Methods for separating particles and substances, kits for separating substances, kits for testing diseases and apparatus for separating substances

Abstract

This invention provides particles useful for separating substances having a lipid bilayer, such as exosomes. [Solution] Particles for separating substances having a lipid bilayer from biological samples, wherein the weight increase rate X1 calculated by the following formula (1) is greater than 0% and less than or equal to 100%. X1 = {(M1 - M0) / M0} × 100 (1) [In formula (1) above, M0 is the weight of the particles in an oven-dry state, and M1 is the weight of the particles after standing for 24 hours in an environment of 60°C and 95%RH.]

Description

[Technical Field]

[0001] This invention relates to particles and their applications. [Background technology]

[0002] Extracellular vesicles secreted by cells are vesicles surrounded by a lipid bilayer and containing proteins, nucleic acids, and other substances, functioning as intercellular signaling molecules. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Of these, exosomes (membrane vesicles with a particle size of 30-100 nm) are secreted by various cells and their presence has been confirmed in all bodily fluids throughout the body, suggesting the possibility that they circulate through bodily fluids and transmit information to distant cells. The proteins and nucleic acids contained within exosomes reflect the state of the secreting cell in real time, making them useful as biomarkers for evaluating the original state of the organism and intercellular signaling.

[0003] Methods for recovering exosomes from biological samples include purifying extracellular vesicles using ion exchange methods that utilize cations (see Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2020-528766 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, the method described in Patent Document 1 involves purifying extracellular vesicles by ion exchange or the like, resulting in a problem where the purified solution contains many impurities such as albumin from biological samples, and the concentration of recovered exosomes is insufficient.

[0006] The primary objective of this invention is to provide particles useful for separating substances having a lipid bilayer, such as exosomes. [Means for solving the problem]

[0007] The inventors of this invention arrived at this present invention as a result of diligent research to solve the above problems. In other words, the present invention is as follows: [1] Particles for separating substances having a lipid bilayer from biological samples, wherein the weight increase rate X1 calculated by the following formula (1) is greater than 0% and less than or equal to 100%. X1 = {(M1 - M0) / M0} × 100 (1) [In formula (1) above, M0 is the weight of the particles in an oven-dry state, and M1 is the weight of the particles after standing for 24 hours in an environment of 60°C and 95%RH.] [2] The particle according to [1], wherein the volume-average particle diameter of the particle is 10 to 600 μm. [3] A method for separating a substance having a lipid bilayer from a biological sample, comprising the following steps (1) and (2). (1) A step of contacting a biological sample with the particles described in [1] or [2] to obtain particles containing a substance having a lipid bilayer. (2) A step of mixing the particles with a monovalent or divalent metal salt to recover a substance having a lipid bilayer. [4] A kit for separating a substance having a lipid bilayer from a biological sample, comprising the particles and salt described in [1] or [2], wherein the salt is a monovalent or divalent metal salt. [5] A kit for testing for diseases using extracellular vesicles, comprising particles and a salt as described in [1] or [2], wherein the salt is a monovalent or divalent metal salt. [6] An apparatus for separating a substance having a lipid bilayer from a biological sample, comprising means for carrying out the method described in [3]. [Effects of the Invention]

[0008] According to the present invention, particles useful for separating substances having a lipid bilayer, such as exosomes, can be provided. [Modes for carrying out the invention]

[0009] <Particle> The particles of the present invention are for separating substances having a lipid bilayer from biological samples, and are particles in which the weight increase rate X1 calculated by the following formula (1) is greater than 0% and less than or equal to 100%. X1 = {(M1 - M0) / M0} × 100 (1) [In formula (1) above, M0 is the weight of the particles in an oven-dry state, and M1 is the weight of the particles after standing for 24 hours in an environment of 60°C and 95%RH.] In this invention, "absolutely dry state" means a state in which the particles are dried at 100°C until the weight reduction rate of the particles is 0.001% or less. The weight of the particles before and after drying is measured under a temperature of 25°C. In this specification, "substance having a lipid bilayer" refers to a vesicle or particulate structure surrounded by a lipid bilayer, and includes, for example, extracellular vesicles (exosomes, microvesicles, apoptotic bodies, etc.). The preferred subject of the present invention is extracellular vesicles.

[0010] The particles of the present invention can increase the concentration of the separated lipid bilayer-containing substance when separating the lipid bilayer-containing substance. Therefore, the particles of the present invention are useful for separating the lipid bilayer-containing substance.

[0011] The particles of this invention are particles with a weight increase rate X1 greater than 0% and less than or equal to 100%. If the weight increase rate X1 exceeds 100%, it is not possible to recover exosomes at a sufficient concentration from the absorbed particles during the exosome extraction process. Conversely, if it is 0%, exosomes cannot be recovered at all. The particle weight increase rate X1 is preferably in the range of 90% or less, more preferably 80% or less, from the viewpoint that the amount of sample used for exosome recovery can be reused without adjustment. More preferably it is 50% or less, and particularly preferably 4 to 45%. The weight increase rate X1 can be measured by the method described in the examples below.

[0012] The material of the particles of the present invention is not particularly limited, but preferred particle materials include polysaccharides such as cellulose, hemicellulose, chitin, chitosan, dextran, agarose, and alginate; proteins such as collagen and gelatin; synthetic polymers such as polystyrene, polyacrylamide, polycarbonate, polyethylene, polypropylene, and silicone; and inorganic substances such as glass, silica, alumina, iron oxides (Fe3O4, Fe2O3, etc.), and magnetic ferrite. Polysaccharides are preferred, and cellulose, dextran, and agarose are more preferred. The particles may be used alone as one type, or in combination of two or more types.

[0013] The surface of the particles of the present invention may be functionalized. Examples include introducing functional groups such as acidic groups (carboxyl groups, sulfonic acid groups) and basic groups (amino groups).

[0014] The particles of the present invention may contain a compound selected from at least one group from the group consisting of cationic groups and hydroxyl groups (hereinafter referred to as "compound Z"). That is, the particles of the present invention may be in the form of a coexistence with compound Z. Compound Z may be present on the surface of the particles or may be present inside the particles. In the present invention, particles having compound Z present on the surface are preferred. In the present invention, it is preferable that compound Z is attached or immobilized on the surface of the particles via non-covalent bonds such as hydrogen bonds and ionic bonds, and it is preferable that part or all of the surface of the particles is coated with compound Z. The surface of the particle means on the molecular chain of the particle or the surface of the particle.

[0015] That the particles of the present invention contain compound Z can be identified by immersing the particles in a suitable solvent (a hydrocarbon solvent such as hexane), eluting compound Z into the solvent, isolating compound Z, and subjecting it to NMR and gas chromatography.

[0016] Compound Z preferably has at least one group selected from the group consisting of cationic groups and hydroxyl groups, and a C2-C30 hydrocarbon group which may be substituted, more preferably has at least one group selected from the group consisting of cationic groups and hydroxyl groups, and a C12-C30 hydrocarbon group which may be substituted, and even more preferably has a C12-C30 hydrocarbon group which may be substituted at one end and at least one group selected from the group consisting of cationic groups and hydroxyl groups at the other end. In compound Z, the number of hydrocarbon groups, cationic groups, or hydroxyl groups may be one or two or more. In this specification, if compound Z has a cationic group and a hydroxyl group is included in the cationic group, compound Z shall be said to have a cationic group and a hydroxyl group, and if compound Z has a C2-C30 (or C12-C30) hydrocarbon group which may be substituted, and a hydroxyl group is included as a substituent in the hydrocarbon group, compound Z shall be said to have the hydrocarbon group and a hydroxyl group.

[0017] The hydrocarbon group having 12 to 30 carbon atoms may be any of the following: an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group; an alicyclic hydrocarbon group such as a cycloalkyl group or a cycloalkenyl group; or an aromatic hydrocarbon group. Examples of substituents on the hydrocarbon group include a hydroxyl group, an oxo group (=O), an alkoxy group, an alkenyloxy group, and combinations thereof. The number of substituents on the hydrocarbon group may be one, two, three, four, or five.

[0018] Aliphatic hydrocarbon groups having 12 to 30 carbon atoms may be either saturated or unsaturated, and examples include aliphatic hydrocarbon groups having 12 to 20 carbon atoms such as lauryl, myristyl, palmityl, stearyl, oleyl, linoleyl, and arachidyl groups.

[0019] For example, lecithin has a C12-C30 aliphatic hydrocarbon group which may be substituted, specifically an acyl group having 12-30 carbon atoms, and more specifically, an oleiroyloxy group and a palmitoyloxy group. More specifically, lecithin has a diacylglycerol skeleton having the aforementioned acyl group.

[0020] Examples of alicyclic hydrocarbon groups having 12 to 30 carbon atoms include groups having a steroid skeleton. Examples of steroid skeletons include structures represented by the following formula (1).

[0021] [ka]

[0022] In formula (1), the dashed line in ring A indicates that any carbon-carbon single bond may also be a carbon-carbon double bond, and the dashed line in ring B indicates that the carbon-carbon bond may be either a carbon-carbon single bond or a carbon-carbon double bond. The hydrogen atoms bonded to the carbon atoms constituting rings A to D may each be substituted. The numbers on the rings are added for convenience to indicate the substitution positions of the substituents.

[0023] In formula (1), possible substituents that can be bonded to rings A to D are, independently, a hydroxyl group, an oxo group (=O), and an alkyl group (a C1-C4 alkyl group such as a methyl group). The substitution position of the substituent is not particularly limited, but preferred positions include the 3rd, 7th, 10th, 11th, 12th, 13th, and 17th positions.

[0024] The skeleton represented by equation (1) encompasses the skeletons represented by equations (1-1) to (1-5) below.

[0025] [ka]

[0026] The alicyclic hydrocarbon group having 12 to 30 carbon atoms is preferably an alicyclic hydrocarbon group having 20 to 30 carbon atoms, more preferably a group in which an optionally substituted alkylene group (an alkylene group having 2 to 4 carbon atoms, such as an ethylene group or a propylene group) is linked to the 17th position of the skeleton represented by formula (1), and even more preferably a group in which an optionally substituted alkylene group (an alkylene group having 2 to 4 carbon atoms, such as an ethylene group or a propylene group) is linked to the 17th position of the skeleton represented by any of formulas (1-1) to (1-5).

[0027] Aromatic hydrocarbon groups with 12 to 30 carbon atoms include fluorene, anthracene, phenanthrene, tetracene, pyrene, triphenylene, chrysene, and tetraphenylene.

[0028] The substituted hydrocarbon group having 12 to 30 carbon atoms is preferably a group having a C12-30 acylglycerol skeleton or a group having a steroid skeleton.

[0029] In this specification, "cationic group" includes groups that form a cation by themselves, and groups that do not form a cation by themselves but can form a cation through proton bonding. Examples of cationic groups include -N(R 1 )2(in the formula, each R 1 Each of these independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms that may be substituted), -N + (R 2 )3(in the formula, each R 2 Examples include a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted.

[0030] R in the above formula 1 and R 2 Examples of hydrocarbon groups having 1 to 6 carbon atoms include alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, and hexyl groups. Examples of substituents that can be substituted for hydrocarbon groups having 1 to 6 carbon atoms include hydroxyl, oxo, carboxylic acid, and sulfonic acid groups.

[0031] The aforementioned group represented by 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 a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, and a hydroxypentyl group (including a pentahydroxypentyl group).

[0032] The aforementioned group represented by formula: -N+(R 2 )3 is preferably -N + (CH3)3, -N + (CH3)2(C2H5), or -N + (CH3)2(C3H7), and these groups may optionally have a substituent such as -CO2 - or -SO3 - .

[0033] Preferred examples of Compound Z include: compounds having an aliphatic hydrocarbon group having 12 to 30 carbon atoms and a cationic group (such as lecithin and hexadecyltrimethylammonium chloride), compounds having a hydrocarbon group having 12 to 30 carbon atoms and a hydroxyl group (such as lauryl alcohol and N,N-bis(3-D-gluconamidopropyl)cholamide (BIGCHAP)), compounds having an alicyclic hydrocarbon group having 12 to 30 carbon atoms, a cationic group and a hydroxyl group (such as 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS)), compounds having a cationic group and a hydroxyl group (such as triethanolamine), and polymers having a cationic group (such as polylysine and polyethyleneimine).

[0034] The lower limit of the molar concentration of cationic groups per mole of Compound Z (particularly when Compound Z is a polymer having a cationic group) is preferably 1 mole or more, and from the viewpoint of improving the recovery amount of a substance having a lipid bilayer, it is preferably 5 moles or more, and more preferably 10 moles or more. Further, the upper limit of the molar concentration of cationic groups in Compound Z is preferably 100 moles or less.

[0035] The lower limit of the chemical formula weight or number average molecular weight of Compound Z is preferably 300 or more, and more preferably 500 or more, from the viewpoint of improving the recovery amount of a substance having a lipid bilayer. The upper limit of the chemical formula weight or number average molecular weight of Compound Z is preferably 10000 or less. The number average molecular weight of Compound Z can be measured using gel permeation chromatography (GPC) under the following conditions. <GPC Measurement Conditions> [1] Apparatus: Gel permeation chromatography [model number "HLC-8120GPC", manufactured by Tosoh Corporation] [2] Columns: "TSKgel G6000PWxl" and "TSKgel G3000PWxl" (both manufactured by Tosoh Corporation) connected in series. [3] Eluent: a solution obtained by dissolving 0.5 wt% sodium acetate in methanol / water = 30 / 70 (volume ratio). [4] Reference substance: polyethylene glycol (hereinafter abbreviated as PEG) [5] Injection conditions: sample concentration 0.25 wt%, column temperature 40°C

[0036] The particles of the present invention can be obtained by a production method including a step of washing particles of the materials exemplified in "Material of Particles of the Present Invention" with water (such as ion-exchanged water) or the like, followed by drying. When the particles comprise Compound Z, particles comprising Compound Z can be obtained by carrying out a step of bringing the particles obtained in the drying step into contact with Compound Z. The contacting step can be performed by mixing the particles and Compound Z, or the like.

[0037] In the separation of substances having a lipid bilayer, the particles of the present invention are preferably particles with a volume-average particle diameter in the range of 10 μm or more, 30 μm or more, 50 μm or more, 70 μm or more, 90 μm or more, or 100 μm or more, in order to reduce impurities (such as albumin). Furthermore, from the viewpoint of improving the recovery amount of the substance having a lipid bilayer, the particles of the present invention are preferably particles with a volume-average particle diameter in the range of 600 μm or less, 500 μm or less, 400 μm or less, or 300 μm or less. The volume-average particle diameter can be measured by the method described in the examples below. In other words, the volume-average particle diameter of the particles is preferably 10 to 600 μm, and more preferably 30 to 500 μm.

[0038] <Method for separating substances containing a lipid bilayer from biological samples> The present invention provides a method for separating a substance having a lipid bilayer from a biological sample, comprising the following steps (1) and (2). (1) A step of contacting a biological sample with the particles of the present invention to obtain particles containing a substance having a lipid bilayer. (2) A step of mixing the particles with a monovalent or divalent metal salt to recover a substance having a lipid bilayer.

[0039] The biological sample used in step (1) is not particularly limited as long as it contains a substance having a lipid bilayer. Examples of biological samples include body fluids such as human or animal blood, plasma, serum, tears, saliva, breast milk, pleural fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, and urine; liquefied tissues or cells such as organs, hair, nails, skin, muscles, and nerves (including cell culture media and their supernatants); and extracts from plants. The biological sample may be a single type or a combination of two or more types. The biological sample is preferably a body fluid, and more preferably blood, plasma, serum, or urine.

[0040] The particles used in step (1) are the same as those described in "Particles" above. The amount of particles used is preferably 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, per 100 parts by mass of biological sample.

[0041] The temperature at which the particles are brought into contact with the biological sample is preferably in the range of 1 to 35°C, and more preferably in the range of 5 to 30°C. Furthermore, it is preferable to allow the sample to stand for a predetermined time after contact (for example, 1 hour or more, or 2 hours or more, and 5 hours or less, or 4 hours or less).

[0042] Step (1) allows for the separation of a substance having a lipid bilayer into particles. These particles are then subjected to step (2).

[0043] The particles used in step (2) are particles containing the lipid bilayer obtained in step (1). The salt used in process (2) is a metal salt (monovalent or divalent metal salt).

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

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

[0046] The salt may be a single type or a combination of two or more types. Preferably, the salt is at least one selected from the group consisting of sodium chloride and magnesium chloride.

[0047] The amount of salt used may be 1 part by mass or more, 5 parts by mass or more, or 10 parts by mass or more per 100 parts by mass of particles, and may be 2000 parts by mass or less, 1500 parts by mass or less, or 1000 parts by mass or less.

[0048] The mixing temperature of the particles and salt is preferably within the range of 1 to 35°C or 5 to 30°C. Furthermore, it is preferable to let the mixture stand for a predetermined time after mixing (for example, 1 hour or more, or 2 hours or more, and 5 hours or less, or 4 hours or less). The mixture of particles and salt may be dialyzed if necessary.

[0049] Step (2) allows for the recovery of lipid bilayer-containing substances in the particles with a high recovery rate. Examples of lipid bilayer-containing substances that can be recovered include extracellular vesicles such as exosomes.

[0050] The method of the present invention allows for a higher concentration of the recovered lipid bilayer-containing substance compared to conventional ultracentrifugation methods. Furthermore, since the separation method of the present invention uses the particles of the present invention, the same particles can be reused repeatedly. The lipid bilayer-containing substance separated by the method of the present invention can be used for evaluation (tests or diagnoses, etc.) of the health status of the organism before separation. In addition, the separated lipid bilayer-containing substance can be used as a carrier for cell-targeted therapeutics and the like.

[0051] <Kit for separating substances containing a lipid bilayer from biological samples> The kit of the present invention is for separating a substance having a lipid bilayer from a biological sample, and comprises the particles of the present invention and a monovalent or divalent metal salt. The configuration of the kit is preferably for use in the "method for separating a substance having a lipid bilayer from a biological sample." The kit of the present invention may further include instructions including a description of the method for separating a substance having a lipid bilayer from a biological sample, and equipment for the separation.

[0052] The present invention provides a kit for testing (or diagnosing) a disease using extracellular vesicles, comprising the aforementioned particles and a monovalent or divalent metal salt.

[0053] The subjects of the examination may be healthy individuals or patients. Diseases that can be examined include lifestyle-related diseases, chronic kidney disease, neurological disorders, immune disorders, cancer, infectious diseases, and degenerative diseases.

[0054] The kit may separate extracellular vesicles from a sample derived from the subject to be tested (body fluids such as blood, plasma, serum, tears, saliva, breast milk, pleural fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, and urine) and test for diseases based on the contents of the extracellular vesicles. The kit may be a known kit for testing for diseases using extracellular vesicles, and may include particles and salts as reagents for separating extracellular vesicles from a sample derived from the subject to be tested.

[0055] The particles and salts in the kit may be the same as those described in the "Method for Separating Substances Having a Lipid Bilayer from a Biological Sample." The kit may further include instructions explaining how to separate extracellular vesicles from the test subject, and equipment for the separation.

[0056] <Apparatus for separating substances containing a lipid bilayer from biological samples> The apparatus of the present invention is an apparatus for separating a substance having a lipid bilayer from a biological sample, and includes an apparatus that provides means for performing the "method for separating a substance having a lipid bilayer from a biological sample" (the method of the present invention). [Examples]

[0057] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. Examples and comparative examples relating to particle production are represented by single-digit numbers (Examples 1-7, Comparative Example 1, etc.), and examples and comparative examples relating to exosome recovery operations using the particles are represented by double-digit numbers (Examples 11-19, Comparative Example 11, Comparative Example 12, etc.).

[0058] <Method for measuring weight increase rate (X1)> For the particles used in the examples and comparative examples, M0 and M1 were measured as follows, and the weight increase rate was calculated using the following formula (1). [Weight increase rate (X1)] = {(M1-M0) / M0} × 100 (1) In equation (1) above, M0 is the weight of the particles in an oven-dry state, and M1 is the weight of the particles after standing for 24 hours in an environment of 60°C and 95% RH. An analytical balance [Shimadzu Corporation, AUX220] was used as the measuring device for M0 and M1. In the examples and comparative examples, when measuring M0, the particles were dried at 100°C until the weight reduction rate of the particles before and after drying was 0.001% or less. The weight of the particles before and after drying was measured under a temperature of 25°C.

[0059] <Manufacturing Example 1: Manufacturing of Particle (X-1)> 10 g of agarose particles {product name "CM Sepharose Fast Flow", manufactured by Global Life Science Technologies Japan Co., Ltd., surface functional group is carboxymethyl group} were mixed with 40 mL of deionized water and washed twice by suction removal. Subsequently, the particles were dried under reduced pressure in a vacuum dryer {40°C, 0.1 kPa or less} for 4 hours to obtain particle (X-1).

[0060] <Manufacturing Example 2: Manufacturing of Particles (X-2)> In Production Example 1, the process was carried out in the same manner as in Production Example 1, except that agarose particles {product name "SP Sepharose XL", manufactured by Global Life Science Technologies Japan Co., Ltd., with a sulfonic acid group as the surface functional group} were used instead of agarose particles {product name "CM Sepharose FastFlow", manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-2) were obtained.

[0061] <Manufacturing Example 3: Manufacturing of Particles (X-3)> In Production Example 1, the process was carried out in the same manner as in Production Example 1, except that cellulose particles {product name "Cellufine MAX CM", manufactured by JNC Corporation, surface functional group is a carboxymethyl group} were used instead of agarose particles {product name CM Sepharose Fast Flow, manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-3) were obtained.

[0062] <Manufacturing Example 4: Manufacturing of Particles (X-4)> In Production Example 1, the process was carried out in the same manner as in Production Example 1, except that cellulose particles {product name "Cellufine MAX Sr", manufactured by JNC Corporation, surface functional group is sulfonic acid group} were used instead of agarose particles {product name CM Sepharose Fast Flow, manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-4) were obtained.

[0063] <Manufacturing Example 5: Manufacturing of Particles (X-5)> In Production Example 1, the process was carried out in the same manner as in Production Example 1, except that magnetic ferrite particles {product name "FF Eu COOH beads", manufactured by Tamagawa Seiki Co., Ltd., surface functional group is a carboxyl group} were used instead of agarose particles {product name CM Sepharose Fast Flow, manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-5) were obtained.

[0064] <Manufacturing Example 6: Manufacturing of Particles (X-6)> In Production Example 1, the process was carried out in the same manner as in Production Example 1, except that polystyrene particles {product name "Corning®", untreated microcarrier, manufactured by Corning} were used instead of agarose particles {product name CM Sepharose Fast Flow, manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-6) were obtained.

[0065] <Manufacturing Example 7: Manufacturing of Particles (X-7)> In Production Example 1, the procedure was carried out in the same manner as in Production Example 1, except that dextran particles {trade name "Cytodex-1", manufactured by Global Life Science Technologies Japan Co., Ltd.} were used instead of agarose particles {trade name CM Sepharose Fast Flow, manufactured by Global Life Science Technologies Japan Co., Ltd.}, and particles (X-7) were obtained.

[0066] <Manufacturing Example 8: Manufacturing of Particles (B-1)> In a 1-liter beaker, 116.5 g of acrylic acid, 272.2 g of deionized water, and 1.5 g of ethylene glycol diglycidyl ether (1.3 wt% / acrylic acid) as a crosslinking agent were added and mixed to dissolve the crosslinking agent. While cooling the beaker in an ice bath, 96.1 g of 48.5 wt% sodium hydroxide aqueous solution was added to neutralize a portion of the acrylic acid (72 mol%). After cooling the neutralized monomer solution to 5°C, 9.3 g of 2 wt% potassium persulfate aqueous solution was added as a polymerization initiator to obtain an aqueous monomer solution. In a 2-liter separable flask equipped with a stirrer and a condenser, 1434 g of cyclohexane and 7.1 g of Rheodol SP-S10V (Kao Corporation, sorbitan monostearate) as a dispersant were placed. The mixture was heated to an internal temperature of 60°C using a water bath and stirred to dissolve the dispersant in the cyclohexane. After reducing the dissolved oxygen in cyclohexane to 0.1 ppm or less by passing nitrogen through the solution in a separable flask, 350 g of monomer aqueous solution was added dropwise using a dropping funnel while stirring with a stirrer, and reverse-phase suspension polymerization was carried out at a polymerization temperature of 80°C. After the addition of the monomer aqueous solution was completed, the mixture was heated for another 2 hours to complete the suspension polymerization, and a spherical hydrated gel was obtained in cyclohexane. After stopping the rotation of the stirrer and allowing the generated hydrated gel to settle, the cyclohexane was removed by decantation, and the remaining hydrated gel was washed several times with cyclohexane to remove the dispersant adhering to the hydrated gel. The obtained spherical water-containing gel was spread on release paper and dried for 1 hour in a vacuum dryer at 130°C (vacuum level: 10,000-20,000 Pa). After that, it was crushed, and then the particle size was adjusted to 90-150 μm using sieves with mesh sizes of 90 and 150 μm to obtain particles (B-1).

[0067] <Example 1: Production of particles (X-1-1)> 10 g of particle (X-1) was mixed with 2 g of a 5 wt% lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, derived from soybeans) cyclohexane solution by dropping with a dropper. After homogeneous mixing, the mixture was heated at 130°C for 30 minutes and then cooled to room temperature to obtain particle (X-1-1). The volume-average particle diameter of particle (X-1-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and found to be 90 μm. The weight increase rate of particle (X-1-1) was 23%.

[0068] <Example 2: Production of particles (X-2-1)> In Example 1, the procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-2), and particle (X-2-1) was obtained. The volume-average particle diameter of particle (X-2-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 92 μm. The weight increase rate of particle (X-2-1) was 28%.

[0069] <Example 3: Production of particles (X-3-1)> In Example 1, the procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-3), and particle (X-3-1) was obtained. The volume-average particle diameter of particle (X-3-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 95 μm. The weight increase rate of particle (X-3-1) was 40%.

[0070] <Example 4: Production of particles (X-4-1)> In Example 1, the particle was changed from (X-1) to (X-4), and 1 g of 5 wt% BIGCHAP (manufactured by Dojin Chemical Laboratories, N,N-bis(3-D-gluconamidopropyl)coramide) cyclohexane solution was used instead of 2 g of 5 wt% lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from soybeans) solution. The procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-4), and 1 g of 5 wt% BIGCHAP (manufactured by Dojin Chemical Laboratories, N,N-bis(3-D-gluconamidopropyl)coramide) cyclohexane solution was used. The particle (X-4-1) was obtained. The volume-average particle diameter of particle (X-4-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 94 μm. The weight increase rate of particle (X-4-1) was 45%.

[0071] <Example 5: Production of particles (X-5-1)> In Example 1, the particle was changed from (X-1) to (X-5), and 1 g of 5% by weight hexadecyltrimethylammonium chloride (HDTMA-Cl) cyclohexane solution was used instead of 2 g of 5% by weight lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from soybeans) solution. The procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-5), and 1 g of 5% by weight hexadecyltrimethylammonium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., HDTMA-Cl) cyclohexane solution was used. The particle (X-5-1) was obtained. The volume-average particle diameter of particle (X-5-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be less than 1 μm. The weight increase rate of particle (X-5-1) was 4%.

[0072] <Example 6: Production of particles (X-6-1)> In Example 1, the particle was changed from (X-1) to (X-6), and 2 g of 5 wt% CHAPS (manufactured by Fujifilm Wako Pure Chemical Industries, 3-[(3-collamidopropyl)dimethylammonio]propanesulfonate) cyclohexane solution was used instead of 2 g of 5 wt% lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, derived from soybeans) cyclohexane solution. The procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-6), and 2 g of 5 wt% CHAPS (manufactured by Fujifilm Wako Pure Chemical Industries, 3-[(3-collamidopropyl)dimethylammonio]propanesulfonate) cyclohexane solution was used. The particle (X-6-1) was obtained. The volume-average particle diameter of particle (X-6-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 181 μm. The weight increase rate of particle (X-6-1) was 5%.

[0073] <Example 7: Production of particles (X-7-1)> In Example 1, the particle was changed from (X-1) to (X-7), and 2 g of 5 wt% lauryl alcohol (manufactured by Tokyo Chemical Industry Co., Ltd.) cyclohexane solution was used instead of 2 g of 5 wt% lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from soybeans) cyclohexane solution. The procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (X-7), and 2 g of 5 wt% lauryl alcohol (manufactured by Tokyo Chemical Industry Co., Ltd.) cyclohexane solution was used instead of 2 g of 5 wt% lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from soybeans). The particle (X-7-1) was obtained. The volume-average particle diameter of particle (X-7-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 185 μm. The weight increase rate of particle (X-7-1) was 40%.

[0074] <Comparative Example 1: Manufacturing of Particles (B-1-1)> In Example 1, the procedure was carried out in the same manner as in Example 1, except that the particle was changed from (X-1) to (B-1), and particle (B-1-1) was obtained. The volume-average particle diameter of particle (B-1-1) was measured with a particle analyzer (Retsch CAMSIZER XT) and was found to be 130 μm. The weight increase rate of particle (B-1-1) was 152%.

[0075] <Examples 11-17, Comparative Example 11: Exosome recovery using particles obtained in Examples 1-7 and Comparative Example 1> Using the particles (X-1-1) to (X-7-1) obtained in Examples 1 to 7 and the particles (B-1-1) obtained in Comparative Example 1, exosomes were recovered from cell culture supernatant samples using the following method, and the concentration of the recovered exosomes was evaluated. The results are shown in Table 1 as Examples 11 to 17 and Comparative Example 11.

[0076] [Evaluation test: Exosome recovery from cell culture supernatant samples using particles] (1) Preparation of biological samples The cell culture supernatant was prepared by subculturing HEK293 cells in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) by volume. Once the cells reached 80% confluence, the medium was replaced with FBS-free DMEM. The conditioned medium was then collected after 48 hours of incubation, and 1 mL of the medium was centrifuged at 2000 G for 10 minutes to remove debris and prepare the cell culture supernatant sample. The exosome concentration of the sample was measured using the following method. The results are shown in Table 1.

[0077] (2) Preparation of the spin column Exosome recovery columns were prepared by placing 0.1 mL of the particles (X-1-1) to (X-7-1) obtained in Examples 1 to 7, or the particles (B-1-1) obtained in Comparative Example 1, into a spin column {product name "Microbio Spin Column", manufactured by BIORAD Co., Ltd.}.

[0078] (3) First exosome retrieval process 1 mL of cell culture supernatant, from which debris had been removed, was added to a spin column containing particles and allowed to stand at 25°C for 30 minutes. Next, the spin column was spun down at 2000 G for 10 seconds in a benchtop centrifuge (Chibitan-R), and the separated liquid (liquid not absorbed by the particles) was discarded. 0.5 mL of physiological saline (0.9 wt%) was added to the spin column containing particles, and after further mixing by pipetting, the spin column was immediately spun down at 2000 G for 10 seconds in a benchtop centrifuge (Chibitan-R), and the separated liquid (liquid not absorbed by the particles) was discarded to wash the particles (washing process). After repeating the same washing procedure a total of three times, 0.1 mL of saline solution (20.5 wt%) was added and allowed to stand at 25°C for 30 minutes. The spin column was then spun down at 2000 G for 10 seconds using a benchtop centrifuge (Chibitan-R), and the recovered solution was sealed in a dialysis tube (Fujifilm Wako Pure Chemical Industries, Dialysis Membrane, Size 20 MWCO:14000). Desalting was performed at 25°C for 2 hours to recover the exosomes. The volume of the recovered liquid was measured using a graduated cylinder. The exosome recovery concentration was measured using the method described below, and the exosome concentration ratio was calculated using the method described below. The results are shown in Table 1. (Method for measuring exosome concentration) Exosome concentration was measured using an ELISA kit [Cosmo Bio Co., Ltd., CD9 / CD63 Exosome ELISA Kit, Human]. (Method for calculating concentration ratio) The concentration ratio was calculated using the following formula (2A). [Concentration ratio (%)] = [Exosome concentration after the first recovery step] / [Exosome concentration before the first recovery step] × 100 (2A)

[0079] (4) Second exosome retrieval process For Examples 11-17, 1 mL of the sample from which debris had been removed was added using the spin column used in "(3) First exosome recovery step," and the mixture was allowed to stand at 25°C for 30 minutes. After standing, the same procedure as in "(3) First exosome recovery step" was performed to recover the exosomes. For Comparative Example 11, the second exosome recovery was performed in the same manner as in Examples 11-17, except that the amount of sample with debris removed was set to 0.5 mL because the particle volume increased due to "(3) First exosome recovery step". The volume of liquid collected from each sample was measured using a graduated cylinder. The exosome concentration was measured using the same method as in "(3) First exosome collection step," and the exosome concentration ratio was calculated using the following method. (Method for calculating concentration ratio) The concentration ratio was calculated using the following formula (2B). [Concentration ratio (%)] = [Exosome concentration after the second recovery step] / [Exosome concentration before the first recovery step] × 100 (2B)

[0080] <Examples 18-19: Exosome recovery using particles obtained in Example 1> For both healthy individual urine and healthy individual serum samples, 1 mL of each sample was centrifuged at 1200 G for 10 minutes to remove debris and prepare samples for exosome extraction. Except for changing the sample used in Example 11 to the aforementioned sample (a sample obtained by centrifuging a healthy person's urine sample and a healthy person's serum sample), exosomes were recovered from the healthy person's urine sample and healthy person's serum sample, respectively, in the same manner as in Example 11. The recovered exosome concentrations were measured, and the results are shown in Table 1.

[0081] <Comparative Example 12: Exosome recovery by conventional method (ultracentrifugation)> Exosomes were isolated and recovered by ultracentrifugation of 1 mL of the sample. Specifically, centrifugation was performed continuously under 4°C conditions as follows. One mL of the sample was centrifuged at 1200 × g for 10 minutes, the supernatant was collected, and it was centrifuged again at 10000 × g for 30 minutes. The supernatant was then collected again and centrifuged at 70000 × g for 1 hour, and the supernatant was discarded. The settled pellet was resuspended in 5 mL of 0.25 M sucrose solution (20 mM HEPES pH 7.2), and the resulting suspension was added to a 40 PA tube containing 30 mL of 0.25 M to 2 M sucrose gradient solution (20 mM HEPES pH 7.2), and centrifuged at 100000 × g for 20 hours. The liquid from the 40 PA tube was aspirated from the bottom, and fractions of 8 mL to 12 mL were divided into 2 mL portions each and placed into 5 PA tubes containing 3 mL of PBS(-). The separated liquids were centrifuged at 200,000 × g for 1 hour, and the settled pellet was resuspended in 0.1 mL of PBS(-) to recover the exosomes. The volume of the recovered liquid was measured using a graduated cylinder. The exosome recovery concentration was measured by the following method, and the exosome enrichment ratio was calculated by the following method. The results are shown in Table 1. (Method for measuring exosome concentration) Exosome concentration was measured using an ELISA kit [Cosmo Bio Co., Ltd., CD9 / CD63 Exosome ELISA Kit, Human]. (Method for calculating concentration ratio) The concentration ratio was calculated using the following formula (2C). [Concentration ratio (%)] = [Exosome concentration after recovery process] / [Exosome concentration before recovery process] × 100 (2C)

[0082] [Table 1] [Industrial applicability]

[0083] As is clear from the above results, when exosomes are separated using the particles of the example, the concentration of separated exosomes can be increased compared to the comparative example. From this, it was found that the particles of the present invention can be suitably used for separating substances having a lipid bilayer from biological samples. Furthermore, the separation method of the present invention allows for a higher concentration of the separated lipid bilayer-containing substance. Additionally, the separation method of the present invention allows for the repeated use of the same particles. Moreover, the lipid bilayer-containing substance separated using the particles of the present invention can be used for evaluating (testing or diagnosing) the state of the original organism. Furthermore, the lipid bilayer-containing substance separated using the particles of the present invention can be used as a carrier in cell-targeted therapeutics and the like.

Claims

1. Particles for separating substances having a lipid bilayer from biological samples, wherein the weight increase rate X is calculated by the following formula (1). １ A particle whose concentration is greater than 0% and less than or equal to 100%. X １ ={(M １ -M ０ ) / M ０ }×100 (1) [In the above formula (1), M ０ This is the weight of the particles in a completely dry state, M １ This is the weight of the particles after standing for 24 hours in an environment of 60°C and 95% RH.

2. The particle according to claim 1, wherein the volume-average particle diameter of the particle is 10 to 600 μm.

3. A method for separating a substance having a lipid bilayer from a biological sample, comprising the following steps (1) and (2). (1) A step of contacting a biological sample with the particles described in claim 1 or 2 to obtain particles containing a substance having a lipid bilayer. (2) A step of mixing the particles with a monovalent or divalent metal salt to recover a substance having a lipid bilayer.

4. A kit for separating a substance having a lipid bilayer from a biological sample, comprising the particles and salt described in claim 1 or 2, wherein the salt is a monovalent or divalent metal salt.

5. A kit for testing for diseases using extracellular vesicles, comprising the particles and salt described in claim 1 or 2, wherein the salt is a monovalent or divalent metal salt.

6. An apparatus for separating a substance having a lipid bilayer from a biological sample, comprising means for performing the method described in claim 3.

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