushed material may be used immediately after preparation or after storage. If stored, they may be stored at room temperature, refrigerated, or frozen, or frozen and then returned to refrigerated or room temperature, or refrigerated and then returned to room temperature.

[0023] The liquid fraction may be the dried juice or a mixture of the dried extract and a solvent. The dried product can be prepared by drying the juice or the extract. Examples of drying methods include freeze-drying, and a powder form is preferred from a handling standpoint.

[0024] In the present invention, a method for purifying extracellular vesicles derived from plants is preferably a method that includes the steps of: obtaining a liquid fraction from a plant containing extracellular vesicles; adjusting the electrical conductivity of the liquid fraction obtained from the plant; contacting the liquid fraction obtained from the plant with a positively charged carrier; removing impurities by contacting the carrier with a buffer solution; and recovering the extracellular vesicles by contacting the carrier with a buffer solution containing a metal salt.

[0025] In the present invention, the step of obtaining a liquid fraction containing extracellular vesicles derived from plants may include a step of removing unwanted large debris and impurities such as fibers. Examples of steps for removing impurities include separation by standing, centrifugation and filtration, microfiltration, ultrafiltration, and dialysis. These steps may be performed individually or in combination. Centrifugation is performed, for example, at 4°C, 1,000 × g, for 5 to 30 minutes. The supernatant from which debris has been removed by centrifugation is collected. Additional centrifugation may be performed to further remove impurities. When additional centrifugation is performed, the centrifugal acceleration (g) may be increased to remove debris of medium size or smaller. If the target of purification is exosomes or microvesicles, vacuoles may be removed.

[0026] Furthermore, filtration can be performed using a filter medium. When removing impurities from the liquid fraction using the filter medium, any residue remaining on the filter medium is removed as impurities, and the filtered fraction is recovered. The filter medium can be a nonwoven fabric filter or diatomaceous earth, and the size of the pores is not particularly limited; any pores that can allow extracellular vesicles to pass through are acceptable.

[0027] Furthermore, microfiltration is a process that uses a membrane filter with a pore size of 0.4 to 1 μm, and by recovering the filtrate, it is possible to remove medium-sized residues and other particles in a similar manner to centrifugation.

[0028] Alternatively, the supernatant obtained by centrifugation and / or the filtrate obtained by microfiltration may be treated by ultrafiltration and / or dialysis using a membrane filter with a pore size of 0.1 μm or less, more preferably 0.05 μm or less. Ultrafiltration and / or dialysis can remove impurities smaller than extracellular vesicles and concentrate extracellular vesicles.

[0029] The present invention may include a step in obtaining a liquid fraction containing extracellular vesicles derived from plants, which may include ultracentrifugation. In the ultracentrifugation step, for example, by applying a centrifugal force of 50,000 to 150,000 × g, the extracellular vesicles can be precipitated, allowing for the removal of impurities smaller than the extracellular vesicles and the concentration of the extracellular vesicles.

[0030] 2. Process for adjusting electrical conductivity In this step, the electrical conductivity of the liquid fraction containing plant-derived extracellular vesicles prepared in the previous step 1 is adjusted to between 4 mS / cm and 12 mS / cm. The electrical conductivity is preferably adjusted by adjusting the salt concentration. Generally, cells have an osmotic pressure close to that of physiological saline, which has a concentration and osmotic pressure of 0.154 mmol / L and 308 mOsm / L, respectively. The osmotic pressure of extracellular vesicles in the liquid fraction largely originates from the ionic components in the liquid; therefore, a positive correlation is observed between the electrical conductivity of the liquid fraction and the salt concentration, or between electrical conductivity and osmotic pressure. By adjusting the electrical conductivity to 12 mS / cm or less, it is possible to increase the adsorption efficiency of extracellular vesicles to positively charged carriers, as extracellular vesicles are thought to have a relatively weak negative charge. On the other hand, by adjusting the electrical conductivity to 4 mS / cm or higher, the alteration of extracellular vesicles can be suppressed. A preferred electrical conductivity is between 6 mS / cm and 10 mS / cm. If the electrical conductivity is 12 mS / cm or less, the concentration of negatively charged ions such as chloride ions in the buffer component containing salts in the liquid fraction is reduced. This reduces the proportion of ion exchange groups on the carrier that are ionically bound to negatively charged ions such as chloride ions, and increases the number of ion exchange groups that are effective in adsorbing negatively charged extracellular vesicles. The increased density of effective ion exchange groups on the carrier surface generates a zeta potential on the carrier surface that can attract extracellular vesicles, which have a lower diffusion coefficient compared to low-molecular-weight ions, to the carrier surface. As a result, particles with a particularly small degree of negative charge among extracellular vesicles are more easily adsorbed to the carrier. On the other hand, if the electrical conductivity is 4 mS / cm or higher, the osmotic pressure of the aqueous solution component in the sample does not decrease excessively, and damage to extracellular vesicles due to the osmotic pressure difference between the inside and outside of the lipid bilayer membrane of the extracellular vesicles can be suppressed. Therefore, it is important to adjust the electrical conductivity to the appropriate range described above. If the electrical conductivity is between 6 mS / cm and 12 mS / cm, the NaCl concentration in an aqueous NaCl solution (containing 10 mM Tris-HCl) is between 49 mmol / L and 110 mmol / L.

[0031] The process of reducing the electrical conductivity and salt concentration of the liquid fraction containing extracellular vesicles derived from plants is not particularly limited, but can be carried out by diluting the plant-derived sample with a buffer solution that has lower electrical conductivity and salt concentration than the plant-derived sample. In this case, examples of buffer solutions that can be used include 10 mM Tris-HCl, phosphate-buffered saline (PBS), and Tris-buffered saline (TBS). In another embodiment, the electrical conductivity and salt concentration of the plant-derived sample containing extracellular vesicles can be adjusted by replacing the buffer solution by ultrafiltration or by desalting by dialysis.

[0032] 3. Contact process with carrier In this step, the liquid fraction whose electrical conductivity was adjusted in the previous step 2 is brought into contact with a positively charged carrier. In this step, the carrier is not particularly limited, but cellulosic polymers such as cellulose acetate and cellulose triacetate, and polysulfone polymers are preferred. The carrier may also be a sheet-like separation membrane such as a flat membrane or a hollow fiber separation membrane (hollow fiber membrane), or it may be a particulate porous body or a non-porous body.

[0033] In the present invention, the method for imparting a positive charge to the support is not particularly limited, but one method is to impart a cationic compound after the support has been molded. More specifically, one method is to impart a positive charge to a support having hydroxyl groups by contacting it with glycidyltrimethylammonium or glycidyldiethylamine. Alternatively, a column packed with commercially available diethylaminoethyl (DEAE) cellulose resin may be used. DEAE cellulose is an anion exchanger in which diethylaminoethyl groups are introduced into cellulose, and can be obtained from companies such as Sigma-Aldrich.

[0034] In the present invention, it is preferable to use a cellulose-based ion exchange membrane as the positively charged carrier, in which at least some of the hydroxyl groups or acetyl groups at the 2nd, 3rd, and 6th positions of a porous substrate membrane containing a cellulose-based polymer are replaced with a positively charged compound.

[0035] Porous substrate films containing cellulose polymers can be manufactured by a wet process. The wet process involves mixing the cellulose polymer with a solvent and a non-solvent to prepare a film-forming solution, then extruding it in a hollow form and guiding it into a solidification bath for phase separation. Alternatively, dry processes, heat-induced phase separation methods, stretching methods, etc., may also be used. On the other hand, flat films can be manufactured by dissolving the cellulose polymer in a solvent, uniformly applying the solution to a substrate such as glass, immersing it in a solidification solution to solidify it, and then washing and drying it if necessary.

[0036] Polar solvents such as γ-butyrolactone, N-methylpyrrolidone, and dimethylacetamide are preferred as solvents for preparing the film-forming solution. These solvents may be used individually or in combination. If necessary, non-solvents such as water, glycerin, ethylene glycol, triethylene glycol, polyethylene glycol 200, and polyethylene glycol 400 may also be added. The mixing ratio of solvent to non-solvent in the film-forming solution (solvent / non-solvent ratio) is preferably 90 / 10 to 10 / 90.

[0037] The coagulation solution is preferably a mixture of a solvent and a non-solvent such as water. Furthermore, the solvent / non-solvent ratio in the coagulation solution is preferably matched to the solvent / non-solvent ratio in the film-forming solution. Matching the solvent / non-solvent ratio of the film-forming solution and the coagulation solution suppresses compositional fluctuations of the coagulation solution even during continuous film formation. In the case of flat membranes, the weight ratio of N-methylpyrrolidone to water in the mixed solution of N-methylpyrrolidone and water is preferably in the range of 30:70 to 50:50. By using such a composition for the coagulation bath, the pore size of the membrane surface on the side that first contacts the coagulation bath becomes larger than the pore size of the membrane surface on the side that contacts the substrate, forming an asymmetric structure with respect to the thickness direction. When the concentration of N-methylpyrrolidone in the coagulation bath is 25% by mass or more and less than 30% by mass, the pore size of the membrane surface on the side that first contacts the coagulation bath becomes equal to the pore size of the membrane surface on the side that contacts the substrate, and the asymmetric structure of the membrane tends to be mitigated. Furthermore, when the concentration of N-methylpyrrolidone in the coagulation bath is less than 25% by mass, a dense layer without distinct pores is more likely to form on the membrane surface that first comes into contact with the coagulation bath. The temperature of the coagulation solution is preferably 5 to 60°C.

[0038] The resulting porous substrate film is washed with warm water to remove excess solvent and other substances. After washing, the porous substrate film may be stored in water, or it may be dried after filling the pores with a pore-retaining agent such as an aqueous glycerin solution.

[0039] The porous substrate film obtained above is then subjected to a treatment to introduce ion exchange groups. Specifically, after deacetylation, a charge transfer treatment is performed on the hydroxyl groups. As an example, a method for producing a cationic cellulose film, which is an ion exchange film, by substituting positively charged groups for the hydroxyl groups of a cellulose-based porous substrate film is to immerse the cellulose-based porous substrate film in an insoluble solvent in the presence of alkali, add a solution of a positively charged compound dropwise, react under heating conditions, and then quench with alcohol.

[0040] The aforementioned insoluble solvent is not particularly limited as long as it is a solvent that does not dissolve the cellulose-based porous substrate film and is capable of dissolving positively charged compounds. For example, water, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc., are good examples and may be used individually or in combination depending on the solubility of the raw materials.

[0041] As alkalis used for deacetylation, hydroxides such as lithium hydroxide, potassium hydroxide, sodium hydroxide, and cesium hydroxide, as well as carbonates and organic amines, can be used. Among these, sodium hydroxide, which is industrially inexpensive, is preferred. The alkali concentration used is preferably in the range of 0.05% to 5.0% by mass. If the alkali concentration is 0.05% by mass or higher, the amount of charged groups introduced can be increased, and the amount of extracellular vesicles adsorbed per unit volume can be more reliably increased. On the other hand, if the alkali concentration is 5.0% by mass or lower, the degree of membrane swelling can be suppressed, so the structure (pore size) and physical properties (membrane strength) of the base membrane can be more reliably maintained in the ion exchange membrane after the introduction of charged groups. Therefore, an alkali concentration of 0.1 to 2.5% by mass is more preferable, and 0.2 to 2.0% by mass is even more preferable.

[0042] The amount of charged groups added to the cellulose membrane can be controlled by adjusting the amount of the positively charged compound described above. For example, when using a tertiary amine compound containing epoxy groups, the concentration of the tertiary amine compound in the insoluble solvent is preferably in the range of 0.01 ml / L to 3.0 ml / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.9 meq / g per cellulose membrane mass. When using a quaternary ammonium compound containing epoxy groups, the concentration of the quaternary amine compound in the insoluble solvent is preferably in the range of 0.01 ml / L to 1.0 ml / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.4 meq / g per cellulose membrane mass. If too many positively charged groups are added, the content of positively charged groups per polymer molecule increases, which can lead to increased water solubility and weakened membrane strength in water. Conversely, if too few positively charged groups are added, the capture efficiency of extracellular vesicles may decrease.

[0043] In the present invention, the positively charged carrier, such as an ion exchange membrane, is preferably in the form of a device housed in a container equipped with an inlet for introducing the liquid to be treated and an outlet for discharging the plant-derived sample, which is the liquid to be treated using ion exchange. This device has a first chamber and a second chamber separated by an ion exchange membrane, and the plant-derived sample, which is the liquid to be treated, introduced into the first chamber moves to the second chamber by permeating through the ion exchange membrane. At this time, extracellular vesicles in the plant-derived sample, which is the liquid to be treated, are adsorbed onto the surface and pore surface of the ion exchange membrane.

[0044] In the present invention, the ion exchange membrane, which is a positively charged carrier, may be a cellulose-based ion exchange membrane in which at least a portion of the hydroxyl groups or acetyl groups at the 2nd, 3rd, and 6th positions of the cellulose-based polymer are substituted with a compound having a positive charge.

[0045] The ion exchange membrane, which is the positively charged carrier, may be combined with a cation exchange membrane, either one or both, and the order does not matter.

[0046] In the present invention, examples of cellulosic polymers include cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate laurate, cellulose acetate oleate, and cellulose acetate stearate. However, cellulose acetate is preferred due to the ease with which positively charged compounds can be introduced. Cellulosic acetates with different degrees of acetate and molecular weights are commercially available, and it is more preferable to use cellulose acetate and / or cellulose triacetate with a degree of acetate of about 52 to 62. Examples of polysulfone polymers include polysulfone and polyethersulfone.

[0047] In the present invention, it is preferable to use a tertiary amine represented by the following formula (I) or a quaternary ammonium compound represented by the following formula (II) as the positively charged compound. In the formula, R 1 ~R 3 X represents the same or different hydrogen atoms, or a linear or branched alkyl group having 1 to 10 carbon atoms, and n of the methylene group represents an integer from 1 to 5. In the formula, X represents one or more selected from the group consisting of halogen atoms such as fluorine, chlorine, bromine, and iodine; leaving groups such as sulfonic acid esters such as tosylate, triflate, and mesylate; silyl groups such as alkoxysilyl and silanol; epoxide groups; isocyanate groups; and carboxylic acid groups. Any substituent X may be used for a positively charged compound, but from the standpoint of ease of obtaining raw materials, compounds in which X is chlorine, a silyl group, or an epoxide group are preferred.

[0048] [ka]

[0049] [ka]

[0050] In the present invention, the cellulose-based ion exchange membrane may be a so-called homogeneous membrane in which the pore diameter is substantially constant from one surface to the other, or it may be a so-called asymmetric membrane in which the pore diameter changes continuously or discontinuously from one surface to the other. Furthermore, the minimum pore diameter of the ion exchange membrane is preferably 50 nm to 1000 nm. In an asymmetric membrane, the minimum pore diameter layer is preferably located near one of the surfaces. If the minimum pore diameter is large, the probability of contact between the membrane surface and / or the pore surface of extracellular vesicles such as exosomes decreases, which may result in a low adsorption rate of extracellular vesicles in the treated solution (plant-derived sample). Also, it may not be possible to remove impurities. On the other hand, if the minimum pore diameter is small, the recovery rate of extracellular vesicles that have entered the inside of the membrane may decrease, or recovery may take a long time. The minimum pore diameter of the ion exchange membrane is defined as the polystyrene particle diameter at which the rejection rate is 80% or more when measured using a polystyrene particle dispersion.

[0051] Furthermore, the average pore size of the surface of the ion exchange membrane, for example, the surface on the side without the minimum pore size layer, is preferably between 100 nm and 5000 nm. If the average pore size of the surface is 100 nm or more, the liquid fraction can be processed at a sufficiently fast rate. If the average pore size of the surface is 5000 nm or less, the adsorption of extracellular vesicles to the carrier can be sufficiently promoted, and the recovery rate can be maintained. In addition, if the average pore size of the surface falls within the above range, the effect of depth filtration can be more reliably achieved. The average pore size of the surface can be calculated using the image processing software Image J based on images taken of both sides of the membrane at a magnification of 1000 to 20,000 times using a scanning electron microscope (SEM).

[0052] In this process, the liquid to be treated may be treated by cross-flow or dead-end treatment. Furthermore, when using an asymmetric membrane, the liquid to be treated may be introduced to either the large-pore side or the small-pore side. For example, in a centrifuged liquid fraction with a low solid component concentration and a high extracellular vesicle concentration, by using the large-pore side as the primary side and the small-pore side as the secondary side, it is possible to prevent the membrane pores from becoming clogged with adsorbed extracellular vesicles when loading a plant-derived sample onto the membrane and allowing the extracellular vesicles to be electrostatically adsorbed onto the membrane. On the other hand, in an uncentrifuged liquid fraction with a relatively high solid component concentration relative to the extracellular vesicle concentration, by using the small-pore side as the primary side and the large-pore side as the secondary side, impurities can be removed by surface filtration, preventing membrane clogging due to complete pore blockage.

[0053] In the present invention, the ion exchange capacity of the positively charged carrier is preferably 0.05 meq / g or more and 1.5 meq / g or less. If the ion exchange capacity is 0.05 meq / g or more, the zeta potential of the membrane is stable, and even substances to be purified with a small diffusion coefficient, such as extracellular vesicles, can be sufficiently attracted to the membrane, and the adsorption amount is sufficiently large, so a large-capacity device is not necessary. On the other hand, if the ion exchange capacity is 1.5 meq / g or less, the strength of the membrane is sufficient, and the adsorption amount per unit volume of the membrane becomes appropriate, suppressing membrane clogging. When using a tertiary amine compound, the ion exchange capacity is preferably 0.03 meq / g or more and 0.9 meq / g or less per mass of carrier. When using a quaternary ammonium compound, the ion exchange capacity is preferably 0.03 meq / g or more and 0.4 meq / g or less per mass of carrier.

[0054] In the present invention, the form of the carrier may be a flat membrane or a hollow fiber membrane, but the membrane thickness is preferably 10 μm to 1000 μm. If the membrane thickness is 10 μm or more, the membrane strength is sufficiently high, and handling convenience in membrane formation and device fabrication is high. On the other hand, if the membrane thickness is 1000 μm or less, the recovery time of extracellular vesicles can be suppressed and the recovery rate can be maintained. In the case of a hollow fiber membrane, the inner diameter is preferably 50 μm to 1000 μm. If the inner diameter is 50 μm or more, the shear stress of the liquid flowing through the hollow portion is sufficiently small, and damage to extracellular vesicles can be sufficiently suppressed. On the other hand, if the inner diameter is 1000 μm or less, the membrane area per device can be sufficiently large.

[0055] In the present invention, the zeta potential of the carrier is preferably in the range of 10 mV to 70 mV under pH conditions of 6.5 to 7.5. If the zeta potential is 10 mV or higher, the adsorption force to extracellular vesicles with low diffusion coefficients is sufficiently high, ensuring adsorption. If the zeta potential is 70 mV or lower, the membrane strength is sufficiently high, and when particles such as extracellular vesicles are to be purified, the amount of adsorption per unit volume of membrane becomes appropriate, suppressing membrane clogging. The zeta potential of a sheet-like carrier can be measured using a plate zeta potential measurement cell.

[0056] In this invention, the specific surface area of ​​the carrier is the sum of the specific surface areas of pores with a pore diameter of 30 nm or more, which is 3 m². 2 Preferably 8m / g or more, 2 A value of 3 m² or more is more preferable. Specific surface area of ​​3 m² is also preferable. 2A specific surface area of ​​30 nm or more ensures sufficient adsorption of extracellular vesicles. Specific surface area is generally measured by gas adsorption or mercury intrusion. Gas adsorption can detect the specific surface area of ​​micropores of approximately 2 nm or less and mesopores of 2 nm to 50 nm, while mercury intrusion can detect mesopores and macropores of 50 nm or more. A larger specific surface area is desirable to ensure sufficient adsorption of extracellular vesicles, but since extracellular vesicles cannot enter pores with a diameter of less than 30 nm, a sufficiently large specific surface area consisting of mesopores and macropores with a diameter of 30 nm or more is necessary to increase the adsorption amount of extracellular vesicles per unit volume of membrane. Note that an excessively large specific surface area of ​​pores is not problematic, but 150 nm is not a problem. 2 Preferably less than / g, 120m 2 Less than / g is preferable.

[0057] Furthermore, the porosity of the carrier is preferably between 40% and 90%. If the porosity is 40% or higher, sufficient space for extracellular vesicles to adsorb can be secured. If the porosity is 90% or lower, sufficient strength of the carrier can be secured. A porosity of 45% to 90% is more preferable.

[0058] When using the carrier of the present invention for the separation and purification of extracellular vesicles, it is preferable to adjust the zeta potential, specific surface area, porosity, etc. to specific ranges. Among extracellular vesicles, exosomes are extracellular vesicles with a size of 30 nm to 400 nm and a negative charge on the surface. For example, when efficiently adsorbing and desorbing plant-derived exosomes, if the pores are too small and there are too many micropores, exosomes cannot enter the pores, so even if the specific surface area of the carrier is increased, the adsorption area and adsorption capacity cannot be obtained. Also, if the pores are too large and there are too many macropores, the distance between the membrane surface and exosomes is too far, making it difficult for the electrical attraction to work, or extracellular vesicles with a diameter exceeding 400 nm may enter the pores, resulting in a decrease in the adsorption efficiency of the target exosomes. On the other hand, the ion exchange membrane may be better with a certain degree of micropores. The liquid fraction contains many various small molecular weight substances, and many of these substances have charges. By adsorbing such small molecular weight substances into the micropores, it becomes possible to effectively utilize the adsorption sites of mesopores and macropores for the adsorption of exosomes. The specific surface area of pores with a pore diameter of less than 30 nm is preferably 30 m 2 / g or more and 80 m 2 / g or less.

[0059] The ion exchange capacity of the carrier having a positive charge is preferably 0.08 meq / g or more and 0.3 meq / g or less. If the ion exchange capacity is 0.3 meq / g or less, the content of positive charge groups per polymer molecule constituting the carrier is not too high, and the water solubility of the carrier is not excessively high, so the carrier strength in water can be sufficiently ensured. Also, if the ion exchange capacity is 0.08 meq / g or more, the capture efficiency of extracellular vesicles can be more reliably maintained at a high level. The positive charge group is preferably a tertiary amine group or a quaternary ammonium group. As the tertiary amine, diethylamine can be used, and as the quaternary ammonium, trimethylammonium can be used.

[0060] In this invention, "contact" refers to immersing the carrier in a liquid fraction. Furthermore, if the carrier has a porous membrane structure, this also includes creating a pressure difference between the primary and secondary sides of the membrane so that the liquid fraction enters the interior of the porous membrane, thereby causing the liquid fraction to pass through the porous membrane from the primary to the secondary side. Additionally, if the carrier is a particulate resin, this also includes passing the liquid fraction through a resin-filled column from the inlet to the outlet using a pressure difference. The amount of extracellular vesicles that a carrier can process depends on the plant species, etc., but when the carrier's anion exchange capacity is 0.08~0.3 meq / g, it is approximately 10 9 ~10 12 The concentration is 1 / mL, and it is necessary to adjust the amount of liquid fraction processed so as not to exceed this amount relative to the amount of carrier.

[0061] 4. Washing process In the present invention, it is preferable to contact a positively charged carrier with a liquid fraction to adsorb extracellular vesicles onto the carrier, and then wash the carrier with a buffer solution. The buffer solution is not particularly limited, but Tris-HCl is preferred. Other buffer solutions include TBS and PBS. The salt concentration of the buffer solution is preferably adjusted to be less than or equal to the salt concentration of the liquid fraction in order to prevent the target extracellular vesicles from detaching from the carrier and eluting together with other contaminants. Specifically, it is preferable to have a salt concentration of 50 mmol / L to 150 mmol / L and a salt concentration of 6.08 mS / cm to 16.01 mS / cm. The pH is not particularly limited, but a range of 7 to 8 is preferred. The amount of buffer solution used for washing is preferably in the range of 5 to 100 times the volume of the column or porous body.

[0062] 5. Recovery Process In this step, the extracellular vesicles adsorbed to the carrier in steps 3 and 4 are brought into contact with a buffer containing a metal salt to dissociate and recover the extracellular vesicles from the carrier. In this invention, in order to elute and recover extracellular vesicles such as exosomes adsorbed and bound to the carrier, it is sufficient to bring them into contact with a buffer containing a monovalent or divalent metal salt. The salt concentration of the buffer is preferably 300 mmol / L or more and 800 mmol / L or less, and 30.91 mS / cm or more and 80.58 mS / cm or less. Depending on the purpose, such as separating and recovering extracellular vesicles by type based on differences in the charge of the extracellular vesicles, it may be preferable to bring them into contact while increasing the salt concentration, such as by using a stepwise elution method or a linear gradient elution method. The monovalent or divalent metal salt is not particularly limited, but NaCl is preferred. In other embodiments, the metal salt may be MgCl2, Na2SO4, or MgSO4. Preferred buffers include Tris-HCl, PBS, and TBS, with Tris-HCl being more preferred.

[0063] In the present invention, the salt concentration and electrical conductivity of the sample containing the extracellular vesicles may be adjusted to enhance the storage stability of the recovered extracellular vesicles. The salt concentration and electrical conductivity should be within a range close to the electrolyte concentration of living organisms, preferably between 140 mmol / L and 160 mmol / L, and between 15 mS / cm and 17.01 mS / cm. A more preferable electrical conductivity is 15.01 mS / cm or higher. One method for measuring the salt concentration is to measure the electrical conductivity of the recovered fraction and calculate the NaCl concentration (Figure 1). Based on the obtained salt concentration, dilution can be performed by adding 10 mmol / L Tris-HCl (pH 7.5). Other methods include buffer replacement by ultrafiltration or desalting by dialysis.

[0064] In the present invention, "concentration" refers to reducing the volume of a solution containing the target extracellular vesicles and increasing the particle concentration of the target extracellular vesicles. [Examples]

[0065] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. The evaluation methods for physical properties, etc., in the following examples are as follows.

[0066] Test Example 1: Measurement of the ion exchange capacity of a support material The ion exchange capacity of the support was measured as follows. A 0.1% by mass aqueous solution of bromophenol blue (BPB) was prepared, and a positively charged support was stained by immersing it in the solution at room temperature for 1 hour. After staining, the support was washed with water and then immersed in an 8% by mass aqueous solution of sodium chloride to elute the BPB adsorbed on the support. The absorbance of the sodium chloride aqueous solution at 590 nm was measured, and the number of moles of BPB adsorbed per unit mass of support was calculated based on a calibration curve obtained in an 8% by mass sodium chloride aqueous solution of BPB, and this was used as the ion exchange capacity.

[0067] Test Example 2: Evaluation of Particle Rejection Rate The rejection rates of porous substrate membranes and ion exchange membranes were evaluated using dispersions of polystyrene particles. A Tween 20 aqueous solution, containing 0.01% by mass of polystyrene particles of various sizes, was permeated through the membrane at an operating pressure of 10 kPa to obtain a liquid fraction. The concentration of polystyrene particles in the original solution and the liquid fraction was measured by absorbance at 250 nm, and the rejection rate was calculated using the following formula. Rejection rate (%)=(Ca-Cb) / Ca×100 Ca: Concentration of polystyrene particles in the original solution Cb: Concentration of polystyrene particles in the liquid fraction The rejection rate was measured using polystyrene with particle sizes of 79, 132, 208, 262, 313, 420, 460, and 616 nm, and the minimum pore size was defined as the polystyrene particle size at which the rejection rate was 80%.

[0068] Test Example 3: Observation of Membrane Structure The film to be evaluated was placed on a sample stage for SEM, dried at room temperature, and then platinum was deposited onto it. It was then observed using a scanning electron microscope ("SU-1500," manufactured by Hitachi High-Technologies Corporation).

[0069] Test Example 4: Measurement of the average pore diameter on the large pore side of the membrane The average pore diameter on the large pore side of the aforementioned surface SEM image of the film observed at 10,000x magnification was measured using the following procedure. The image was opened in the image processing software ImageJ, and a line was drawn within the range of the scale bar using the line tool from the toolbar. Analyze>Set Scale was selected, and the length of the scale bar was entered in "Known Distance" and the unit in "Unit of length". Image>Adjust>Auto Threshold was set to Default for Method mode, and OK was selected to binarize the image. Analyze>Set Measurements was confirmed to have "Feret's diameter" checked, and OK was pressed. Analyze>Analayze Particles was set to 0-Imfinity for "Size (pixel^2)", and it was confirmed that the boxes for "Display results", "Exclude on edges", and "Include holes" were checked, and OK was selected to obtain the measurement data for the ferret diameter on the Results screen. To remove noise smaller than 15 pixels, the length equivalent to 15 pixels was calculated in the same units as the ferret diameter, and values ​​smaller than that were removed to calculate the average ferret diameter.

[0070] Test Example 5: Measurement of Zeta Potential The zeta potential of the membrane was calculated by attaching a planar zeta potential measurement cell ("ZEN1020," manufactured by Malvern Panalytical) to a Zetasizer Nano ZS and measuring the mobility of aluminum oxide particles in ion-exchanged water with a pH of 6.5 to 7.5.

[0071] Test Example 6: Measurement of Specific Surface Area The following measurements were performed using a pore distribution analyzer ("Autopore IV9520," manufactured by Shimadzu Corporation). The sample was cut into strips of approximately 12.5 mm × 25 mm, and 0.04 to 0.15 g of each strip was placed in a 5 mL powder cell (stem volume 0.4 mL). Measurements were performed under an initial pressure of approximately 3.7 kPa (equivalent to a pore diameter of approximately 340 μm). The mercury parameters were set to the instrument's default settings: a mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. The pore distribution was obtained by plotting the change in the log differential volume of mercury that penetrated the sample against the pore diameter to obtain a pore distribution curve. The specific surface area was calculated by measuring the pore volume V for each mode diameter D, determining the specific surface area S for each mode diameter D using the following formula, and calculating the total specific surface area from the sum of the ranges from 0.03 to 500 μm. S = 4V / D

[0072] Test Example 7: Nanoparticle Tracking Analysis The average number and diameter of recovered extracellular vesicles were measured using nanoparticle tracking analysis (NTA) with a nanoparticle analysis system ("NanoSight NS300," Malvern Panalytical). The camera level was set to 14 and the detection threshold to 7. When the salt concentration of the culture supernatant was changed, the number of particles was measured at least 24 hours after the change.

[0073] Example 1 (1) Steps to obtain a liquid fraction containing extracellular vesicles derived from plants. As a plant-derived sample, we used the juice of fresh tomatoes. Fresh tomatoes were cut into 1 cm cubes and placed in a polyethylene nonwoven fabric filter to obtain tomato juice. The obtained tomato juice was centrifuged at 25°C at 1,000 × g for 10 minutes, and supernatant 1 was collected. Supernatant 1 was centrifuged at 3,000 × g for 10 minutes, and supernatant 2 was collected. Supernatant 2 was centrifuged at 10,000 × g for 10 minutes, and supernatant 3 was collected. Supernatant 3 was filtered through a 0.45 μm filter and a 0.22 μm filter (both manufactured by Merck Millipore) to prepare the tomato concentrate.

[0074] (2) Preparation of anion exchange membrane As a positively charged support, a cellulose acetate membrane filter (ADVANTEC) with a nominal pore size of 0.2 μm was deacetylated by immersion in a 0.1 M sodium hydroxide aqueous solution at room temperature for 4 hours. The resulting deacetylated membrane was then immersed in a 0.4 M glycidyl diethylamine aqueous solution at 65°C for 4 hours to obtain an ion exchange membrane. The performance of the obtained ion exchange membrane is shown in Table 1.

[0075] [Table 1]

[0076] (3) Equilibration process NaCl was dissolved in 10 mmol / L Tris-HCl buffer, and a calibration curve showing the relationship between NaCl concentration and electrical conductivity was created. Electrical conductivity was measured using an electrical conductivity meter ("LAQUAtwin EC-33," manufactured by HORIBA). Before measurement, the measurement section was rinsed twice with the sample before each measurement. An anion exchange membrane with a diameter of 25 mm was equilibrated by passing 5 mL of 10 mmol / L Tris-HCl (pH 7.5) (equilibrium buffer) containing 50 mmol / L (6.6 mS / cm) NaCl through it.

[0077] (4) Steps to adjust the electrical conductivity of the liquid fraction A liquid fraction with an electrical conductivity of 6.6 mS / cm was obtained by adding 12 mL of 10 mmol / L Tris-HCl (pH 7.5) containing 50 mmol / L (6.6 mS / cm) NaCl to 6 mL of prepared tomato concentrate. (5) Step of bringing into contact with a positively charged carrier The obtained liquid fraction was passed through the anion exchange membrane prepared in the previous step (2) at a rate of 2 mL / min.

[0078] (6) Washing process The membrane was washed by passing 10 mL of 10 mmol / L Tris-HCl buffer containing 50 mmol / L (6.6 mS / cm) NaCl through it. (7) Elution process 6 mL of 200 mmol / L (21.0 mS / cm) NaCl was passed through the membrane to elute the extracellular vesicles bound to the membrane, and a purified fraction of extracellular vesicles was obtained.

[0079] (8) Isotonicity process Next, the electrical conductivity of the fraction from which extracellular vesicles were eluted was measured. After calculating the NaCl concentration from the electrical conductivity values ​​using a calibration curve (Figure 1), 10 mmol / L Tris-HCl (pH 7.5) was immediately added to isotonicize the solution to 10 mmol / L Tris-HCl (pH 7.5) containing 0.15 mol / L NaCl, thereby obtaining the extracellular vesicle solution. The equilibration and isotonicization steps were both performed while the solution was cooled to 4-10°C.

[0080] (9) Characterization Samples containing fractionated extracellular vesicles were subjected to NTA analysis, and the average particle number and diameter were measured. NTA analysis was performed using a nanoparticle analysis system ("NanoSight NS300," Malvern Panalytical) at camera level 14 and detection threshold (DT) 7.

[0081] Example 2 In the permeation step, 12 mL of 10 mmol / L Tris-HCl (pH 7.5) containing 100 mmol / L (11.0 mS / cm) NaCl was added to 6 mL of the prepared tomato stock to obtain a liquid fraction with an electrical conductivity of 9.5 mS / cm. This fraction was then permeated through the obtained anion exchange membrane. In the elution step, the membrane was washed by passing 10 mL of 10 mmol / L Tris-HCl buffer containing 100 mmol / L (11.0 mS / cm) NaCl through the membrane. Otherwise, an extracellular vesicle solution was obtained in the same manner as in Example 1.

[0082] Comparative Example 1 In the permeation step, 12 mL of 10 mmol / L Tris-HCl (pH 7.5) containing 200 mmol / L (21.0 mS / cm) NaCl was added to 6 mL of the prepared tomato stock to obtain a liquid fraction with an electrical conductivity of 16.0 mS / cm. The obtained liquid fraction was permeated through an anion exchange membrane. In the elution step, the membrane was washed by passing 10 mL of 10 mmol / L Tris-HCl buffer containing 200 mmol / L (21.0 mS / cm) NaCl through the membrane. Otherwise, an extracellular vesicle solution was obtained in the same manner as in Example 1.

[0083] Comparative Example 2 An extracellular vesicle solution was obtained in the same manner as in Example 2, except that a cellulose acetate membrane filter (ADVANTEC) with a nominal pore size of 0.2 μm was used instead of an anion exchange membrane.

[0084] [Table 2]

[0085] As shown in Table 2, the greatest number of particles were recovered under the conditions of Example 1, where the electrical conductivity of the liquid fraction was 6.6 mS / cm. This indicates that increasing the electrical conductivity of the liquid fraction prevents the extracellular vesicles from being sufficiently adsorbed onto the carrier, causing them to flow out into the permeation (flow-through) fraction. Furthermore, in Comparative Example 2, where an anion exchange membrane was not used, the number of recovered particles was reduced. This suggests that the extracellular vesicles contained in the tomato extract have a weak electrical charge, and that adsorption to the anion exchange membrane can increase the amount of extracellular vesicles recovered. [Industrial applicability]

[0086] According to the present invention, it is possible to purify and recover extracellular vesicles derived from plants in a simple manner with a high recovery rate. The extracellular vesicles obtained by the present invention can be suitably used in fields such as drug delivery, pharmaceuticals, food, and cosmetics.

Claims

1. A method for producing extracellular vesicles derived from plants, Step (a) Step of obtaining a liquid fraction containing extracellular vesicles from the plant. Step (b) A step of adjusting the electrical conductivity of the liquid fraction to 4 mS / cm or more and 12 mS / cm or less. Step (c) A step of bringing the prepared liquid fraction into contact with a positively charged carrier to adsorb the extracellular vesicles onto the carrier. A method characterized by comprising step (d) contacting the carrier with a buffer containing a metal salt to recover extracellular vesicles.

2. The method according to claim 1, characterized in that between step (a) and step (b), there is a step of removing at least a portion of impurities from the liquid fraction obtained from the plant.

3. The method according to the previous invention, characterized in that the step of removing at least a portion of the impurities is one or more selected from the group consisting of centrifugation, filtration, microfiltration, ultrafiltration, and dialysis.

4. The method according to claim 1, characterized in that, in step (c), the carrier is a separation membrane having anion exchange ability.

5. The method according to 4, characterized in that, in step (c), the carrier has a tertiary amine and / or a quaternary ammonium group.

6. The method according to claim 1, characterized in that between step (c) and step (d), a step of contacting the carrier with a buffer solution to remove impurities is included.

7. The method according to claim 1, characterized in that, in step (d), the buffer containing the metal salt is any of Tris-HCl buffer, phosphate-buffered saline, or Tris-buffered saline.

8. The method according to claim 1, characterized in that the metal salt in step (d) is NaCl.

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