Method for purifying extracellular vesicles and / or membrane vesicles in feces

Abstract

The purpose of the present invention is to provide a method for easily and efficiently purifying extracellular vesicles and / or membrane vesicles in feces at low cost. A method for purifying extracellular vesicles and / or membrane vesicles in feces according to the present invention comprises: a step for filtering a sample of the feces with a filter; and a step for bringing the filtered sample into contact with a scavenger, wherein the scavenger contains a crosslinked polymer that contains a monomer unit having an acidic group and / or a monomer unit having a neutralizing base of the acidic group, and satisfies a predetermined (i) and / or (ii).

Description

Method for purifying extracellular vesicles and / or membrane vesicles from feces

[0001] The present invention relates to a method for purifying extracellular vesicles and / or membrane vesicles in feces.

[0002] Extracellular vesicles are lipid bilayer membrane vesicles secreted by cells. They encapsulate proteins, nucleic acids, and other substances, transport these substances, and are involved in various physiological phenomena. Extracellular vesicles are present in most body fluids, but they are also present in large quantities in feces. In addition to extracellular vesicles, feces also contain membrane vesicles released by intestinal bacteria and other organisms, which have functions similar to extracellular vesicles. Extracellular vesicles and membrane vesicles have recently attracted attention in the medical field, for example, due to their potential application in the diagnosis of diseases. Therefore, there is a need for the development of methods for purifying extracellular vesicles and / or membrane vesicles from biological samples. However, there has been little research on extracellular vesicles and membrane vesicles in feces.

[0003] Feces contain many impurities compared to urine, blood, etc., making it difficult to purify extracellular vesicles and membrane vesicles from feces. Conventionally, purification by a method combining high-speed filtration and sucrose density gradient ultracentrifugation has been reported (e.g., Non-Patent Document 1). However, this method, particularly because of the use of ultracentrifugation, takes a long time to process and requires expensive and large equipment.

[0004] Meanwhile, methods for isolating exosomes or lipid bilayers from biological samples other than feces are known. For example, Patent Document 1 discloses a method for separating exosomes from a biological sample, comprising the steps of contacting the biological sample with a superabsorbent polymer to obtain a superabsorbent polymer gel containing exosomes, and mixing the superabsorbent polymer gel with a salt to recover exosomes. It also describes that the superabsorbent polymer preferably has a repeating unit represented by a specific structural formula. Patent Document 2 also discloses a method for separating a substance having a lipid bilayer from a biological sample, comprising the steps of contacting the biological sample with a specific crosslinked polymer to obtain a polymer gel containing a substance having a lipid bilayer, and mixing the polymer gel with a salt to recover the substance having a lipid bilayer. It also describes that the specific crosslinked polymer is a crosslinked polymer containing a monomer unit having an acidic group and / or a neutralizing base thereof, and containing a compound having at least one group selected from the group consisting of a cationic group and a hydroxyl group. However, neither Patent Document 1 nor Patent Document 2 describes or suggests that extracellular vesicles and membrane vesicles can be purified from feces.

[0005] International Publication No. 2021 / 251462 International Publication No. 2022 / 211006

[0006] Yang C, et al., “Isolation and Characterization of Exosomes from Mouse Feces.” Bio Protoc.2020 Apr 20;10(8):e3584.

[0007] An object of the present invention is to provide a method for purifying extracellular vesicles and / or membrane vesicles in feces simply, efficiently, and at low cost.

[0008] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that the efficiency of capturing extracellular vesicles can be greatly improved by filtering the supernatant of a fecal suspension and capturing the filtered sample using a specific capturing agent, leading to the completion of the present invention.

[0009] That is, the present invention relates to, for example, the following inventions. [1] A method for purifying extracellular vesicles and / or membrane vesicles in feces, comprising the steps of filtering a fecal sample through a filter and contacting the filtered sample with a capture agent, wherein the capture agent contains a crosslinked polymer including a monomer unit having an acidic group and / or a monomer unit having a neutralized base of the acidic group, and satisfies the following (i) and / or (ii): (i) the crosslinked polymer contains at least a monomer unit having a neutralized base of the acidic group and a first cationic surfactant, and (ii) the capture agent further contains a second cationic surfactant (excluding cases where the second cationic surfactant is a crosslinked polymer including a monomer unit having a neutralized base of the acidic group). [2] The method according to [1], wherein the monomer unit having an acidic group is an unsaturated monocarboxylic acid having 3 to 10 carbon atoms, and the monomer unit having a neutralized base of the acidic group is a salt of an unsaturated monocarboxylate having 3 to 10 carbon atoms. [3] The method according to [1] or [2], wherein the monomer unit having an acidic group is acrylic acid, and the monomer unit having a neutralized salt of the acidic group is an acrylate. [4] The cationic surfactant has a cationic group of -N ＋ R ３ (wherein each R independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms). [5] The method according to any one of [1] to [4], wherein the cationic surfactant has an optionally substituted aliphatic hydrocarbon group having 12 to 30 carbon atoms. [6] The method according to any one of [1] to [5], wherein the cationic surfactant is hexadecyltrimethylammonium chloride. [7] The method according to any one of [1] to [6], wherein the pore size of the filter is 0.8 μm or less.

[0010] According to the present invention, a method for purifying extracellular vesicles and / or membrane vesicles in feces can be provided simply, at low cost, and efficiently.

[0011] Graphs showing CD9 / CD63 luminescence values ​​in the supernatant before and after reaction (flow-through) when urine supernatant is reacted with a capture agent (A), or when fecal suspension supernatant is reacted with a capture agent (B). Graphs showing CD9 / CD63 luminescence values ​​in the supernatant of fecal suspension before and after reaction (flow-through) when fecal suspension supernatant is reacted with a capture agent without filtering (A), or when fecal suspension supernatant is filtered and then reacted with a capture agent (B). Graphs showing CD9 / CD63 luminescence values ​​in the supernatant before and after treatment (flow-through) when serum supernatant is treated with a specified kit (A), or when fecal suspension supernatant is treated with the same kit (B).

[0023] Figure 1 is a graph showing CD9 / CD63 luminescence values ​​in the fecal suspension filtrate (flow-through) before and after reaction when the filtrate of a fecal suspension filtered through a PVDF filter (A) with a pore size of 0.45 μm, an MCE filter (B) with a pore size of 0.8 μm, an MCE filter (C) with a pore size of 1.2 μm, or a PVDF filter (D) with a pore size of 5.0 μm was reacted with a capture agent.

[0024] Figure 1 is a graph showing the results of Western blot of LPS / Lipid A in the culture supernatant when a specific bacterium was cultured, in the culture supernatant in which the bacterium was not cultured, or in the eluate obtained by reacting these supernatants with a capture agent and eluting membrane vesicles captured by the capture agent.

[0025] Figure 1 is a graph showing the results of analyzing the bacterial species profile of feces or samples obtained when feces was subjected to each purification method (ultracentrifugal purification, capture agent purification).

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0013] The method for purifying extracellular vesicles and / or membrane vesicles in feces according to this embodiment (hereinafter also referred to as the "method according to this embodiment") comprises a step of filtering the fecal sample through a filter (filtration step), and a step of contacting the filtered sample with a capture agent (contact step), wherein the capture agent contains a crosslinked polymer including a monomer unit having an acidic group and / or a monomer unit having a neutralizing base of the acidic group, and satisfies the specified (i) and / or (ii).

[0014] In the contact step, extracellular vesicles and / or membrane vesicles are specifically captured by the capture agent, thereby purifying the extracellular vesicles and / or membrane vesicles. Here, "capture" means that the extracellular vesicles and / or membrane vesicles specifically bind to the surface of the capture agent through physical interactions such as electrostatic attraction. The extracellular vesicles and / or membrane vesicles captured by the capture agent can be used as is (e.g., for quantification or content analysis), or can be used after separation from the capture agent. Therefore, the method according to this embodiment may further include a step of separating the extracellular vesicles and / or membrane vesicles from the capture agent (a separation step or a recovery step).

[0015] As used herein, "extracellular vesicles" refer to vesicles with a lipid bilayer membrane that contain intracellular components and are secreted extracellularly from cells, and refer to those secreted from cells of organisms other than bacteria. Extracellular vesicles contain nucleic acids such as microRNA, messenger RNA (mRNA), and DNA; proteins such as growth factors and enzymes; lipids, and the like, and are involved in many important functions in the body, such as intercellular communication, regulation of gene transcription rates, and induction and adjustment of immune responses. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

[0016] The organism other than bacteria may be any organism from which feces can be collected, such as mammals, birds, reptiles, or fish, and is preferably a mammal. Examples of mammals include humans, chimpanzees, mice, hamsters, cats, dogs, cows, horses, and rats.

[0017] As used herein, "membrane vesicles (MVs)" refer to vesicles secreted by bacteria that contain bacterial components. Membrane vesicles contain nucleic acids such as DNA, pathogenic toxins, signal proteins, proteins such as enzymes, lipids, polysaccharides such as peptidoglycans, and the like, and are involved in various functions at their destination, such as interactions between microorganisms, propagation of pathogenicity, transformation, survival under stress conditions, and inflammation control. The membrane vesicles to which the method according to this embodiment is applied are preferably those secreted by bacteria present in the intestine, particularly in the large intestine (enteric bacteria).

[0018] The bacteria may be gram-negative or gram-positive bacteria. The membrane vesicles secreted by gram-negative bacteria are also called outer membrane vesicles (OMVs).

[0019] Examples of Gram-negative bacteria include bacteria belonging to the genera Bacteroides, Escherichia, Salmonella, and Pseudomonas. Examples of bacteria belonging to the genus Bacteroides include Bacteroides vulgatus. Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Examples of bacteria belonging to the genus Salmonella include Salmonella enterica. Examples of bacteria belonging to the genus Pseudomonas include Pseudomonas aeruginosa.

[0020] Examples of Gram-positive bacteria include bacteria belonging to the genus Lactobacillus, Bacillus, Streptococcus, and Clostridium. Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus acidophilus. Examples of bacteria belonging to the genus Bacillus include Bacillus cereus. Examples of bacteria belonging to the genus Streptococcus include Streptococcus thermophilus. Examples of bacteria belonging to the genus Clostridium include Clostridium inoculum.

[0021] Examples of bacteria include bacteria belonging to the genus Prevotella, bacteria belonging to the genus Clostridia UCG-014, bacteria belonging to the genus Alistipes, bacteria belonging to the genus Bacteroides, bacteria belonging to the genus Faecalibacterium, bacteria belonging to the genus Barnesiella, bacteria belonging to the genus Turicibacter, bacteria belonging to the genus Rhombutzia, Prevotella copri, Alistipes inopus, bacteria belonging to the family Oscillospiraceae, Alistipes shahii, Clostridium leptum, Alistipes indicatinctus, Bacteroides coprocola, bacteria belonging to the genus Subdoligranurum, and bacteria belonging to the family Ruminococcaceae.

[0022] As used herein, the term "fecal sample" refers to a sample containing feces, and may be prepared using feces for the filtration step described below. Examples of fecal samples include fecal suspensions in which feces is suspended in an aqueous medium, or the supernatants of such fecal suspensions, which may contain extracellular vesicles and / or membrane vesicles. Examples of aqueous media include physiological saline solutions such as phosphate-buffered saline (PBS), sterile water, and pH buffer solutions such as Good's buffer. The supernatant of a fecal suspension can be obtained by centrifuging the fecal suspension to remove solid matter.

[0023] The filtration step is a step of filtering a fecal sample through a filter to obtain a filtrate. By including the filtration step in the method according to this embodiment, the capture efficiency of extracellular vesicles and / or membrane vesicles in the contact step described below is improved.

[0024] From the viewpoint of further improving the capture efficiency of extracellular vesicles and / or membrane vesicles in the contacting step described below, the pore size of the filter in the filtration step may be, for example, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, or 0.45 μm or less. Furthermore, the pore size of the filter in the filtration step may be, for example, 0.2 μm or more, or 0.3 μm or more.

[0025] The material of the filter used in the filtration step is not particularly limited, and examples thereof include polyvinylidene fluoride (PVDF), cellulose mixed ester (a mixture of cellulose acetate and nitrocellulose; MCE), polyethersulfone (PES), and glass fiber.

[0026] In the contacting step, the fecal sample that has undergone the filtration step is brought into contact with a capturing agent, thereby reacting the extracellular vesicles and / or membrane vesicles in the feces with the capturing agent and capturing the extracellular vesicles and / or membrane vesicles in the capturing agent. Contacting the fecal sample that has undergone the filtration step with the capturing agent may involve mixing the two.

[0027] The scavenger in the contacting step contains a crosslinked polymer containing a monomer unit having an acidic group and / or a monomer unit having a neutralized salt group of the acidic group, and the scavenger satisfies the following (i) and / or (ii): (i) the crosslinked polymer contains at least a monomer unit having the acidic group and a neutralized salt group of a first cationic surfactant, and (ii) the scavenger further contains a second cationic surfactant (with the exception of a case where the second cationic surfactant is a crosslinked polymer containing a monomer unit having a neutralized salt group of the acidic group).

[0028] When a scavenger satisfies only (i), the scavenger contains both a monomer unit having an acidic group and a monomer unit having a neutralizing salt group formed by the acidic group and a first cationic surfactant, or contains only a crosslinked polymer containing a monomer unit having a neutralizing salt group formed by the acidic group and a first cationic surfactant. That is, when a scavenger satisfies only (i), the scavenger does not contain a first cationic surfactant that does not form a neutralizing salt group with the acidic group. When a scavenger satisfies only (ii), the phrase "the scavenger further contains a second cationic surfactant (excluding those in which the second cationic surfactant is a crosslinked polymer containing a monomer unit having a neutralizing salt group formed by the acidic group)" means that the second cationic surfactant is present in the scavenger without reacting with the crosslinked polymer, i.e., the second cationic surfactant does not form a neutralizing salt group with the acidic group of the crosslinked polymer. That is, when a scavenger satisfies only (ii), the crosslinked polymer contained in the scavenger does not contain a monomer unit having a neutralizing salt group formed by the second cationic surfactant. In this case, the second cationic surfactant is not a crosslinked polymer containing a monomer unit having a neutralized base of the acidic group. When the scavenger satisfies (i) and (ii), the scavenger may contain a crosslinked polymer containing a monomer unit having a neutralized base of the acidic group and the first cationic surfactant, and a second cationic surfactant that does not form a neutralized base with the acidic group of the crosslinked polymer, or may contain a crosslinked polymer containing a monomer unit having a neutralized base of the acidic group and the first cationic surfactant, and a second cationic surfactant that does not form a neutralized base with the acidic group of the crosslinked polymer. Note that the types of the first cationic surfactant in (i) and the second cationic surfactant in (ii) may be the same or different.

[0029] Acidic groups are groups that can react with protons (H ＋ The acidic group is not particularly limited, but is preferably an organic acid group, such as a carboxylic acid group (—COOH), a sulfonic acid group [—S(═O)], ２(-OH)], sulfate group [-OS(=O) ２ (—OH)], phosphonic acid group [—P(═O)(—OH) ２ ], a phosphate group [—O—P(═O)(—OH)], and the like.

[0030] The neutralizing base of an acidic group is a compound having any cationic group capable of neutralizing with an acidic group, and the acidic group reacts with the compound to release a proton (H ＋ ) refers to a functional group formed by an ionic bond between a cation derived from a compound having a cationic group and the acidic group. For example, when the acidic group is a carboxylic acid group (—COOH) and the cation derived from the compound having a cationic group is XY ＋ (Y ＋ is a cationic group, and X is Y ＋ Specific examples of cations include metals (such as monovalent or divalent metals), cations of the formula: NR ４ ＋ (wherein each R independently represents a hydrogen atom or an optionally substituted hydrocarbon group, and two or three R may form a ring together with the adjacent nitrogen atom). The cation may also be, for example, a cation derived from a cationic surfactant. A "cationic surfactant" is a surfactant having a cationic group. In this specification, the term "cationic group" includes a group that itself forms a cation, and a group that does not itself form a cation but can form a cation by bonding with a proton. The neutralizing base of an acidic group and a cationic surfactant is a functional group (acidic group + cationic surfactant residue) in which the acidic group and the cationic surfactant react via the cationic group in the cationic surfactant.

[0031] Examples of monovalent metals include alkali metals such as lithium, sodium, and potassium. Examples of divalent metals include alkaline earth metals such as magnesium, calcium, and barium; lead; zinc; and tin.

[0032] Formula:NR ４ ＋In the cation represented by the formula (I), when R is an optionally substituted hydrocarbon group, the hydrocarbon group may be, for example, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. Examples of the alkyl group include C groups such as a methyl group, an ethyl group, a propyl group (n-propyl group or isopropyl group), and a butyl group (n-butyl group, isobutyl group, sec-butyl group, or t-butyl group). １－６ Examples of the cycloalkyl group include a C alkyl group such as a cyclopentyl group and a cyclohexyl group. ５－１４ Examples of the aryl group include a C aryl group such as a phenyl group and a naphthyl group. ６－１４ Examples of the aralkyl group include a C aryl group such as a benzyl group and a phenethyl group. ７－１４ aralkyl groups, etc. Formula: NR ４ ＋ The cation represented by the formula (I) may be a cation derived from a cationic surfactant, or may not be a cation derived from a cationic surfactant.

[0033] Examples of substituents that can be substituted on the hydrocarbon group include halogen atoms, hydroxyl groups, and mercapto groups.

[0034] Formula:NR ４ ＋ In the cation represented by the formula (I), when two or three R's form a ring together with the adjacent nitrogen atom, the ring may be a monocyclic ring such as a pyridine ring or an imidazole ring, or a fused ring such as a quinoline ring. The ring may have one or more substituents, and examples of the substituents include a halogen atom, a hydroxyl group, an amino group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, and an N,N-dialkylamino group. The number of the substituents may be, for example, one, two, or three.

[0035] The cationic surfactant preferably has a cationic group and an optionally substituted hydrocarbon group having 2 to 30 carbon atoms, and more preferably has a cationic group and an optionally substituted hydrocarbon group having 12 to 30 carbon atoms. The number of the hydrocarbon group and cationic group in the cationic surfactant may be one or two or more.

[0036] The hydrocarbon group having 12 to 30 carbon atoms may be any of aliphatic hydrocarbon groups such as alkyl groups and alkenyl groups, alicyclic hydrocarbon groups such as cycloalkyl groups and cycloalkenyl groups, and aromatic hydrocarbon groups, but is preferably an aliphatic hydrocarbon group. Examples of substituents on the hydrocarbon group include a hydroxyl group, an oxo group (=0), an alkoxy group, an alkenyloxy group, and combinations thereof. The number of substituents on the hydrocarbon group may be, for example, 1, 2, 3, 4, or 5.

[0037] The aliphatic hydrocarbon group having 12 to 30 carbon atoms may be either saturated or unsaturated, and examples thereof include aliphatic hydrocarbon groups having 12 to 20 carbon atoms such as lauryl, myristyl, palmityl, stearyl, oleyl, linoleyl, and arachidyl groups.

[0038] For example, hexadecyltrimethylammonium chloride has a palmityl group as the optionally substituted aliphatic hydrocarbon group having 12 to 30 carbon atoms.

[0039] Examples of the alicyclic hydrocarbon group having 12 to 30 carbon atoms include a group having a steroid skeleton. The steroid skeleton is represented by, for example, the following formula (1): (In the formula, the dashed line of ring A indicates that any carbon-carbon single bond may be a carbon-carbon double bond, the dashed line of ring B indicates that the carbon-carbon single bond may be a carbon-carbon double bond, and rings A to D are each optionally substituted. Note that the numbers on the rings are written for convenience to indicate the substitution positions of the substituents.)

[0040] Examples of substituents that can be substituted on rings A to D include a hydroxyl group, an oxo group (=0), an alkyl group (for example, an alkyl group having 1 to 4 carbon atoms such as a methyl group), a combination thereof (for example, a hydroxyalkyl group), etc. The substitution position of the substituent is not particularly limited, and examples include the 3-position, 7-position, 10-position, 11-position, 12-position, 13-position, and 17-position.

[0041] The skeleton represented by formula (1) includes skeletons represented by the following formulas (1-1) to (1-5).

[0042] The alicyclic hydrocarbon group having 12 to 30 carbon atoms is preferably an alicyclic hydrocarbon group having 20 to 30 carbon atoms, more preferably a group in which an optionally substituted alkylene group (for example, an alkylene group having 2 to 4 carbon atoms such as an ethylene group or a propylene group) is linked to the 17th position of a skeleton represented by formula (1), and even more preferably a group in which an optionally substituted alkylene group (for example, an alkylene group having 2 to 4 carbon atoms such as an ethylene group or a propylene group) is linked to the 17th position of a skeleton represented by any of formulas (1-1) to (1-5).

[0043] Examples of aromatic hydrocarbon groups having 12 to 30 carbon atoms include fluorene, anthracene, phenanthrene, tetracene, pyrene, triphenylene, chrysene, and tetraphenylene.

[0044] The optionally substituted hydrocarbon group having 12 to 30 carbon atoms is a diC １２－３０ The group may be a group having an acylglycerol skeleton or a group having a steroid skeleton.

[0045] Examples of the cationic group in the cationic surfactant include -N(R １ ) ２ (In the formula, each R １ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms), -N ＋ (R ２ ) ３ (In the formula, each R ２ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms), and -N ＋(R ２ ) ３ It is preferable that:

[0046] R in the above formula １ and R ２ In the formula (I), examples of the hydrocarbon group having 1 to 6 carbon atoms include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group. Examples of the substituent that can be substituted on the hydrocarbon group having 1 to 6 carbon atoms include a hydroxyl group and an oxo group.

[0047] The formula: -N(R １ ) ２ is preferably —NH ２ or -N(R １１ ) ２ (In the formula, R １１ represents a hydroxyalkyl group having 1 to 5 carbon atoms. Examples of the hydroxyalkyl group having 1 to 5 carbon atoms include a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, and a hydroxypentyl group (including a pentahydroxypentyl group).

[0048] The above formula: -N ＋ (R ２ ) ３ is preferably —N ＋ (CH ３ ) ３ , -N ＋ (CH ３ ) ２ (C ２ H ５ ), -N ＋ (CH ３ ) ２ (C ３ H ７ ) etc.

[0049] The cationic surfactant may be a compound having an aliphatic hydrocarbon group having 12 to 30 carbon atoms and a cationic group (e.g., hexadecyltrimethylammonium chloride), a compound having an alicyclic hydrocarbon group having 12 to 30 carbon atoms and a cationic group, a compound having a cationic group (e.g., triethanolamine), a polymer having a cationic group (e.g., polylysine, polyethyleneimine), etc. The cationic surfactant is preferably a compound having an aliphatic hydrocarbon group having 12 to 30 carbon atoms and a cationic group, and more preferably hexadecyltrimethylammonium chloride (HDTMA-Cl).

[0050] The lower limit of the molar concentration of cationic groups per mole of cationic surfactant is preferably 1 mole or more, more preferably 5 moles or more, and even more preferably 10 moles or more, and the upper limit of the molar concentration of cationic groups per mole of cationic surfactant is preferably 100 moles or less.

[0051] The lower limit of the chemical formula weight or number average molecular weight of the cationic surfactant is preferably 300 or more, and more preferably 500 or more. The upper limit of the chemical formula weight or number average molecular weight of the cationic surfactant is preferably 10,000 or less. The number average molecular weight of the cationic surfactant can be measured using gel permeation chromatography (GPC) under the following conditions. <GPC Measurement Conditions> [1] Apparatus: Gel permeation chromatography [Model number "HLC-8120GPC", manufactured by Tosoh Corporation] [2] Column: "TSKgel G6000PWx I" and "TSKgel G3000PWxl" [both manufactured by Tosoh Corporation] connected in series. [3] Eluent: Methanol / water = 30 / 70 (volume ratio) with 0.5 wt % sodium acetate dissolved therein. [4] Reference substance: polyethylene glycol (hereinafter abbreviated as PEG) [5] Injection conditions: sample concentration 0.25 wt%, column temperature 40°C

[0052] Examples of monomers having an acidic group include unsaturated carboxylic acids. Examples of unsaturated carboxylic acids include unsaturated monocarboxylic acids and unsaturated dicarboxylic acids. Examples of unsaturated monocarboxylic acids include unsaturated monocarboxylic acids having 3 to 10 carbon atoms, such as acrylic acid, methacrylic acid, and crotonic acid, and acrylic acid is more preferred. Examples of unsaturated dicarboxylic acids (including anhydrides) include unsaturated dicarboxylic acids having 4 to 10 carbon atoms, such as maleic acid, fumaric acid, citraconic acid, and itaconic acid, or anhydrides thereof.

[0053] Examples of other monomers having an acidic group include (meth)acrylic monomers having a sulfonic acid group (for example, (meth)acrylic acid sulfoalkyl esters such as 2-sulfoethyl (meth)acrylate, and N-sulfoalkyl (meth)acrylic acid amides such as 2-(meth)acrylamido-2-methylpropanesulfonic acid), and (meth)acrylic monomers having a phosphoric acid group (for example, (meth)acrylic acid phosphonooxyalkyl esters such as 2-((meth)acryloyloxy)ethyl phosphate). In this specification, (meth)acrylic means acryl and / or methacryl, and (meth)acryloyloxy means acryloyloxy and / or methacryloyloxy.

[0054] The monomer having an acidic group may be used alone or in combination of two or more kinds.

[0055] Examples of monomers having a neutralizing base of an acidic group include unsaturated carboxylates. Examples of unsaturated carboxylates include alkali metal salts of unsaturated carboxylic acids (e.g., sodium salts, potassium salts), alkylamine salts (e.g., trialkylamine salts such as triethylamine salts), alkanolamine salts (e.g., dialkanolamine salts such as diethanolamine salts, trialkanolamine salts such as triethanolamine salts), ammonium salts, or tetraalkylammonium salts (e.g., tetramethylammonium salts, tetraethylammonium salts). Examples of unsaturated carboxylates include unsaturated monocarboxylates and unsaturated dicarboxylates. Examples of unsaturated monocarboxylates include unsaturated monocarboxylates having 3 to 10 carbon atoms, with acrylates, methacrylates, and crotonates being preferred, and acrylates being more preferred. Examples of unsaturated dicarboxylates (including anhydrous salts) include unsaturated dicarboxylates having 4 to 10 carbon atoms, with maleates, fumarates, citraconates, itaconates, and anhydrous salts thereof being preferred.

[0056] Other monomers having a neutralizing salt of an acidic group include, for example, neutralized salts of the above-mentioned (meth)acrylic monomers having a sulfonic acid group, neutralized salts of the above-mentioned (meth)acrylic monomers having a phosphate group, etc. More specific examples of these neutralized salts include the above-mentioned alkali metal salts, etc.

[0057] The monomer having a neutralizing salt group of an acidic group may be used alone or in combination of two or more kinds.

[0058] The crosslinked polymer preferably contains both a monomer unit having an acidic group and a monomer unit having a neutralized salt of the acidic group. That is, in the crosslinked polymer containing a monomer unit having an acidic group, it is preferable that a portion of the monomer unit having the acidic group is neutralized and replaced with a monomer unit having a neutralized salt of the acidic group. The degree of neutralization [100 x (molar number of neutralized salts of acidic groups) / (total mole number of acidic groups and neutralized salts of the acidic groups)] is not particularly limited, and may be, for example, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, or 50 mol% or more, or 90 mol% or less, 85 mol% or less, or 80 mol% or less.

[0059] The crosslinked polymer may further contain, in addition to the monomer unit having an acidic group and / or the monomer unit having a neutralized salt group of the acidic group, other monomer units different from the above-mentioned monomers. Examples of the other monomers include the following monofunctional ethylenically unsaturated monomers.- Esters of unsaturated carboxylic acids (for example, acrylic acid, methacrylic acid, etc.) (for example, alkyl esters such as methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, heptyl ester, octyl ester, 2-ethylhexyl ester, etc.; hydroxyalkyl esters such as 2-hydroxyethyl ester; haloalkyl esters such as 2,2,2-trifluoroethyl ester; aminoalkyl esters such as 2-aminoethyl ester; (mono- or dialkylamino)alkyl esters such as 2-(N,N-dimethylamino)ethyl ester; cycloalkyl esters such as cyclohexyl ester; aryl esters such as phenyl ester and naphthyl ester; aralkyl esters such as benzyl ester and phenethyl ester; glycidyl ester; polyethylene glycol ester, etc.); - Unsaturated carboxylic acid amides (for example, free amides; N-substituted amides such as N-monoalkylamide and N,N-dialkylamide, etc.); - Unsaturated dicarboxylic acid imides (for example, maleimide, citraconic imide, itaconimide, N-alkyl-substituted, N-cycloalkyl-substituted, or N-aryl-substituted versions of these, etc.) - Alkenyl esters (e.g., vinyl esters, allyl esters, etc.) of saturated carboxylic acids (e.g., acetic acid, propionic acid, etc.); - Esters or amides of unsaturated sulfonic acids (e.g., esters or amides of unsaturated sulfonic acids corresponding to the above-mentioned esters or amides of unsaturated carboxylic acids); - Unsaturated alcohols (e.g., allyl alcohol, propenyl alcohol, etc.); - Unsaturated ethers (e.g., alkyl vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; alkyl allyl ethers such as methyl allyl ether and ethyl allyl ether; cycloalkyl vinyl ethers such as cyclohexyl vinyl ether; glycidyl vinyl ether; - Unsaturated nitriles (e.g., acrylonitrile, methacrylonitrile, etc.); - Olefins (e.g., ethylene, propylene, butene, pentene, hexene, etc.); - Aromatic vinyl compounds (e.g., styrene, α-methylstyrene, vinyltoluene, hydroxystyrene, etc.); - Heterocyclic vinyl compounds (e.g., N-vinylpyrrolidone, etc.).

[0060] When the crosslinked polymer contains another monomer other than the monomer having an acidic group and / or a neutralized salt group of the acidic group, the other monomer may be one type alone or a combination of two or more types.

[0061] The monomer constituting the crosslinked polymer preferably has a solubility in water at 25° C. (g / 100 g of water) of 1 g or more, more preferably 5 g or more. The monomer constituting the crosslinked polymer may be miscible with water.

[0062] A crosslinked polymer is a polymer having a crosslinked structure (a structure in which the constituent atoms of a plurality of linear macromolecules (polymers of monomers having acidic groups and / or monomers having neutralized salts of the acidic groups) are covalently bonded to each other directly or via other atoms). When the monomers constituting the crosslinked polymer have reactive groups (for example, a combination of a monomer having a carboxyl group and a monomer having an amino group), the crosslinked structure may be self-crosslinked, or may be crosslinked with any crosslinking agent as needed.

[0063] Examples of crosslinking methods using a crosslinking agent include the following: 1) A method of polymerizing the above-mentioned monomer with an internal crosslinking agent to directly obtain a crosslinked polymer having a crosslinked structure (e.g., the methods described in JP-A-2003-225565 and JP-A-2005-075982). 2) A method of crosslinking a polymer containing the above-mentioned monomer as an essential constituent monomer using a surface crosslinking agent to obtain a crosslinked polymer (e.g., the methods described in JP-A-3648553, JP-A-2003-165883, JP-A-2003-225565, JP-A-2005-75982, JP-A-2005-95759).

[0064] The crosslinking agent (including an internal crosslinking agent and a surface crosslinking agent) is not particularly limited, but examples thereof include the following difunctional or higher ethylenically unsaturated monomers. Alkenyl ethers (for example, vinyl ethers, allyl ethers, etc.) of dihydric or higher alcohols (for example, alkylene glycols or polyalkylene glycols (the alkylene group preferably has 2 to 4 carbon atoms, and the alkylene oxide preferably has 10 to 50 repeating atoms) such as ethylene glycol, polyethylene glycol, propylene glycol, butylene glycol, and neopentyl glycol, glycerin, polyglycerin, trimethylolethane, trimethylolpropane, pentaerythritol, dipentaerythritol, and sorbitan); esters of unsaturated carboxylic acids (for example, acrylic acid, methacrylic acid, etc.) and dihydric or higher alcohols (for example, those exemplified as ethers); alkenyl esters (for example, vinyl esters, allyl esters, etc.) of unsaturated carboxylic acids (for example, acrylic acid, methacrylic acid, etc.); and alkenyl esters (for example, vinyl esters, allyl esters, etc.) of polycarboxylic acids (for example, tartaric acid, citric acid, adipic acid, etc.) Alkenyl esters of isocyanuric acid (for example, triallyl isocyanate, etc.) Alkylenebisacrylamides (for example, methylenebisacrylamide, etc.) Alkylenebismethacrylamides (for example, methylenebismethacrylamide, etc.) Di- or trialkenylamines (for example, diallylamine, triallylamine, etc.) Aromatic polyvinyl compounds (for example, divinylbenzene, etc.)

[0065] The crosslinking agent may be one having at least two functional groups capable of reacting with the substituents (carboxyl group, hydroxyl group, etc.) of the monomer. Examples of such crosslinking agents include polyhydric alcohol glycidyl ethers (e.g., alkylene glycol diglycidyl ethers such as ethylene glycol diglycidyl ether, and alkanetriol di- or triglycidyl ethers such as glycerin diglycidyl ether).

[0066] The crosslinking agent may be used alone or in combination of two or more.

[0067] The amount of crosslinking agent can be appropriately selected depending on the desired crosslink density, and may be, for example, 0.005 mol % or more, or 0.01 mol % or more, or 0.5 mol % or less, or 0.4 mol % or less, relative to the monomers constituting the crosslinked polymer.

[0068] When the scavenger satisfies (ii), the second cationic surfactant may be any of the cationic surfactants described above (provided that the second cationic surfactant is not a crosslinked polymer containing a monomer unit having a neutralized salt group of the acidic group). When the "cationic group" of the second cationic surfactant is a "group that itself forms a cation," the counter anion may be a halide ion (F － , Cl － ,Br － , I － etc.)

[0069] Whether a scavenger satisfies (i) and / or (ii) can be identified, for example, by immersing the scavenger in an appropriate solvent (e.g., a hydrocarbon solvent such as hexane), liberating the cationic surfactant in the solvent, isolating the cationic surfactant, and subjecting it to NMR and gas chromatography.

[0070] The scavenger is preferably particles having a volume average particle diameter in the range of 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, or 150 μm or more. The scavenger is preferably particles having a volume average particle diameter in the range of 2000 μm or less, 1500 μm or less, 1000 μm or less, 500 μm or less, or 300 μm or less. The volume average particle diameter can be measured, for example, by the method described in the Examples below.

[0071] The absorbency of the sequestering agent of the present invention in physiological saline (absorption capacity in physiological saline) is preferably 1 g / g or more, more preferably 3 g / g or more, even more preferably 5 g / g or more, even more preferably 10 g / g or more, and most preferably 15 g / g or more. If the absorbency of the sequestering agent in physiological saline (absorption capacity in physiological saline) is too low, the absorbency tends to increase by increasing the amount of the crosslinker. There is no particular limitation on the upper limit of the absorbency of the sequestering agent in physiological saline (absorption capacity in physiological saline), but for example, it is preferably 120 g / g or less, more preferably 100 g / g or less, and particularly preferably 50 g / g or less. The absorbency in physiological saline can be measured, for example, by the method described in the Examples below.

[0072] When the scavenger satisfies (i) and / or (ii), the molar concentration of the cationic group in the scavenger is 6×10 based on the weight of the scavenger after drying. －６ mol / g to 1×10 －４ The weight of the scavenger after drying can be measured, for example, by the following method: 1 g of the scavenger is placed on a petri dish, covered with filter paper, and heated and dried at 130°C for 60 minutes in a circulating air dryer, and the weight of the residue after that can be taken as the weight of the crosslinked polymer after drying.

[0073] The scavenger of the present invention can be produced, for example, via the production of a crosslinked polymer, as follows. The crosslinked polymer of the present invention can be produced, for example, by a method including the steps of polymerizing (solution polymerization, emulsion polymerization, suspension polymerization, etc.) a composition containing a monomer, a crosslinking agent, a solvent, an initiator, and, if necessary, a neutralizing agent, drying the polymer, and, if necessary, classifying the polymer. The scavenger of the present invention can be produced, for example, by a method including the steps of mixing the above-mentioned crosslinked polymer with a cationic surfactant (preferably mixing at 20 to 30°C for 1 to 2 minutes), and, if necessary, subsequently aging the mixture at 100 to 150°C for 10 to 60 minutes.

[0074] In the contacting step, the amount of the capturing agent brought into contact with the fecal sample that has been subjected to the filtration step may be, for example, 0.01 mg or more, 0.05 mg or more, 0.1 mg or more, 0.5 mg or more, 1 mg or more, 5 mg or more, 10 mg or more, 15 mg or more, 20 mg or more, or 25 mg or more relative to 1 g of the mass of the feces used in preparing the fecal sample. In the contacting step, the amount of the capturing agent brought into contact with the fecal sample that has been subjected to the filtration step may be, for example, 100 mg or less, 90 mg or less, 80 mg or less, 70 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, or 10 mg or less relative to 1 g of the mass of the feces used in preparing the fecal sample.

[0075] The contacting step may be performed, for example, by contacting the stool sample that has undergone the filtration step with the capturing agent and holding the sample for, for example, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. The contacting step may be performed, for example, by contacting the stool sample that has undergone the filtration step with the capturing agent and holding the sample for, for example, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, or 50 minutes or less. The contacting step may be performed at, for example, 4°C to 40°C, 10°C to 35°C, or 15°C to 30°C, but is not particularly limited thereto.

[0076] The method according to this embodiment may further include a step (recovery step) of recovering the extracellular vesicles and / or membrane vesicles captured by the capture agent in the contact step. In this recovery step, the extracellular vesicles and / or membrane vesicles captured by the capture agent are separated from the capture agent. Therefore, the recovery step is also called a separation step. By including the recovery step in the method according to this embodiment, purified extracellular vesicles and / or membrane vesicles can be obtained.

[0077] The recovery step can be carried out by a conventional method. For example, after the contact step, the salt concentration or pH may be changed to elute the extracellular vesicles and / or membrane vesicles from the capture agent.

[0078] As described above, the method according to this embodiment can purify extracellular vesicles and / or membrane vesicles in feces by performing a filtration step on a fecal sample and then performing a contact step. Therefore, according to one embodiment of the present invention, there is also provided a kit for purifying extracellular vesicles and / or membrane vesicles in feces, comprising a filter and the above-described capture agent.

[0079] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. <Preparation of Scavenger> In this example, a scavenger of the present invention was prepared by the following procedure. First, 116.5 g of acrylic acid, 272.2 g of ion-exchanged water, and 0.58 g of ethylene glycol diglycidyl ether (0.5 wt % / acrylic acid) as a crosslinking agent were placed in a 1-liter beaker and mixed to dissolve the crosslinking agent. While the beaker was cooled in an ice bath, 96.1 g of a 48.5 wt % aqueous sodium hydroxide solution was added to neutralize a portion (72 mol %) of the acrylic acid. The neutralized monomer solution was then cooled to 5°C, and 9.3 g of a 2 wt % aqueous potassium persulfate solution was added as a polymerization initiator to prepare an aqueous monomer solution. A 2-liter separable flask equipped with a stirrer and condenser (cooler) was charged with 1,434 g of cyclohexane and 7.1 g of Rheodol SP-S10V (Kao Corporation, sorbitan monostearate) as a dispersant. The flask was heated to an internal temperature of 60°C using a hot water bath and stirred to dissolve the dispersant in the cyclohexane. Nitrogen was then introduced into the solution in the separable flask to reduce the dissolved oxygen content of the cyclohexane to 0.1 ppm or less. Then, while stirring with a stirrer, 350 g of aqueous monomer solution was added dropwise using a dropping funnel. Reverse-phase suspension polymerization was carried out at a polymerization temperature of 80°C. After the addition of the aqueous monomer solution was completed, the mixture was heated for an additional 2 hours to complete the suspension polymerization, yielding a spherical hydrogel in cyclohexane. The stirrer was stopped, the resulting hydrogel was allowed to settle, and the cyclohexane was removed by decantation. The remaining hydrogel was washed several times with cyclohexane to remove any dispersant adhering to the hydrogel. The obtained spherical hydrogel was spread on a release paper and dried for 1 hour in a vacuum dryer at 130° C. (vacuum degree: 10,000 to 20,000 Pa) to obtain a crosslinked polymer A.

[0080] Next, this crosslinked polymer A was sieved using sieves with openings of 300 μm and 500 μm to have a particle size of 300 μm to 500 μm, thereby obtaining a crosslinked polymer B.

[0081] Subsequently, 20 g of a 50 wt % aqueous potassium carbonate solution was sprayed into 50 g of Crosslinked Polymer B through a spray nozzle, and after uniform mixing, the mixture was heated at 130° C. for 30 minutes and cooled to room temperature to obtain Crosslinked Polymer C.

[0082] Finally, 1 g of a 5 wt % hexadecyltrimethylammonium chloride (HDTMA-Cl, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) cyclohexane solution was added dropwise with a dropper to 10 g of crosslinked polymer C, and after uniform mixing, the mixture was heated at 130°C for 30 minutes and cooled to room temperature to obtain a scavenger.

[0083] The volume average particle size of the scavenger was measured with a particle analyzer (CAMSIZER XT, manufactured by Retsch) and was found to be 420 μm.

[0084] The saline absorption capacity of the scavenger was measured as follows. First, 1 g of a measurement sample was placed in a tea bag (20 cm long, 10 cm wide) made of nylon mesh with a mesh size of 63 μm (JIS Z8801-1:2006), and the bag was immersed in 1000 ml of saline (salt concentration 0.9 wt%) for 1 hour without stirring, and then hung for 15 minutes to drain. Thereafter, the weight (h1) of the tea bag was measured, and the saline absorption capacity was calculated using the following formula. The saline used and the temperature of the measurement atmosphere were 25°C ± 2°C. The weight of the tea bag was measured in the same manner as above, except that no measurement sample was used, and this was taken as (h2). Saline absorption capacity (g / g) = (h1) - (h2)

[0085] As a result, the absorbency of the scavenger for saline was 32 g / g, and the measurement result of the pH of the saline solution was 7.0. The pH measurement was performed according to the method described below. In addition, the weight ratio of the scavenger after heat drying to the weight of the scavenger was 97.4%, and the molar concentration of the cationic groups relative to the weight of the scavenger after drying was 1.60 × 10 －５ mol / g.

[0086] <Method for measuring pH of physiological saline solution> 0.5 g of a measurement sample was added to a 100 mL cylindrical beaker with a diameter of 50 mm to make up a total volume of 100 g with physiological saline (salt concentration: 0.9 wt %), and the mixture was stirred at 25° C. for 30 minutes at 60 rpm with a stirrer tip (length: 30 mm) and then allowed to stand for 1 minute at 25° C. After standing, the pH of the supernatant at 25° C. was measured with a pH meter, and the value was taken as the pH of the physiological saline solution.

[0087] Reference Example 1: Evaluation of capture of exosomes in urine or feces by capture agents Comparative Example 1 (1. Reaction of urine supernatant with capture agent) 1.1 mL of a human urine sample was centrifuged at 500 × g for 10 minutes, and 1 mL of supernatant was collected. Subsequently, the collected 1 mL of supernatant was added to a microbiospin chromatography column (Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 × g for 1 minute, and the urine supernatant (flow-through) after reaction with the capture agent was obtained.

[0088] (2. Measurement of exosome quantity) The quantity of exosomes captured by the capture agent was evaluated by measuring the quantity of exosomes in the urine supernatant before reaction with the capture agent and the quantity of exosomes in the urine supernatant (flow-through) after the reaction. The quantity of exosomes was measured by a sandwich immunoassay using antibodies against CD9 and CD63, which are representative surface antigens of extracellular vesicles. The sandwich immunoassay was performed using a fully automated analyzer BLEIA (registered trademark)-1200 (manufactured by Eiken Chemical Co., Ltd.) constructed using a BLEIA measurement system based on bioluminescence (BLEIA bioluminescence enzyme immunoassay). For the primary reaction, magnetic particles on which anti-human CD9 antibody (Cosmo Bio, SHI-EXO-M01) was immobilized, 50 μL of sample, and biotin-labeled anti-human CD63 antibody (Cosmo Bio, SHI-EXO-M02-B) were mixed and reacted at 37°C for 15 minutes. Thereafter, unreacted substances and biotin-labeled antibodies were removed by washing, and 80 μL of Tris buffer containing streptavidin and biotin-labeled luciferase was added, followed by a secondary reaction for an additional 15 minutes at 37° C. After the secondary reaction, washing was again carried out, and 100 μL of luminescent substrate solution (manufactured by Eiken Chemical Co., Ltd.) was added to detect the luminescent signal, which was then quantified as a luminescence value.

[0089] Comparative Example 2 (1. Reaction of Fecal Suspension Supernatant with Capture Agent) Approximately 3 g of a human fecal specimen was suspended in 50 mL of PBS to obtain a fecal suspension. The fecal suspension was centrifuged at 3,000 × g for 10 minutes, and the supernatant of the fecal suspension was collected. Subsequently, 1 mL of the collected supernatant was added to a microbiospin chromatography column (Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 × g for 1 minute, and the supernatant (flow-through) of the fecal suspension after reaction with the capture agent was obtained.

[0090] (2. Measurement of exosome amount) The amount of exosomes captured by the capturing agent was measured as described above in "Measurement of exosome amount".

[0091] <Results> The results are shown in Figure 1. Figure 1(A) shows the CD9 / CD63 luminescence values ​​in the urine supernatant and flow-through before reacting the urine supernatant with the capture agent. Figure 1(B) shows the CD9 / CD63 luminescence values ​​in the fecal suspension supernatant and flow-through before reacting the fecal suspension supernatant with the capture agent.

[0092] As shown in Figure 1(A), when urine supernatant was reacted with the capture agent, the CD9 / CD63 luminescence value in the flow-through was low, indicating that the amount of exosomes in the flow-through was very low. This suggests that most of the exosomes in the urine supernatant were captured by the capture agent. Furthermore, as shown in Figure 1(B), when fecal suspension supernatant was reacted with the capture agent, the flow-through showed a higher CD9 / CD63 luminescence value than the fecal suspension supernatant before the reaction, confirming that the amount of exosomes in the flow-through was high. This suggests that exosomes in the fecal suspension supernatant were not captured by the capture agent.

[0093] Because the capture agent is water-absorbent, the volume of the flow-through obtained after the reaction between the supernatant of the fecal suspension and the capture agent is smaller than that of the supernatant of the fecal suspension before the reaction. Therefore, if exosomes are not captured by the capture agent, the exosomes in the flow-through will be more concentrated than those in the supernatant of the fecal suspension before the reaction, and the CD9 / CD63 luminescence value in the flow-through will be higher than that in the supernatant of the fecal suspension before the reaction.

[0094] The results of Reference Example 1 showed that exosomes in feces cannot be captured simply by reacting them with a capturing agent, and that extracellular vesicles in feces are more difficult to purify than those in urine.

[0095] Test Example 1: Evaluation of exosome capture in feces by filter filtration and capture agent Example 1 (1. Reaction of the filtrate from the supernatant of a fecal suspension with a capture agent) Approximately 3 g of a human fecal specimen was suspended in 50 mL of PBS to obtain a fecal suspension. The fecal suspension was centrifuged at 3,000 × g for 10 minutes, and the supernatant was collected. The collected supernatant was then filtered through a 0.45 μm pore size PVDF filter (Whatman) to obtain a filtrate, which was then added to a microbiospin chromatography column (Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 × g for 1 minute, and the filtrate (flow-through) after reaction with the capture agent was obtained.

[0096] (2. Measurement of exosome amount) The amount of exosome was measured as described above in "Measurement of exosome amount".

[0097] Comparative Example 3 (1. Reaction of the supernatant of a fecal suspension with a capturing agent) Approximately 3 g of a human fecal specimen was suspended in 50 mL of PBS to obtain a fecal suspension. The fecal suspension was centrifuged at 3,000 × g for 10 minutes, and the supernatant was collected. Thereafter, the supernatant after reaction with the capturing agent (flow-through) was obtained in the same manner as in Example 1, except that the supernatant was not filtered.

[0098] (2. Measurement of exosome amount) The amount of exosome was measured as described above in "Measurement of exosome amount".

[0099] <Results> The results are shown in Figure 2. Figure 2(A) shows the CD9 / CD63 luminescence values ​​in the supernatant and flow-through of the fecal suspension before reaction when the supernatant was reacted with the capture agent without filtering. Figure 2(B) shows the CD9 / CD63 luminescence values ​​in the supernatant and flow-through of the fecal suspension before reaction when the supernatant was filtered and then reacted with the capture agent.

[0100] As shown in Figure 2(A), when the supernatant of the fecal suspension was reacted with the capture agent without filtering, the amount of exosomes in the flow-through was higher than that in the supernatant of the fecal suspension before the reaction, suggesting that the exosomes in the supernatant of the fecal suspension were not captured by the capture agent (Figure 2(A)). In contrast, as shown in Figure 2(B), when the supernatant of the fecal suspension was filtered and then reacted with the capture agent, the amount of exosomes in the flow-through was significantly lower than that in the supernatant of the fecal suspension before the reaction, suggesting that the exosomes in the supernatant of the fecal suspension were captured by the capture agent (Figure 2(B)).

[0101] The results of Test Example 1 showed that filtering the supernatant of a fecal suspension through a filter significantly improved the capture efficiency of extracellular vesicles in the supernatant of the fecal suspension by the capture agent.

[0102] Reference Example 2: Evaluation of exosome capture rate by filter filtration and magnetic beads One purification method used for extracellular vesicle purification is a method using MagCapture™ Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). This method is excellent in both speed and recovery rate, allowing extracellular vesicle purification to be completed in approximately 3 hours. The above method was used to examine the purification of extracellular vesicles from serum or feces.

[0103] Comparative Examples 4 and 5 (1. Pretreatment) 1200 μL of a human serum sample was centrifuged at 1,000 × g for 10 minutes, and the supernatant was used as input for treatment with the above kit. Approximately 0.2 g of a human fecal sample was suspended in 3.84 mL of Tris buffer to obtain a fecal suspension. The fecal suspension was centrifuged at 3,000 × g for 10 minutes, and the supernatant was recovered. Subsequently, the recovered supernatant was filtered through a 0.45 μm filter (Whatman), and the filtrate was used as input for treatment with the above kit.

[0104] (2. Exosome Purification) Exosomes were purified from the supernatant of serum or the filtrate of the supernatant of fecal suspension using MagCapture™ Exosome Isolation Kit PS. Purification was performed according to the attached instructions.

[0105] (3. Measurement of exosome amount) The amount of exosomes in the filtrate of the serum supernatant or fecal suspension supernatant, and the filtrate of the serum supernatant or fecal suspension supernatant were reacted with the exosome-capturing magnetic particles of the kit, and then the amount of exosomes in the residual sample liquid (flow-through) separated from the magnetic particles was measured to evaluate the amount of exosomes captured by the kit. The measurement of exosome amount was performed as described above in "Measurement of exosome amount."

[0106] <Results> The results are shown in Figure 3. Figure 3(A) shows the CD9 / CD63 luminescence values ​​in the serum supernatant and flow-through before treatment when the serum supernatant was treated with the MagCapture™ Exosome Isolation Kit PS. Figure 3(B) shows the CD9 / CD63 luminescence values ​​in the filtrate and flow-through when the filtrate of the supernatant of a fecal suspension was treated with the same kit.

[0107] As shown in Figure 3(A), when serum supernatant was reacted with the magnetic beads in the kit, the amount of exosomes in the flow-through was lower than that in the serum supernatant before treatment, suggesting that the exosomes in the serum supernatant were captured by the MagCapture magnetic beads.On the other hand, as shown in Figure 3(B), when the filtrate of the supernatant of the fecal suspension was reacted with the magnetic beads in the kit, the amount of exosomes in the flow-through was higher than that in the filtrate, suggesting that the exosomes in the filtrate of the supernatant of the fecal suspension were not captured by the MagCapture magnetic beads.

[0108] Test Example 2: Evaluation of filter pore size and material in the capture of exosomes in feces by filter filtration and capture agent Examples 2 to 5 (1. Reaction between the supernatant of a fecal suspension and a capture agent) Approximately 3 g of a human fecal specimen was suspended in 50 mL of PBS to obtain a fecal suspension. Subsequently, the fecal suspension was filtered through a 0.45 μm pore size PVDF filter (manufactured by Whatman), a 0.8 μm pore size MCE filter (manufactured by Millipore), a 1.2 μm pore size MCE filter (manufactured by Millipore), or a 5.0 μm pore size PVDF filter (manufactured by Millipore), and the filtrate was added to a microbiospin chromatography column (manufactured by Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 xg for 1 minute to obtain the filtrate (flow-through) after reaction with the capture agent.

[0109] (2. Measurement of exosome amount) The amount of exosome was measured as described above in "Measurement of exosome amount".

[0110] <Results> The results are shown in Figure 4. Figures 4(A) to (D) show the results of calculating the CD9 / CD63 luminescence values ​​in the filtrate before the reaction and in the flow-through when the filtrate obtained by filtering through a PVDF filter with a pore size of 0.45 µm, an MCE filter with a pore size of 0.8 µm, an MCE filter with a pore size of 1.2 µm, or a PVDF filter with a pore size of 5.0 µm was reacted with a capture agent.

[0111] As shown in Figures 4(A) and 4(B), when fecal suspensions were filtered through filters with pore sizes of 0.8 μm or less, almost no exosomes were present in the flow-through, suggesting that most of the exosomes in the fecal suspension were captured by the capture agent. On the other hand, as shown in Figures 4(C) and 4(D), when fecal suspensions were filtered through filters with pore sizes of 1.2 μm to 5.0 μm, exosomes were present in the flow-through. This suggests that some exosomes in the supernatant of the fecal suspension passed through without being captured by the capture agent. Furthermore, the filter material did not affect exosome capture by the capture agent.

[0112] Test Example 3: Evaluation of OMV Capture by Capture Agent (1) Example 6 (1. Culturing of Bacteroides vulgatus) Bacteroides vulgatus, stored frozen at Eiken Chemical Co., Ltd., was seeded onto Poremedia sheep blood agar medium (manufactured by Eiken Chemical Co., Ltd.) and cultured overnight in an anaerobic environment at 37°C. Subsequently, bacteria were picked from the colony and seeded onto a sterilized medium prepared by dissolving 100 mL of clinical thioglycollate medium "Eiken" (manufactured by Eiken Chemical Co., Ltd.), followed by overnight culture in an anaerobic environment at 37°C. As a control, a medium without picked bacteria was also cultured at the same time. After culturing, the supernatant was centrifuged at 3,000 x g for 30 minutes, and further centrifuged at 40,000 x g for 1.5 hours to remove bacterial cells.

[0113] (2. Reaction of bacterial culture supernatant with capture agent) 1 mL of the supernatant of a culture medium containing cultured bacteria or a culture medium not containing cultured bacteria (culture medium supernatant_B. vulguta or culture medium supernatant_control) centrifuged at 40,000 × g was added to a microbiospin chromatography column (Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 × g for 1 minute to remove unreacted medium. Furthermore, 500 μL of saline was added, and the column was centrifuged at 1,000 × g for 1 minute to remove the unreacted medium. This process was repeated three times. Then, 300 μL of 0.5% Triton X-100 solution was added to the column and allowed to stand at room temperature for 10 minutes. The column was then centrifuged at 1,000 × g for 1 minute to obtain an eluate. This was repeated once more to obtain an eluate (elution_B. vulgutas or elution_control).

[0114] (3. Western Blot Analysis of Eluates) 17.5 μL of each eluate was mixed with SDS and DTT and heated at 95°C for 10 minutes to prepare Western blot samples. 20 μL of the Western blot sample was electrophoresed on a 10-20% acrylamide gel, transferred to a PVDF membrane, and reacted with anti-Lipid A LPS antibody (Invitrogen) overnight in a refrigerator. Subsequently, the membrane was reacted with HRP-labeled anti-goat antibody for 1 hour, and then developed with ECL prime (GE Healthcare). Specific bands reacting with the anti-Lipid A LPS antibody were detected using iBright Imaging Systems (Thermo Fisher Scientific).

[0115] <Results> The results are shown in Figure 5. Figure 5 shows the results of Western blotting of LPS / Lipid A in the culture medium supernatant and eluate. As shown in Figure 5, a ladder-like band thought to be LPS protein was detected in the eluate as well as in the culture medium supernatant, suggesting that the capture agent had captured membrane vesicles.

[0116] Test Example 4: Evaluation of Capture of Bacterial Membrane Vesicles by Capture Agent (2) The ability of the capture agent of the present invention to capture membrane vesicles in culture medium was demonstrated in Example 6 using membrane vesicles of Bacteroides vulgatus. To confirm whether this capture method can capture a wide range of bacterial membrane vesicles, feces and bacterial membrane vesicles purified from feces were evaluated. The evaluation was performed by comparing with ultracentrifugation, which is considered the gold standard for extracellular vesicle purification.

[0117] Example 7 (1. Preparation of pooled feces and purification of bacterial DNA in feces) Feces from 20 healthy individuals were mixed to prepare pooled feces from healthy individuals. Bacterial DNA in the feces was purified by treating 200 mg of the pooled feces with a QIAamp PowerFecal Pro DNA Kit (Qiagen) according to the method described in the package insert.

[0118] (2. Purification of bacterial membrane vesicles from feces by ultracentrifugation) Pooled feces were suspended in PBS to a concentration of 5%, yielding a 5% fecal suspension. The suspension was centrifuged at 3,000 × g for 30 minutes, and the resulting supernatant was filtered through a 0.45 μm filter. The suspension was then centrifuged at 150,000 × g for 120 minutes in an ultracentrifuge (Beckman Coulter). The supernatant was then discarded, an appropriate amount of PBS was added, and the mixture was centrifuged again at 150,000 × g for 60 minutes. After removing the supernatant, CD1 buffer included with the QIAamp PowerFecal Pro DNA Kit (Qiagen) was added, and the mixture was treated according to the method described in the package insert to obtain DNA.

[0119] (3. Purification of bacterial membrane vesicles from feces using a capture agent) 1 mL of a 5% fecal suspension was added to a microbiospin chromatography column (Bio-Rad, 7326204) packed with 40 mg of capture agent and allowed to stand at room temperature for 30 minutes. The column was then centrifuged at 1,000 × g for 1 minute to remove unreacted solution. 500 μL of saline was added, and the column was centrifuged at 1,000 × g for 1 minute to remove the unreacted solution. This process was repeated three times. CD1 buffer included in the QIAamp PowerFecal Pro DNA Kit (Qiagen) was then added to the column and allowed to stand at room temperature for 10 minutes. The column was then centrifuged at 1,000 × g for 1 minute to obtain an eluate. The eluate was treated according to the method described in the kit's package insert to obtain DNA.

[0120] (4. Amplicon sequencing analysis of DNA in bacteria in feces and membrane vesicles derived from the bacteria) The DNA obtained by each method (feces, ultracentrifugal purification, capture agent purification) was subjected to amplicon sequencing analysis (16S ribosomal DNA V3-V4 region) using the analysis contracted by Seibu Giken Co., Ltd.

[0121] <Results> The results are shown in Figure 6 and Table 1. Figure 6 shows the top 20 bacterial species profiles obtained by ultracentrifugal purification, as a result of amplicon sequencing analysis of DNA obtained from feces or DNA obtained from bacterial-derived membrane vesicles obtained by each purification method (ultracentrifugal purification, capture agent purification). Table 1 shows the total reads analyzed by amplicon sequencing and the number of reads. As shown in Figure 6 and Table 1, the bacterial species profiles in feces and the profiles of bacterial-derived membrane vesicles obtained by ultracentrifugal purification were significantly different. On the other hand, the profiles of bacterial-derived membrane vesicles obtained by ultracentrifugal purification and capture agent purification were very similar, suggesting that capture agent purification can comprehensively capture bacterial-derived membrane vesicles in feces, similar to ultracentrifugal purification.

[0122]

Claims

1. A method for purifying extracellular vesicles and / or membrane vesicles in feces, comprising the steps of filtering a fecal sample through a filter and contacting the filtered sample with a capture agent, wherein the capture agent contains a crosslinked polymer containing a monomer unit having an acidic group and / or a monomer unit having a neutralized base of the acidic group, and satisfies the following (i) and / or (ii): (i) the crosslinked polymer contains at least a monomer unit having a neutralized base of the acidic group and a cationic group in a first cationic surfactant, and (ii) the capture agent further contains a second cationic surfactant (with the exception of a case where the second cationic surfactant is a crosslinked polymer containing a monomer unit having a neutralized base of the acidic group).

2. The method according to claim 1, wherein the monomer unit having an acidic group is an unsaturated monocarboxylic acid having 3 to 10 carbon atoms, and the monomer unit having a neutralizing salt of the acidic group is an unsaturated monocarboxylate having 3 to 10 carbon atoms.

3. The method of claim 1, wherein the monomer unit having an acidic group is acrylic acid and the monomer unit having a neutralizing salt of the acidic group is an acrylate.

4. The cationic group in the cationic surfactant is -N ＋ R ３ (wherein each R is independently a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms).

5. The method according to claim 1, wherein the cationic surfactant has an optionally substituted aliphatic hydrocarbon group having 12 to 30 carbon atoms.

6. The method of claim 1, wherein said cationic surfactant is hexadecyltrimethylammonium chloride.

7. The method according to any one of claims 1 to 6, wherein the pore size of the filter is 0.8 μm or less.

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