Superabsorbent polymer for classifying particles and classification method using the same

Abstract

Provided is a method for easily obtaining a desired size fraction from an aqueous sample containing particles. The method includes the following steps: (1) a step for bringing the aqueous sample containing the particles into contact with the superabsorbent polymer and obtaining a superabsorbent polymer gel containing some of the particles; and (2) a step for mixing the superabsorbent polymer gel with salt and retrieving some of the particles.

Description

Technical Field

[0001] The present invention relates to a superabsorbent polymer for classifying particles and a classification method using the same.

Background Art

[0002] A biological sample is collected from an organism, cells or their secretions are extracted from the biological sample, and the proteins, nucleic acids, etc. contained in the extract are analyzed to evaluate the state of the original organism.

[0003] Cell secretions include, for example, exosomes, microvesicles, and apoptotic bodies. Among these, exosomes (membrane vesicles with a particle size of 30 to 100 nm) are secreted from various cells and their presence has been confirmed in all body fluids throughout the body, suggesting that they may circulate in body fluids and transmit information to distant cells. The proteins, nucleic acids, etc. encapsulated in exosomes reflect the state of the cells that secreted them in real time and are useful as biomarkers for evaluating the state of the original organism, intercellular information transmission, etc.

[0004] As a method for recovering exosomes from a biological sample, a size fractionation method using an ultracentrifuge is widely used. This method is a method of separating relatively low-density exosomes from other particles using a sucrose density gradient or the like.

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the size fractionation method using an ultracentrifuge has problems that the purity of the recovered exosomes is not sufficient and the operation takes a long time.

[0006] A first object of the present invention is to provide a method (classification method) for easily obtaining a desired size fraction from an aqueous sample containing particles. A second object of the present invention is to provide a method for easily separating exosomes from a biological sample.

Means for Solving the Problem

[0007] As a result of intensive studies to achieve the above object, the present inventors have found that by bringing an aqueous sample containing particles into contact with a superabsorbent polymer to form a gel and mixing this gel with a salt, a desired size fraction can be easily obtained from the aqueous sample. The present invention has been completed through further studies based on this finding.

[0008] The present invention includes the following aspects. Item 1. A method for classifying particle A, comprising the following steps: (1) A step of bringing an aqueous sample containing particle A into contact with a superabsorbent polymer to obtain a superabsorbent polymer gel containing a part of particle A, and (2) A step of mixing the superabsorbent polymer gel with a salt to recover a part of particle A The method comprising the above steps. Item 2. The method according to Item 1, wherein particle A consists of particle A1 having a particle diameter of 200 nm or less and particle A2 having a particle diameter exceeding 200 nm, and step (2) is a step of recovering particle A1. Item 3. A method for separating exosomes from a biological sample, comprising the following steps: (3) A step of bringing a biological sample into contact with a superabsorbent polymer to obtain a superabsorbent polymer gel containing exosomes, and (4) A step of mixing the superabsorbent polymer gel with a salt to recover exosomes The method comprising the above steps. Item 4. The method according to Item 3, wherein the biological sample is a body fluid. Item 5. The method according to Item 3 or 4, wherein the biological sample is urine. Item 6. The method according to any one of Items 1 to 5, wherein the salt is a metal salt. Item 7. The method according to any one of Items 1 to 6, wherein the salt is a monovalent or divalent metal salt. Item 8. The method according to any one of items 1 to 7, further comprising a step of washing the superabsorbent polymer gel. Item 9. A method for increasing the cell density in a urine sample, comprising the following steps: (5) A step of bringing the urine sample into contact with a superabsorbent polymer to obtain a superabsorbent polymer gel, and (6) A step of recovering the urine sample not absorbed by the superabsorbent polymer gel The method comprising the steps. Item 10. The superabsorbent polymer is a three-dimensional crosslinked polymer having a constitutional repeating unit represented by the following formula:

Chemical formula

Chemical formula

Chemical Formula

Advantages of the Invention

[0009] According to the present invention, a desired size fraction of particles can be easily obtained. Further, according to the present invention, cells or their secretions can be easily separated from various biological samples. The separation method of the present invention can, for example, increase the purity of the separated substance and shorten the operation time as compared with the conventional ultracentrifugation method and the like. Further, the separation method of the present invention can reduce the number of steps and has excellent reproducibility as compared with the conventional ultracentrifugation method and the like. The separated substance can be used for evaluating the state of the original organism (for example, examination or diagnosis). Further, the separated substance can be used as a carrier for a cell-targeted therapeutic agent.

Brief Description of the Drawings

[0010]

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Modes for Carrying Out the Invention

[0011] [Method for Classifying Particle A] In one embodiment, a method for classifying particle A (or classified substance A) comprises the following steps: (1) A step of contacting an aqueous sample containing particle A with a superabsorbent polymer to obtain a superabsorbent polymer gel containing a part of particle A, and (2) Mixing the superabsorbent polymer gel with a salt and recovering a part of Particle A is included.

[0012] Particle A Particle A is not particularly limited and may be either synthetic particles or biological particles.

[0013] Examples of the synthetic particles include polymer particles. Specific examples thereof include polyvinyl chloride particles, polymethyl methacrylate particles, polystyrene particles, styrene-butadiene copolymer particles, acrylonitrile-butadiene copolymer particles, methyl methacrylate-butadiene copolymer particles, and silicone particles.

[0014] Examples of the biological particles include cells (such as germ cells, blood cells, fibroblasts, epithelial cells, stem cells, cancer cells, and cultured cells), cell organelles (such as nuclei, mitochondria, Golgi apparatuses, endoplasmic reticulums, and ribosomes), bacteria, viruses, and extracellular vesicles (such as exosomes, microvesicles, and apoptotic bodies).

[0015] The particle diameter and average particle diameter of Particle A are not particularly limited, and may be, for example, 0.3 nm or more, 0.4 nm or more, or 0.5 nm or more, and may also be 10 μm or less, 5 μm or less, or 1 μm or less.

[0016] The particle size distribution of Particle A may be unimodal or multimodal. In the particle size distribution of Particle A, for example, one or more peaks may exist in the range of 30 nm or more, 40 nm or more, or 50 nm or more, and one or more peaks may also exist in the range of 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, or 200 nm or less. The particle diameter, average particle diameter, and particle size distribution of Particle A can be measured, for example, by the dynamic light scattering method.

[0017] In one embodiment, particle A preferably consists of particle A1 with a particle diameter of 200 nm or less and particle A2 with a particle diameter exceeding 200 nm. In this embodiment, "a part of particle A" in step (1) and / or step (2) is preferably particle A1.

[0018] The aqueous sample containing particle A may be, for example, a latex containing synthetic particles or a biological sample (or bio-sample) containing biological particles. The biological sample may be a biological sample derived from an animal (e.g., human, non-human mammal) or a sample derived from a plant (e.g., extract). Also, the sample may be a sample derived from a healthy individual or a sample derived from a patient (e.g., a patient with a lifestyle disease, a patient with chronic kidney disease, a patient with a neurological disease, a patient with an immune disease, a cancer patient, an infectious disease patient, a degenerative disease patient). Examples of the biological sample include body fluids such as blood, plasma, serum, tears, saliva, breast milk, pleural effusion, ascites, amniotic fluid, cerebrospinal fluid, and urine; tissues such as hair, nails, skin, muscle, and nerve, or liquidized products of cells. These biological samples may contain exosomes.

[0019] The aqueous sample containing particle A may contain any additive. Examples of the additive include surfactants. The surfactant may be anionic, cationic, nonionic, or amphoteric, but is preferably nonionic. Examples of nonionic surfactants include polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, and polyoxyethylene oleyl ether; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene octyl phenyl ether and polyoxyethylene nonyl phenyl ether; and polyhydric alcohol fatty acid esters such as glycerin fatty acid ester, sorbitan fatty acid ester, and pentaerythritol fatty acid ester. These additives may be used alone or in combination of two or more.

[0020] The concentration of Particle A in the aqueous sample is preferably, for example, 1 particle / mL or more, or 2 particles / mL or more, and preferably 50 particles / mL or less, 40 particles / mL or less, or 30 particles / mL or less.

[0021] Superabsorbent polymer A superabsorbent polymer (SAP) is usually a polymer that absorbs water 100 times or more its own weight (for example, 100 to 3000 times, 200 to 2000 times, or 300 to 1000 times). In the present invention, it is preferable to use a superabsorbent polymer, but it is not limited thereto, and any water-absorbing substance can be similarly applied. The superabsorbent polymer may be a three-dimensional crosslinked polymer based on natural polymers (for example, natural polysaccharides such as starch, cellulose, alginic acid, chitin, chitosan, etc.), but preferably, it is a three-dimensional crosslinked polymer having at least one selected from the group consisting of a monomer having an acidic group (for example, an unsaturated carboxylic acid), a salt thereof, and an amide thereof as a polymerization component.

[0022] In a monomer having an acidic group, examples of the acidic group 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)2], and the like. The monomer may have only one type of acidic group, or may have two or more types of acidic groups (for example, a carboxylic acid group and a sulfonic acid group, a carboxylic acid group and a phosphoric acid group). For a salt or amide of a monomer having two or more types of acidic groups, it is sufficient that at least one type of acidic group forms a salt or amide. Representative examples of the monomer having an acidic group include ethylenically unsaturated monomers having an acidic group such as unsaturated carboxylic acids. Unsaturated carboxylic acids include, for example, unsaturated monocarboxylic acids and unsaturated dicarboxylic acids. Examples of the unsaturated monocarboxylic acid include acrylic acid, methacrylic acid, crotonic acid, and the like. Examples of the unsaturated dicarboxylic acid (including anhydrides herein) include maleic acid, fumaric acid, citraconic acid, itaconic acid, and their anhydrides. The unsaturated carboxylic acids may be used alone or in combination of two or more types.

[0023] In the salt of an unsaturated carboxylic acid, the cation is not particularly limited, and examples thereof include a metal (such as a monovalent or divalent metal), and a cation represented by the formula: NR4 + (wherein each R independently represents a hydrogen atom or a hydrocarbon group which may have one or more substituents, and two Rs may form a ring together with the adjacent nitrogen atom).

[0024] Examples of the monovalent metal include alkali metals (such as lithium, sodium, potassium). Examples of the divalent metal include alkaline earth metals (such as magnesium, calcium, barium), lead, zinc, and tin.

[0025] The formula: NR4 +In the cation represented by , when R is a hydrocarbon group which may have one or more substituents, 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). Examples of the cycloalkyl group include C 5-14 cycloalkyl groups such as cyclopentyl group, cyclohexyl group. Examples of the aryl group include C 6-14 aryl groups such as phenyl group, naphthyl group. Examples of the aralkyl group include C 7-14 aralkyl groups such as benzyl group, phenethyl group.

[0026] Examples of the substituent which the hydrocarbon group may have include a halogen atom, a hydroxyl group.

[0027] In the cation represented by the formula: NR4 + when two Rs form a ring together with the adjacent nitrogen atom, the ring may be a monocyclic ring such as a pyridine ring, 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 a halogen atom, a hydroxyl group, an amino group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, an N,N-dialkylamino group.

[0028] The salt of the unsaturated carboxylic acid is preferably an alkylamine salt (for example, a trialkylamine salt such as a triethylamine salt), an alkanolamine salt (for example, a dialkanolamine salt such as a diethanolamine salt, a trialkanolamine salt such as a triethanolamine salt), an ammonium salt, or a tetraalkylammonium salt (for example, a tetramethylammonium salt).

[0029] The amide of an unsaturated carboxylic acid is used in the sense that it includes not only the free amide but also N-substituted amides (for example, N-monoalkylamide, N,N-dialkylamide).

[0030] The superabsorbent polymer is preferably a three-dimensional crosslinked polymer having a constitutional repeating unit derived from a salt of a monomer having an acidic group, more preferably a three-dimensional crosslinked polymer having a constitutional repeating unit derived from a salt of an unsaturated carboxylic acid, and is represented by the following formula: [Chemical formula] (In the formula, M represents a cation.) It is even more preferably a three-dimensional crosslinked polymer having a constitutional repeating unit represented by the formula.

[0031] Examples of the cation represented by M include the same ones as those exemplified in the above-mentioned "salt of an unsaturated carboxylic acid".

[0032] The superabsorbent polymer may further be a three-dimensional crosslinked polymer having a constitutional repeating unit (preferably a constitutional repeating unit derived from an unsaturated carboxylic acid) derived from a monomer having an acidic group. That is, the superabsorbent polymer is a three-dimensional crosslinked polymer having a monomer having an acidic group XH (preferably an unsaturated carboxylic acid) as a polymerization component, and a part of the acidic group XH (preferably COOH) is a neutralizing base X - M + (preferably COO - M + )(for example, neutralized with a neutralizing agent (such as sodium hydroxide when M is sodium)) may be a three-dimensional crosslinked polymer. The degree of neutralization (100×(number of moles of neutralizing base of acidic group) / (total number of moles of acidic group and its neutralizing base)) 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 be 90 mol% or less, 85 mol% or less, or 80 mol% or less.

[0033] The superabsorbent polymer may contain, as polymerization components, a monomer having an acidic group, a salt thereof, and a monomer other than its amide, and may contain an unsaturated carboxylic acid, a salt thereof, and an ethylenically unsaturated monomer other than its amide. Examples of the ethylenically unsaturated monomer include the following monofunctional ethylenically unsaturated monomers. · Esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid) (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; 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) · Unsaturated dicarboxylic acid imides (e.g., maleimide, citraconimide, itaconimide, N-alkyl-substituted products, N-cycloalkyl-substituted products, or N-aryl-substituted products thereof) · Alkenyl esters of saturated carboxylic acids (e.g., acetic acid, propionic acid) (e.g., vinyl ester, allyl ester) · Unsaturated sulfonic acids (e.g., vinyl sulfonic acid, allyl sulfonic acid), salts thereof, esters thereof, or amides thereof · Unsaturated alcohols (e.g., allyl alcohol, propenyl alcohol) · Unsaturated ethers (e.g., alkyl vinyl ethers such as methyl vinyl ether, ethyl vinyl ether; alkyl allyl ethers such as methyl allyl ether, ethyl allyl ether; cycloalkyl vinyl ethers such as cyclohexyl vinyl ether; glycidyl vinyl ether) · Unsaturated nitriles (e.g., acrylonitrile, methacrylonitrile) · Olefins (e.g., ethylene, propylene, butene, pentene, hexene, etc.) · Aromatic vinyl compounds (e.g., styrene, α-methylstyrene, vinyltoluene, hydroxystyrene) · Heterocyclic vinyl compounds (e.g., N-vinylpyrrolidone) These may be used alone or in combination of two or more.

[0034] The superabsorbent polymer is preferably a three-dimensional cross-linked polymer cross-linked with a cross-linking agent. There is no particular limitation on the cross-linking agent, and examples thereof include the following ethylenically unsaturated monomers having two or more functional groups. · Alkenyl ethers of dihydric or higher alcohols (e.g., alkylene glycols or polyalkylene glycols such as ethylene glycol, polyethylene glycol, propylene glycol, butylene glycol, neopentyl glycol, etc., glycerin, polyglycerin, trimethylolethane, trimethylolpropane, pentaerythritol, dipentaerythritol, sorbitan), (e.g., vinyl ether, allyl ether) · Esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid) and dihydric or higher alcohols (e.g., those exemplified for the above ether) · Alkenyl esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid) (e.g., vinyl ester, allyl ester) · Alkenyl esters of polycarboxylic acids (e.g., tartaric acid, citric acid, adipic acid) (e.g., vinyl ester, allyl ester) · Alkenyl esters of isocyanuric acid (e.g., triallyl isocyanate) · Alkylene bisacrylamide (e.g., methylene bisacrylamide) · Alkylene bismethacrylamide (e.g., methylene bismethacrylamide) · Di- or tri-alkenylamine (e.g., diallylamine, triallylamine) · Aromatic vinyl compound (e.g., divinylbenzene) These may be used singly or in combination of two or more.

[0035] As the crosslinking agent, a monomer having an acidic group, its salt, or a crosslinking agent having at least two or more functional groups capable of reacting with its amide can also be used. Examples of such crosslinking agents include polyhydric alcohol glycidyl ethers, and specific examples thereof include alkylene glycol diglycidyl ethers such as ethylene glycol diglycidyl ether; and alkane triol di- or triglycidyl ethers such as glycerin diglycidyl ether and glycerin triglycidyl ether.

[0036] 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, and may be 0.5 mol% or less, or 0.4 mol% or less, based on the polymerization components (total of unsaturated carboxylic acid, its salt, its amide, and any other monofunctional ethylenically unsaturated monomer).

[0037] The superabsorbent polymer preferably comprises particles having an average particle diameter (e.g., 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, 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, 0.4 mm or more, 0.5 mm or more, or 1 mm or more, and preferably comprises particles having an average particle diameter (e.g., volume average particle diameter) in the range of 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, or 1 mm or less. The average particle diameter can be measured, for example, by dynamic light scattering method or image analysis method using a conventional apparatus (e.g., "CAMSIZER (trademark)" manufactured by Retsch).

[0038] A superabsorbent polymer can be produced, for example, by a method including a step of polymerizing (solution polymerization, emulsion polymerization, suspension polymerization, etc.) a composition containing a polymerization component, a solvent, and an initiator, a step of drying the polymer, and a step of classifying the polymer if necessary. Also, a commercially available product can be used as the superabsorbent polymer.

[0039] The amount of the superabsorbent polymer used is preferably, for example, 1 part by mass or more, 5 parts by mass or more, or 10 parts by mass or more, and preferably 2000 parts by mass or less, 1500 parts by mass or less, or 1000 parts by mass or less, based on 100 parts by mass of particle A.

[0040] The temperature at which the aqueous sample is brought into contact with the superabsorbent polymer is preferably, for example, within the range of 1 to 35°C or within the range of 5 to 30°C. Also, after the contact, it is preferable to let it stand for a predetermined time (for example, 1 hour or more or 2 hours or more, or 5 hours or less or 4 hours or less).

[0041] By step (1), particle A can be sieved into the inside and outside of the superabsorbent polymer gel according to its size. For example, in the case of a biological sample, it can be separated so that extracellular vesicles (such as exosomes) are contained inside the superabsorbent polymer gel and cells are contained outside the superabsorbent polymer gel.

[0042] The method for classifying Particle A preferably further includes a step of washing the superabsorbent polymer gel. This step is preferably carried out after Step (1) and before Step (2). By including this step, the particle recovery rate can be further improved. As the cleaning liquid, pure water may be used, but water containing a surfactant is preferred, and deionized water containing a surfactant is particularly preferred. As the surfactant, for example, the same surfactant as that which may be contained in the aqueous sample containing Particle A can be used, and a nonionic surfactant is preferred. In the water containing a surfactant, the content of the surfactant can be, for example, 0.1 part by mass or more, 0.5 part by mass, or 1 part by mass or more with respect to 100 parts by mass of water, and can be 1 part by mass or less, 2 parts by mass or less, 3 parts by mass or less, 4 parts by mass or less, or 5 parts by mass or less.

[0043] Salt The salt is not particularly limited as long as it can discharge the particles contained in the superabsorbent polymer gel. Examples of the salt include metal salts (such as monovalent or divalent metal salts).

[0044] Examples of the monovalent metal salt include alkali metal salts (such as sodium salts and potassium salts). Examples of the divalent metal salt include alkaline earth metal salts (such as magnesium salts, calcium salts, and barium salts).

[0045] Examples of the counter anion of the salt include, but are not limited to, halide ions such as chloride ions and bromide ions.

[0046] The salt may be a single type or a combination of two or more types.

[0047] The salt is preferably at least one selected from the group consisting of sodium chloride and magnesium chloride. The salt may be used in the form of an aqueous solution.

[0048] The amount of salt used can be, for example, 1 part by mass or more, 5 parts by mass or more, or 10 parts by mass or more, and can be 2000 parts by mass or less, 1500 parts by mass or less, or 1000 parts by mass or less, based on 100 parts by mass of the superabsorbent polymer gel.

[0049] The mixing temperature of the superabsorbent polymer gel and the salt is preferably, for example, within the range of 1 to 35 °C, or within the range of 5 to 30 °C. Further, after mixing, it is preferable to let it stand for a predetermined time (for example, 1 hour or more or 2 hours or more, or 5 hours or less or 4 hours or less).

[0050] By step (2), particles (partially or entirely) contained in the superabsorbent polymer gel can be discharged (or recovered). In the classification method of the present invention, particles can be recovered with a high recovery rate.

[0051] In one embodiment, the classification method of particle A is preferably a method for separating (or purifying) exosomes from biological particles, and the following steps: (3) A step of bringing a biological sample into contact with a superabsorbent polymer to obtain a superabsorbent polymer gel containing exosomes, and (4) A step of mixing the superabsorbent polymer gel with salt to recover exosomes are preferably included.

[0052] In this embodiment, the types, amounts used, etc. of the biological sample, superabsorbent polymer, and salt can be those described above. The biological sample is preferably a body fluid, and more preferably urine.

[0053] In this embodiment, the mixture of the superabsorbent polymer gel and salt may be dialyzed.

[0054] In this embodiment, compared with the conventional ultracentrifugation method, the purity of the recovered exosomes can be increased (the amount of urinary protein as an impurity can be reduced), and the operation time can be shortened.

[0055] [Method for increasing cell density in urine sample] The present invention relates to a method for increasing the cell density in a urine sample, comprising the following steps: (5) a step of bringing the urine sample into contact with a superabsorbent polymer to obtain a superabsorbent polymer gel, and (6) a step of recovering the urine sample not absorbed by the superabsorbent polymer gel is included.

[0056] The superabsorbent polymer and its usage amount may each be the same as those exemplified in the above-mentioned "method for classifying particles A".

[0057] The recovered urine sample contains cells (for example, red blood cells, white blood cells, epithelial cells (such as cells shed from the kidney, renal tubules, bladder), columnar substances containing these) at a higher density than the original urine sample, and can be suitably used for the examination of various diseases.

[0058] This method can omit the step of centrifuging a conventional urine sample.

[0059] [Superabsorbent polymer for classifying particles] The present invention includes a superabsorbent polymer for classifying particles. Examples of the particles and the superabsorbent polymer may be the same as particles A and the superabsorbent polymer described in the above-mentioned "method for classifying particles A".

[0060] The superabsorbent polymer for classifying particles is preferably the superabsorbent polymer used for the above-mentioned "method for classifying particles A", and more preferably the superabsorbent polymer for separating exosomes from a biological sample.

[0061] [Kit for classifying particles] The present invention relates to a kit for classifying particles, which includes a kit containing a superabsorbent polymer and a salt. As the particles, superabsorbent polymer, and salt, those same as the particles A, superabsorbent polymer, and salt described in the "Method for Classifying Particles A" can be mentioned respectively. The kit may further contain a cleaning solution. As the cleaning solution, those same as the cleaning solution described in the step of cleaning the superabsorbent polymer gel can be mentioned. The kit may further contain an instruction manual including an explanation regarding the method for classifying particles.

[0062] [Kit for Examining Diseases Using Exosomes] The present invention relates to a kit for examining (or diagnosing) diseases using exosomes, which includes a kit containing a superabsorbent polymer and a salt.

[0063] The subject to be examined may be a healthy person or a patient. Examples of diseases include lifestyle-related diseases, chronic kidney diseases, neurological diseases, immune diseases, cancer, infectious diseases, degenerative diseases, and the like. Exosomes can be collected from, for example, body fluids (such as blood, plasma, serum, tears, saliva, breast milk, pleural effusion, ascites, amniotic fluid, cerebrospinal fluid, urine, etc.), cells, or the liquefied matter of tissues of the subject to be examined. The kit may be a kit containing a superabsorbent polymer and a salt as a reagent for collecting exosomes from the subject to be examined in a known kit for examining diseases using exosomes.

[0064] As the superabsorbent polymer and the salt, those same as the superabsorbent polymer and the salt described in the "Method for Classifying Particles A" can be mentioned respectively. The kit may further contain a cleaning solution. As the cleaning solution, those same as the cleaning solution described in the step of cleaning the superabsorbent polymer gel can be mentioned. The kit may further contain an instruction manual including an explanation regarding the method for collecting exosomes from the subject to be examined.

[0065] [Device for Classifying Particles] The present invention includes an apparatus for classifying particles, the apparatus comprising means for performing the "method for classifying Particle A".

[0066] [Apparatus for separating exosomes from a biological sample] The present invention includes an apparatus for separating exosomes from a biological sample, the apparatus comprising means for performing the "method for separating exosomes from biological particles".

Example

[0067] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[0068] [Example 1] The following experiment was conducted to demonstrate the size fractionation effect in the absorption - excretion process of nanoparticles by a superabsorbent polymer.

[0069] To polystyrene latex with an average particle size of 100 nm (LB1 manufactured by Sigma - Aldrich) and polystyrene latex with an average particle size of 300 nm (LB3 manufactured by Sigma - Aldrich), deionized water containing 0.20% nonionic surfactant Tween20 (Leodol TW - L120 manufactured by Kao Chemicals) was added, and after diluting each by 1000 times, the two were mixed in equal amounts. Hereinafter, this will be referred to as the latex mixed solution.

[0070] Four commercially available superabsorbent polymer beads ( "Vixker Puyopuyo Ball", a cross - linked polymer mainly composed of sodium polyacrylate, spherical, particle diameter: 2.66 ± 0.11 mm, particle size distribution: normal distribution) (60 mg) used in household hydroponics, etc. were placed in a glass sample tube (capacity 50 mL), and 5.868 g [a] of the latex mixed solution was added thereto, and the mixture was allowed to stand at room temperature for 3 hours. After removing 2.170 g [b] of the solution remaining unabsorbed by the polymer beads, 1.003 g [c] of sodium chloride was added, and the mixture was stirred with a vortex mixer for 10 seconds. After standing at room temperature for 2 hours, 2.909 g [d] of the solution discharged from the polymer beads was recovered. The recovery rate [100×d / (a - b + c)] was 61.9%.

[0071] The results of measuring the particle size distribution of the polystyrene latex contained in the recovered solution by dynamic light scattering are shown in Fig. 1A. For the measurement, a ZetaSizer Nano ZSP type device manufactured by Malvern and a 10 mm square glass cell were used, and the analysis was performed by the non - negative least squares method under the conditions of a measurement temperature of 30°C, a solvent refractive index of 1.367, and a solvent viscosity of 1.394 cP (equivalent to a 24% aqueous sodium chloride solution). For comparison, the particle size distribution measurement under the same conditions was performed on a solution in which 24% sodium chloride was dissolved in the original latex mixed solution (Fig. 1B). Since the average particle size of the particles in the original latex mixed solution is 164.5 ± 67.7 nm, while the average particle size of the particles in the effluent is 123.5 ± 13.5 nm, it can be seen that small - sized particles (particle size of 137 nm or less) were fractionally recovered during the discharge process from the polymer beads.

[0072] The average particle size of the particles in the mixed solution (b) remaining without being absorbed by the polymer beads is 196.6 ± 83.5 nm (Fig. 2A), while the average particle size of the particles in the original latex mixed solution is 172.7 ± 72.0 nm (Fig. 2B). Therefore, it is considered that size fractionation also occurs during the absorption process into the polymer beads. The particle size distributions shown in Fig. 2 were all analyzed under the conditions of the refractive index (1.330) and viscosity (0.792 cP) of pure water.

[0073] [Example 2A] When an aqueous magnesium chloride solution is used for the nanoparticle discharge operation (an alternative method of Example 1)

[0074] For the nanoparticle discharge operation from the superabsorbent polymer gel, an aqueous magnesium chloride solution can also be used instead of sodium chloride powder (Example 1). Due to the chelation effect between the carboxyl groups in the superabsorbent polymer and divalent magnesium ions, the degree of shrinkage of the polymer gel increases, and the nanoparticles are discharged more efficiently.

[0075] To polystyrene latex with an average particle size of 100 nm and polystyrene latex with an average particle size of 600 nm (LB6 manufactured by Sigma-Aldrich), deionized water containing 0.20% Tween 20 was added, and after diluting each by 1000 times, the two were mixed in equal amounts. Hereafter, this is referred to as latex mixed solution B.

[0076] Four superabsorbent polymer beads (60 mg) were placed in a glass sample tube (capacity 50 mL), and 5.864 g [a] of latex mixed solution B was added thereto, followed by standing at room temperature for 3 hours. After removing 2.267 g [b] of the solution remaining without being absorbed by the polymer beads, 4.738 g [c] of a 54% magnesium chloride aqueous solution was added, and the mixture was stirred with a vortex mixer for 10 seconds. After standing at room temperature for 2 hours, 7.254 g [d] of the solution discharged from the polymer beads was recovered. The recovery rate [100×d / (a - b + c)] was 87.0%.

[0077] The particle size distribution of the polystyrene latex contained in the recovered solution is shown in Fig. 3A. The dynamic light scattering measurement was carried out using the same apparatus as in Example 1, and the analysis was performed under the conditions of a measurement temperature of 30°C, a solvent refractive index of 1.376, and a solvent viscosity of 2.170 cP (equivalent to a 20% magnesium chloride aqueous solution). For comparison, the particle size distribution measurement under the same conditions was performed on a solution obtained by mixing a 54% magnesium chloride aqueous solution with the original latex mixed solution B (equivalent to a 20% magnesium chloride aqueous solution) (Fig. 3B). While the particle sizes in the latex mixed solution B show a bimodal distribution of 115.7±42.3 nm and 566.2±258 nm (Fig. 3B), the particle size distribution in the discharge liquid is unimodal, and the average particle size is 178.5±13.1 nm (Fig. 3A). It can be seen that small-sized particles (particle size of 192 nm or less) were fractionally recovered during the discharge process from the polymer beads.

[0078] The particle size of the particles in the mixed solution (b) remaining without being absorbed by the polymer beads showed a bimodal distribution of 149.7 ± 42.1 nm and 965.7 ± 351 nm (Fig. 4A), while the particle size of the particles in the latex mixed solution B showed a bimodal distribution of 149.7 ± 51.0 nm and 774.2 ± 328 nm (Fig. 4B). Comparing the latex particle sizes in both solutions, no difference was observed in the average particle size on the small diameter side, but on the large diameter side, since the average particle size of the former (965.7 nm) was larger than that of the latter (774.2 nm), it is considered that size fractionation also occurs during the absorption process into the polymer beads. The particle size distributions shown in Fig. 4 were all analyzed using the refractive index and viscosity values of pure water.

[0079] [Example 2B] When washed with deionized water before the nanoparticle discharge operation (alternative method of Example 1)

[0080] To polystyrene latex with an average particle size of 100 nm (LB1 manufactured by Sigma-Aldrich) and polystyrene latex with an average particle size of 600 nm (LB6 manufactured by Sigma-Aldrich), deionized water containing 0.20% Tween 20 was added, and after diluting each by 1000 times, the two were mixed in equal amounts. Hereafter, this is referred to as latex mixed solution B.

[0081] Four high water-absorbing polymer beads (60 mg) were placed in a glass sample tube (capacity 50 mL), and 5.970 g [a] of the latex mixed solution B was added thereto, followed by standing at room temperature for 3 hours. After removing 1.961 g [b] of the solution remaining without being absorbed by the polymer beads, the surface of the polymer beads was washed 3 times with 1 mL of deionized water containing 1% Tween 20. Subsequently, 8.077 g [c] of an approximately 26% aqueous sodium chloride solution was added to the polymer beads, and the mixture was stirred with a vortex mixer for 10 seconds. After stirring at room temperature for 2 hours, 10.795 g [d] of the solution discharged from the polymer beads was recovered. The recovery rate [100×d / (a - b + c)] was 89%.

[0082] To the recovered solution, 1% of Tween 20 was added to prevent the aggregation of latex particles. The particle size distribution of the polystyrene latex contained in the recovered solution is shown in Fig. 5A. Dynamic light scattering (DLS) measurements were performed using the same apparatus as in Example 1 and analyzed by the non-negative least squares method under the conditions of a measurement temperature of 30 °C, a solvent refractive index of 1.358, and a solvent viscosity of 1.1723 cP (equivalent to 18% aqueous sodium chloride solution). For comparison, the particle size distribution measurement under the same conditions was performed on a solution obtained by mixing an aqueous sodium chloride solution (equivalent to 18% aqueous sodium chloride solution) with the original latex mixed solution B (Fig. 5B). The particle size distribution of the particles in the original latex mixed solution B is bimodal (peak top particle sizes of 142.8 and 594.1 nm), while the particle size distribution of the particles in the effluent is unimodal, and the peak top particle size is 264.5 nm (average value of three DLS measurements). In addition, a peak of micelles (average particle size 8.1 nm) formed by Tween 20 added for anti-aggregation is also observed in the particle size distribution of Fig. 5A.

[0083] [Example 3] The following steps were performed in sequence to recover exosomes from urine samples. 1. Superabsorbent polymer beads (“Vixker Puyopuyo Ball”, a cross-linked polymer mainly composed of sodium polyacrylate, spherical, particle diameter: 2.66 ± 0.11 mm, particle size distribution: normal distribution) were placed in a 100 mL urine collection cup so that the surface area was 100 mm 2 was achieved. 2. 7 mL of urine sample was added to the urine collection cup containing the superabsorbent polymer beads. 3. It was left standing at room temperature for 3 hours. 4. The urine sample not absorbed by the superabsorbent polymer gel was collected. 5. The surface of the superabsorbent polymer gel was washed 3 times with 1 mL of pure water. 6. The superabsorbent polymer gel was collected into a centrifuge tube, and NaCl with a volume of 1 / 10 of the urine sample was added. 7. It was vortexed for 10 seconds and left standing at room temperature for about 1 to 2 hours. 8. The solution discharged from the superabsorbent polymer gel was recovered. 9. The recovered solution was placed in a dialysis membrane (SPECTRUMLABS.COM, Spectra / Por, MWCO: 3500) and dialyzed with PBS (2 hours, room temperature). 10. The protein concentration of the solution after dialysis was measured by the Bradford method. 11. Silver staining and Western blotting were performed.

[0084] The silver staining in Step 11 was performed according to the following procedure. Electrophoresis (SDS-PAGE) was performed on 2 - 15 μL of exosome-derived protein samples using a 10% polyacrylamide gel, and silver staining was performed using a silver staining kit (product name EzStain Silver) manufactured by ATTO.

[0085] The Western blotting in Step 11 was performed according to the following procedure. <Electrophoresis (SDS-PAGE)> Electrophoresis (SDS-PAGE) was performed on 2 - 15 μL of exosome-derived protein samples using a 10% polyacrylamide gel. <Blotting> Each of Extra Thick Blot Paper (Bio-Rad) and Hybond ECL nitrocellulose membrane (GE Healthcare) was immersed in MilliQ water and then immersed in transfer buffer (3.03 g of Tris, 14.41 g of glycine, 0.1 g of SDS, 200 mL of methanol, MilliQ water to make 1 L) for 20 - 40 minutes. The polyacrylamide gel after electrophoresis was also immersed in transfer buffer for 20 minutes. Using these, a laminate: Extra Thick Blot Paper / Hybond ECL nitrocellulose membrane / polyacrylamide gel / Extra Thick Blot Paper was prepared and placed on the platinum anode at the bottom of the blotting apparatus. Air bubbles between the layers were removed, the upper stainless steel cathode and safety cover were attached, and blotting was performed. <Washing of the membrane> For the membrane after transcription, wash twice for 5 minutes each using 20 mL of autoclaved MilliQ water, immerse in 10 mL of blocking solution, and incubate on an orbital shaker for 60 minutes. Rinse for 5 minutes using 20 mL of TBS-T [1 mL of Tween 20 and 1 L of TBS (8 g of NaCl, 20 mL of Tris-HCl (pH 7.6), MilliQ water to make 1 L)], and repeat this rinse one or more times. <Primary antibody reaction> Immerse the washed membrane in 10 mL of primary antibody solution (about 0.2 - 1 μg / mL; anti-CD9 antibody, anti-CD63 antibody, or anti-CD81 antibody), and incubate at room temperature for 1 hour or more. Rinse for 5 minutes using 20 mL of TBS-T, and repeat this rinse one or more times. After washing with 200 mL of TBS-T for 15 minutes, rinse again for 5 minutes using 20 mL of TBS-T, and repeat this rinse two or more times. <Secondary antibody reaction> Immerse the membrane after the primary antibody reaction in 10 mL of HRP-labeled secondary antibody solution, and incubate on an orbital shaker at room temperature for 60 minutes. Rinse for 5 minutes using 20 mL of TBS-T, and repeat this rinse two or more times. After washing with 200 mL of TBS-T for 15 minutes, rinse again for 5 minutes using 20 mL of TBS-T, and repeat this rinse two or more times. <Detection> Immerse the membrane after the secondary antibody reaction in the detection solution (1.5 mL of ECL Advance Solution A, 1.5 mL of ECL Advance Solution B, 1.5 mL of autoclaved MilliQ water), and allow the reaction to proceed on a seesaw shaker for 5 minutes. Place the membrane, protein side up, on a transparent plastic sheet, cover the membrane with another transparent plastic sheet, and irradiate with X-rays for 15 seconds to 1 hour.

[0086] [Comparative Example 1] Exosomes were recovered from urine samples using ultracentrifugation, and silver staining and Western blotting were performed in the same manner as in Example 3.

[0087] [Comparative Example 2] Exosomes were recovered from urine samples using a commercially available kit (Exo-Urine EV Isolation Kit, manufactured by SBI), and silver staining and Western blotting were performed in the same manner as in Example 3. TM

[0088] The results of silver staining and Western blotting for Example 3, Comparative Example 1, and Comparative Example 2 are shown in Fig. 6. As is clear from Fig. 6, in Example 3, exosomes were recovered with higher purity compared to Comparative Examples 1 and 2.

[0089] Micrographs of the original urine sample and the urine sample obtained in Step 4 of Example 3 are shown in Fig. 7. As is clear from Fig. 7, the urine sample obtained in Step 4 of Example 3 has a higher cell density and is useful for the examination of various diseases compared to the original urine sample.

Claims

1. A method for classifying particle A, comprising the following steps: (1) contacting an aqueous sample containing particle A with a superabsorbent polymer to obtain a superabsorbent polymer gel containing a part of particle A; and (2) mixing the superabsorbent polymer gel with a salt to recover a part of particle A A method comprising the steps of.

2. The method according to claim 1, wherein particle A consists of particle A1 having a particle diameter of 200 nm or less and particle A2 having a particle diameter exceeding 200 nm, and step (2) is a step of recovering particle A1.

3. A method for separating exosomes from a biological sample, comprising the following steps: (3) contacting a biological sample with a superabsorbent polymer to obtain a superabsorbent polymer gel containing exosomes; and (4) mixing the superabsorbent polymer gel with a salt to recover exosomes A method comprising the steps of.

4. The method according to claim 3, wherein the biological sample is a body fluid.

5. The method according to claim 3 or 4, wherein the biological sample is urine.

6. The method according to any one of claims 1 to 5, wherein the salt is a metal salt.

7. The method according to any one of claims 1 to 6, wherein the salt is a monovalent or divalent metal salt.

8. The method according to any one of claims 1 to 7, further comprising a step of washing the superabsorbent polymer gel.

9. The superabsorbent polymer has the following formula: 【Chemical Formula 1】 (In the formula, M represents a cation.) The method according to any one of claims 1 to 8, which is a three-dimensional crosslinked polymer having a constitutional repeating unit represented by the formula.

10. The method according to any one of claims 1 to 9, wherein the superabsorbent polymer is particles having an average particle diameter of 0.1 to 5 mm.

11. A superabsorbent polymer for use in the method according to any one of claims 1 to 8.

12. An apparatus for classifying particles, comprising means for performing the method according to claim 1 or 2.

13. An apparatus for separating exosomes from a biological sample, comprising means for performing the method according to any one of claims 3 to 5.

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