WO2024185655A1 - Particules magnétiques pour purification de vésicules extracellulaires, leur procédé de production et procédé de purification de vésicules extracellulaires - Google Patents

Particules magnétiques pour purification de vésicules extracellulaires, leur procédé de production et procédé de purification de vésicules extracellulaires Download PDF

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WO2024185655A1
WO2024185655A1 PCT/JP2024/007608 JP2024007608W WO2024185655A1 WO 2024185655 A1 WO2024185655 A1 WO 2024185655A1 JP 2024007608 W JP2024007608 W JP 2024007608W WO 2024185655 A1 WO2024185655 A1 WO 2024185655A1
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extracellular vesicles
magnetic particles
particles
magnetic
metal oxide
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Japanese (ja)
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豪士 久野
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東ソー株式会社
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G49/00—Compounds of iron
    • C01G49/02—Oxides; Hydroxides
    • C01G49/08—Ferroso-ferric oxide [Fe3O4]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the present invention relates to magnetic particles used in the purification of extracellular vesicles, a method for producing the same, and a method for purifying extracellular vesicles.
  • Magnetic particles are used to recover biological substances such as antibodies and proteins from samples by utilizing their magnetic properties.
  • biological substances such as antibodies are bound to the surface of magnetic particles, they are sometimes reacted in an acidic aqueous solution, which can cause problems such as the detachment of the magnetic material and the elution of iron ions, resulting in the leakage of substances derived from the magnetic material components.
  • Extracellular vesicles are generally purified by centrifugal procedures such as ultracentrifugation or spin columns.
  • centrifugal procedures such as ultracentrifugation or spin columns.
  • magnetic particles with antibodies that can specifically recognize extracellular vesicles bound to their surface have been disclosed.
  • the mechanism by which these magnetic particles recognize extracellular vesicles is a specific antibody, it is not possible to recover all extracellular vesicles, and there is a problem that the extracellular vesicles are biased toward a specific population.
  • Patent Document 1 As a technology for adsorbing extracellular vesicles, a device in which zinc oxide protrusions are formed within a flow channel has been disclosed (Patent Document 1). However, this device requires the formation of protrusions within the flow channel, which poses issues with mass production of the device.
  • Magnetic particles in which a magnetic body is dispersed within a metal oxide particle have been disclosed for use in separating biological substances other than extracellular vesicles (Patent Document 2).
  • Patent Document 2 Magnetic particles in which a magnetic body is dispersed within a metal oxide particle have been disclosed for use in separating biological substances other than extracellular vesicles.
  • these magnetic particles have a problem in that the specific gravity of the particles is large because the magnetic particles are encapsulated within the metal oxide, and they tend to settle out easily, making them poorly dispersible in aqueous solvents.
  • a large amount of magnetic particles is required to recover biological substances because the specific surface area of the particles cannot be increased.
  • the present invention has been made in consideration of the above problems, and aims to provide magnetic particles that have excellent acid resistance, that cause little leaching of iron from the micromagnetic particles even when treated with an acidic aqueous solution, that have good dispersibility in aqueous solvents, that adsorb a large amount of extracellular vesicles per particle, and that have detachment and recovery properties.
  • a magnetic particle for purifying extracellular vesicles comprising a polymer core particle and a coating layer formed on the surface of the polymer core particle, the coating layer containing a fine magnetic material and a metal oxide.
  • the metal oxide is a single or multiple component selected from the group consisting of zinc oxide, titanium oxide, silicon dioxide, nickel oxide, yttria oxide, tin oxide, indium oxide and indium tin oxide.
  • [6] The magnetic particles for extracellular vesicle purification according to any one of [1] to [5], wherein the specific surface area of the magnetic particles is 2 to 100 m 2 /g.
  • the method according to [8] further comprising a pH adjustment step of adjusting the pH of the liquid containing the extracellular vesicles to make it an acidic liquid prior to the adsorption step.
  • the magnetic particles according to one embodiment of the present invention have excellent acid resistance, and even when treated with an acidic aqueous solution, there is little elution of iron from the micromagnetic particles. In addition, they have good dispersibility in aqueous solvents. In addition, the specific surface area of the magnetic particles is large, and they can carry a large amount of extracellular vesicles, making it possible to purify the carried extracellular vesicles by detaching and recovering them.
  • FIG. 1 is a schematic diagram showing the structure of a magnetic particle according to one embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the configuration of a magnetic particle according to another embodiment of the present invention.
  • 3 is a histogram showing the particle size distribution of the extracellular vesicles of Example 21.
  • A shows the particle size distribution of the extracellular vesicles before purification
  • B shows the particle size distribution of the extracellular vesicles in recovery solution 1
  • C shows the particle size distribution of the extracellular vesicles in recovery solution 2.
  • present embodiment provides a detailed description of an embodiment of the present invention (hereinafter, simply referred to as the "present embodiment").
  • present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content.
  • present invention can be implemented with appropriate modifications within the scope of its intent.
  • a numerical range indicated using “ ⁇ ” indicates a range that includes the numerical values before and after “ ⁇ ” as the minimum and maximum values, respectively. Furthermore, unless specifically stated otherwise, the units of the numerical values before and after “ ⁇ ” are the same.
  • the upper or lower limit of a numerical range of a certain stage may be replaced with the upper or lower limit of a numerical range of another stage.
  • the upper or lower limit of the numerical range may be replaced with a value shown in the examples.
  • the upper and lower limits described individually can be combined in any way.
  • the magnetic particles according to one embodiment of the present invention have a polymer core particle (hereinafter, also referred to as a "core particle") and a coating layer formed on the surface of the polymer core particle.
  • the coating layer contains a fine magnetic material and a metal oxide.
  • the magnetic particles are for extracellular vesicle purification.
  • FIG. 1 is a schematic diagram (cross-sectional view) of a magnetic particle according to one embodiment.
  • the magnetic particle 10 shown in FIG. 1 comprises a core particle 1 and a coating layer 4 that coats the surface of the core particle 1.
  • the coating layer 4 is a metal oxide layer that contains a micro-magnetic material 2 and a metal oxide 3.
  • the micro-magnetic material 2 is dispersed in the metal oxide layer.
  • FIG. 2 is a schematic diagram (cross-sectional view) of a magnetic particle according to another embodiment.
  • the coating layer 4 coats the surface of the core particle and includes a micro-magnetic material layer that contains a micro-magnetic material 2, and a metal oxide layer that coats the micro-magnetic material layer and contains a metal oxide 3.
  • the core particle used in the magnetic particles is the particle located at the very center of the magnetic particle.
  • the shape of the core particle can be selected as appropriate, and examples of the shape include a nearly perfect sphere, a non-spherical shape such as an ellipsoid, cube, cylinder, or polygonal prism, and particles with large specific surface area and irregularities such as a porous shape or a shape with protrusions. Particles with large specific surface area and irregularities such as a porous shape or a shape with protrusions are preferred because they are suitable for coating many fine magnetic bodies on the surface of the core particle.
  • the particle size of the core particles is preferably 0.1 ⁇ m or more, more preferably 0.5 ⁇ m or more, particularly preferably 1 ⁇ m or more or 1.5 ⁇ m or more, and most preferably 2 ⁇ m or more.
  • the particle size of the core particles is preferably 100 ⁇ m or less, more preferably 80 ⁇ m or less, 60 ⁇ m or less, 40 ⁇ m or less, 20 ⁇ m or less, or 10 ⁇ m or less, particularly preferably 5 ⁇ m or less, and most preferably 3 ⁇ m or less.
  • the particle size of the core particles may be 0.1 ⁇ m or more and 100 ⁇ m or less, 0.5 ⁇ m or more and 10 ⁇ m or less, 1.0 ⁇ m or more and 5.0 ⁇ m or less, or 2.0 ⁇ m or more and 3.0 ⁇ m or less.
  • the particle size of the core particles can be calculated by determining the mode diameter from the particle size distribution measured by dynamic light scattering.
  • the specific gravity of the magnetic particles can be reduced compared to core particles whose main component is a component with a high specific gravity (magnetic material or metal oxide), improving the dispersibility of the particles in the solvent.
  • the improved dispersibility makes it possible to disperse the particles uniformly with less stirring force, improving the reproducibility of extracellular vesicle purification.
  • the material of the core particles can be, for example, polymer particles containing styrene-based monomer units, acrylate-based monomer units, or methacrylate-based monomer units.
  • Styrenic monomers include styrene, ⁇ -methylstyrene, vinyltoluene, p-methylstyrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, 4-tert-butylstyrene, 3,4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4-chloro-3-methylstyrene, divinylbenzene, and sodium p-styrenesulfonate.
  • Acrylate-based monomers or methacrylate-based monomers include (cyclo)alkyl (meth)acrylates such as (meth)acrylate, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, and cyclohexyl (meth)acrylate; alkoxy (cyclo)alkyl (meth)acrylates such as 2-methoxyethyl (meth)acrylate and p-methoxycyclohexyl (meth)acrylate; p) Acrylates; polyvalent (meth)acrylates such as trimethylolpropane tri(meth)acrylate; vinyl esters such as vinyl acetate, vinyl propionate, and vinyl versatate; cyanoacrylates such as 2-cyanoethyl (meth)acrylate, 2-cyanopropyl (meth)acrylate,
  • the core particles may contain micromagnetic material.
  • a paramagnetic thin film coating layer may be provided between the core particles and the coating layer containing the micromagnetic material.
  • the thickness of the paramagnetic coating layer is preferably 0.001 to 1 ⁇ m, more preferably 0.001 to 0.1 ⁇ m, particularly preferably 0.001 to 0.01 ⁇ m, and most preferably 0.001 to 0.005 ⁇ m, since it is suitable for reducing residual magnetization in the core particles and suppressing aggregation between the core particles.
  • the thickness of the paramagnetic coating layer can be calculated by measuring the thickness of the layer for 10 or more particles in an image of the cross section of the core particle observed with a transmission electron microscope and calculating the average value.
  • the surface of the core particles may be coated with a substance having a positive or negative charge.
  • a substance having a positive or negative charge there are no particular limitations on the method for coating the surface of the core particles with a substance having a positive or negative charge, but examples include a method of forming a polymer layer on the surface of the core particles using a monomer having a charge on the side chain, and a method of coating the surface of the core particles with a charged polymer by layer-by-layer method.
  • the coating layer formed on the surface of the core particle contains a micromagnetic material and a metal oxide.
  • the micromagnetic material is a particle smaller in diameter than the core particle and has magnetic responsiveness.
  • the particle diameter of the micromagnetic material may be 1 ⁇ m or less, 0.80 ⁇ m or less, 0.60 ⁇ m or less, 0.40 ⁇ m or less, 0.30 ⁇ m or less, 0.20 ⁇ m or less, or 0.15 ⁇ m or less, and may be 0.001 ⁇ m or more, 0.005 ⁇ m or more, or 0.008 ⁇ m or more.
  • the particle diameter of the micromagnetic material is preferably 0.001 to 1 ⁇ m, more preferably 0.001 to 0.5 ⁇ m, particularly preferably 0.001 to 0.1 ⁇ m, and most preferably 0.001 to 0.05 ⁇ m.
  • the micromagnetic material is preferably a nano-sized magnetic material (nanomagnetic material).
  • the particle size of micromagnetic particles can be calculated by measuring the maximum particle diameter of 10 or more particles in an image observed with a transmission electron microscope and calculating the average value.
  • Examples of materials for micro-magnetic bodies include iron oxides such as magnetite.
  • the nano-magnetic particles may contain inorganic surface modifiers such as lipids and silane coupling agents on their surfaces.
  • silane coupling agent include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, tris
  • the micromagnetic material can be a commercially available product.
  • An example of a commercially available micromagnetic material is EMG607 (product name, manufactured by Ferrotec Corporation).
  • the micro-magnetic bodies 2 are dispersed in a metal oxide layer formed by a metal oxide 3 as shown in FIG. 1, or that the surface of the first coating layer formed by the micro-magnetic bodies 2 is completely coated with a metal oxide layer formed by a metal oxide 3 as a second coating layer as shown in FIG. 2.
  • the acid resistance of the magnetic particles can be increased.
  • the content of the micro-magnetic material is suitable for making magnetic particles with good magnetic response, so the content of the micro-magnetic material per 1 g of magnetic particles is preferably 0.1 g/g or more, more preferably 0.2 g/g or more, particularly preferably 0.5 g/g or more, and most preferably 0.7 g/g or more. Also, since it is suitable for increasing the dispersibility of the magnetic particles in aqueous solvents, the content of the micro-magnetic material per 1 g of magnetic particles is preferably 0.9 g/g or less, more preferably 0.8 g/g or less, particularly preferably 0.7 g/g or less, and most preferably 0.6 g/g or less.
  • the weight ratio of the micromagnetic material contained in the magnetic particles is preferably 5 to 70 wt%, more preferably 10 to 60 wt%, particularly preferably 15 to 50 wt%, and most preferably 20 to 40 wt%.
  • the content of the micromagnetic material can be measured by ICP (Inductively Coupled Plasma) emission analysis.
  • the metal oxide layer is formed by coating the surface of the core particle with a metal oxide.
  • metal oxides include silicon dioxide (silica), glass, zinc oxide, titanium oxide, nickel oxide, alumina oxide, yttria oxide, tin oxide, indium oxide, and indium tin oxide, as well as ceramics such as zirconia and hydroxyapatite.
  • silica is preferred for the purpose of increasing acid resistance.
  • zinc oxide, titanium oxide, nickel oxide, yttria oxide, tin oxide, indium oxide, and indium tin oxide are preferred because they are suitable for adsorbing extracellular vesicles, with zinc oxide, nickel oxide, and yttria oxide being even more preferred.
  • silica, indium oxide, and indium tin oxide are preferred because they are suitable for desorbing extracellular vesicles at a pH near neutral, where denaturation of extracellular vesicles is unlikely to occur.
  • tin oxide, indium oxide, and indium tin oxide are preferred because they increase electrical conductivity and are suitable for peeling off adsorbed extracellular vesicles by passing electricity through the particles.
  • silica and titanium oxide are preferred because they are suitable for adsorbing extracellular vesicles in blood and samples containing serum or plasma.
  • Tin oxide, indium oxide, and indium tin oxide are preferred because they are easy to remove phenol red contained in the culture medium and are suitable for recovering extracellular vesicles with high purity.
  • Silica is preferred because it is suitable for recovering extracellular vesicles with a high content of microRNA.
  • Indium oxide and indium tin oxide are preferred because they are suitable for recovering extracellular vesicles with a high content of tetraspanins such as CD63.
  • Titanium oxide is preferred because it is suitable for recovering extracellular vesicles with a high content of proteins.
  • the metal oxide layer may be surface-treated with a compound having one or a combination of structures selected from the group consisting of anionic functional groups, cationic functional groups, hydrophobic functional groups, and polyethylene glycol groups.
  • the above compounds are preferably provided on the surface of the metal oxide layer by electrostatic interactions, hydrophobic interactions, hydrogen bonds, coordinate bonds, or the like. From the viewpoint of stable dispersibility, it is preferable that the various functional groups possessed by the above compounds are provided on the surface of the metal oxide layer by forming bonds such as coordinate bonds.
  • the surface of the magnetic particles is more likely to be negatively charged, which further suppresses the adsorption of negatively charged proteins, genes, phenol red, and other impurities to the magnetic particles. In addition, it becomes easier to detach extracellular vesicles from the magnetic particles, further improving the detachment rate.
  • the anionic functional group carboxylic acid (carboxy group), sulfonic acid (sulfo group), or phosphoric acid (phosphate group) can be suitably used.
  • Methods for binding the anionic functional group to the surface of the metal oxide layer include a method of modifying the surface of the metal oxide layer with 3-trimethoxysilylpropyl succinic acid, 3-trimethoxysilylpropyl sulfonate, and 3-trimethoxysilylpropyl phosphonate compounds, and a method of reacting the hydroxyl group of the metal oxide with succinic anhydride to introduce a carboxylic acid (carboxy group).
  • the surface of the magnetic particles is easily positively charged, and it is possible to further suppress the adsorption of impurities such as positively charged proteins to the magnetic particles.
  • impurities such as positively charged proteins
  • an amino group unsubstituted amino group or substituted amino group
  • a guanidinium group, a pyridinium group, or an imidazolium group can be suitably used.
  • a method for bonding the cationic functional group to the surface of the metal oxide layer there is a method of modifying the surface of the metal oxide layer with (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-trimethoxysilylpropyl)benzyldimethylammonium chloride, (3-glycidoxypropyl)trimethoxysilane, [(2-aminoethyl)aminoethyl]triethoxysilane, and 1-imidazolylpropyltriethoxysilane, etc.
  • the surface of the magnetic particles becomes hydrophobic and easily interacts with the hydrophobic portion of the lipids that make up the extracellular vesicles, further improving the adsorption rate of the extracellular vesicles.
  • the hydrophobic functional group alkyl groups having 1 to 20 carbon atoms such as methyl groups, ethyl groups, propyl groups, and butyl groups, phenyl groups, alkenyl groups, and alkynyl groups can be suitably used.
  • Methods for bonding the hydrophobic functional group to the surface of the metal oxide layer include a method of modifying the surface of the metal oxide layer with methyltrimethoxysilane, methyltriethoxysilane, hexyltrimethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, octyltrimethoxysilane, octyltriethoxysilane, isobutyltrimethoxysilane, hexadecyltrimethoxysilane, dimethyldiethoxysilane, and diphenyldimethoxysilane, etc.
  • polyethylene glycol groups By bonding polyethylene glycol groups to the surface of the metal oxide layer, it is possible to further suppress the adsorption of contaminants such as proteins, genes, and phenol red to the magnetic particles. In addition, when polyethylene glycol groups are bonded to the surface of the metal oxide layer, it becomes easier to detach extracellular vesicles from the magnetic particles, further improving the detachment rate. Examples of methods include modifying the surface of the metal oxide layer with methoxypolyethylene glycol, polyethylene glycol distearate, polyethylene glycol monomethyl ether, and polyethylene glycol sorbitan monooleate, etc.
  • a metal oxide layer with an uneven surface structure can be formed, resulting in magnetic particles with a large specific surface area.
  • the specific surface area of the magnetic particles By increasing the specific surface area of the magnetic particles, the amount of extracellular vesicles that can be carried can be increased.
  • the thickness of the metal oxide layer can be, for example, 0.001 ⁇ m or more and 0.1 ⁇ m or less.
  • the thickness of the metal oxide layer is preferably 0.001 ⁇ m or more, more preferably 0.005 ⁇ m or more, particularly preferably 0.01 ⁇ m or more, and most preferably 0.05 ⁇ m or more, because it is suitable for increasing acid resistance.
  • the thickness of the metal oxide layer is preferably 0.1 ⁇ m or less, more preferably 0.05 ⁇ m or less, particularly preferably 0.02 ⁇ m or less, and most preferably 0.01 ⁇ m or less, because it is suitable for making magnetic particles with a large specific surface area.
  • the thickness of the metal oxide layer can be calculated by measuring the thickness of the layer for 10 or more particles in an image of the cross section of the particle observed with a transmission electron microscope and calculating the average value.
  • the metal oxide content in the magnetic particles is preferably 0.1 to 10 wt%, more preferably 0.5 to 5 wt%, particularly preferably 1 to 5 wt%, and most preferably 1 to 3 wt%.
  • the metal oxide content can be measured by ICP emission spectrometry.
  • the specific surface area of the magnetic particles according to one embodiment of the present invention is preferably 2 m 2 /g or more.
  • the specific surface area of the magnetic particles is more preferably 5 m 2 /g or more, particularly preferably 10 m 2 /g or more, and most preferably 20 m 2 /g or more.
  • the specific surface area of the magnetic particles is preferably 100 m 2 /g or less, 90 m 2 /g or less, 80 m 2 /g or less, 70 m 2 /g or less, or 60 m 2 /g or less, more preferably 50 m 2 /g or less, particularly preferably 40 m 2 /g or less, and most preferably 30 m 2 /g or less.
  • the specific surface area of the magnetic particles can be calculated from the amount of adsorption of an inert gas by the BET method of JIS Z8830:2013.
  • the magnetic particles can be manufactured by a method including forming a metal oxide layer containing a micromagnetic body on the surface of a core particle.
  • the method of forming a metal oxide layer containing a micromagnetic body on the surface of a core particle is not particularly limited, but examples include a method of forming a metal oxide layer after adsorbing and accumulating a micromagnetic body on the surface of a core particle, and a method of adding a micromagnetic body when forming a metal oxide layer. Since the reaction is easy to control, the method of forming a metal oxide layer after adsorbing and accumulating a micromagnetic body on the surface of a core particle is preferred.
  • One embodiment of the method for manufacturing magnetic particles includes a step of adsorbing minute magnetic bodies onto the surface of a core particle (magnetization step), and a step of coating the core particle with the minute magnetic bodies adsorbed thereon with a metal oxide (coating step).
  • the core particles and the micro-magnetic bodies may be brought into contact with each other to physically adsorb the micro-magnetic bodies onto the surfaces of the core particles.
  • the method for bringing the core particles and the micro-magnetic bodies into contact with each other is not particularly limited, but the core particles and the micro-magnetic bodies can be brought into contact with each other by stirring in a liquid phase or a gas phase.
  • the amount of the micromagnetic material used may be, for example, 0.5 g or more, 0.6 g or more, 0.7 g or more, 0.8 g or more, 0.9 g or more, 1.0 g or more, or 1.1 g or more per 1 g of core particles, and may be 2.0 g or less, 1.9 g or less, 1.8 g or less, 1.7 g or less, 1.6 g or less, or 1.5 g or less.
  • Methods for adsorbing the micromagnetic material to the core particle surface include a method in which the core particle is brought into contact with a micromagnetic material having a surface charge opposite to that of the core particle, and the micromagnetic material is spontaneously adsorbed to the core particle surface by electrostatic interaction, a method in which the micromagnetic material is bonded to the core particle surface by a chemical reaction between the functional group on the core particle surface and the micromagnetic material, a method in which the core particle and the micromagnetic material are combined by applying mechanical energy, a method in which a hydrophobic core particle is brought into contact with a hydrophobic micromagnetic material, and a method in which a paramagnetic core particle is brought into contact with a micromagnetic material.
  • the method for adsorbing the micromagnetic material to the core particle surface may be a combination of multiple methods.
  • the micromagnetic material may be spontaneously adsorbed to the core particle surface by electrostatic interaction, and then the dried particles may be used, and mechanical energy may be applied to firmly adsorb the micromagnetic material to the core particle.
  • the core particle and the micromagnetic material in an aqueous solution, as this is suitable for increasing the amount of the micromagnetic material adsorbed to the core particle.
  • the aqueous solution contains an electrolyte such as sodium chloride.
  • the electrolyte concentration in the aqueous solution is preferably a concentration at which the micromagnetic material does not aggregate with each other, and for example, a concentration of about 0.01 to 0.5 M can be suitably used.
  • reaction temperature and reaction time when adsorbing the micromagnetic material to the core particle surface by electrostatic interaction can be set appropriately depending on the type of material used, etc.
  • the reaction temperature may be, for example, 1 to 50°C, 5 to 40°C, 10 to 30°C, or 15 to 25°C.
  • the reaction time of the core particles may be, for example, 1.0 hours or more, 1.5 hours or more, 2.0 hours or more, 3.0 hours or more, or 4.0 hours or more, and may be 5.0 hours or less, 4.0 hours or less, or 3.0 hours or less.
  • a method capable of processing particles in a dry manner using a device that performs compounding by swirling force in a high-speed air stream, a device that performs mixing and stirring using a rotation-revolution mixer or ball mill, or a device that performs surface coating using a spray dryer, etc. is preferred.
  • Examples of devices for compounding by applying mechanical energy include the Hybridization System NHS Series (manufactured by Nara Machinery Works, Ltd.), Nobilta NOB (manufactured by Hosokawa Micron Corporation), High-Speed Mixing Type Powder Spheroidizer NSM Series (manufactured by Seishin Enterprise Co., Ltd.), and Mini Spray Dryer B-290 Model (Shibata Scientific Co., Ltd.).
  • the rotation speed is preferably 8000 min -1 or more, more preferably 9000 min -1 or more, particularly preferably 10000 min -1 or more, and most preferably 11000 min -1 or more, because it is suitable for firmly fixing the micromagnetic material to the core particle. Also, the rotation speed is preferably 15000 min -1 or less, more preferably 14000 min -1 or less, particularly preferably 13000 min - 1 or less, and most preferably 12000 min -1 or less, because it is suitable for forming the particle shape into a sphere.
  • the processing time is preferably 1 to 60 minutes, more preferably 1 to 20 minutes, particularly preferably 1 to 10 minutes, and most preferably 3 to 10 minutes, because it is suitable for forming the particle shape into a sphere.
  • the treatment temperature is preferably 25 to 80°C, more preferably 25 to 60°C, particularly preferably 25 to 50°C, and most preferably 25 to 40°C.
  • the drying conditions can be appropriately set according to the type of material used, compounding conditions, etc.
  • the drying temperature may be, for example, 40°C or more, 45°C or more, 50°C or more, 55°C or more, or 60°C or more, and may be 80°C or less, 70°C or less, 65°C or less, 60°C or less, or 55°C or less.
  • the drying time may be, for example, 0.5 hours or more, 1 hour or more, 3 hours or more, 5 hours or more, or 10 hours or more, and may be 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less.
  • the core particles with the micro-magnetic material adsorbed thereto may be dispersed in a high-velocity air current after drying.
  • the particle aggregates can be dissociated by the impact force.
  • the shape of the particles can be made spherical.
  • the high-velocity air current treatment can be carried out using the device described above that performs compounding by swirling force in a high-velocity air current.
  • the coating step the surface of the core particle to which the micro-magnetic material is adsorbed is coated with a metal oxide to form a metal oxide layer on the surface of the particle.
  • the method of forming the metal oxide layer is not particularly limited, but examples include a method in which core particles with micro-magnetic bodies adsorbed thereto are dispersed in a solution containing a reagent and a reaction catalyst that are the raw materials for the metal oxide layer, and a metal oxide layer is formed on the particle surface by allowing a reaction to proceed; a method in which core particles with micro-magnetic bodies adsorbed thereto and metal oxide powder are dispersed in a high-velocity air stream to form a metal oxide layer on the particle surface; and a method in which a metal oxide layer or its raw materials are coated on the particle surface.
  • the sol-gel method can be suitably used, in which core particles to which minute magnetic material has been adsorbed are dispersed in a solvent, and then a metal oxide precursor and a reaction catalyst are added.
  • Metal alkoxides can be cited as examples of metal oxide precursors.
  • the precursor of the metal oxide for example, in the case of silica, tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate; in the case of zinc oxide, zinc methoxide, zinc ethoxide, zinc propoxide, zinc butoxide; in the case of titanium oxide, tetramethyl orthotitanate, tetraethyl orthotitanate, tetrapropyl orthotitanate, tetrabutyl orthotitanate; in the case of yttria oxide, yttrium methoxide, yttrium ethoxide, yttrium propoxide, yttrium butoxide; in the case of tin oxide, tin methoxide, tin ethoxide, tin propoxide, tin butoxide; in the case of indium oxide, indium me
  • indium tin oxide a mixture of a precursor of tin oxide and a precursor of indium oxide can be used.
  • water or ammonia water can be used as the reaction catalyst.
  • the amount of the metal oxide precursor added during the sol-gel reaction is preferably 0.05 g or more per 1 g of core particles, more preferably 0.1 g or more, more preferably 0.2 g or more, and most preferably 0.5 g or more, because it is suitable for forming a sufficient amount of metal oxide on the core particle surface.
  • the amount of the reaction catalyst added during the reaction is preferably 20 mL or more per 1 L of solvent, more preferably 30 mL or more, particularly preferably 40 mL or more, and most preferably 50 mL or more, because it is suitable for suppressing the aggregation of the core particles during the reaction.
  • the amount of the reaction catalyst added during the reaction is preferably 100 mL or less per 1 L of solvent, more preferably 80 mL or less, particularly preferably 70 mL or less, and most preferably 60 mL or less.
  • Metal oxides can also be formed by dispersing particles in a solution containing metal ions and then precipitating the metal oxide to coat the particle surfaces.
  • a metal salt and a precipitating agent for precipitation are used.
  • the metal salt a salt of a metal ion with a nitrate ion, an acetate ion, a sulfate ion, or the like can be used.
  • zinc nitrate, zinc sulfate, and zinc acetate can be used for zinc oxide; titanium nitrate, titanium sulfate, and titanium acetate can be used for titanium oxide; yttrium nitrate, yttrium sulfate, and yttrium acetate can be used for yttria oxide; tin nitrate, tin sulfate, and tin acetate can be used for tin oxide; and indium nitrate, indium sulfate, and indium acetate can be used for indium oxide.
  • indium tin oxide a mixture of a metal salt that gives tin oxide and a metal salt that gives indium oxide can be used.
  • the precipitating agent an alkali metal hydroxide, an aqueous ammonia solution, or an alkali such as hexamethylenetetramine can be used.
  • a reaction solvent for forming the metal oxide ethanol, 2-propanol, or butanol are preferred, ethanol or 2-propanol are more preferred, and 2-propanol is particularly preferred, as these are suitable for uniformly coating the metal oxide while suppressing aggregation of the core particles.
  • the metal oxide As a method for coating the metal oxide, powder sputtering can also be used, in which the metal oxide is sputtered while the dried particles are stirred in a drum or the like.
  • the coating time is preferably 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.
  • the magnetic particles according to one embodiment of the present invention can be produced, for example, by a method including the following steps (i) to (iii). (i) a step of mixing core particles and micromagnetic bodies in a solution to physically adsorb the micromagnetic bodies onto the surfaces of the core particles; (ii) a step of drying the particles to which the micromagnetic bodies have been adsorbed, dispersing the particles in a high-velocity air stream, and dissociating the particle aggregates by a collision force; and (iii) a step of forming a crosslinked inorganic layer on the surfaces of the particles treated in the high-velocity air stream.
  • the method for purifying extracellular vesicles includes a step of contacting a liquid containing extracellular vesicles with magnetic particles to adsorb the extracellular vesicles to the surface of the magnetic particles (adsorption step), and a step of separating the magnetic particles to which the extracellular vesicles are adsorbed from the liquid (separation step).
  • the method for purifying extracellular vesicles may include a step of adjusting the liquid containing the extracellular vesicles to an acidic pH (to make the liquid acidic) (pH adjustment step) before the adsorption step.
  • liquids that contain extracellular vesicles include culture media (culture supernatants) in which cells are cultured, blood, serum, plasma, urine, sweat, saliva, breast milk, etc.
  • the pH of the liquid containing the extracellular vesicles is adjusted to make it an acidic liquid.
  • the pH acidic hydrogen ion exponent
  • the magnetic particles and proteins tend to be positively charged, while the extracellular vesicles are negatively charged even in an acidic environment, making it easier for the extracellular vesicles to be selectively adsorbed onto the surface of the magnetic particles.
  • acids such as phosphoric acid, sodium dihydrogen phosphate, disodium phosphate, tris(tris(hydroxymethyl)aminomethane), hydrochloric acid, acetic acid, sodium acetate, 2-morpholinoethanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, trishydroxymethylaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3-tris(hydroxymethyl)methylaminopropanesulfonic acid, boric acid, sodium borate, 2-(N-morpholino)ethanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), acetamidoimidole, N-(2-acetamido)-2-aminoethanesulfonic acid, and 2-(N-cyclohexylamino)ethan
  • the pH is preferably 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more.
  • the pH is preferably less than 7, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.
  • inorganic salts such as sodium chloride and calcium chloride, and chaotropic salts such as guanidine thiocyanate and urea may be added as necessary.
  • the magnetic particles are brought into contact with the liquid, and the extracellular vesicles are adsorbed onto the surface of the magnetic particles.
  • the magnetic particles are brought into contact with the liquid, for example, by putting the magnetic particles into the liquid, stirring, and dispersing them in the liquid.
  • the adsorption time be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, and 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more.
  • the adsorption time is preferably 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, and preferably 30 minutes or less, 20 minutes or less, or 10 minutes or less.
  • the amount of magnetic particles added is preferably 1 mg or more, 2 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, or 50 mg or more per 1 mL of liquid.
  • the amount of magnetic particles added is preferably 100 mg or less, 75 mg or less, 50 mg or less, 30 mg or less, 20 mg or less, 10 mg or less, 5 mg or less, 3 mg or less, or 2 mg or less per 1 mL of liquid.
  • the magnetic particles are separated from the liquid.
  • separation method may be a combination of magnetic separation and, if necessary, centrifugation, filtration, natural sedimentation, etc.
  • the method for purifying extracellular vesicles preferably further includes, after the separation step, a step of contacting the magnetic particles separated from the liquid with a liquid having a pH of 5 or higher to detach the extracellular vesicles from the surface of the magnetic particles (detachment step).
  • the liquid used in the desorption step is preferably an alkaline liquid. In this case, it is easy to desorb the extracellular vesicles from the magnetic particles.
  • the pH of the liquid used in the desorption step may be 6 or more, and in order to further increase the desorption rate of the extracellular vesicles, it is preferable that the pH be 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 or more. In order to suppress the denaturation of the extracellular vesicles, it is preferable that the pH of the liquid used in the desorption step be 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, or 8 or less.
  • a base such as sodium hydroxide, sodium phosphate, sodium citrate, sodium carbonate, or sodium bicarbonate.
  • the desorption time is not particularly limited, but in order to fully desorb the extracellular vesicles in the liquid from the surface of the magnetic particles, it is preferably 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, and preferably 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more.
  • the desorption time is preferably 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, and preferably 30 minutes or less, 20 minutes or less, or 10 minutes or less.
  • the desorption time for each recovery operation can be selected arbitrarily from the desorption times described above. For example, by setting the desorption time for the first operation to 1 minute to 1 hour and the desorption time for the second operation to 1 hour to 72 hours, it is possible to separate extracellular vesicles with a particle size of more than 150 nm from extracellular vesicles with a particle size of around 50 to 150 nm.
  • the method for purifying extracellular vesicles may further include a step of removing proteins from the liquid containing extracellular vesicles (protein removal step) prior to the pH adjustment step.
  • protein removal step a step of removing proteins from the liquid containing extracellular vesicles
  • Methods for removing proteins include, for example, salting out, a polymer flocculant, a method of precipitating and removing proteins using an organic solvent, and a method of adding a protein adsorbent.
  • the method for purifying extracellular vesicles is preferably capable of adsorbing 30% or more of the extracellular vesicles contained in the sample, more preferably 50% or more, particularly preferably 70% or more, and most preferably 80% or more.
  • a serum-containing sample it is preferably capable of adsorbing 20% or more of the extracellular vesicles contained in the sample, more preferably 40% or more, particularly preferably 60% or more, and most preferably 70% or more.
  • 10% or more of the extracellular vesicles captured by the magnetic particles can be detached, more preferably 30% or more, particularly preferably 50% or more, and most preferably 70% or more.
  • 10% or more of the extracellular vesicles captured by the magnetic particles can be detached, more preferably 20% or more, particularly preferably 40% or more, and most preferably 60% or more.
  • the concentration of extracellular vesicles was similarly measured using a CD63-Capture human exosome ELISA kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the adsorption rate was calculated from the following formula. (concentration before addition of magnetic particles ⁇ concentration after addition of magnetic particles) ⁇ 100/concentration before addition of magnetic particles [%]
  • the concentration of the extracellular vesicles was determined for either the microRNA concentration or the CD63 concentration, and the desorption rate was calculated from the following formula. (amount of extracellular vesicles contained in solution) x 100 / amount of extracellular vesicles adsorbed to magnetic particles [%]
  • amount of extracellular vesicles contained in solution x 100 / amount of extracellular vesicles adsorbed to magnetic particles [%]
  • 10% dilute hydrochloric acid was diluted with pure water and the concentration was adjusted using a pH meter.
  • 1M aqueous sodium hydroxide solution was diluted with pure water and the concentration was adjusted using a pH meter.
  • microRNA content The amount of microRNA contained in the extracellular vesicles collected by the same procedure as in the evaluation of the detachment rate of extracellular vesicles was determined by Qubit. The content was determined by creating a calibration curve using the microRNA standard included in the Qubit microRNA Assay Kits.
  • Step (i) (fine magnetic material adsorption) 1 g of polydivinylbenzene particles (particle diameter 2.2 ⁇ m) was dispersed in 50 mL of 0.1 M sodium chloride pure water at room temperature at a rotation speed of 100 rpm. 1.2 g of a magnetic material (material: magnetite, particle diameter: 10 nm, product name: EMG607, manufactured by Ferrotec Corporation) having a cationic dispersant on the surface was added to this particle dispersion, and the mixture was reacted at room temperature at a rotation speed of 100 rpm for 2 hours. The solid content (particles) was filtered and washed with pure water.
  • a magnetic material material: magnetite, particle diameter: 10 nm, product name: EMG607, manufactured by Ferrotec Corporation
  • Step (ii) (Drying and High Velocity Air Flow Treatment)
  • the particles washed with pure water were dried for 15 hours at 50° C. to obtain 2.18 g of magnetized dried particles.
  • the magnetized particles were placed in a hybridization system NHS-0 (manufactured by Nara Machinery Co., Ltd.) and treated for 5 minutes at a rotation speed of 11,300 min ⁇ 1 and an average temperature during treatment of 35° C.
  • NHS-0 manufactured by Nara Machinery Co., Ltd.
  • Step (iii) Metal Oxide Coating on Magnetized Particles
  • 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, and 0.8 g of tetraethyl orthotitanate was added and stirred at room temperature at 180 rpm.
  • 0.4 mL of 25% aqueous ammonia was added, and the mixture was reacted at 60° C. and 180 rpm for 4 hours.
  • the mixture was washed with methanol and dried under reduced pressure.
  • Example 2 The magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1, and the following step (iii) was further carried out using the synthesized particles.
  • Step (iii) Metal Oxide Coating on Magnetized Particles
  • the magnetized particles were sputtered for 8 hours using zinc oxide as a target to form a coating layer of zinc oxide on the surface of the particles.
  • the sputtering was performed at a power of 1000 W and a gas pressure of 1.5 Pa in the presence of a mixed gas of argon and oxygen.
  • Example 3 The magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1, and the following step (iii) was further carried out using the synthesized particles.
  • Step (iii) Metal Oxide Coating on Magnetized Particles
  • 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, 0.4 g of tetraethyl orthosilicate was added, and the mixture was stirred at room temperature at 180 rpm. 0.35 mL of 25% aqueous ammonia was added, and the mixture was reacted at 60° C. and 180 rpm for 4 hours.
  • the particles were washed with methanol and dried under reduced pressure.
  • the cross-sectional shape of the particles was confirmed by a transmission electron microscope, and the thickness of the metal oxide layer was about 0.02 to 0.05 ⁇ m.
  • Example 4 The magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1, and the following step (iii) was further carried out using the synthesized particles.
  • Step (iii) Metal Oxide Coating on Magnetized Particles
  • a coating layer made of indium oxide and indium tin oxide was formed on the surface of the particles by sputtering the magnetized particles for 8 hours using a mixture of 95% by mass of indium oxide and 5% by mass of tin oxide as a target.
  • the sputtering was performed at a power of 1000 W and a gas pressure of 1.5 Pa in the presence of a mixed gas of argon and oxygen.
  • the cross-sectional shape of the particles was confirmed by a transmission electron microscope, and the thickness of the metal oxide layer was about 0.06 to 0.11 ⁇ m.
  • Example 5 Magnetic particles were synthesized in the same manner as in Example 2.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 6).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.008.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 23.1 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good, at 83.2%.
  • the desorption rate of extracellular vesicles was 6.3%.
  • the microRNA contained in the recovered extracellular vesicles was 77 ng/mL.
  • Example 6 Magnetic particles were synthesized in the same manner as in Example 2.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and pure water (pH 7).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.008.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 23.1 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 80%.
  • the desorption rate of extracellular vesicles was 5.6%.
  • the microRNA contained in the recovered extracellular vesicles was 68 ng/mL.
  • Example 7 Magnetic particles were synthesized in the same manner as in Example 2.
  • Example 8 Magnetic particles were synthesized in the same manner as in Example 2.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 12).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.008.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 23.1 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 77.5%.
  • the desorption rate of extracellular vesicles was also good at 41.4%.
  • the microRNA contained in the recovered extracellular vesicles was 506 ng/mL.
  • Example 9 Magnetic particles were synthesized in the same manner as in Example 3.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 6).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.003.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 64.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was good at 87.9%.
  • the desorption rate of extracellular vesicles was good at 43.4%.
  • the microRNA contained in the recovered extracellular vesicles was 519 ng/mL.
  • Example 10 Magnetic particles were synthesized in the same manner as in Example 3.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and pure water (pH 7).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.003.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 64.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was good at 83.2%.
  • the desorption rate of extracellular vesicles was good at 96.7%.
  • the microRNA contained in the recovered extracellular vesicles was 1155 ng/mL.
  • Example 11 Magnetic particles were synthesized in the same manner as in Example 3.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 8).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.003.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 64.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 86.0%.
  • the desorption rate of extracellular vesicles was also good at 79.6%.
  • the microRNA contained in the recovered extracellular vesicles was 952 ng/mL.
  • Example 13 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 6).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 75.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was good at 82.7%.
  • the desorption rate of extracellular vesicles was good at 20.3%.
  • the microRNA contained in the recovered extracellular vesicles was 222 ng/mL.
  • Example 14 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 8).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 75.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 70.8%.
  • the desorption rate of extracellular vesicles was also good at 30.8%.
  • the microRNA contained in the recovered extracellular vesicles was 336 ng/mL.
  • Example 16 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 12).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 75.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 74.4%.
  • the desorption rate of extracellular vesicles was also good at 84.6%.
  • the microRNA contained in the recovered extracellular vesicles was 924 ng/mL.
  • Example 17 Magnetic particles were synthesized in the same manner as in Example 3.
  • Example 18 Magnetic particles were synthesized in the same manner as in Example 4.
  • Example 19 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption rate of extracellular vesicles was evaluated using a medium containing 10% serum (pH 2) and CD63 as the measurement target.
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 75.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 65.8%.
  • Example 20 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption rate of extracellular vesicles was evaluated using a medium containing 10% serum (pH 4) and CD63 as the measurement target.
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the specific surface area was 75.6 m 2 /g.
  • the adsorption rate of extracellular vesicles was also good at 38.9%.
  • Example 21 Magnetic particles were synthesized in the same manner as in Example 3.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 3) and aqueous sodium hydroxide solution (pH 12). After the extracellular vesicles were adsorbed to the magnetic particles, they were contacted with an aqueous sodium hydroxide solution (pH 12) for 15 minutes, and the supernatant was recovered by magnetic separation. This supernatant was designated as recovered solution 1. Next, the magnetic particles were contacted with an aqueous sodium hydroxide solution (pH 12) for 65 hours, and the supernatant was recovered by magnetic separation. This supernatant was designated as recovered solution 2.
  • Figure 3 shows the results of comparing the particle size distribution of extracellular vesicles before purification with recovered solution 1 and recovered solution 2.
  • the average particle size was 203 nm before purification, 224 nm for recovered solution 1, and 134 nm for recovered solution 2.
  • Example 22 Magnetic particles were synthesized in the same manner as in Example 4.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 3) and aqueous sodium hydroxide solution (pH 12). After the extracellular vesicles were adsorbed to the magnetic particles, they were contacted with an aqueous sodium hydroxide solution (pH 12) for 15 minutes, and the supernatant was recovered by magnetic separation. This supernatant was designated as recovered solution 1. Next, the magnetic particles were contacted with an aqueous sodium hydroxide solution (pH 12) for 17 hours, and the supernatant was recovered by magnetic separation. This supernatant was designated as recovered solution 2.
  • Figure 3 shows the results of comparing the particle size distribution of extracellular vesicles before purification with recovered solution 1 and recovered solution 2.
  • the average particle size was 203 nm before purification, 215 nm for recovered solution 1, and 170 nm for recovered solution 2.
  • Example 23 Amino groups were introduced into the magnetic particles of Example 1 by the following method. 10 mg of magnetic particles were dispersed in 200 ⁇ L of methanol, and 3-aminopropyltrimethoxysilane was added at a concentration of 500 mg/mL. The reaction solution containing the magnetic particles and 3-aminopropyltrimethoxysilane was left to stand overnight at room temperature. After the reaction, the magnetic particles were washed with methanol to obtain magnetic particles having amino groups on the surface.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 74.2%.
  • the content of phenol red, an impurity, was 0.082.
  • the desorption rate of extracellular vesicles was good at 51.4%.
  • the microRNA contained in the recovered extracellular vesicles was 463 ng/mL.
  • Example 24 Amino groups were introduced into the magnetic particles of Example 2 in the same manner as in Example 23.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 72.5%.
  • the desorption rate of extracellular vesicles was good at 68.3%.
  • the microRNA contained in the recovered extracellular vesicles was 601 ng/mL.
  • the content of phenol red, an impurity, was 0.078.
  • Example 25 Amino groups were introduced into the magnetic particles of Example 3 in the same manner as in Example 23.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 78.9%.
  • the desorption rate of extracellular vesicles was good at 72.1%.
  • the content of phenol red, an impurity, was 0.071.
  • the microRNA contained in the recovered extracellular vesicles was 691 ng/mL.
  • the introduction of amino groups improved the adsorption rate of extracellular vesicles compared to before the introduction.
  • Example 26 Amino groups were introduced into the magnetic particles of Example 4 in the same manner as in Example 23.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 75.3%.
  • the desorption rate of extracellular vesicles was good at 61.0%.
  • the microRNA contained in the recovered extracellular vesicles was 558 ng/mL.
  • the content of phenol red, an impurity, was 0.098.
  • the introduction of amino groups improved the adsorption and desorption rates of extracellular vesicles compared to before the introduction.
  • Example 27 An alkyl group having 6 carbon atoms was introduced into the magnetic particles of Example 4 by the following method. 10 mg of magnetic particles were dispersed in 200 ⁇ L of methanol, and hexyltrimethoxysilane was added at a concentration of 500 mg/mL. The reaction solution containing the magnetic particles and hexyltrimethoxysilane was left to stand overnight at room temperature. After the reaction, the magnetic particles were washed with methanol to obtain magnetic particles having an alkyl group with 6 carbon atoms on the surface.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 45.1%.
  • the desorption rate of extracellular vesicles was good at 22.9%.
  • the microRNA contained in the recovered extracellular vesicles was 125 ng/mL.
  • the content of phenol red, an impurity, was 0.013.
  • the introduction of the hexyl group improved the adsorption rate of extracellular vesicles and reduced the content of impurities compared to before the introduction.
  • Example 28 An alkyl group having 12 carbon atoms was introduced into the magnetic particles of Example 4 by the following method. 10 mg of magnetic particles were dispersed in 200 ⁇ L of methanol, and dodecyltriethoxysilane was added at a concentration of 500 mg/mL. The reaction solution containing the magnetic particles, methanol, and dodecyltriethoxysilane was left to stand overnight at room temperature. After the reaction, the magnetic particles were washed with methanol to obtain magnetic particles having an alkyl group with 12 carbon atoms on the surface.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 62.3%.
  • the desorption rate of extracellular vesicles was good at 18.2%.
  • the microRNA contained in the recovered extracellular vesicles was 137 ng/mL.
  • the content of phenol red, an impurity, was 0.012.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 35.8%.
  • the desorption rate of extracellular vesicles was good at 89.3%.
  • the microRNA contained in the recovered extracellular vesicles was 388 ng/mL.
  • the content of phenol red, an impurity, was 0.008.
  • the introduction of the carboxyl group improved the desorption rate of extracellular vesicles and reduced the content of impurities compared to before the introduction.
  • Example 30 Polyethylene glycol groups were introduced into the magnetic particles of Example 4 by the following method. 10 mg of magnetic particles were dispersed in 200 ⁇ L of tetrahydrofuran, and 2 mg of succinic anhydride and 4-dimethylaminopyridine were added at a concentration of 0.2 mg/mL. The mixture was stirred at 60° C. for 3 hours. After the reaction, the magnetic particles were washed with tetrahydrofuran to obtain magnetic particles having carboxyl groups on the surface. A buffer in which 10% CE210 (PEG blocking agent) was dissolved was added to the particles, and 1 mg/mL of DMT-MM was added. The solution was stirred overnight at 37° C. After the reaction, the magnetic particles were washed with pure water to obtain magnetic particles having polyethylene glycol groups.
  • 10 mg of magnetic particles were dispersed in 200 ⁇ L of tetrahydrofuran, and 2 mg of succinic anhydride and 4-dimethylaminopyridine were added at a concentration of 0.2 mg
  • the adsorption and desorption rates of extracellular vesicles were evaluated using a medium containing 10% serum (pH 7) and an aqueous solution of sodium hydroxide (pH 12).
  • the adsorption rate of extracellular vesicles was good at 31.7%.
  • the desorption rate of extracellular vesicles was good at 83.4%.
  • the microRNA contained in the recovered extracellular vesicles was 321 ng/mL.
  • the content of phenol red, an impurity, was 0.005.
  • the introduction of polyethylene glycol groups improved the desorption rate of extracellular vesicles and reduced the content of impurities compared to before the introduction.
  • Example 31 The magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1, and the following step (iii) was further carried out using the synthesized particles.
  • Step (iii) Polymer and Metal Oxide Coating on Magnetized Particles
  • 0.1 g of particles was dispersed in 18 mL of pure water, 4 g of methyl methacrylate and 0.02 g of potassium persulfate were added, and the mixture was degassed and reacted at 65° C. for 3 hours under a nitrogen atmosphere. The mixture was washed with pure water and methanol, and dried under reduced pressure.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 10).
  • the magnetic particles produced had a high iron ion elution rate, with an absorbance of 0.065.
  • the magnetic particles had poor dispersibility in water.
  • the adsorption rate of extracellular vesicles was 99.9%.
  • the desorption rate of extracellular vesicles was 0.0%, which was poor.
  • Magnetic particles were synthesized in the same manner as in Example 1, except that the metal oxide coating was not performed and the surface was coated with a polymer in the following manner.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 10).
  • the magnetic particles produced had an absorbance of 0.004 for the elution of iron ions.
  • the dispersibility of the magnetic particles in water was good.
  • the adsorption rate of the extracellular vesicles was 29.2%, which was a poor adsorption rate.
  • the desorption rate of the extracellular vesicles was 68.5%.
  • the microRNA contained in the recovered extracellular vesicles was 393 ng/mL, CD63 was 0.015, and protein was 12 ⁇ g/mL.
  • Step (iii) Metal Coating on Magnetized Particles
  • the magnetized particles were sputtered for 8 hours using an iron target to form an iron coating layer on the surface of the particles, with a sputtering power of 1000 W and a gas pressure of 1.5 Pa in the presence of a mixed gas of argon and oxygen.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 10).
  • the magnetic particles produced had a high iron ion elution rate of 0.072 absorbance.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was 98.2%.
  • the desorption rate of extracellular vesicles was 0.0%, which was poor.
  • Step (iii) Metal Coating on Magnetized Particles
  • the magnetized particles were sputtered for 8 hours using zinc as a target to form a zinc coating layer on the surface of the particles, with a sputtering power of 1000 W and a gas pressure of 1.5 Pa in the presence of a mixed gas of argon and oxygen.
  • the adsorption and desorption rates of extracellular vesicles were evaluated using serum-free medium (pH 7) and sodium hydroxide aqueous solution (pH 10).
  • the magnetic particles produced had a high iron ion elution rate of 0.012 absorbance.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was 99.1%.
  • the desorption rate of extracellular vesicles was 3.0%, which was poor.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 2).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.008.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good at 78.2%.
  • the desorption rate of extracellular vesicles was 0%.
  • the adsorption/desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 4).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.008.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good, at 82.1%.
  • the desorption rate of extracellular vesicles was 0%.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 2).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.003.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good, at 85.2%.
  • the desorption rate of extracellular vesicles was 0.0%.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 4).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.003.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good, at 81.4%.
  • the desorption rate of extracellular vesicles was 0.0%.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 2).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good, at 81.1%.
  • the desorption rate of extracellular vesicles was 0.0%.
  • the adsorption and desorption rate of extracellular vesicles was evaluated using serum-free medium (pH 7) and hydrochloric acid (pH 4).
  • the magnetic particles produced had low iron ion elution, with an absorbance of 0.005.
  • the magnetic particles had good dispersibility in water.
  • the adsorption rate of extracellular vesicles was also good at 73.2%.
  • the desorption rate of extracellular vesicles was 0.0%.
  • Table 7 shows the results of an evaluation of the effect of the pH of the liquid when desorbing extracellular vesicles. It was confirmed that the desorption rate decreased when the pH of the liquid during desorption was 2 or 4. This confirmed that the pH of the liquid used in the desorption process is preferably 5 or higher (Compare Examples 2, 5-8 in Table 1 with Reference Examples 1-2 in Table 7, compare Examples 3, 9-12 in Table 1 with Reference Examples 3-4 in Table 7, and compare Examples 4, 13-16 in Table 1 with Reference Examples 5-6 in Table 7).

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Abstract

La présente invention concerne des particules magnétiques pour la purification de vésicules extracellulaires. Les particules magnétiques comprennent chacune une particule centrale polymère et une couche de revêtement formée sur la surface de la particule centrale polymère. La couche de revêtement contient des microparticules magnétiques et un oxyde métallique.
PCT/JP2024/007608 2023-03-06 2024-02-29 Particules magnétiques pour purification de vésicules extracellulaires, leur procédé de production et procédé de purification de vésicules extracellulaires WO2024185655A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02501753A (ja) * 1987-10-26 1990-06-14 デイド、インターナショナル、インコーポレイテッド 磁気応答ポリマー粒子の製造法およびその応用
JP2016184703A (ja) * 2015-03-26 2016-10-20 東ソー株式会社 磁性微粒子の製造方法
JP2019521322A (ja) * 2016-05-13 2019-07-25 エクソサム ダイアグノスティクス，インコーポレイティド 生体液からの細胞外小胞の単離及びセルフリーｄｎａの同時単離のための自動及び手動方法
WO2021122846A1 (fr) * 2019-12-16 2021-06-24 Qiagen Gmbh Procédé d'enrichissement
WO2021226589A1 (fr) * 2020-05-08 2021-11-11 The University Of Kansas Compositions immunomagnétiques pour la capture spécifique au ph de vésicules extracellulaires

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02501753A (ja) * 1987-10-26 1990-06-14 デイド、インターナショナル、インコーポレイテッド 磁気応答ポリマー粒子の製造法およびその応用
JP2016184703A (ja) * 2015-03-26 2016-10-20 東ソー株式会社 磁性微粒子の製造方法
JP2019521322A (ja) * 2016-05-13 2019-07-25 エクソサム ダイアグノスティクス，インコーポレイティド 生体液からの細胞外小胞の単離及びセルフリーｄｎａの同時単離のための自動及び手動方法
WO2021122846A1 (fr) * 2019-12-16 2021-06-24 Qiagen Gmbh Procédé d'enrichissement
WO2021226589A1 (fr) * 2020-05-08 2021-11-11 The University Of Kansas Compositions immunomagnétiques pour la capture spécifique au ph de vésicules extracellulaires

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