Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/10—Esters
  • C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere

Definitions

• the present invention relates to an extracellular vesicle peeling agent for separating extracellular vesicles from a crude solution containing the extracellular vesicles.
• Extracellular vesicles are secreted from a variety of cells and are found in blood (plasma, serum), urine, saliva, breast milk, etc. They are also released into the culture supernatant of many cell lines. Extracellular vesicles include exosomes with diameters of 30-150 nm and microvesicles with diameters of 30-1,000 nm, and have characteristic antigens and lipids such as phosphatidylserine (PS) on their membranes.
• PS phosphatidylserine
• extracellular vesicles contain nucleic acids such as proteins, messenger RNA (mRNA), microRNA (miRNA), small double-stranded RNA (siRNA), and DNA that reflect the characteristics of the cells from which they originate, and are said to be involved in intercellular information transmission by being taken up by other cells.
• nucleic acids such as proteins, messenger RNA (mRNA), microRNA (miRNA), small double-stranded RNA (siRNA), and DNA that reflect the characteristics of the cells from which they originate, and are said to be involved in intercellular information transmission by being taken up by other cells.
• extracellular vesicles reflecting the characteristics of the cells from which they are derived has been exploited for their use as biomarkers for disease.
• extracellular vesicles themselves as carriers for drug delivery systems and the use of useful proteins contained in extracellular vesicles for tissue regeneration have also been investigated. When using extracellular vesicles for these purposes, it is important that the separated and collected extracellular vesicles are in an intact, undamaged state.
• Known methods for separating and isolating extracellular vesicles from body fluids include ultracentrifugation, density gradient centrifugation, column methods, and immunoprecipitation using magnetic particles.
• immunoprecipitation using magnetic particles is a method that produces high purity and recovery rates of separated extracellular vesicles. It is also a method that can separate the specific extracellular vesicles mentioned above.
• Non-Patent Document 1 describes a method of recovering extracellular vesicles via Tim4 protein, which recognizes phosphatidylserine (PS) on extracellular vesicles.
• Tim4 protein bound to magnetic particles recognizes PS on extracellular vesicles and recovers the extracellular vesicles from crude solutions such as body fluids.
• a chelating agent is used to sever the bond between Tim4 protein and PS, thereby detaching the extracellular vesicles from the magnetic particles.
• Patent Document 1 also describes a method for recovering extracellular vesicles using a peptide that has high affinity for phosphatidylserine (PS) on extracellular vesicles.
• PS phosphatidylserine
• a PS-affinity peptide bound to magnetic particles recognizes the PS on the extracellular vesicles and recovers the extracellular vesicles from the crude solution.
• the extracellular vesicles are then detached from the magnetic particles by cleaving the bond between the peptide and PS using a 500 mM aqueous solution of inorganic salt.
• a commonly known method for isolating extracellular vesicles that contain specific antigens is to use magnetic particles bound to antibodies. Although this method can be used to isolate extracellular vesicles that contain specific antigens, it requires high-temperature treatment at approximately 70°C or high-concentration ion treatment to sever the antigen-antibody bond, which can damage the extracellular vesicles.
• the objective of the present invention is to provide an extracellular vesicle release agent capable of recovering specific extracellular vesicles without damage from a crude solution containing the extracellular vesicles to be separated, and a method for purifying extracellular vesicles using the extracellular vesicle release agent.
• the inventors have found that the expansion and contraction of polymer chains due to the temperature response of a temperature-responsive polymer can be used as a driving force for detaching the separated extracellular vesicles from the separation carrier, and that an extracellular vesicle release agent capable of binding to the separation carrier is useful. Furthermore, they have found that the above-mentioned problems can be solved by optimizing the method for purifying extracellular vesicles using the above-mentioned extracellular vesicle release agent.
• an agent for releasing extracellular vesicles comprising a polymer having a number average molecular weight of 20,000 to 1,000,000 and further having a separation carrier binding site at an end thereof, said polymer comprising a structural unit A represented by the following formula (1) and a structural unit B represented by the following formula (2) in a molar ratio of 0.8 ⁇ A/(A+B):
• R 1 represents a hydrogen atom or a methyl group.
• R 2 represents a hydrogen atom or a methyl group.
• a method for purifying extracellular vesicles comprising the following steps A to E in this order: [Step A] A step of binding a substance for recognizing extracellular vesicles to be separated, which has a binding site to a separation carrier, to obtain a separation carrier for recognizing extracellular vesicles to be separated [Step B] A step of adding a separation carrier that recognizes extracellular vesicles to be separated to a crude solution containing the extracellular vesicles to be separated and reacting the carrier with the extracellular vesicles to be separated to obtain a complex of the extracellular vesicles to be separated and the separation carrier [Step C] Step D: Separating only the separated extracellular vesicles-separation carrier complex from the crude solution A step of adding the extracellular vesicle release agent according to [1] or [2] to the extracellular vesicle-separation carrier complex to be separated, reacting the mixture, and then stirring the mixture to release the extracellular ve
• a step of recovering the separation carrier and extracting the separated extracellular vesicles [4] The method for purifying extracellular vesicles according to [3], wherein the separated extracellular vesicle recognition substance is an antibody, an antibody fragment, a peptide, an aptamer or a charged polymer. [5] The method for purifying extracellular vesicles described in [3], wherein the separated extracellular vesicle recognition substance is an antibody. [6] The method for purifying extracellular vesicles described in any one of [3] to [5], wherein the separation carrier is a magnetic particle. [7] The method for purifying extracellular vesicles described in any one of [3] to [6], characterized in that the stirring is performed by pipetting. [8] The method for purifying extracellular vesicles described in any one of [3] to [6], characterized in that the stirring is performed by pipetting 6 to 20 times.
• the present invention provides an extracellular vesicle release agent capable of recovering specific extracellular vesicles without damage from a crude solution containing extracellular vesicles, and an extracellular vesicle purification method using the agent.
• the extracellular vesicle release agent includes a polymer containing a structural unit A represented by the following formula (1) and a structural unit B represented by the following formula (2), and the polymer has a separation carrier binding site at its end.
• R 1 represents a hydrogen atom or a methyl group.
• R 2 represents a hydrogen atom or a methyl group.
• structural unit refers to not only continuous repetition of the structural unit, but also intermittent repetition.
• the upper critical solution temperature (UCST) is the boundary temperature between the temperature at which the polymer is insolubilized in water or a medium to form an insoluble phase, and the temperature at which the polymer is dissolved in water or a medium to form a dissolved phase.
• the upper critical solution temperature (UCST) is not particularly limited, but since it must be a temperature suitable for handling extracellular vesicles, it is preferably in the range of 2 to 60°C, more preferably 4 to 50°C, and even more preferably 4 to 40°C.
• the temperature is 60°C or lower, there is no risk of damage to the extracellular vesicles, and if the temperature is 4°C or higher, there is no risk of the solution containing the extracellular vesicle release agent freezing, causing freezing damage to the extracellular vesicles.
• the upper critical solution temperature can be determined by dissolving the polymer in water or a culture medium at a concentration of at least 0.01 mg/mL, measuring the transmittance of visible light at 500 nm in a quartz cell while lowering the temperature, and assuming that the transmittance is 100% when the polymer is completely dissolved, as the temperature is lowered, as the temperature at which the transmittance begins to decrease.
• the molar ratio of the structural unit A represented by the above formula (1) and the structural unit B represented by the above formula (2) can be appropriately determined so as to exhibit the required function, but in order to set the upper critical solution temperature of the above polymer in the range of 2 to 60°C, it is preferable that the molar ratio of the above structural unit A and the above structural unit B is in the range of 0.3 ⁇ A/(A+B).
• the relationship is in the range of 0.4 ⁇ A/(A+B), even more preferably, the relationship is in the range of 0.5 ⁇ A/(A+B), even more preferably, the relationship is in the range of 0.6 ⁇ A/(A+B), particularly preferably, the relationship is in the range of 0.7 ⁇ A/(A+B), and particularly preferably, the relationship is in the range of 0.8 ⁇ A/(A+B).
• the polymer may be a copolymer with other monomers in addition to the monomer from which the structural unit A represented by the above formula (1) is derived and the monomer from which the structural unit B represented by the above formula (2) is derived, and the upper critical solution temperature (UCST) can be adjusted depending on the type, molecular weight combination, etc. of the other monomers.
• UST upper critical solution temperature
• the polymer may be a copolymer with the other monomers listed below.
• a hydrophilic monomer for example, glycerol (meth)acrylate, (meth)acryloyloxyethyl phosphate, N-methyl carboxybetaine (meth)acrylate, N-methyl sulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, N,N'-dimethylacrylamide, S-methylsulfonium carboxylic acid (meth)acrylate, ethylene glycol (meth)acrylate, etc.
• (meth)acryloyloxyethyl phosphate N-methyl carboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, S-methylsulfonium carboxylic acid (meth)acrylate, or ethylene glycol (meth)acrylate.
• the molecular weight of the polymer can be appropriately determined by adjusting the polymerization conditions etc. so that the required performance can be achieved, but is usually about 1,000 to 5,000,000 in number average molecular weight, and in order to increase the efficiency of separation of extracellular vesicles by the extracellular vesicle release agent of the present invention, a molecular weight of 10,000 to 2,000,000 is preferred, and 20,000 to 1,000,000 is more preferred.
• the molecular weight of the polymer is 5,000,000 or less, the solubility of the polymer in water is increased, making it easier to use as an extracellular vesicle release agent, which is preferable.
• the separation carrier binding site is a site capable of binding to a separation carrier, and is not particularly limited as long as it is one that is normally used in this field, but examples include hydrophobic substituents, biotin residues, desthiobiotin residues, avidin residues, streptavidin residues, amino groups, carboxyl groups, mercapto groups, glutathione residues, glutathione transferase residues, lectin residues, polysaccharide residues such as cyclodextrin residues, adamantyl groups, phenylboronic acid residues, and the like, preferably biotin residues, streptavidin residues, amino groups, carboxyl groups, mercapto groups, and the like.
• the content of the separation carrier binding site is not particularly limited as long as the polymer binds to the separation carrier, but one molecule of the polymer may contain 0.1 to 2 molecules of the separation carrier binding site, preferably 0.2 to 1.5 molecules, and more preferably 0.5 to 1 molecule. If there are 2 molecules or less, cross-linking between separation carriers is less likely to occur, and if there are 0.1 molecules or more, the polymer is more likely to bind to the separation carrier, which is more preferable.
• the portion of the polymer other than the separation carrier binding site can be produced, for example, by radical polymerization of 2-ureidoethyl methacrylate represented by the following formula (4) and 2-methacryloyloxyethyl phosphorylcholine represented by the following formula (5).
• the radical polymerization of the above 2-ureidoethyl methacrylate and the above 2-methacryloyloxyethyl phosphorylcholine also allows copolymerization with other monomers.
• the radical polymerization of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers) can be carried out as a bulk polymerization, but a solvent can also be added to carry out the polymerization.
• the solvent is not particularly limited as long as it dissolves the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers), and any common solvent can be used.
• the solvent is selected from polar aprotic solvents such as acetone, dioxane, N,N-dimethylformamide, dimethylsulfoxide, and tetrahydrofuran, polar protic solvents such as methanol, ethanol, and water, and mixtures thereof.
• polar aprotic solvents such as acetone, dioxane, N,N-dimethylformamide, dimethylsulfoxide, and tetrahydrofuran
• polar protic solvents such as methanol, ethanol, and water, and mixtures thereof.
• the radical polymerization of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers) can be carried out by thermal polymerization or photopolymerization.
• the thermal polymerization can be carried out, for example, using a thermal polymerization initiator.
• thermal polymerization initiator examples include peroxide radical initiators (benzoyl peroxide, ammonium persulfate, etc.), azo radical initiators (azobisisobutyronitrile (AIBN), 2,2'-azobis-dimethylvaleronitrile (ADVN), etc.), 2,2'-azobiscyanovaleric acid (ACVA), azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), and water-soluble or oil-soluble redox radical initiators (made of dimethylaniline and benzoyl peroxide).
• peroxide radical initiators benzoyl peroxide, ammonium persulfate, etc.
• azo radical initiators azobisisobutyronitrile (AIBN), 2,2'-azobis-dimethylvaleronitrile (ADVN), etc.
• ACVA 2,2'-azobiscyanovaleric acid
• VA-044 azobis[2-(2-imida
• the amount of radical initiator used is usually preferably 0.01 to 10 parts by mass per 100 parts by mass of the total of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers).
• the polymerization temperature and polymerization time can be appropriately selected and determined depending on the type of radical initiator and the presence or absence and type of other monomers. For example, when radical polymerization of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers) is carried out using ACVA, the polymerization temperature is preferably 40 to 90°C and the polymerization time is preferably about 2 to 48 hours.
• the photopolymerization can be carried out, for example, by irradiation with ultraviolet light (UV) at a wavelength of 254 nm or with an electron beam (EB) at an acceleration voltage of 150 to 300 kV.
• UV ultraviolet light
• EB electron beam
• the use of a photopolymerization initiator is optional, but it is preferable to use one in terms of reaction time.
• photopolymerization initiators include 2-hydroxy-2-methyl-1-phenyl-1-propanone and 1-hydroxy-cyclohexyl phenyl ketone, with 2-hydroxy-2-methyl-1-phenyl-1-propanone being preferred in terms of solubility, etc.
• a chain transfer agent can also be used.
• the chain transfer agent include 2-mercaptoethanol, 1-mercapto-2-propanol, 3-mercapto-1-propanol, p-mercaptophenol, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, and 2-mercaptonicotinic acid.
• the radical polymerization of the above 2-ureidoethyl methacrylate and the above 2-methacryloyloxyethyl phosphorylcholine (and the above other monomers) can also be carried out by a living radical polymerization method, specifically, atom transfer radical polymerization method (ATRP method), reversible addition-fragmentation chain transfer polymerization method (RAFT polymerization method), polymerization method via nitroxide (NMP method), etc. can be used.
• ATRP method atom transfer radical polymerization method
• RAFT polymerization method reversible addition-fragmentation chain transfer polymerization method
• NMP method nitroxide
• RAFT polymerization method reversible addition-fragmentation chain transfer polymerization method
• RAFT polymerization method is preferred because it does not use metals and does not reduce enzyme activity.
• a publicly known method can be used as the RAFT polymerization method, and for example, the methods described in WO99/31144, WO98/01478, and US Patent No. 6,153,705 are effective.
• radical polymerization of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers) is carried out using the RAFT polymerization method, it can be carried out by adding a RAFT agent to normal radical polymerization.
• RAFT agent examples include, in addition to the biotin group-containing RAFT agent represented by the following formula (6), 4-cyanopentanoic acid dithiobenzoate, 2-cyano-2-propyl benzodithioate, benzyl benzodithioate, 2-phenyl-2-propyl benzodithioate, methyl 2-phenyl-2-(phenyl-carbonothioylthio)acetate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecyl sulfuryl ester, etc.) 4-Cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, 2-cyano-2-propyld
• RAFT agents 4-cyanopentanoic acid dithiobenzoate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, or 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester are preferred.
• the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine can form random copolymers or block copolymers with the other monomers, but a random copolymer is preferred to bring out the performance of the extracellular vesicle release agent.
• Methods for incorporating the separation carrier binding site into the end of the portion of the polymer other than the separation carrier binding site include addition, click reaction, amidation, esterification, thioesterification, carbonation, Schiff base formation reaction, reductive amination, radical-mediated coupling reaction, Diels-Alder reaction, disulfidation, Michael addition reaction, ene-thiol reaction, aromatic boron compound-mediated coupling reaction, tertiary sulfonium formation reaction, quaternary ammonium formation reaction, etc., but click reaction, amidation, or esterification are preferred due to their high versatility.
• the compound for constituting the separation carrier binding site is not particularly limited, and examples thereof include the compound represented by formula (6) described below.
• the polymer having the above-mentioned separation carrier binding site at the end can also be produced in one step by using a living radical polymerization method.
• a living radical polymerization method Specifically, atom transfer radical polymerization (ATRP method), reversible addition-fragmentation chain transfer polymerization (RAFT polymerization method), and polymerization via nitroxide (NMP method) can be used.
• RAFT polymerization method reversible addition-fragmentation chain transfer polymerization
• a publicly known method can be used as the RAFT polymerization method, and for example, the methods described in WO99/31144, WO98/01478, and US Patent No. 6,153,705 are effective.
• the RAFT polymerization method to carry out radical polymerization of the 2-ureidoethyl methacrylate and the 2-methacryloyloxyethyl phosphorylcholine (and the other monomers) and simultaneously introduce the separation carrier binding site, this can be carried out by adding a compound for constituting the separation carrier binding site and a RAFT agent similar to those described above to normal radical polymerization.
• the RAFT agent also serves as a compound for forming a binding site on a separation carrier similar to that described above.
• the polymer may be selected from the group consisting of 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester (N3-DTPA), benzyl 1H-pyrrole-1-carbodithioate, 2-
• biotin represented by the following formula (6) is preferred.
• Group-containing RAFT agents 4-cyanopentanoic acid dithiobenzoate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol or 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester are preferred.
• the extracellular vesicle release agent in the present invention is a material for releasing the extracellular vesicles to be separated from the separation carrier, and contains at least the above-mentioned polymer.
• the agent usually contains water or a medium, and may contain additives such as a chelating agent or a protein for more efficient separation of extracellular vesicles.
• the water is preferably purified water, pure water, ion-exchanged water, etc., or may be various buffer solutions containing water.
• any buffer solution normally used in this field may be used, so long as it does not destroy the enzyme activity, antigenicity, and other physiological activities of the protein. Examples include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, etc., and these may be used in combination.
• the above medium is not particularly limited as long as it is suitable for the extracellular vesicles to be isolated, and general cell culture media can be used.
• Examples include Dulbecco's modified Eagle's MEM medium (DMEM), ⁇ -MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, and GMEM medium. These may be mixed, and supplements such as fetal bovine serum (FBS), glutamine, bovine serum albumin, human serum albumin, and even antibiotics may be added as necessary.
• the above-mentioned additives include other reagents that are typically used in this field for the purpose of improving the efficiency of separation of extracellular vesicles, such as chelating agents, sugars, polysaccharides, proteins, salts, vitamins, surfactants, etc.
• chelating agents examples include ethylenediaminetetraacetic acid (EDTA), 1,2-bis(O-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid (BAPTA), bicine, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid (DTPA), iminodiacetic acid, and triethylenetetraminehexaacetic acid.
• EDTA ethylenediaminetetraacetic acid
• BAPTA 1,2-bis(O-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid
• CyDTA trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid
• DTPA diethylenetriaminepentaacetic acid
• iminodiacetic acid and triethylenet
• sugars refer to monosaccharides, disaccharides, oligosaccharides, etc.
• monosaccharides include allose, glucose (grape sugar), altrose, mannose, galactose, psicose, fructose (fruit sugar), sorbose, ribose, xylose, arabinose, etc.
• disaccharides include xylulose, trehalose, isotrehalose, maltose (malt sugar), cellobiose, isomaltose, etc.
• oligosaccharides include fructooligosaccharides, galactooligosaccharides, lactose oligosaccharides, deoxyribose, fucose, rhamnose, glucuronic acid, galacturonic acid, glucosamine, galactosamine xylitol, sorbitol, ascorbic acid (vitamin C), glucuronolactone,
• polysaccharides examples include chitosan, dextran, cyclodextrin, hyaluronic acid, carboxymethylcellulose, hydroxypropylcellulose, hydroxyethylstarch, starch, and pullulan.
• proteins examples include fetal bovine serum (FBS), antifreeze peptides, bovine serum albumin, collagen, gelatin, casein, etc.
• FBS fetal bovine serum
• antifreeze peptides examples include bovine serum albumin, collagen, gelatin, casein, etc.
• the above salts include, for example, amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, and histidine; peptides such as glycylglycine; inorganic salts such as phosphates, borates, sulfates, and tris salts; flavins; organic acids and salts of organic acids such as acetic acid, citric acid, malic acid, maleic acid, and gluconic acid.
• amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, and histidine
• peptides such as glycylglycine
• inorganic salts such as phosphates, borates, sulfates, and tris salts
• vitamins include, for example, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin-like substances, etc.
• surfactants examples include Triton, polyoxyethylene alkyl ether, Tween 20, and Pluronic.
• the additives contained in the extracellular vesicle release agent of the present invention are not particularly limited as long as they can be used to separate extracellular vesicles, but are preferably 0.01% by mass or more, and more preferably 0.05% by mass or more, relative to the extracellular vesicle release agent.
• the shape of the separation carrier may be, for example, plate-like, particulate, or fibrous, or may be porous with holes, grooves, or protrusions provided in a plate-like or particulate base material.
• plate-like, particulate, fibrous, or magnetic particulate are preferred for ease of handling during separation of extracellular vesicles, with magnetic particles being particularly preferred.
• the separation carrier is a magnetic particle
• the average particle size is 10 nm or more and less than 5000 nm, and in order to improve the recognition of the target object, it is particularly preferable that the average particle size is 20 nm or more and less than 3000 nm.
• materials for magnetic particles include magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet, and hematite.
• the combination of the surface of the separation carrier and the binding site of the separation carrier is not particularly limited as long as binding is possible, and examples include a combination of streptavidin residues and biotin residues, a combination of streptavidin residues and desthiobiotin residues, a combination of a tosyl group and an amino group, a combination of an epoxy group and an amino group, a combination of a carboxyl group and an amino group, a combination of a cyclodextrin residue and an adamantyl group, and a combination of a polyhydric alcohol residue and a phenylboronic acid residue.
• a combination of streptavidin residues and biotin residues, a combination of streptavidin residues and desthiobiotin residues, a combination of a tosyl group and an amino group, a combination of an epoxy group and an amino group, or a combination of a carboxyl group and an amino group is preferred, and a combination of streptavidin residues and biotin residues, a combination of streptavidin residues and desthiobiotin residues, or a combination of a tosyl group and an amino group is most preferred.
• examples of the above-mentioned cells include established cells for culture, fertilized eggs and egg cells of animals including humans.
• Other examples include stem cells such as sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and umbilical cord blood cells, as well as animal cells including humans or plant cells such as liver cells, nerve cells, cardiac muscle cells, vascular endothelial cells, vascular smooth muscle cells, and blood cells.
• examples of the state of the cells include a suspension state, a planar culture state, as well as a sheet state, a spheroid, an organoid, and organs with higher-order structures.
• ⁇ Extracellular vesicle purification method> There are two known methods for purifying extracellular vesicles using a separation carrier: the direct method and the indirect method.
• the direct method a substance that recognizes the extracellular vesicles to be separated and has a separation carrier binding site is bound to the separation carrier, and then
• the indirect method the separated extracellular vesicles are reacted with the separated extracellular vesicles in a crude solution containing the separated extracellular vesicles (hereinafter simply referred to as the "crude solution").
• the direct method is recommended for the separation of extracellular vesicles. More preferred.
• Separation of extracellular vesicles to be separated from a crude solution containing the extracellular vesicles to be separated by using the extracellular vesicle release agent of the present invention can be carried out by an extracellular vesicle purification method comprising the following steps [Step A] to [Step F] in this order.
• Step A is a step in which a substance that recognizes extracellular vesicles to be separated, which has a binding site for the separation carrier, is bound to the separation carrier to obtain a separation carrier that recognizes extracellular vesicles to be separated.
• the above-mentioned separated extracellular vesicles refer to a group of extracellular vesicles obtained as a result of separating extracellular vesicles using the extracellular vesicle release agent of the present invention, and may be a single type or a group consisting of multiple types of extracellular vesicles.
• the above-mentioned separated extracellular vesicle recognition substance is a substance that exhibits specific affinity for antigens or membrane components expressed inside or outside the extracellular vesicles, and examples of such substances include any antibody, antibody derivative, fragmented antibody, or fragmented antibody derivative against an antigen, peptide, cell adhesion receptor molecule, receptor for costimulatory molecule, charged polymer, nucleic acid, artificially engineered binding molecule, protein A, lectin, aptamer, etc.
• the reaction between the separated extracellular vesicle recognition substance having the above separation carrier binding site and the above separation carrier is not particularly limited as long as it does not impair the activity of the separated extracellular vesicle recognition substance, but the reaction temperature is generally in the range of 2°C to 35°C, preferably in the range of 2°C to 30°C, and more preferably in the range of 5°C to 25°C.
• the reaction time is also not particularly limited, but is generally in the range of 10 minutes to 2 hours, preferably in the range of 10 minutes to 1 hour, and more preferably in the range of 10 minutes to 30 minutes.
• a separation carrier that recognizes extracellular vesicles to be separated can be obtained.
• a specific example of the substance that recognizes extracellular vesicles to be separated and has a binding site to the separation carrier in [Step A] is a biotinylated antibody, and a specific example of the separation carrier is streptavidin magnetic particles.
• a specific example of [Step A] is the step of reacting a biotinylated antibody with streptavidin magnetic particles to obtain antibody-modified magnetic particles.
• Step B is a step in which a separation carrier that recognizes extracellular vesicles to be separated is added to a crude solution containing the extracellular vesicles to be separated, and reacted with the extracellular vesicles to obtain a complex of the extracellular vesicles to be separated and the separation carrier.
• the above-mentioned crude solutions include cell culture supernatant, blood, plasma, serum, saliva, urine, tears, sweat, breast milk, amniotic fluid, cerebrospinal fluid (spinal fluid), bone marrow fluid, pleural fluid, ascites, synovial fluid, aqueous humor, vitreous fluid, etc. From the viewpoint of ease of collection, cell culture supernatant, blood, plasma, serum, saliva, urine, etc. are preferably used.
• the reaction between the above-mentioned extracellular vesicles-recognizing separation carrier and the above-mentioned extracellular vesicles to be separated is not particularly limited as long as it does not adversely affect the extracellular vesicles to be separated, but the reaction temperature is generally in the range of 2°C to 35°C, preferably in the range of 2°C to 30°C, and more preferably in the range of 5°C to 25°C.
• the reaction time is also not particularly limited, but is generally in the range of 10 minutes to 2 hours, preferably in the range of 10 minutes to 1 hour, and more preferably in the range of 10 minutes to 30 minutes.
• the reaction between the above-mentioned separated extracellular vesicle-recognizing separation carrier and the above-mentioned separated extracellular vesicles can be carried out, for example, in a buffer for extracellular vesicle purification.
• a buffer for extracellular vesicle purification There are no particular limitations as long as it does not adversely affect the separated extracellular vesicles, but buffers and media usually used in this field can be used.
• buffers and media usually used in this field can be used.
• phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, etc. can be mentioned, and these may be mixed and used.
• Phosphate buffer is preferable.
• DMEM Dulbecco's modified Eagle's MEM medium
• ⁇ -MEM Roswell Park Memorial Institute
• RPMI Roswell Park Memorial Institute
• F12 medium F12 medium
• TC199 medium F12 medium
• GMEM medium etc.
• supplements such as fetal bovine serum (FBS), glutamine, and antibiotics may be added as necessary, but serum-free media are preferable in order not to impair the reactivity of the separated extracellular vesicle-recognizing substance.
• FBS fetal bovine serum
• glutamine glutamine
• antibiotics antibiotics
• various chelating agents can be used to prevent aggregation of extracellular vesicles.
• the chelating agent examples include the same ones as those exemplified as the chelating agent as an additive in the extracellular vesicle release agent, but ethylenediaminetetraacetic acid (EDTA) is preferred.
• the concentration of the chelating agent in the buffer for extracellular vesicle purification is in the range of 0.1 mM to 20 mM, preferably in the range of 0.5 mM to 10 mM, and more preferably in the range of 1 mM to 5 mM.
• the concentration of the chelating agent is 0.1 mM or more, the effect of inhibiting aggregation of extracellular vesicles is more likely to be exhibited, while when the concentration is 20 mM or less, damage to the extracellular vesicles is less likely to occur.
• polysaccharides, proteins, surfactants, etc. may be added to promote the reaction between the above-mentioned extracellular vesicles recognition and separation carrier and the above-mentioned extracellular vesicles to be separated.
• polysaccharides include those listed as examples of polysaccharides as additives in the above-mentioned extracellular vesicles release agent
• examples of the above-mentioned proteins include those listed as examples of proteins as additives in the above-mentioned extracellular vesicles release agent
• examples of the above-mentioned surfactants include those listed as examples of surfactants as additives in the above-mentioned extracellular vesicles release agent, but dextran, bovine serum albumin, or Tween 20 is preferred, and bovine serum albumin is more preferred.
• the polysaccharides, proteins, surfactants, etc. in the extracellular vesicles purification buffer are not particularly limited as long as they can be used for extracellular vesicles purification, but are preferably 0.01% by mass or more, and more preferably 0.05% by mass or more. When the polysaccharides, proteins, surfactants, etc. in the extracellular vesicles purification buffer are within the above-mentioned ranges, the above-mentioned additives are more likely to exert their full effect.
• [Step B] By carrying out the above [Step B], a complex of separated extracellular vesicles and separation carrier can be obtained.
• a specific example of [Step B] is the reaction between antibody-modified magnetic particles and separated extracellular vesicles.
• Step C is a step in which only the separated extracellular vesicles-separation carrier complexes are separated from the crude solution.
• the method for separating only the above-mentioned extracellular vesicles-separation carrier complexes from the crude solution containing the extracellular vesicles to be separated varies depending on the separation carrier.
• the separation carrier is a magnetic particle
• a method of collecting the complexes by magnetic force is preferable.
• the separation carrier is a particle such as latex particles, precipitation due to time changes or sedimentation by centrifugation is possible.
• the separation carrier is a flat plate, it is sufficient to simply remove the complexes from the above-mentioned extracellular vesicle purification buffer.
• the washing method As long as it does not adversely affect the separated extracellular vesicles.
• the above-mentioned extracellular vesicle purification buffer can be used.
• the separated extracellular vesicles-separation carrier complex can be separated.
• a specific example of [Step C] is a step of removing the separated extracellular vesicles-separation carrier complex from a crude solution containing the separated extracellular vesicles by magnetic force, centrifugation, or the like.
• Step D is a step in which the above-mentioned extracellular vesicle release agent is added to the separated extracellular vesicles-separation carrier complex, reacted, and then stirred to release the separated extracellular vesicles from the separated extracellular vesicles-separation carrier complex.
• the extracellular vesicle release agent can be added directly to the extracellular vesicle-separation carrier complex to be separated, or the extracellular vesicle release agent can be mixed with the extracellular vesicle purification buffer and added as an extracellular vesicle release agent solution.
• reaction time There are no particular limitations on the reaction time, but it is generally in the range of 10 minutes to 2 hours, preferably in the range of 10 minutes to 1 hour, and more preferably in the range of 10 minutes to 30 minutes.
• the above stirring method is not particularly limited as long as it does not adversely affect the extracellular vesicles.
• stirring using a vortex, pipetting, inversion mixing, or a sample mixer is preferred, and pipetting is even more preferred from the viewpoint of less damage to the extracellular vesicles.
• the above-mentioned pipetting is an operation of aspirating and discharging a liquid containing the complex of the extracellular vesicles to be separated and the separation carrier using a measuring device such as a pipetteman, syringe, or electric pipetter.
• the number of times is not particularly limited as long as it is a method that does not adversely affect the extracellular vesicles, but for example, a range of 6 to 50 times is preferable, and a range of 6 to 20 times is more preferable. If it is 50 times or less, there is less risk of damage such as rupture or crushing of the extracellular vesicles, and if it is more than 5 times, the separated extracellular vesicles are more likely to detach from the separation carrier.
• the temperature when the extracellular vesicle release agent is added to the extracellular vesicle-separation carrier complex to be separated is preferably equal to or lower than the upper critical solution temperature (UCST) of the polymer, which is a component of the extracellular vesicle release agent used, more preferably in the range of 2°C to the upper critical solution temperature (UCST) of the polymer, and even more preferably in the range of 5°C to the upper critical solution temperature (UCST) of the polymer as a temperature that does not adversely affect the extracellular vesicles. Furthermore, it is preferable to change the temperature of the liquid obtained by the reaction to a temperature equal to or higher than the upper critical solution temperature (UCST) of the polymer before stirring.
• UST upper critical solution temperature
• changing the temperature means heating from a temperature equal to or lower than the upper critical solution temperature (UCST) of the polymer, which is a component of the extracellular vesicle release agent, to a temperature equal to or higher than the upper critical solution temperature (UCST).
• the temperature after heating is equal to or higher than the temperature at which the extracellular vesicle release agent is added in the reaction and equal to or lower than 60°C, preferably equal to or higher than the temperature at which the extracellular vesicle release agent is added in the reaction and equal to or lower than 50°C, more preferably equal to or higher than the temperature at which the extracellular vesicle release agent is added in the reaction and equal to or lower than 45°C.
• the temperature of the liquid obtained in the reaction is changed to a temperature equal to or higher than the upper critical solution temperature (UCST) of the polymer, it is preferable to further react the obtained liquid before stirring.
• the reaction time before stirring is not particularly limited, but is generally in the range of 10 minutes to 2 hours, preferably in the range of 10 minutes to 1 hour, and more preferably in the range of 10 minutes to 30 minutes.
• the separated extracellular vesicles can be separated from the separated extracellular vesicles-separation carrier complex.
• a specific example of [Step D] is a step in which the separated extracellular vesicles-separation carrier complex is reacted with an extracellular vesicle release agent, and then the mixture is stirred to sever the interaction between the separation carrier and the separated extracellular vesicles, thereby releasing the separated extracellular vesicles.
• Step E is a step of recovering the separation carrier and extracting the separated extracellular vesicles.
• the method of recovering the separation carrier and extracting the separated extracellular vesicles varies depending on the separation carrier.
• the separation carrier is a magnetic particle
• a method of collecting them by magnetic force is common.
• the separation carrier is a particle such as latex particle, sedimentation due to time change or sedimentation by centrifugation is possible.
• the separation carrier is a flat plate, it is sufficient to simply remove them from the extracellular vesicle purification buffer.
• the washing method As long as it does not adversely affect the separated extracellular vesicles, but for example, the above-mentioned extracellular vesicle purification buffer can be used.
• the separation carrier can be recovered and the separated extracellular vesicles can be extracted.
• a specific example of [Step E] is a process in which the separation carrier is collected by magnetic force or centrifugation, the separation carrier is recovered, and the separated extracellular vesicles are extracted.
• the number average molecular weight of the obtained polymer A was determined by gel permeation chromatography using a multi-angle light scattering detector (MALS) manufactured by WYATT.
• MALS multi-angle light scattering detector
• the pump used was an LC-720AD manufactured by Shimadzu Corporation
• the detector (differential refractometer) was an RID10A manufactured by Shimadzu Corporation
• the detector (UV) was an SPD-20A.
• the column used was a combination of two columns, a TSKGel G3000PWXL and a TSKGel G5000PWXL (column size 4.6 mm x 25 cm) manufactured by Tosoh Corporation.
• the developing solvent was distilled water/acetonitrile (5/5 v/v) with 100 mM sodium nitrate dissolved therein.
• the measurement conditions were a flow rate of 0.6 ml/min, a column temperature of 40°C, a sample concentration of 2 mg/mL, and an injection volume of 100 ⁇ L.
• Gel permeation chromatography showed that the number average molecular weight of polymer A was 309,000.
• the upper critical solution temperature (UCST) of the obtained polymer A was measured using an ultraviolet-visible spectrophotometer.
• Polymer A was dissolved in a buffer for extracellular vesicle purification (phosphate-buffered saline (PBS) + 0.1% bovine serum albumin (BSA) + 2 mM EDTA) to a concentration of 2.5 mg/mL.
• PBS phosphate-buffered saline
• BSA bovine serum albumin
• the transmittance (%) of the solution was calculated using the following formula.
• the upper critical solution temperature (UCST) of polymer A was 20°C.
• Preparation Example 1 Preparation of extracellular vesicle release agent solution A
• Polymer A was dissolved in an extracellular vesicle purification buffer (phosphate buffered saline (PBS) + 0.1% bovine serum albumin (BSA) + 2 mM EDTA) to a concentration of 0.8 mg/mL to prepare an extracellular vesicle release agent solution A.
• PBS phosphate buffered saline
• BSA bovine serum albumin
• 2 mM EDTA 2 mM EDTA
• Example 1-1> (Preparation of separation carrier for recognizing extracellular vesicles to be separated) Dynabeads (registered trademark) M-280 Streptavidin (0.5 mg) manufactured by Thermo Fisher was used as a separation carrier, and biotinylated CD9 antibody (5 ⁇ g) manufactured by GeneTex was used as a substance that recognizes extracellular vesicles to be separated. The mixture was reacted at room temperature for 20 minutes to obtain CD9 antibody-carrying magnetic particles as a separation carrier that recognizes extracellular vesicles to be separated.
• Human serum from Merck was centrifuged at 10,000 ⁇ g for 10 minutes at 4° C.
• the human serum supernatant was diluted 5-fold with PBS( ⁇ ) to prepare a human serum solution.
• the number of extracellular vesicles in the human serum solution and the measurement sample was analyzed using a Malvern Nanosite LM10.
• the number of extracellular vesicles was calculated by counting the number of particles with a particle diameter of 50 to 150 nm, and the number of extracellular vesicles in the human serum solution before the separation operation was set to 1.
• the separated extracellular vesicles were observed under an electron microscope using a JEOL transmission microscope after fixation with glutaraldehyde. Evaluation using an electron microscope was performed by observing the shape of the extracellular vesicles, and an analysis was performed to determine whether they were close to spherical and whether they were damaged.
• the analysis results of the extracellular vesicles are shown in Table 1.
• Example 1-2 The extracellular vesicle purification test was carried out in the same manner as in Example 1-1, except that pipetting was performed five times in the extracellular vesicle purification test from human serum solution. The analysis results of the extracellular vesicles are shown in Table 1.
• Example 1-3 The extracellular vesicle purification test was carried out in the same manner as in Example 1-1, except that pipetting was performed 50 times in the extracellular vesicle purification test from human serum solution. The analysis results of the extracellular vesicles are shown in Table 1.
• Example 1-1 An extracellular vesicle purification test was carried out in the same manner as in Example 1-1, except that no extracellular vesicle release agent was used. The analysis results of the extracellular vesicles are shown in Table 1.
• Example 1-2 The extracellular vesicle purification test was performed in the same manner as in Example 1-1, except that pipetting was not performed in the extracellular vesicle purification test from human serum solution. The analysis results of the extracellular vesicles are shown in Table 1.
• Example 1-4 Pretreatment of human serum
• the human serum was pretreated in the same manner as in Example 1-1.
• the inventors have found that the expansion and contraction of polymer chains due to the temperature response of a temperature-responsive polymer can be used as a driving force for detaching the separated extracellular vesicles from the separation carrier, and that an extracellular vesicle release agent capable of binding to the separation carrier is useful. Furthermore, they have found that the above-mentioned problems can be solved by optimizing the extracellular vesicle purification method using the above-mentioned extracellular vesicle release agent.

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