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

Technical Field

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

Background Art

[0002] Extracellular vesicles are vesicles with a lipid bilayer membrane secreted by cells, which contain proteins, nucleic acids, etc., transport these substances, and are involved in various physiological phenomena. Extracellular vesicles are present in most body fluids, and are also present in large amounts in feces. In addition to extracellular vesicles, feces also contain membrane vesicles released by intestinal bacteria, etc., which have functions similar to those of extracellular vesicles. Since extracellular vesicles and membrane vesicles may be applicable to the diagnosis of diseases, etc., they have attracted attention in recent years, for example, in the medical field. Therefore, the development of a method for purifying extracellular vesicles and / or membrane vesicles from biological samples is desired. However, there is little research on extracellular vesicles and membrane vesicles in feces.

[0003] Feces contain more contaminants compared to urine, blood, etc., and it is 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 (for example, Non-Patent Document 1). However, in this method, since ultracentrifugation is particularly used, the treatment takes time, and expensive and large equipment is required.

[0004] Methods for separating exosomes or lipid bilayers from biological samples that are not feces are known. For example, Patent Document 1 discloses a method for separating exosomes from a biological sample, which includes a step of contacting the biological sample with a superabsorbent polymer to obtain a superabsorbent polymer gel containing exosomes, and a step of mixing the superabsorbent polymer gel with a salt to recover the exosomes. It is described that the superabsorbent polymer preferably has repeating units represented by a predetermined structural formula. Further, Patent Document 2 discloses a method for separating a substance having a lipid bilayer from a bio-derived sample, which includes a step of contacting the bio-derived sample with a predetermined crosslinked polymer to obtain a polymer gel containing the substance having a lipid bilayer, and a step of mixing the polymer gel with a salt to recover the substance having a lipid bilayer. It is described that the predetermined crosslinked polymer is a crosslinked polymer containing monomer units having an acidic group and / or its neutralized base, and is a crosslinked polymer 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 2 describes or suggests that extracellular vesicles and membrane vesicles can be purified from feces.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

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

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that by filtering the supernatant of a fecal suspension with a filter and capturing the filtered sample using a predetermined capture agent, the capture efficiency of extracellular vesicles is greatly improved, 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: a step of filtering a sample of the feces with a filter, and a step of bringing the filtered sample into contact 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 a monomer unit having at least a neutralized base of the acidic group and a first cationic surfactant. (ii) The capture agent further contains a second cationic surfactant (however, excluding those in which 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 [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 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 base of the acidic group is acrylate. [4] The cationic group in the above cationic surfactant is -N + R3 (wherein each R independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms), the method according to any one of [1] to [3]. [5] The above cationic surfactant has an optionally substituted aliphatic hydrocarbon group having 12 to 30 carbon atoms, the method according to any one of [1] to [4]. [6] The above cationic surfactant is hexadecyltrimethylammonium chloride, the method according to any one of [1] to [5]. [7] The pore size of the above filter is 0.8 μm or less, the method according to any one of [1] to [6].

Advantages of the Invention

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

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

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Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0012] Hereinafter, the mode for carrying out the present invention will be described in detail. However, 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 the present embodiment (hereinafter, also referred to as "the method according to the present embodiment") includes a step of filtering the above fecal sample with a filter (filtering step), and a step of bringing the filtered above sample into contact with a capture agent (contact step), and the capture agent contains a crosslinked polymer containing 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).

[0014] In the contacting step, extracellular vesicles and / or membrane vesicles are purified by specifically capturing them with a capture agent. Here, "capturing" means that extracellular vesicles and / or membrane vesicles specifically bind onto the surface of the capture agent by physical interactions such as electrostatic attraction. The extracellular vesicles and / or membrane vesicles captured by the capture agent can be used as they are (for example, for quantification or analysis of the contents), but can also be used after separation from the capture agent. Therefore, the method according to this embodiment may further include a step of separating extracellular vesicles and / or membrane vesicles from the capture agent (separation step or recovery step).

[0015] As used herein, "extracellular vesicle" is a vesicle having a lipid bilayer membrane that contains intracellular components and is secreted from a cell to the extracellular space, and means those secreted from cells of organisms other than bacteria. Extracellular vesicles contain nucleic acids such as microRNA, messenger RNA (mRNA), DNA; proteins such as growth factors, enzymes; lipids, etc., and are involved in many important functions in vivo, such as intercellular communication, regulation of gene transcription rate, induction and regulation of immune response. Examples of extracellular vesicles include exosomes, microvesicles, apoptotic vesicles, etc.

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

[0017] As used herein, "membrane vesicle (MV)" refers to vesicles secreted by bacteria and containing components within the bacteria. Membrane vesicles contain nucleic acids such as DNA, pathogenic toxins, signal proteins, proteins such as enzymes, lipids, polysaccharide peptidoglycans, etc., and are involved in various functions at the destination of transport, such as interactions between microorganisms, transmission of pathogenicity, transformation, survival under stress conditions, regulation of inflammation, etc. The membrane vesicles to which the method according to this embodiment is applied are preferably those secreted by bacteria (intestinal bacteria) present in the intestine, particularly in the large intestine.

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

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

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

[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 Romboutsia, Prevotella copri, Alistipes inops, bacteria belonging to the Oscillospiraceae family, Alistipes shahii, Clostridium leptum, Alistipes indistinctus, Bacteroides coprocola, bacteria belonging to the genus Subdoligranulum, bacteria belonging to the Lachnospiraceae family, and the like.

[0022] In this specification, the "fecal sample" means a sample containing feces, and it may be prepared using feces for carrying out the filtration step described below. Examples of the fecal sample include a fecal suspension in which feces are suspended in an aqueous medium, or the supernatant of the fecal suspension, etc., and it may contain extracellular vesicles and / or membrane vesicles. Examples of the aqueous medium include physiological saline such as phosphate buffered saline (PBS), sterilized water, pH buffer solutions such as Good buffer, and the like. The supernatant of the fecal suspension can be obtained by centrifuging the fecal suspension to remove solids.

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

[0024] From the viewpoint of further improving the capture efficiency of extracellular vesicles and / or membrane vesicles in the contact 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. Also, 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 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), glass fiber, and the like.

[0026] In the contact step, the fecal sample that has undergone the filtration step is brought into contact with a capture agent, so that extracellular vesicles and / or membrane vesicles in the feces react with the capture agent, and the extracellular vesicles and / or membrane vesicles are captured by the capture agent. Bringing the fecal sample that has undergone the filtration step into contact with the capture agent may be achieved by mixing the two.

[0027] The capture agent in the contact step contains a crosslinked polymer containing a monomer unit having an acidic group and / or a monomer unit having a neutralizing base of the acidic group, and the capture agent satisfies the following (i) and / or (ii). (i) The crosslinked polymer contains a monomer unit having at least a neutralizing base of the acidic group and a first cationic surfactant. (ii) The capture agent further contains a second cationic surfactant (however, excluding those in which the second cationic surfactant is a crosslinked polymer containing a monomer unit having a neutralizing base of the acidic group).

[0028] When the scavenger satisfies only (i), the scavenger contains both a monomer unit having an acidic group and a monomer unit having a neutralization base of the acidic group and a first cationic surfactant, or contains only a crosslinked polymer containing a monomer unit having a neutralization base of the acidic group and a first cationic surfactant. That is, when the scavenger satisfies only (i), the scavenger does not contain a first cationic surfactant that does not form a neutralization base with the acidic group. Further, when the scavenger satisfies only (ii), "the scavenger further contains a second cationic surfactant (provided that the second cationic surfactant is not a crosslinked polymer containing a monomer unit having a neutralization base of the acidic group)" means that the second cationic surfactant exists in the scavenger in a state where it has not reacted with the crosslinked polymer, that is, the second cationic surfactant in the scavenger does not form a neutralization base with the acidic group of the crosslinked polymer. That is, when the scavenger satisfies only (ii), the crosslinked polymer contained in the scavenger does not contain a monomer unit having a neutralization base with the second cationic surfactant. At this time, the second cationic surfactant is not a crosslinked polymer containing a monomer unit having a neutralization base of the acidic group. When the scavenger satisfies (i) and (ii), the scavenger may contain a monomer unit having the acidic group, a crosslinked polymer containing a monomer unit having a neutralization base of the acidic group and a first cationic surfactant, and a second cationic surfactant that does not form a neutralization base with the acidic group of the crosslinked polymer, and may contain a crosslinked polymer containing a monomer unit having a neutralization base of the acidic group and a first cationic surfactant, and a second cationic surfactant that does not form a neutralization 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] The acidic group means a proton (H in an aqueous solution +releases and represents a functional group showing acidity (pH < 7.0). The acidic group is not particularly limited, but is preferably an organic acid group. Examples thereof include a carboxylic acid group (-COOH), a sulfonic acid group [-S(=O)2(-OH)], a sulfuric acid group [-O-S(=O)2(-OH)], a phosphonic acid group [-P(=O)(-OH)2], a phosphoric acid group [-O-P(=O)(-OH)], and the like.

[0030] The neutralization base of the acidic group means a functional group formed by ionic bonding of a cation derived from a compound having any cationic group capable of reacting with the acidic group with the acidic group, replacing the proton (H + ) of 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 + (where Y + is a cationic group and X is any group covalently bonded to Y + ), the neutralization base is -COOYX. Specific examples of the cation include a metal (such as a monovalent or divalent metal), a cation represented by the formula: NR4 + (wherein each R independently represents a hydrogen atom or an optionally substituted hydrocarbon group, and two or three Rs may form a ring together with adjacent nitrogen atoms), and the like. Further, as the cation, for example, a cation derived from a cationic surfactant may be used. The "cationic surfactant" is a surfactant having a cationic group. In the present specification, the "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 of a proton. The neutralization base of the acidic group and the 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; tin; etc.

[0032] Formula: NR4 + In the cation represented by the formula: NR4, when R is a hydrocarbon group which may be substituted, 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 1-6 alkyl groups such as methyl group, ethyl group, propyl group (n-propyl group or isopropyl group), butyl group (n-butyl group, isobutyl group, sec-butyl group, or t-butyl group), etc. Examples of the cycloalkyl group include C 5-14 cycloalkyl groups such as cyclopentyl group, cyclohexyl group, etc. Examples of the aryl group include C 6-14 aryl groups such as phenyl group, naphthyl group, etc. Examples of the aralkyl group include C 7-14 aralkyl groups such as benzyl group, phenethyl group, etc. Formula: NR4 + The cation represented by the formula: NR4 may be a cation derived from a cationic surfactant, or may not be a cation derived from a cationic surfactant.

[0033] Examples of the substituent that can substitute the hydrocarbon group include halogen atom, hydroxyl group, mercapto group, etc.

[0034] Formula: NR4 + In the cation represented by the formula: NR4, when two or three Rs 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 condensed ring such as a quinoline ring. The ring may have one or more substituents, and examples of the substituent include halogen atom, hydroxyl group, amino group, alkyl group, haloalkyl group, hydroxyalkyl group, N,N-dialkylamino group, etc. The number of the substituents may be, for example, 1, 2, or 3.

[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. In the cationic surfactant, the number of the hydrocarbon group and the cationic group may be 1 or 2 or more.

[0036] The hydrocarbon group having 12 to 30 carbon atoms may be any of an aliphatic hydrocarbon group such as an alkyl group and an alkenyl group, a cycloaliphatic hydrocarbon group such as a cycloalkyl group and a cycloalkenyl group, and an aromatic hydrocarbon group, but is preferably an aliphatic hydrocarbon group. Examples of the substituent of the hydrocarbon group include a hydroxyl group, an oxo group (=0), an alkoxy group, an alkenyloxy group, and combinations thereof. The number of the substituents of 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 a lauryl group, a myristyl group, a palmityl group, a stearyl group, an oleyl group, a linoleyl group, and an arachidyl group.

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

[0039] Examples of the cycloaliphatic 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).

Chemical formula

[0040] Examples of the substituents that can be substituted on rings A to D include, for example, a hydroxyl group, an oxo group (=O), an alkyl group (for example, an alkyl group having 1 to 4 carbon atoms such as a methyl group), combinations thereof (for example, a hydroxyalkyl group), and the like. 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, 17-position, and the like.)

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

Chemical formula

[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 alkylene group (for example, an alkylene group having 2 to 4 carbon atoms such as an ethylene group or a propylene group) which may be substituted is linked to the 17-position of the skeleton represented by formula (1), and even more preferably a group in which an alkylene group (for example, an alkylene group having 2 to 4 carbon atoms such as an ethylene group or a propylene group) which may be substituted is linked to the 17-position of the skeleton represented by any one of formulas (1-1) to (1-5).

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

[0044] The optionally substituted hydrocarbon group having 12 to 30 carbon atoms is diC 12-30It may also 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, for example, -N(R 1 )2 (wherein each R 1 independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms), -N + (R 2 )3 (wherein each R 2 independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 6 carbon atoms), etc., and -N + (R 2 )3 is preferred.

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

[0047] The group represented by the above formula: -N(R 1 )2 is preferably -NH2 or -N(R 11 )2 (wherein R 11 represents a hydroxyalkyl group having 1 to 5 carbon atoms). Examples of the hydroxyalkyl group having 1 to 5 carbon atoms include hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, hydroxy pentyl group (including pentahydroxy pentyl group, etc.).

[0048] The group represented by the above formula: -N + (R 2 )3 is preferably -N + (CH3)3, -N + (CH3)2(C2H5), -N + (CH3)2(C3H7), etc.

[0049] Examples of the cationic surfactant include compounds having an aliphatic hydrocarbon group with 12 to 30 carbon atoms and a cationic group (e.g., hexadecyltrimethylammonium chloride), compounds having an alicyclic hydrocarbon group with 12 to 30 carbon atoms and a cationic group, compounds having a cationic group (e.g., triethanolamine), polymers having a cationic group (e.g., polylysine, polyethyleneimine), etc. The cationic surfactant is preferably a compound having an aliphatic hydrocarbon group with 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 the cationic group per mole of the cationic surfactant is preferably 1 mol or more, more preferably 5 mol or more, and even more preferably 10 mol or more. The upper limit of the molar concentration of the cationic group per mole of the cationic surfactant is preferably 100 mol 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, etc. <GPC measurement conditions> [1] Apparatus: Gel permeation chromatography [Model number "HLC-8120GPC", manufactured by Tosoh Corporation] [2] Columns: "TSKgeI G6000PWx I" and "TSKgelG3000PWxl" [both manufactured by Tosoh Corporation] are connected in series. [3] Eluent: A solution prepared by dissolving 0.5 wt% of sodium acetate in methanol / water = 30 / 70 (volume ratio). [4] Standard substance: Polyethylene glycol (hereinafter abbreviated as PEG) [5] Injection conditions: Sample concentration 0.25 wt%, column temperature 40 °C

[0052] Examples of the monomer having an acidic group include unsaturated carboxylic acids and the like. Unsaturated carboxylic acids include, for example, unsaturated monocarboxylic acids and unsaturated dicarboxylic acids. Preferred examples of the unsaturated monocarboxylic acid 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 the unsaturated dicarboxylic acid (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, sulfalkyl esters of (meth)acrylic acid such as 2-sulfoethyl (meth)acrylate, and N-sulfoalkyl (meth)acrylamides such as 2-(meth)acrylamido-2-methylpropanesulfonic acid), and (meth)acrylic monomers having a phosphoric acid group (for example, phosphonooxyalkyl esters of (meth)acrylic acid such as 2-((meth)acryloyloxy)ethyl phosphate). In this specification, (meth)acrylic means acrylic and / or methacrylic, 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.

[0055] Examples of monomers having a neutralizing base for an acidic group include, for example, unsaturated carboxylates. Examples of unsaturated carboxylates include alkali metal salts of unsaturated carboxylic acids (for example, sodium salts, potassium salts), alkylamine salts (for example, trialkylamine salts such as triethylamine salts), alkanolamine salts (for example, dialkanolamine salts such as diethanolamine salts, trialkanolamine salts such as triethanolamine salts), ammonium salts, or tetraalkylammonium salts (for example, tetramethylammonium salts, tetraethylammonium salts). Further, examples of unsaturated carboxylates include unsaturated monocarboxylates and unsaturated dicarboxylates. Examples of unsaturated monocarboxylates include unsaturated monocarboxylates having 3 to 10 carbon atoms, and acrylate, methacrylate, and crotonate are preferred, and acrylate is more preferred. Examples of unsaturated dicarboxylates (including anhydrous salts) include unsaturated dicarboxylates having 4 to 10 carbon atoms, and maleate, fumarate, citraconate, itaconate, or anhydrous salts thereof are preferred.

[0056] Examples of other monomers having a neutralizing base for an acidic group include, for example, neutralized salts of (meth)acrylic monomers having the above sulfonic acid group, neutralized salts of (meth)acrylic monomers having a phosphoric acid group, and the like. More specific examples of these neutralized salts include the above alkali metal salts and the like.

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

[0058] The crosslinked polymer preferably contains both a monomer unit having an acidic group and a monomer unit having a neutralizing base for the acidic group. That is, in a crosslinked polymer containing a monomer unit having an acidic group, it is preferable that a part of the monomer unit having an acidic group is neutralized and substituted with a monomer unit having a neutralizing base for the acidic group. The degree of neutralization [100×(number of moles of neutralizing base for acidic group) / (total number of moles of acidic group and neutralizing base for the acidic group)] is not particularly limited, but 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, and may also be 90 mol% or less, 85 mol% or less, or 80 mol% or less.

[0059] In addition to the monomer unit having an acidic group and / or the monomer unit having a neutralizing base for the acidic group, the crosslinked polymer may further contain other monomer units different from the monomer. Examples of other monomers include the following monofunctional ethylenically unsaturated monomers. · Esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) (e.g., 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, naphthyl ester; aralkyl esters such as benzyl ester, phenethyl ester; glycidyl ester; polyethylene glycol ester, etc.) · Unsaturated carboxylic acid amides (e.g., free amide; N-substituted amides such as N-monoalkylamide, N,N-dialkylamide, etc.) · Unsaturated dicarboxylic acid imides (e.g., maleimide, citraconimide, itaconimide, their N-alkyl substituted products, N-cycloalkyl substituted products, or N-aryl substituted products, etc.) · Alkenyl esters of saturated carboxylic acids (e.g., acetic acid, propionic acid, etc.) (e.g., vinyl esters, allyl esters, etc.) · Esters or amides of unsaturated sulfonic acids (e.g., esters or amides of unsaturated sulfonic acids corresponding to the esters or amides of the above unsaturated carboxylic acids, etc.) · Unsaturated alcohols (e.g., allyl alcohol, propenyl alcohol, etc.) · Unsaturated ethers (e.g., alkyl vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, etc.; alkyl allyl ethers such as methyl allyl ether, ethyl allyl ether, etc.; cycloalkyl vinyl ethers such as cyclohexyl vinyl ether, etc.; glycidyl vinyl ether, etc.) · 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 monomers other than the monomers having an acidic group and / or a neutralizing base of the acidic group, the other monomers may be a single kind or a combination of two or more kinds.

[0061] In addition, the monomers constituting the crosslinked polymer preferably have a solubility (g / 100 g of water) in water at 25 ° C of 1 g or more, and more preferably 5 g or more. Further, the monomers constituting the crosslinked polymer may be water-miscible.

[0062] The crosslinked polymer is a polymer having a crosslinked structure (a structure in which the constituent atoms of each of a plurality of linear polymers (polymers of monomers having an acidic group and / or monomers having a neutralizing base of the acidic group) are covalently bonded directly or via other atoms). When the monomers constituting the crosslinked polymer have reactive groups (for example, in the case of using a combination of a monomer having a carboxyl group and a monomer having an amino group), the crosslinked structure may be self-crosslinked, but if necessary, it may be crosslinked with any crosslinking agent.

[0063] Examples of the method of crosslinking using a crosslinking agent include the following methods. 1) A method of obtaining a crosslinked polymer having a crosslinked structure directly by subjecting the above monomers and an internal crosslinking agent to a polymerization reaction (for example, the methods described in JP-A-2003-225565 and JP-A-2005-075982). 2) A method of obtaining a crosslinked polymer by crosslinking a polymer having the above monomers as essential constituent monomers using a surface crosslinking agent (for example, the methods described in Japanese Patent No. 3648553, JP-A-2003-165883, JP-A-2003-225565, JP-A-2005-75982, and JP-A-2005-95759).

[0064] The crosslinking agent (including an internal crosslinking agent and a surface crosslinking agent) is not particularly limited, and examples thereof include the following ethylenically unsaturated monomers having two or more functional groups. · Alkenyl ethers of dihydric or higher alcohols (for example, alkylene glycols or polyalkylene glycols such as ethylene glycol, polyethylene glycol, propylene glycol, butylene glycol, neopentyl glycol (preferably, the alkylene group has 2 to 4 carbon atoms and the alkylene oxide has a preferred repeating number of 10 to 50), glycerin, polyglycerin, trimethylolethane, trimethylolpropane, pentaerythritol, dipentaerythritol, sorbitan) (for example, vinyl ether, allyl ether, etc.) · Esters of unsaturated carboxylic acids (for example, acrylic acid, methacrylic acid, etc.) and dihydric or higher alcohols (for example, those exemplified in the above ethers). · Alkenyl esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) (e.g., vinyl esters, allyl esters, etc.) · Alkenyl esters of polycarboxylic acids (e.g., tartaric acid, citric acid, adipic acid, etc.) (e.g., vinyl esters, allyl esters, etc.) · Alkenyl esters of isocyanuric acid (e.g., triallyl isocyanate, etc.) · Alkylene bisacrylamide (e.g., methylene bisacrylamide, etc.) · Alkylene bismethacrylamide (e.g., methylene bismethacrylamide, etc.) · Di- or tri-alkenylamine (e.g., diallylamine, triallylamine, etc.) · Aromatic polyvinyl compounds (e.g., divinylbenzene, etc.)

[0065] As the crosslinking agent, a crosslinking agent having at least two or more functional groups capable of reacting with the substituents (such as carboxy groups, hydroxyl groups, etc.) possessed by the above monomers can also be used. As such a crosslinking agent, polyhydric alcohol glycidyl ethers (e.g., alkylene glycol diglycidyl ethers such as ethylene glycol diglycidyl ether, alkane triol di- or triglycidyl ethers such as glycerin diglycidyl ether) etc. can be used.

[0066] The crosslinking agent may be a single type alone, or a combination of two or more types.

[0067] The amount of the crosslinking agent can be appropriately selected according to the desired crosslinking density. For example, it may be 0.005 mol% or more, or 0.01 mol% or more with respect to the monomers constituting the crosslinked polymer, and may be 0.5 mol% or less, or 0.4 mol% or less.

[0068] When the scavenger satisfies (ii), as the second cationic surfactant, the cationic surfactants described above can be used (however, the second cationic surfactant excludes those that are crosslinked polymers containing monomer units having a neutralizing base for the acidic group). When the "cationic group" of the second cationic surfactant is a "group that forms a cation by itself", examples of its counter anion include halide ions (F - , Cl - , Br - , I - , etc.).

[0069] Whether the scavenger satisfies (i) and / or (ii) can be identified, for example, by immersing the scavenger in a suitable solvent (e.g., a hydrocarbon solvent such as hexane), releasing the cationic surfactant into 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. Also, 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 water absorption ratio (physiological saline absorption ratio) of the scavenger of the present invention in physiological saline is preferably 1 g / g or more, more preferably 3 g / g or more, still more preferably 5 g / g or more, even more preferably 10 g / g or more, or most preferably 15 g / g or more. When the water absorption ratio (physiological saline absorption ratio) of the scavenger in physiological saline is too low, increasing the amount of the above crosslinking agent tends to increase the ratio. The upper limit of the water absorption ratio (physiological saline absorption ratio) of the scavenger is not particularly limited, but for example, 120 g / g or less is preferable, 100 g / g or less is more preferable, and 50 g / g or less is particularly preferable. The physiological saline absorption ratio 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 -6 mol / g to 1×10 -4 mol / g, preferably based on the weight of the scavenger after drying. The weight of the scavenger after drying can be measured, for example, by the following method. 1 g of the scavenger can be placed in a petri dish, covered with filter paper, and heated and dried in a circulating air dryer at 130 °C for 60 minutes, and the remaining weight can be taken as the weight of the crosslinked polymer after drying.

[0073] The scavenger of the present invention can be produced, for example, through the production of a crosslinked polymer as follows. The crosslinked polymer in the present invention can be produced, for example, by a method including a step 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, a step of drying the polymer, and a step of classifying the polymer if necessary. The scavenger of the present invention can be produced, for example, by a method including a step of mixing the above crosslinked polymer with a cationic surfactant (preferably mixing at 20 to 30 °C for 1 to 2 minutes), and, if necessary, a subsequent step of aging at 100 to 150 °C for 10 to 60 minutes.

[0074] In the contacting step, the amount of the capture agent to be contacted with the fecal sample that has undergone 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 per 1 g of the mass of the feces used when preparing the above fecal sample. In the contacting step, the amount of the capture agent to be contacted with the fecal sample that has undergone 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 per 1 g of the mass of the feces used when preparing the above fecal sample.

[0075] In the contacting step, for example, after contacting a fecal sample that has undergone the filtration step with the capture agent, it may be held for, for example, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. Further, in the contacting step, for example, after contacting a fecal sample that has undergone the filtration step with the capture agent, it may be held 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 carried out, for example, at 4°C or higher and 40°C or lower, 10°C or higher and 35°C or lower, or 15°C or higher and 30°C or lower, and is not particularly limited.

[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 contacting 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 referred to as a separation step. By providing 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 contacting step, the extracellular vesicles and / or membrane vesicles may be eluted from the capture agent by changing the salt concentration or pH, etc.

[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 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, which comprises a filter and the above-mentioned capture agent.

Example

[0079] Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited thereto. <Production of capture agent> In this example, the scavenger of the present invention was produced by the following procedure. First, 116.5 g of acrylic acid, 272.2 g of ion-exchanged water, and 0.58 g (0.5 wt% / acrylic acid) of ethylene glycol diglycidyl ether as a cross-linking agent were placed in a 1-liter beaker and mixed to dissolve the cross-linking agent. While cooling the beaker in an ice bath, 96.1 g of a 48.5 wt% aqueous sodium hydroxide solution was added to neutralize a part (72 mol%) of the acrylic acid. After cooling the neutralized monomer solution to 5°C, 9.3 g of a 2 wt% aqueous potassium persulfate solution was added as a polymerization initiator to obtain an aqueous monomer solution. 1434 g of cyclohexane and 7.1 g of Leodol SP-S10V (manufactured by Kao Corporation, sorbitan monostearate) as a dispersant were placed in a 2-liter separable flask equipped with a stirrer and a condenser (cooler), and heated to an internal temperature of 60°C using a hot water bath and stirred to dissolve the dispersant in cyclohexane. After passing nitrogen through the solution in the separable flask to reduce the dissolved oxygen in cyclohexane to 0.1 ppm or less, while stirring using a stirrer, 350 g of the aqueous monomer solution was added dropwise using a dropping funnel, and inverse suspension polymerization was carried out at a polymerization temperature of 80°C. Further, after the addition of the aqueous monomer solution was completed, heating was continued for another 2 hours to complete the suspension polymerization, and a spherical water-containing gel was obtained in cyclohexane. After stopping the rotation of the stirrer and allowing the generated water-containing gel to settle, cyclohexane was removed by decantation, and the remaining water-containing gel was washed several times with cyclohexane to remove the dispersant adhering to the water-containing gel. The obtained spherical water-containing gel was spread on a release paper and dried in a vacuum dryer at 130°C (degree of vacuum: 10000 - 20000 Pa) for 1 hour to obtain a cross-linked polymer A.

[0080] Next, this cross-linked polymer A was adjusted to a particle size of 300 μm to 500 μm using sieves with apertures of 300 μm and 500 μm to obtain a cross-linked polymer B.

[0081] Subsequently, 20 g of a 50 wt% aqueous potassium carbonate solution was spray-added to 50 g of the cross-linked polymer B from a spray nozzle, uniformly mixed, then heated at 130°C for 30 minutes, and cooled to room temperature to obtain a cross-linked polymer C.

[0082] Finally, 1 g of a 5 wt% hexadecyltrimethylammonium chloride (manufactured by FUJIFILM Wako Pure Chemical Corporation, HDTMA-Cl) cyclohexane solution was added dropwise to 10 g of the crosslinked polymer C10 with a dropper, and after thorough mixing, it was heated at 130 °C for 30 minutes and then cooled to room temperature to obtain a scavenger.

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

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

[0085] As a result, the absorption ratio of the scavenger in physiological saline was 32 g / g, and the pH measurement result of the physiological saline solution was 7.0. The pH measurement was performed according to the method described below. Also, the weight ratio after heat drying with respect to the weight of the scavenger was 97.4%, and the molar concentration of the cationic group with respect to the weight of the scavenger after drying was 1.60×10 -5 mol / g.

[0086] <Method for measuring pH of physiological saline solution> To a 100 mL cylindrical beaker with a diameter of 50 mm, physiological saline (salt concentration 0.9 wt%) was added to 0.5 g of the measurement sample so that the total amount was 100 g, and after stirring with a stirrer chip (length 30 mm) at 60 rpm for 30 minutes at 25 °C, it was allowed to stand at 25 °C for 1 minute. After standing, the pH of the supernatant at 25 °C measured with a pH meter was taken as the pH of the physiological saline solution.

[0087] [Reference Example 1: Evaluation of Capture of Exosomes in Urine or Feces by a Capture Agent] [Comparative Example 1] (1. Reaction between Urine Supernatant and Capture Agent) 1.1 mL of a human urine sample was centrifuged at 500 × g for 10 minutes, and 1 mL of the supernatant was collected. Subsequently, the collected 1 mL of the supernatant was added to a column for microbiospin chromatography (manufactured by Bio-Rad, 7326204) filled with 40 mg of the capture agent, and left standing at room temperature for 30 minutes. Then, the column was centrifuged at 1,000 × g for 1 minute to obtain the urine supernatant (flow-through) after reaction with the capture agent.

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

[0089] [Comparative Example 2] (1. Reaction between Fecal Suspension Supernatant and Capture Agent) Approximately 3 g of human fecal samples were 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 column for microbiospin chromatography (manufactured by Bio-Rad, 7326204) filled with 40 mg of a capture agent, and left standing at room temperature for 30 minutes. Thereafter, the column was centrifuged at 1,000×g for 1 minute to obtain the supernatant (flow-through) of the fecal suspension after reaction with the capture agent.

[0090] (2. Measurement of exosome amount) The measurement of the amount of exosomes captured by the capture agent was carried out as described in the above "Measurement of exosome amount".

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

[0092] As shown in Fig. 1(A), when the urine supernatant was reacted with the capture agent, the luminescence value of CD9 / CD63 in the flow-through was low, indicating that the amount of exosomes in the flow-through was very low. That is, it was suggested that almost all exosomes in the urine supernatant were captured by the capture agent. Also, as shown in Fig. 1(B), when the supernatant of the fecal suspension was reacted with the capture agent, the flow-through showed a higher luminescence value of CD9 / CD63 compared to the supernatant of the fecal suspension before the reaction, confirming that the amount of exosomes in the flow-through was high. That is, it was suggested that exosomes in the supernatant of the fecal suspension were not captured by the capture agent.

[0093] Since the scavenger is water-absorbent, the volume of the flow-through obtained after the reaction between the supernatant of the fecal suspension and the scavenger decreases compared to the volume of the supernatant of the fecal suspension before the reaction. Therefore, when exosomes are not captured by the scavenger, the exosomes in the flow-through are concentrated compared to the exosomes in the supernatant of the fecal suspension before the reaction, and the luminescence values of CD9 / CD63 in the flow-through become higher than those in the supernatant of the fecal suspension before the reaction.

[0094] From the results of Reference Example 1, it was shown that exosomes in feces are not captured only by reacting with a scavenger, and that extracellular vesicles in feces are more difficult to purify compared to those in urine.

[0095] [Test Example 1: Evaluation of Capture of Exosomes in Feces by Filter Filtration and a Scavenger] <Example 1> (1. Reaction between the filtrate of the supernatant of the fecal suspension and the scavenger) Approximately 3 g of human fecal specimens were 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. Subsequently, the collected supernatant was filtered through a PVDF filter (manufactured by Whatman) with a pore size of 0.45 μm to obtain a filtrate, and then the filtrate was added to a micro-biospin chromatography column (manufactured by Bio-Rad, 7326204) filled with 40 mg of the scavenger and allowed to stand at room temperature for 30 minutes. Thereafter, the column was centrifuged at 1,000 × g for 1 minute to obtain a filtrate (flow-through) after the reaction with the scavenger.

[0096] (2. Measurement of the amount of exosomes) The measurement of the amount of exosomes was carried out as described in the above "Measurement of the amount of exosomes".

[0097] <Comparative Example 3> (1. Reaction between the supernatant of the fecal suspension and the scavenger) Approximately 3 g of human fecal specimens were 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, except that the supernatant was not filtered through a filter, in the same manner as in Example 1, a supernatant (flow-through) after reaction with a capture agent was obtained.

[0098] (2. Measurement of exosome amount) The measurement of the amount of exosomes was carried out as described in the above "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 of the fecal suspension before reaction and the flow-through when the above supernatant was reacted with a capture agent without being filtered through a filter. Figure 2(B) shows the CD9 / CD63 luminescence values in the supernatant of the fecal suspension before reaction and the flow-through when the above supernatant was filtered through a filter and reacted with a capture agent.

[0100] As shown in Figure 2(A), when the supernatant of the fecal suspension was reacted with a capture agent without being filtered through a filter, the amount of exosomes in the flow-through was higher than that in the supernatant of the fecal suspension before 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 through a filter and reacted with a capture agent, the amount of exosomes in the flow-through was very low compared to that in the supernatant of the fecal suspension before reaction, suggesting that the exosomes in the supernatant of the fecal suspension were captured by the capture agent (Figure 2(B)).

[0101] From the results of Test Example 1, it was shown that by filtering the supernatant of the fecal suspension through a filter, the capture efficiency of extracellular vesicles in the supernatant of the fecal suspension by the capture agent was greatly improved.

[0102] [Reference Example 2: Evaluation of capture rate of exosomes by filter filtration and magnetic beads] As a purification method used for extracellular vesicle purification, there is a method using MagCapture (trademark) Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Corporation). This method is excellent in both rapidity, which can complete the purification of extracellular vesicles in about 3 hours, and recovery rate. The purification of extracellular vesicles from serum or feces was examined using the above method.

[0103] <Comparative Examples 4 - 5> (1. Pretreatment) 1200 μL of human serum specimen was centrifuged at 1,000×g for 10 minutes, and the supernatant was used as the input when processing with the above kit. Approximately 0.2 g of human feces specimen was suspended in 3.84 mL of Tris buffer to obtain a feces suspension. The feces suspension was centrifuged at 3,000×g for 10 minutes, and the supernatant was collected. Subsequently, the filtrate obtained by filtering the collected supernatant through a 0.45 μm filter (manufactured by Whatman) was used as the input when processing with the above kit.

[0104] (2. Exosome Purification) MagCapture (trademark) Exosome Isolation Kit PS was used for the purification of exosomes from the supernatant of serum or the filtrate of the supernatant of feces suspension. The purification was carried out in accordance with the attached instruction manual.

[0105] (3. Measurement of Exosome Amount) The amount of exosomes in the supernatant of serum or the filtrate of the supernatant of feces suspension, and the amount of exosomes in the sample residue (flow - through) separated from the magnetic particles after reacting the supernatant of serum or the filtrate of the supernatant of feces suspension with the magnetic particles for exosome capture in the above kit were measured to evaluate the amount of exosomes captured by the kit. The measurement of the exosome amount was carried out as described in the above "Measurement of Exosome Amount".

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

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

[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 of supernatant of fecal suspension with capture agent) Approximately 3 g of human fecal specimens were suspended in 50 mL of PBS to obtain a fecal suspension. Subsequently, the fecal suspension was filtered through a PVDF filter (manufactured by Whatman) with a pore size of 0.45 μm, an MCE filter (manufactured by Millipore) with a pore size of 0.8 μm, an MCE filter (manufactured by Millipore) with a pore size of 1.2 μm, or a PVDF filter (manufactured by Millipore) with a pore size of 5.0 μm. Then, the filtrate was added to a microbio spin chromatography column (manufactured by Bio-Rad, 7326204) filled with 40 mg of the capture agent and allowed to stand at room temperature for 30 minutes. Thereafter, the column was centrifuged at 1,000 × g for 1 minute to obtain the filtrate (flow-through) after reaction with the capture agent.

[0109] (2. Measurement of exosome amount) The measurement of the amount of exosomes was carried out as described in the above "Measurement of the amount of exosomes".

[0110] <Results> The results are shown in Fig. 4. Figs. 4(A) to (D) respectively show the results of calculating the luminescence values of CD9 / CD63 in the filtrate before reaction and the flow-through when the filtrate filtered 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 Figs. 4(A) to (B), when the fecal suspension was filtered through a filter with a pore size of 0.8 μm or less, almost no exosomes were present in the flow-through, suggesting that the exosomes in the fecal suspension were almost completely captured by the capture agent. On the other hand, as shown in Figs. 4(C) to (D), when the fecal suspension was filtered through a filter with a pore size of 1.2 μm to 5.0 μm, the presence of exosomes was recognized in the flow-through. That is, it was considered that a part of the exosomes in the supernatant of the fecal suspension passed through without being captured by the capture agent. Also, the filter material did not affect the capture of exosomes by the capture agent.

[0112] [Test Example 3: Evaluation of the capture of outer membrane vesicles (OMVs) by a capture agent (1)] <Example 6> (1. Culture of Bacteroides vulgatus) Bacteroides vulgatus stored frozen in the company of Eiken Chemical Co., Ltd. was inoculated into a Pore Media sheep blood agar medium (manufactured by Eiken Chemical Co., Ltd.) and cultured overnight in an anaerobic environment at 37°C. Subsequently, it was inoculated by picking colonies into a sterilized medium prepared by dissolving 100 mL of clinical thioglycollate medium "Eiken" (manufactured by Eiken Chemical Co., Ltd.) and cultured overnight in an anaerobic environment at 37°C. A medium without picking colonies was also cultured simultaneously as a control. After culturing, the supernatant centrifuged at 3,000×g for 30 minutes was further centrifuged at 40,000×g for 1.5 hours to remove the bacterial cells.

[0113] (2. Reaction of the bacterial culture supernatant with the capture agent) 1 mL of the supernatant of the medium in which bacteria centrifuged at 40,000×g were cultured or the medium in which bacteria were not cultured (culture supernatant _B. vulgutas or culture supernatant _control) was added to a column for microbiospin chromatography (manufactured by Bio-Rad, 7326204) filled with 40 mg of a capture agent, and allowed to stand at room temperature for 30 minutes. Then, the column was centrifuged at 1,000×g for 1 minute to remove the unreacted medium. Further, 500 μL of physiological saline was added, and the process of centrifuging at 1,000×g for 1 minute and removing it was repeated 3 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. Then, the column was centrifuged at 1,000×g for 1 minute to obtain an eluate. This was repeated once more to obtain an eluate (eluate _B. vulgutas or eluate _control).

[0114] (3. Western blot analysis of the eluate) After mixing 17.5 μL of each eluate with SDS and DTT, it was heated at 95°C for 10 minutes to prepare a Western blot sample. 20 μL of the Western blot sample was electrophoresed on a 10 - 20% acrylamide gel, then transferred to a PVDF membrane, reacted with an anti-Lipid A LPS antibody (manufactured by Invitrogen) overnight at 4°C, subsequently reacted with an HRP-labeled anti-goat antibody for 1 hour, and then developed with ECL prime (GE Healthcare), and a specific band reacting with the anti-Lipid A LPS antibody was detected using an iBright Imaging Systems (Thermo Fisher Scientific).

[0115] <Results> The results are shown in Figure 5. Figure 5 is a diagram showing the results of Western blot of LPS·Lipid A in the culture supernatant and the eluate. As shown in Figure 5, ladder-like bands considered to be LPS proteins were detected in the eluate as well as in the culture supernatant, suggesting that the capture agent captured the membrane vesicles.

[0116] [Test Example 4: Evaluation of capture of bacterial-derived membrane vesicles by a capture agent (2)] Although Example 6 demonstrated that the capture agent of the present invention can capture membrane vesicles in the culture solution, in order to confirm whether this capture method can widely capture membrane vesicles derived from bacteria, feces and bacterial-derived membrane vesicles purified from feces were evaluated. The evaluation was performed by comparing with the ultracentrifugation method, which is regarded as 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. The bacterial DNA in feces was purified by treating 200 mg of the pooled feces with the QIAamp PowerFecal Pro DNA Kit (Qiagen) according to the method described in the attached document.

[0118] (2. Purification of bacterial-derived membrane vesicles from feces by ultracentrifugation) The pooled feces were suspended in PBS to a concentration of 5% to obtain a 5% fecal suspension. The supernatant obtained by centrifuging the suspension at 3,000×g for 30 minutes was treated with a 0.45 μm filter. Then, after centrifuging at 150,000×g for 120 minutes using an ultracentrifuge (Beckman Coulter), the supernatant was discarded, an appropriate amount of PBS was added, and centrifugation was performed again at 150,000×g for 60 minutes. After removing the supernatant, CD1 buffer attached to the QIAamp PowerFecal Pro DNA Kit (Qiagen) was added and treated according to the method described in the attached document to obtain DNA.

[0119] (3. Purification of bacterial-derived membrane vesicles from feces using the capture agent) 1 mL of a 5% fecal suspension was added to a column for microbiospin chromatography (manufactured by Bio-Rad, 7326204) filled with 40 mg of a capture agent, and the mixture was allowed to stand at room temperature for 30 minutes. Then, the column was centrifuged at 1,000 × g for 1 minute to remove the unreacted solution. Further, 500 μL of physiological saline was added, and the step of centrifuging at 1,000 × g for 1 minute and removing it was repeated 3 times. Then, CD1 buffer attached to the QIAamp PowerFecal Pro DNA Kit (Qiagen) was added to the column and allowed to stand at room temperature for 10 minutes. Then, the column was centrifuged at 1,000 × g for 1 minute to obtain an eluate. The eluate was processed according to the method described in the kit's attached document to obtain DNA.

[0120] (4. Amplicon sequence analysis of bacteria in feces and DNA in bacterial-derived membrane vesicles) For the DNA obtained by each method (feces, ultracentrifugation purification, capture agent purification), amplicon sequence analysis (16S ribosomal DNA V3-V4 region) was performed using the commissioned analysis of Biotechnology Research Co., Ltd.

[0121] <Results> The results are shown in Fig. 6 and Table 1. Fig. 6 is a diagram showing the top 20 bacterial species profiles in the case of performing ultracentrifugation purification, obtained from the results of amplicon sequence analysis of DNA obtained from feces or DNA obtained from bacterial-derived membrane vesicles obtained by each purification method (ultracentrifugation purification, capture agent purification). Table 1 shows all the reads analyzed by amplicon sequence and the number of those reads. As shown in Fig. 6 and Table 1, the bacterial species profiles in feces and the profiles of bacterial-derived membrane vesicles obtained by ultracentrifugation purification were significantly different. On the other hand, the profiles of bacterial-derived membrane vesicles obtained by ultracentrifugation purification and capture agent purification were very similar, suggesting that purification with a capture agent can comprehensively capture bacterial-derived membrane vesicles in feces in the same way as ultracentrifugation purification.

[0122] [Table 1] JPEG0007713604000004.jpg 221149 JPEG0007713604000005.jpg 221149 JPEG0007713604000006.jpg 221149 JPEG0007713604000007.jpg 221149 JPEG0007713604000008.jpg 221149 JPEG0007713604000009.jpg 221149 JPEG0007713604000010.jpg 221149 JPEG0007713604000011.jpg 225149 JPEG0007713604000012.jpg 221149 JPEG0007713604000013.jpg 221149 JPEG0007713604000014.jpg 221149 JPEG0007713604000015.jpg 221149 JPEG0007713604000016.jpg 221149 JPEG0007713604000017.jpg 103149

Claims

Claim 1 A method for purifying extracellular vesicles and / or membrane vesicles in feces, comprising: a step of filtering a sample of the feces through a filter, and a step of bringing the filtered sample into contact with a capturing agent, wherein the capturing agent contains a crosslinked polymer containing a monomer unit having an acidic group and / or a monomer unit having a neutralizing base of the acidic group, and satisfies the following (i) and / or (ii): The monomer unit having the acidic group is an unsaturated monocarboxylic acid having 3 to 10 carbon atoms, and the monomer unit having the neutralizing base of the acidic group is an unsaturated monocarboxylate having 3 to 10 carbon atoms. (i) The crosslinked polymer contains at least a monomer unit having a neutralizing base of the acidic group and a cationic group in a first cationic surfactant. (ii) The capturing agent further contains a second cationic surfactant (however, excluding those in which the second cationic surfactant is a crosslinked polymer containing a monomer unit having a neutralizing base of the acidic group). However, the cationic surfactant has a hydrocarbon group having 12 to 30 carbon atoms, and the cationic group in the cationic surfactant is -N + R3 (wherein each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms). Claim 2 The method according to claim 1, wherein the monomer unit having the acidic group is acrylic acid and the monomer unit having the neutralizing base of the acidic group is acrylate. Claim 3 The method according to claim 1, wherein the cationic surfactant has an aliphatic hydrocarbon group having 12 to 30 carbon atoms. Claim 4 The method according to claim 1, wherein the cationic surfactant is hexadecyltrimethylammonium chloride. Claim 5 The method according to any one of claims 1 to 4, wherein the pore size of the filter is 0.8 μm or less.

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