JP2023149346A - Method for recovering fine particles or extracellular vesicles covered by lipid bilayer membrane, and kit for recovering fine particles or extracellular vesicles covered by lipid bilayer membrane - Google Patents

Abstract

To provide a method for recovering fine particles or extracellular vesicles covered by a lipid bilayer membrane in which extracellular vesicles can be sufficiently separated.SOLUTION: A method for recovering fine particles or extracellular vesicles covered by a lipid bilayer membrane in which fine particles or extracellular vesicles covered by a lipid bilayer membrane are recovered includes: a complex formation process of forming a complex in which fine particles or extracellular vesicles covered by a lipid bilayer membrane are bonded to an amino group carried by a carrier capable of carrying a substance containing the amino group by bringing the sample, the substance and the carrier in contact with each other; and a dissociation process of bringing the complex obtained by the complex formation process in contact with dissociation buffer solution containing metal cation to dissociate fine particles or extracellular vesicles covered by the lipid bilayer membrane from the complex.SELECTED DRAWING: Figure 1

Description

Embodiments relate to a method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, and a kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane. In particular, it relates to an exosome collection method and an exosome collection kit.

Exosomes, which are microparticles or extracellular vesicles covered with a lipid bilayer, are membrane-covered particles with a diameter of 20 to 150 nm that are secreted from cells. Exosomes are present in a wide range of biological fluids such as blood, saliva, urine, and cerebrospinal fluid, and are known to contain various substances such as proteins, mRNA, and microRNA. There is. Recently, it has been suggested that exosomes have the function of transmitting information between cells via the substances they contain, and are attracting attention as biomarkers for various diseases and life phenomena.

Several methods have been reported to date for collecting exosomes, including (1) ultracentrifugation (Patent Document 1), (2) pelleting by centrifugation, and (3) depending on particle size. Fractionation, (4) immunoprecipitation, etc. are known.

JP2017-038566A Drug Delivery System 17-4 2002

However, this method has the problem that only some exosomes in the sample can be recovered. Furthermore, since the binding between exosomes and antibodies in the recovered exosome-antibody complex is strong, it is necessary to dissociate the exosomes and antibodies and recover exosomes without damaging the structure and function of the exosomes. , it is necessary to dissociate under severe conditions that involve denaturation of the protein. For this reason, it is difficult to recover exosomes without damaging their structure and function.
Therefore, an object of the embodiments is to provide an exosome collection method and an exosome collection kit that can efficiently separate exosomes without impairing the structure and function of exosomes.

In order to solve the above problems, we collected microparticles covered with a lipid bilayer membrane or extracellular vesicles from a sample containing microparticles covered with a lipid bilayer membrane or extracellular vesicles. A method for recovering fine particles or extracellular vesicles, the method comprising:
The sample is brought into contact with a substance containing an amino group and a carrier capable of supporting the substance containing an amino group, or the sample is brought into contact with the substance containing an amino group supported on the carrier. a complex forming step in which a complex is formed by bonding the fine particles or extracellular vesicles covered with the lipid bilayer membrane with the amino group supported on the carrier;
The complex obtained by the complex formation step is brought into contact with a dissociation buffer solution containing metal cations, and the fine particles or extracellular vesicles covered with the lipid bilayer membrane are dissociated from the complex. A method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane is used, which includes a dissociation step.

Furthermore, an exosome collection kit is used to carry out the above exosome collection method, which is characterized by containing the above peptide, the above carrier, and the above dissociation buffer. Furthermore, an exosome collection kit is used to collect exosomes from a sample containing exosomes, and includes a peptide containing polyornithine and a carrier on which the peptide is supported.
Further, a kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane for performing the above-mentioned method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane,
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane is used, which includes the above-mentioned substance containing an amino group, the above-mentioned carrier, and the above-mentioned dissociation buffer. Furthermore, microparticles covered with a lipid bilayer membrane or extracellular vesicles are collected from a sample containing microparticles covered with a lipid bilayer membrane or extracellular vesicles. A kit for recovering microparticles or extracellular vesicles covered with a lipid bilayer membrane, which includes a peptide containing polyornithine and a carrier on which the peptide is supported, is used.

With the exosome collection method and exosome collection kit of the present application, exosomes can be sufficiently separated.

A perspective view of a column for separating particles or vesicles according to an embodiment Graph showing the results of Example A Graph showing the results of Example B Graph showing the results of Example B Graph showing the results of Example C Graph showing the results of Example D

(Embodiment 1)
One embodiment of the embodiment will be described below, but the embodiment is not limited to this. The embodiments are not limited to the configurations described below, and various changes can be made within the scope of the claims. The technical scope of the embodiments also includes embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples.

[1. How to collect exosomes]
An exosome collection method according to one embodiment is an exosome collection method that collects exosomes from a sample containing exosomes, the method comprising: a sample, a peptide containing ornithine, and a carrier capable of supporting the peptide. or by contacting the sample with the peptide carried on the carrier, a complex is formed in which the exosome and the peptide carried on the carrier are combined to form a complex. and a dissociation step of bringing the complex obtained by the complex formation step into contact with a dissociation buffer containing a metal cation to dissociate the exosome from the complex.
Since the exosome recovery method according to one embodiment has the above configuration, it achieves the following effects (1) to (3).

(1) Since there is no need to use an ultracentrifuge or the like, exosomes can be collected easily and in a short time.

(2) Since it utilizes the fact that ornithine binds to the membrane of exosomes, it is possible to capture and recover a wide range of exosomes, regardless of the expression state of antigen molecules in exosomes.

(3) Exosomes are dissociated from the complex under mild conditions using a dissociation buffer containing metal cations that has little effect on the membrane structure of exosomes and the structure of proteins present in the membrane. Therefore, exosomes can be recovered without damaging the exosomes (in other words, without damaging the structure and function of the exosome, or with the structure and function of the exosome substantially intact).

By collecting exosomes in a state where the structure and function of exosomes are not impaired (or in a state where the structure and function of exosomes are almost not impaired), for example, (1) functional analysis such as physiological effects of exosomes, and (2) biomarkers (exosome-derived proteins, nucleic acids such as miRNA), etc.

In addition, exosomes are expected to be applied to the treatment of a wide range of diseases, including cancer, Alzheimer's disease, myocardial infarction, cerebral infarction, neurodegenerative diseases, knee osteoarthritis, and infectious diseases. Therefore, by collecting exosomes in a state where the structure and function of exosomes are not impaired (or in a state where the structure and function of exosomes are not substantially impaired), the collected exosomes themselves can be used for (3) therapeutic drugs and (4) drug delivery. It can be used as a system carrier. In particular, the use of exosomes as a therapeutic drug in (3) above includes (A) supplementary use in regenerative medicine, (B) use for the purpose of immune control, and (C) use as a vaccine. It is being

In the following, materials used in the method for collecting exosomes according to one embodiment will be described first, and then each step of the method for collecting exosomes will be described.
[1-1. material〕

(sample)
In the collection method of the embodiment, the "sample containing exosomes" (herein also simply referred to as a sample) is not particularly limited in other compositions as long as it is a mixture containing exosomes, for example, a sample containing exosomes. Examples include biological samples.

As used herein, the term "biological sample" refers to a specimen collected from the body of an organism, an extract from the organism, a culture solution, or a component derived from a microorganism. Biological samples include, for example, blood, plasma, serum, saliva, urine, lacrimal fluid, sweat, breast milk, amniotic fluid, cerebrospinal fluid (cerebrospinal fluid), bone marrow fluid, pleural fluid, ascites fluid, synovial fluid, aqueous humor, Examples include, but are not limited to, the vitreous body, extracts from the living body itself or some tissues, culture fluids, components derived from microorganisms, and the like. The selection of biological samples can be appropriately determined by those skilled in the art depending on the purpose. For example, from the viewpoint of ease of collection, blood, plasma, serum, saliva, urine, etc. are preferably used. Other examples include extracts of various organs, culture fluids after culturing animal and plant cells, microorganisms or microorganism-derived components, and the like.

In one embodiment, the origin of the biological sample is not particularly limited as long as it is derived from a biological species that possesses exosomes. For example, mammals are used as the biological species from which the biological sample is derived. Examples of mammals include mice (Musmusculus), rats (Rattus norvegicus), cats (Felis catus), dogs (Canis lupus familiaris), pigs (Sus scrofa domesticus), sheep (Ovis a ries), cows (Bosaurus), green monkeys ( Cercopithecus aethiops), humans (Homosapiens), and the like. Further, as the culture solution, a culture solution in which stem cells, cancer cells, oral mucosa-derived cells, etc. are cultured can be used.
Other materials that can be used include extracts from leaves and fruits, and plant juice components. Examples of plants include grapes, grapefruit, acerola, dragon fruit, ginger, carrots, citrus fruits such as lemon, broccoli, and apples. Furthermore, the microorganisms include Aspergillus aspergillus, lactic acid bacteria, yeast, and the like. This also includes foods (miso, soy sauce, sake, yogurt, milk, and other fermented foods).

(peptide)
The peptide used in the recovery method of the embodiment (hereinafter referred to as the "peptide of the embodiment" as appropriate) is a peptide containing ornithine, and is capable of binding to exosomes in the sample.

The peptides of the embodiments can be easily produced according to any method known in the art, for example, they can be expressed by a transformant into which a peptide expression vector has been introduced, or they can be chemically synthesized. That is, polynucleotides encoding the peptides of the embodiments are also within the scope of the embodiments. As the chemical synthesis method, a solid phase method or a liquid phase method can be mentioned. In the solid phase method, for example, various commercially available peptide synthesizers (ModelMultiPepRS (Intavis AG), etc.) can be used. Furthermore, commercially available polyornithine can be used as the peptide of the embodiment. Commercially available polyornithines include, but are not particularly limited to, poly-L-ornithine (manufactured by Sigma-Aldrich) used in the examples described below.

Although the upper limit of the molecular weight of the peptide of the embodiment is not particularly limited, it can be said that about 300,000 is the upper limit from the viewpoint of operability such as solubility and viscosity.
The peptide of the embodiment may be composed of a peptide having a single molecular weight, or may be composed of a mixture of peptides each having a different molecular weight.

(carrier)
The carrier used in the recovery method of the embodiment (hereinafter referred to as "carrier of the embodiment" as appropriate) means a carrier capable of supporting the peptide of the embodiment. The carrier according to the embodiment can bind to the peptide according to the embodiment bound to the exosome to form a complex in which the exosome, the peptide according to the embodiment, and the carrier according to the embodiment are bound in this order.

In the recovery method of the embodiment, by using a carrier, a complex containing exosomes can be efficiently and easily recovered, thereby enabling highly efficient recovery of exosomes.

The structure of the carrier is not particularly limited as long as it is a structure that can directly or indirectly support (retain) the peptide of the embodiment. The carrier of the embodiment is preferably a support that does not weaken the function of the peptide of the embodiment that binds to the carrier, such as glass, nylon membrane, semiconductor wafer, latex particles, cellulose particles, μ beads, and silica. Examples include beads, magnetic particles, monolithic carriers, and the like. As the carrier of the embodiment, a monolithic carrier is particularly preferable in that it is possible to easily recover a complex containing exosomes and to recover exosomes. Monolithic carriers are widely used in the separation and purification of peptides, proteins, antibodies, nucleic acids, etc., and are well understood by those skilled in the art.

(Method for binding the peptide of the embodiment to the carrier of the embodiment)
As described above, the peptide of the embodiment can be supported by the carrier of the embodiment by binding the peptide of the embodiment to the carrier.

In one embodiment, the method for binding the peptide to the carrier is not particularly limited, and any known method can be used as appropriate. Further, the binding between the peptide and the carrier may be direct or indirect.

The type of peptide that binds to the carrier may be one type or a combination of multiple types. When multiple types of peptides are used in combination, the combination is not particularly limited.

<Monolith>
Here, the following carriers are particularly suitable. Embodiments include a monolith body whose surface is coated with a polymer and the polymer is modified with an ion exchange group, and a column for separating fine particles or vesicles equipped with such a monolith body. Further, the ion exchange group is not particularly limited, but an anion exchange group can be used.

Microparticles or vesicles are particles with a diameter of 1 nm to several 300 nm, and vesicles are particles such as viruses, exosomes, lysosomes, and liposomes that have a space filled with liquid etc. may or may not contain substances such as proteins and nucleic acids. Microparticles or vesicles include those produced by organisms and those produced industrially. Microparticles or vesicles include, but are not limited to, drug-containing nanomedicines, including liposome formulations, protein-bound formulations, exosomes, and viruses. Note that the surfaces of many nanomedicines are negatively charged to enable stable circulation within blood vessels. Drugs included in nanomedicine include, but are not limited to, low-molecular compounds, high-molecular antibodies, oligonucleic acids, peptides, proteins, DNA, messenger RNA, and μRNA (miRNA). Further, the matrix of the fine particles or vesicles can be made of, but not limited to, gold, silver, carbon, or the like.

Separation of particles or vesicles includes separation for analysis, purification, etc. of particles or vesicles.
A monolith body has a three-dimensional network skeleton structure, is formed by a skeleton, is continuous from one end of the skeleton to the other end, in other words, penetrates from the top end of the skeleton to the bottom end, and communicates with each other. It is a porous body that is a single structure, and is an integrated porous body that is equipped with macropores that are through-holes. In addition, the monolith body has a structure in which the skeleton inside the through-holes (macropores) has mesopores, which are pores that are smaller in diameter than the through-holes and communicate or do not communicate between the through-holes, and a structure without mesopores. There are things. The monolith body preferably has through holes that form a continuous and regular three-dimensional network structure, but is not limited thereto. Further, it is preferable that the through-hole has a circular cross section or a circular cross section in a direction perpendicular to the axis of the monolith body.

Macropores include cultured cells, proteins contained in blood, microparticles or vesicles, low-molecular substances released or released from microparticles or vesicles, nanomedicine, and drugs contained in nanomedicine. Mesopores act as a passageway for released drugs or free drugs from nanomedicine, and mesopores serve as passageways for released drugs or free drugs from nanomedicines, such as small molecules released from microparticles or vesicles, or drugs contained in nanomedicines. and the ability to separate free drugs. In addition, by selecting the diameter of mesopores depending on the size of the particles or vesicles and the size of the substances released or released from the particles or vesicles, the mesopores can be It is possible to separate fine particles or vesicles and substances released or liberated from fine particles or vesicles, from small molecules to substances released or liberated from macromolecules such as antibodies or nucleic acids.

It is thought that the binding force with microparticles or vesicles changes depending on the size of the mesopores, and due to the change in surface area, there are mesopores that give the highest recovery rate. That is, if the mesopore is smaller than the target microparticle or vesicle, it will no longer be able to be captured within the mesopore, whereas if the mesopore is made large enough, the surface area will be reduced and the captured microparticle Or there will be fewer vesicles. In order to maximize the recovery rate, it is preferable that the mesopore size be 0.005 to 200 nm.

On the other hand, it has been found that if the mesopores are made small enough, it becomes difficult to recover large particles or vesicles. This is probably due to the fact that large particles or vesicles are partially trapped in the mesopores, making it impossible for cations in the dissociation solution to access the particles or vesicles and the anionic groups in the mesopores. It will be done. The ability to selectively obtain small particles or vesicles is effective when using the particles or vesicles as shells for drug delivery systems, vaccines, and the like. In other words, the smaller the size, the easier it is to be taken into cells. (Non-Patent Document 1) In this case, the mesopores are preferably 0.005 to 300 nm or less.

In the monolith body, the diameter of macropores, which are through holes through which a liquid containing a sample flows, is not particularly limited, but is preferably 1 to 50 μm. Basically, the smaller the diameter of the macropore, the higher the number of contacts, but this also increases the resistance to flowing liquid containing the sample, so when using the macropore as an HPLC column or solid phase extraction column, it is necessary to The diameter of the pores is preferably 1 to 20 μm.

As the monolith body, a ceramic monolith body or a polymer monolith body including a silica monolith body or a glass monolith body can be used. The raw material for the monolith body is not particularly limited, but inorganic materials such as ceramic and glass are preferable. Furthermore, in the case of biological sample components, adsorption of non-target substances may occur depending on the raw material forming the monolith body, so a silica monolith body made from silicon alkoxide, which has a proven track record in solid phase extraction, is preferable.

Silica monoliths made from silicon alkoxide are not particularly limited, but for example, silica monoliths produced from tetraethoxysilane, tetramethoxysilane, methyltrimethoxysilane, methyltriethoxysilane, etc. are preferred; A silica monolith body produced by sol-gel synthesis using these raw materials alone or after mixing them is preferred.

Production of the silica monolith body is not particularly limited, but can be carried out as follows using a generally known method such as a sol-gel method. First, a water-soluble polymer is dissolved in an acetic acid aqueous solution, the above raw materials are added to this solution, a hydrolysis reaction is carried out, and the resulting solution is solidified after stirring. Then, the solidified gel is immersed in an ammonia aqueous solution, dried, and heated to obtain a porous silica monolith body made of amorphous silica. Incidentally, the higher the temperature of immersion in the ammonia solution, the larger the mesopores obtained, and the size of the mesopores can be freely changed by controlling the temperature during drying and heating. In particular, the silica monolith body can be made non-porous at about 1100°C. Moreover, macropores can also be controlled by the above-mentioned raw material ratio and temperature during solidification.

As a specific example, when producing by a sol-gel method, tetraethoxysilane is polymerized in an acidic aqueous solution containing polyethylene glycol polymer (PEO) to obtain a sol solution. The sol solution is poured into a cylindrical polycarbonate pipe, for example, having an inner diameter of 8 mm and a length of 50 cm, and the pipe is sealed and incubated for 20 hours. In this way, a cylindrical monolithic sol body, for example, a monolithic sol body with an outer diameter of 5 mm and a length of 40 cm can be obtained. Then, after completely replacing the solution in the gel with an ammonia aqueous solution, it is incubated to create mesopores, and washed with methanol to obtain a silica monolith body.

By changing the PEO concentration from 1% to 20%, the macropore diameter of the monolithic porous body can be changed from 0.1 μm to 200 μm. Furthermore, by changing the concentration of the ammonia aqueous solution from 0.01% to 1%, the diameter of the mesopores of the monolithic porous body can be changed from 0.005 to 200 nm.

That is, the silica monolith body is partially hydrolyzed and polycondensed using a metal alkoxide such as tetramethoxysilane or a reactive organic monomer such as trimethoxysilane alone or in combination to form a colloidal oligomer. (generation of sol), and then hydrolyzed to promote polymerization and crosslinking to create a three-dimensional network skeletal structure (generation of gel). Finally, it is fired to completely gel and solidify, and if the firing temperature is 300°C or higher, gelation is completed. Note that the silica monolith body can be made non-porous by performing a high-temperature treatment at a firing temperature of about 1100°C. If gelation is completed, it will not be denatured even by heat such as fusion.

The glass monolith body and the ceramic monolith body are also porous bodies having a structure similar to that of the silica monolith body, although the raw materials and manufacturing methods are different.

Ceramic monolith bodies made from ceramics include, but are not particularly limited to, compounds of Group 4 elements such as silica, titanium, zirconium, and hafnium, alumina-silicate A (sintered hard magnetic particles), silica, and alumina. , alumina silitic B (sintered chamotte particles), porous mullite, diatomaceous earth, etc.

In this case, the manufacturing method may include, but is not limited to, ceramic particles (hard magnetically ground material, silica, alumina, chamotte, etc.) having a particle size within a certain range, a pore-forming material such as crystalline cellulose, and an appropriate dispersion. It can be manufactured by mixing with a solvent, molding, and sintering. More specifically, for example, but not limited to, silica sand, boric acid, soda ash, and various metal oxides such as Al ₂ O ₃ , ZrO ₂ , ZnO ₂ , TiO ₂ , SnO ₂ , and MgO ₂ are mixed. , 1200 to 1400° C., it is possible to produce a ceramic monolith body having a composition such as metal oxide-Na ₂ O-B ₂ O ₃ -SiO ₂ -CaO.

Glass monolith bodies made from glass are not particularly limited, but include those having a composition of NaO-B ₂ O ₃ -SiO ₂ -CaO, such as A1 ₂ O ₃ , ZrO ₂ , ZnO ₂ , TiO ₂ , In some cases, it is manufactured using glass to which various oxides such as SnO ₂ and MgO ₂ are added. In this case, the manufacturing method is not limited to this, but for example, a glass lump is made by mixing silica sand, boric acid, soda ash, and alumina and melting the mixture at 1200 to 1400°C. The composition becomes Na ₂ O--B ₂ O ₃ --SiO ₂ --CaO, resulting in a glass porous body.

After molding this at 800 to 110°C, unseparated silica glass was obtained, which was heat treated to separate into SiO ₂ phase and B ₂ O ₃ -NaO-CaO phase, and acid treated to form glass with the SiO ₂ skeleton remaining. Monolith bodies can be manufactured. When the raw material for the monolith body is inorganic, it is possible to produce a monolith body with a uniform pore distribution by changing the heat treatment conditions.

In ceramic monolith bodies and glass monolith bodies, the diameter of macropores can be controlled by composition and phase separation conditions, and mesopores can be formed simultaneously with macropores.

Further, as the monolith body, a porous polymer monolith body made from a high molecular substance (polymer) can be used, which has high chemical resistance and durability against alkaline solvents, is not affected by silanol, and is made from a high molecular substance (polymer). The monolith body made of a polymer in this manner has a structure in which the surface of the monolith body is coated with a polymer. If the polymer monolith is used as is, the nanomedicine will elute into the voids, making accurate quantification difficult when the concentration of nanomedicine is low. Therefore, it is necessary to introduce an ion exchange group, preferably an anion exchange group. It is.

The material of the polymer monolith body is not particularly limited, but specific examples thereof include styrene-divinylbenzene-based, methacrylic acid ester-based, and acrylamide-based copolymers. The monolith body may be constructed by introducing an anion exchange group into a polymer that is a copolymer of these. The monomers constituting the polymer monolith are not particularly limited as long as they have copolymerizability, and monomers that are suitable for the purpose may be selected. Furthermore, hydrophobicity can be controlled by adjusting the blending ratio of monomers. Furthermore, the uniformity of the macropore diameter and mesopore diameter of the polymer monolith body is controlled by the blending ratio of the porogen that dissolves the monomer, the type of solvent, the monomer concentration, the ratio of the crosslinking agent and polymerization initiator, the polymerization temperature, etc. I can do it.

As an example of producing a polymer monolith body, an example of producing an anion exchange polymer monolith using a ring-opening reaction of an epoxy group will be given. First, 300 μL of glycidyl methacrylate (GMA) and 100 μL of ethylene glycol methacrylate (EDMA) are dissolved in hologen consisting of 350 μL of 1-propanol, 200 μL of 1,4-butanediol, and 50 μL of water. After passing nitrogen through this monomer solution, add 4 mg of 2,2'-azobis(isobutyronitrile), fill the column tube, and react at 60°C for 24 hours to produce a GMA-based polymer monolith. I can do it.

The surface of the monolith body is coated with a polymer, and the surface of the polymer is modified with an ion exchange group. Ion exchange groups are exposed on the surface of the polymer coated on the surface of the monolith body, in other words, on the surfaces of the macropores and mesopores of the monolith body. Ion exchange chromatography, which is used in the embodiments, is a technique that utilizes the charged state of molecules in microparticles or vesicles to reversibly bind and cleave carriers with opposite charges. In addition to reversible interactions with ion-exchange groups, the bonding between molecules of microparticles or vesicles and carriers may also involve, for example, van der Waals forces or hydrophobic interactions, but in general, these is a very weak bond compared to ionic bonds, so it can be ignored by controlling the surface charge of microparticles or vesicle molecules.

However, when using a silica-based monolith as a carrier, not only ionic bonds but also fine particles or vesicles strongly adsorb to the silanol groups remaining during surface treatment of the monolith, and they cannot be eluted by simply breaking the ionic bonds. I can't do that. Therefore, in order to eliminate the influence of residual silanol groups, the surface of the monolith body is coated with a polymer, thereby eliminating the influence of the silanol groups and making it possible to elute fine particles or vesicles. Note that a polymer monolith body can be used as the monolith body. With such a configuration, there is no influence of silanol, and since the surface of the monolith body is coated with a polymer in advance, it is not necessary to separately coat the monolith body with a polymer.

The polymer covering the surface of the monolith may be formed by bonding a polymeric material directly to the surface of the monolith, or may be formed by bonding to the surface of the monolith via a linker. In the case of via a linker, a pre-formed polymer may be bonded to the linker, but it is also possible to bond a monomer to the linker and bond a polymer formed by graft polymerization of the monomer.

As the linker, one having a double bond such as an epoxy group or a vinyl group can be used. For example, linkers derived from 3-methacryloxypropyltriethoxysilane, 3-methacrylamidopropyltriethoxysilane, vinyltrimethoxysilane, allyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane can be used.

The monomer to be polymerized is not particularly limited, but 3-acrylamidopropyltrimethylammonium, 2-acryloyloxyethyltrimethylammonium, 2-methacryloyloxyethyltrimethylammonium, vinylbenzyltrimethylammonium, 3-methacryloylaminopropyltrimethylammonium, etc. can be used. I can do it.

Furthermore, the surface of the monolith body may be coated with the polymer itself, which has an anion exchange ability. Although such a configuration is not particularly limited, it can be configured as follows. The surface of the silica monolith body is modified with a linker containing an epoxy group, isocyanate, isothiocyanate, acid anhydride, etc. that is reactive with amino groups, and polyamine, polyamino acid, polyethyleneimine, polymerized type DEAE, polyallylamine, aminoethylated acrylic polymer, acrylamide diallyldimethylammonium copolymer, poly(diallyldimethylammonium) polymer, and poly(trimethylaminoethyl methacrylate) polymer can be coated. In addition, the polymer having anion exchange ability may be one in which the polymer itself has an anion exchange group, or may be one in which an anion exchange group is introduced into the polymer in advance.

In addition, as linkers targeting silanol groups on silica monolith bodies, 3-glycidoxypropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane, 3-trimethoxysilylpropylsuccinic anhydride, trimethylsilylisothiocinate Silane coupling agents such as the following can be used.

In addition, the silanol group can be replaced with N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane having an amino group. An amino group-introduced silica monolith can be produced by modifying it with a silane coupling agent such as silane or 3-aminopropyltriethoxysilane. Further, after introducing 3-trimethoxysilylpropylsuccinic anhydride into the silanol group, a silica monolith introduced with a carboxyl group can be prepared by carrying out a ring-opening reaction with succinic acid.

Homobifunctional crosslinking agents and heterobifunctional crosslinking agents have functional groups such as isothiocynate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, and carbodiimide that form chemical bonds with the above amino groups and carboxyl groups. Agents can also be used as cross-linkers. As the homodipolar cross-linking agent, for example, DSS (disuccinimidyl suberate), DMP (dimethylpimelimidate), etc. can be used, and immobilization via an amino group is possible. 3-(dimethylaminopropyl)carbodiimide and the like can be used, and crosslinking via a carboxyl group and an amino group is possible.

The anion exchange group used is one that has a positive charge and can bind anions. Anion exchange groups adsorb and release negatively charged microparticles or vesicles. Microparticles or vesicles including, but not limited to, primary amine groups, secondary amine groups, tertiary amine groups, or quaternary amine groups can be used. In addition, as the anion exchange group, quaternary ammonium (Q), which is a strong anion exchange group, diethylaminoethyl (DEAE), diethylaminopropyl (ANX), etc., which are weak anionic functional groups, can be used. It can be selected as appropriate depending on the pH range to be used and the particles or vesicles to be separated. Furthermore, examples of anion exchange groups include strong anion exchange functional groups having quaternary amines such as trimethylammonium, polyamines having primary and secondary amines, polylysine, which is a type of basic polyamino acid, and polyamino acids. Arginine, polyhistidine, polyornithine, and polydiaminobutyric acid are preferred.

The microparticles or vesicles containing the nanomedicine and the released substances containing the released drug and the free substances containing the free drug from the microparticles or vesicles, which are contained in the mobile phase and sent to the monolith body, pass through the macropores. At this time, the fine particles or vesicles bind to the ion exchange groups, including the anion exchange groups, exposed on the sides of the macropores excluding the mesopores, and while the bonds are broken, they do not enter the mesopores, but instead enter the macropores. and is released from the monolith body. On the other hand, substances released or liberated from fine particles or vesicles enter the mesopores, proceed through the macropores, and are released from the monolith body.

In the embodiment, a monolith body is coated with a polymer having an ion exchange group according to the charge characteristics of the particles or vesicles to be separated, thereby eliminating adsorption of the particles or vesicles to silanol and inhibiting interaction with the ion exchange groups. Microparticles or vesicles are retained by action and eluted by ionic strength. On the other hand, the released substances and free substances from fine particles or vesicles pass through the mesopores, and depending on the charge characteristics of the released substances and free substances, the ion exchange groups in the mesopores and the ion exchange groups in the macropores and the fine particles Alternatively, it can be separated by interacting with a strength different from that with vesicles.

Surface coating of the monolithic body with a polymer and modification of the surface of the polymer with an ion exchange group can be carried out as follows. Covering the surface of the monolith body includes cases where the surface is not completely covered and is partially exposed. As a specific example, when a silica monolith body is coated with acrylamide, a linker may be bonded to the silica surface of the silica monolith body, and the silica monolith body may be coated with a polymer by graft polymerization, although this method is not limited to this method. I can do it.

Specifically, a solution of methacryloyl chloride was added dropwise in toluene, and N-(3 - Prepare silane as a linker (spacer) with (triethoxysilylpropyl) methacrylamide, and react the silica monolith with a 1:1:1 mixture of silane, pyridine, and toluene at 80°C for 12 hours under a nitrogen atmosphere. The resulting linker-modified silica monolith was washed with toluene and methanol. Next, the silica monolith modified with a linker is mixed with a monomer solution containing 3-acrylamidopropyltrimethylammonium chloride, which is an acrylamide monomer having a trimethylammonium group (the weight of the monomer reagent is 3 to 30% by weight based on the weight of the reaction solution), and peroxodi A reaction was carried out at 70°C for 15 hours in a methanol aqueous solution containing ammonium sulfate to coat the monolith surface with polymer and introduce ion exchange groups, followed by washing with water and acetone to coat the monolith with polymer and modify anion exchange groups. A silica monolith body was obtained.

As described above, there is a method of coating the surface of a monolith body with a polymer by attaching a linker to the surface of the monolith body and graft polymerizing the monomer to the linker, but it is not possible to bond the polymer material directly to the surface of the monolith body. You can also do it. In addition, as a method for modifying a polymer with an ion exchange group, in addition to a method of modifying a polymer with an ion exchange group, there is a method of polymerizing a monomer modified with an ion exchange group to form a polymer and introducing an ion exchange group. can be adopted. Examples of methods for directly bonding a polymeric material to the surface of a monolith body include the following methods, but the method is not limited to these methods.

As a method for coating the silica monolith body with polylysine, a silane agent having an epoxy group (5 wt %) such as 3-glycidoxypropyltrimethoxysilane was mixed therein, and then nitrogen purging was performed. After reacting at 200°C for 10 hours, the mixture was washed with acetone and dried to prepare an epoxy-modified silica monolith. Polylysine can be introduced into the silica monolith by adding an aqueous solution of polylysine having a molecular weight of 4,000 to 15,000 to the epoxy-modified silica monolith and reacting at 70° C. for 10 hours. The molecular weight of polylysine is not limited to 4,000 to 15,000, and polylysine having a molecular weight of up to about 500,000 can be used. In addition, other basic polyamino acids or polymers in which basic amino acids and other amino acids are combined can be introduced into the silica monolith body by a similar method.

Although the method for introducing anion exchange groups into the polymer monolith body is not limited to these, the following methods can be employed. DEAE groups, which are weak anion exchange groups, can be introduced by adding 10% diethylamine solution in acetonitrile to a GMA-based polymer monolith and reacting at 70° C. for 10 hours. Furthermore, by reacting a DEAE group-modified polymer monolith in chlorotoluene, a quaternary amine type strong anion exchange group can be introduced.

The 3 mm diameter monolith body whose surface is coated with a polymer and which has been modified with an ion exchange group, obtained as described above, is dried, and the monolith body is coated with a first polymer layer such as chemically inert PEEK resin. Cover the sides of the body and then insert it into a stainless steel tube (6mm OD, 1.2mm thickness, 50mm length) and add a second layer of epoxy resin etc. between the monolithic body and the stainless steel tube whose sides are coated with the first polymer layer. A column for separating fine particles or vesicles was obtained by filling the polymer layer.

In the case of this method, it is possible to obtain a column that can be used at a maximum temperature of 50° C. and up to 30 MPa, and a monolithic silica gel column with low liquid feeding pressure can produce a column with sufficient performance. The end of the stainless steel tube can be cut off and used as a cartridge-type HPLC column (3 mm ID x 50 mm) for separation of microparticles or vesicles. A cartridge type column can be used as a column by fitting it into a special holder, which is also a stainless steel tube, and tightening it with an end nut that can be connected to 1/16 inch piping.

The monolith body of the embodiment can also be used in a solid phase extraction column that enables simple separation and purification. For example, as a solid phase extraction column, as shown in Fig. 1, a spin column 1 (collection kit) configured by installing a monolith body 3 in an empty column 2 regardless of its material and diameter may be used. You can take it.
The solid-phase extraction column referred to here is an alternative to conventional methods such as column chromatography packed with large particles or thin layer chromatography, which require complicated and special equipment and techniques. This method was established for filling columns. In addition to suction using a centrifuge, vacuum pump, or aspirator, liquid can be passed using pressurization to easily separate and purify compounds. For example, exosomes in nanomedicine stem cell culture can be passed through, washed, and collected.
In addition to these simple methods, liquid chromatography that uses devices such as pumps and purification devices that feature low-pressure pumps can also be used, and the form of the monolith column can be changed as appropriate depending on the equipment used. It is.

Further, as a conventional method for separating and purifying nanomedicine by solid phase extraction, a column using a size exclusion chromatography packing material is used. However, with this conventional method, the elution time of exosomes takes about 4 to 5 minutes, and since it is size exclusion chromatography, careful column equilibration is required before sample application. This is not a method that can be used in a timely manner. On the other hand, anyone can easily purify exosomes in a short time by using a spin column 1 as a column for separating microparticles or vesicles according to the embodiment, as shown in FIG. 1, and passing the liquid through a centrifuge. It has become possible.

(dissociation buffer)
In the recovery method of the embodiment, in the dissociation step described below, the complex is brought into contact with a dissociation buffer containing metal cations and whose pH is adjusted (hereinafter referred to as "dissociation buffer of the embodiment" as appropriate). The exosomes are dissociated from the complex and recovered. The dissociation buffer of the embodiment does not contain surfactants and/or protein denaturants, or contains them at very low concentrations, and exosomes can be dissociated from the complex under mild conditions. , exosomes can be recovered in a state where the structure and function of exosomes are not impaired (a state where the structure and function of exosomes are almost intact). Further, the effect that exosomes can be easily recovered in a state where the structure and function of exosomes are not impaired (a state where the structure and function of exosomes are almost not impaired) is a remarkable and advantageous effect of the embodiment.

The metal cations contained in the dissociation buffer of the embodiment are not particularly limited, and include, for example, monovalent metal cations such as sodium ions, potassium ions, lithium ions, silver ions, and copper (I) ions, magnesium ions, etc. ions, divalent metals such as calcium ions, zinc ions, nickel ions, barium ions, copper (II) ions, iron (II) ions, tin (II) ions, cobalt (II) ions, and lead (II) ions. Examples include cations and trivalent metal cations such as aluminum ions and iron (III) ions. Among these metal cations, sodium ions, potassium ions, magnesium ions, zinc ions, and nickel ions are preferable as the metal cations contained in the dissociation buffer of the embodiment because of their high water solubility. Furthermore, since exosomes can be sufficiently dissociated from the complex, the dissociation buffer of the embodiment may contain sodium ions, potassium ions, or magnesium ions among the above-mentioned metal cations, either alone or in combination. preferable. Various buffer solutions can be used for pH adjustment. Good buffers such as MES, TRIS, TRISIN, MOPS, BES, BISTRIS, TES, HEPES, TAPSO, HEPSO, POPSO, MOPSO, BICINE, TAPS, CHES, CAPS, phosphate buffer, carbonate buffer, citrate buffer, Various buffer solutions such as can be used. Further, a reagent such as a bactericide can be added to the dissociation liquid depending on the intended use.

The purpose of the embodiment is to easily recover exosomes without impairing the structure and function of exosomes (or with substantially intact structure and function of exosomes). Therefore, in the collection method of the embodiment, "recovering exosomes without damaging the structure and function of exosomes (or with almost no damage to the structure and function of exosomes)" means "recovering exosomes without damaging exosomes (almost without damaging them)" This means separating and recovering exosomes in a sample from substances other than exosomes (e.g. proteins, etc.) in the sample. In other words, in the collection method of the embodiment, "recovering exosomes without impairing the structure and function of exosomes (or in a state with substantially intact structure and function of exosomes)" means that the membrane structure of exosomes (specifically Exosomes in a sample are transferred to the sample while maintaining (almost maintaining) the structure of the lipid bilayer membrane, and while maintaining (almost maintaining) the structure of the proteins present in the membrane. This means separating and recovering substances other than exosomes.

The dissociation buffer may contain substances other than metal cations as long as they do not affect the membrane structure of exosomes and the structure of proteins present in the membrane.

The concentration of metal cations in the dissociation buffer is not particularly limited as long as it can dissociate exosomes from the complex and does not affect the membrane structure of exosomes and the structure of proteins present in the membrane. In other words, the optimal cation concentration may vary depending on the binding strength between the peptide of the embodiment used and the exosome, the type (valence) of the metal cation, etc., so after considering the optimal concentration as appropriate, All you have to do is decide. Note that the concentration of metal cations in the dissociation buffer of the embodiment is not particularly limited, but for example, it is preferably 0.01M to 5M, more preferably 0.05M to 2M, It is more preferably 0.1M to 1M, and particularly preferably 0.3M to 0.7M. If the concentration of salt is low, it is expected that more exosomes will be obtained, but there is a concern that contaminants such as proteins may be mixed in. It is possible to select the salt concentration as appropriate depending on the intended use.

Further, the pH of the dissociation buffer can be set as appropriate. The pH of the dissociation buffer is preferably 5 to 10, more preferably 6 to 9, even more preferably 7 to 8, and particularly preferably 7.3 to 7.5. It is preferable that the pH of the dissociation buffer is within the above range because it does not affect the membrane structure of exosomes and the structure of proteins present in the membrane.
[1-2. Process]

(Complex formation process)
The complex formation step included in the recovery method of the embodiment (hereinafter referred to as the "complex formation step of the embodiment" as appropriate) involves a sample containing an exosome, a peptide of the embodiment, and a carrier of the embodiment. or by contacting the sample with the peptide of the embodiment supported on the carrier of the embodiment, a complex formed by binding the exosome and the peptide supported on the carrier. This is a complex formation step of forming a body (a complex in which exosomes, peptides of embodiments, and carriers of embodiments are bound in this order. Hereinafter, appropriately referred to as "complex of embodiments"). That is, in the complex formation step of the embodiment, the peptide of the embodiment is brought into contact with the carrier of the embodiment and the sample in a state where the peptide of the embodiment is not supported on the carrier of the embodiment, and then the exosomes are A complex may be formed in which the peptide of the embodiment is bound to the carrier of the embodiment in this order, or the peptide of the embodiment is already supported on the carrier of the embodiment, and then the peptide and the sample are combined. This means that the composite of the embodiment may be formed by contacting the two. However, from the viewpoint of forming the composite of the embodiment more efficiently, the latter aspect is more preferable.

A method of contacting a sample containing exosomes, the peptide of the embodiment, and the carrier of the embodiment is such that the exosome in the sample, the peptide of the embodiment, and the carrier of the embodiment are bonded to each other, and the peptide of the embodiment is bonded to the carrier of the embodiment. Other methods are not limited as long as they are carried out under conditions that allow the formation of a complex. For example, a method may be mentioned in which the peptide of the embodiment and the carrier of the embodiment are added to a sample and mixed. The order of addition and mixing when bringing the sample, the peptide of the embodiment, and the carrier of the embodiment into contact with each other is not particularly limited. For example, (1) the peptide of the embodiment and the carrier of the embodiment may be added to a sample at the same time and mixed, or (2) the peptide of the embodiment may be added to the sample and mixed, and then the peptide of the embodiment may be added to the sample and mixed. (3) After adding the carrier of the embodiment to the sample and mixing, the peptide of the embodiment may be added and mixed. However, since the peptide of the embodiment binds to the exosome and the peptide of the embodiment and the carrier of the embodiment bind, the procedure (2) above may be preferable. By contacting the sample, the peptide, and the carrier by the method described above, a complex of the embodiment including the exosome, the peptide, and the carrier can be formed.

In addition, as a method for contacting a sample with the peptide of the embodiment supported on the carrier of the embodiment, a method in which the exosomes in the sample and the peptide of the embodiment supported on the carrier of the embodiment are bonded. , other methods are not limited as long as they are carried out under conditions that allow the formation of the complexes of the embodiments. For example, a method may be mentioned in which the peptide of the embodiment supported on the carrier of the embodiment is added to a sample and mixed.
The contact time between the sample, the peptide of the embodiment, and the carrier of the embodiment, or the contact time of the sample and the peptide of the embodiment supported on the carrier of the embodiment, determines the time when the complex of the embodiment is formed. The period of time is not limited as long as it is sufficient for the purpose of the present invention, and may be determined after considering the optimum conditions as appropriate.

In addition, in the contact between a sample containing exosomes, the peptide of the embodiment, and the carrier of the embodiment, a carrier prepared by, for example, filling a columnar or cylindrical container (column) with the carrier of the embodiment may be used. It is also possible to use a column (referred to herein as a "carrier column"). A method for obtaining the complex of the embodiment by passing a sample-containing solution and a peptide-containing solution through a carrier column is such that the exosome in the sample, the peptide of the embodiment, and the carrier of the embodiment bind, respectively. However, other methods are not limited as long as they are carried out under conditions that allow the formation of the complex of the embodiment. For example, the following (1) to (3) can be mentioned: (1) A solution containing the peptide of the embodiment is added to a carrier column, and the solution is passed through the carrier of the embodiment. After binding the peptide of the form, a solution containing the sample is added to the carrier column, and the solution is passed through to form the complex of the embodiment; (2) The peptide of the embodiment is attached to the sample in advance. By adding and mixing the sample and the peptide of the embodiment, a complex formed by binding the exosomes and the peptide in the sample (exosome-peptide complex) is formed, and then , by adding a solution containing the exosome-peptide complex to a carrier column and passing the solution, the peptide of the embodiment and the carrier of the embodiment are combined to form the complex of the embodiment; (3 ) The complex of the embodiment is formed by simultaneously adding a solution containing a sample and a solution containing the peptide of the embodiment to a carrier column and passing these solutions through.
In (1) to (3) above, the contact time between the carrier column and the peptide and/or sample, or the sample containing the exosome-peptide complex, is determined according to the embodiment of the present invention. Preferably, the time is sufficient to form a complex.

The above time is the rate at which the sample solution (a sample containing exosomes and/or a solution containing a peptide of an embodiment, or a solution containing an exosome-peptide complex) is passed through the carrier column, or the sample solution is added to the carrier column. It can be set as appropriate by adjusting the time from when the liquid is passed until the start of liquid passage.

In addition, in contacting the sample containing exosomes with the peptide of the embodiment supported on the carrier of the embodiment, for example, the peptide of the embodiment supported on a columnar or cylindrical container (column) may be used. It is also possible to use a column prepared by packing a carrier (referred to herein as a "peptide column"). By passing a solution containing a sample through the peptide column, the sample and the peptide of the embodiment supported on the carrier of the embodiment can be brought into contact to obtain the complex of the embodiment. The contact time between the peptide column and the sample is preferably a sufficient time for the exosomes in the sample and the peptide of the embodiment supported on the carrier of the embodiment to form the complex of the embodiment. . The above-mentioned time can be set as appropriate by adjusting the flow rate of the solution containing the sample, the time from the addition of the solution to the start of passage, etc.

A conventionally known physical method can be used for passing the sample solution through the carrier column or the peptide column. Examples include methods (1) to (3) below.

(1) This is a method in which the sample solution is added onto a vertically placed carrier column (or peptide column), and the sample solution is allowed to flow through the carrier column (or peptide column) by gravity.

(2) By adding the above sample solution to one side of the carrier column (or peptide column) and applying pressure from the direction in which the sample solution was added, or by suctioning from the direction opposite to the direction in which the sample solution was added, This is a method in which the sample solution is passed through a carrier column (or peptide column).

(3) After adding the sample solution to one of the carrier columns (or peptide columns), load the carrier column (or peptide column) into a centrifuge tube, and centrifuge the centrifuge tube. This is a method in which the sample solution is passed through a peptide column. Note that the method (3) above is a method using a so-called conventionally known spin column. In the case of (3), it is desirable to proceed to the next step immediately after centrifugation to prevent drying.

(Dissociation step)
The dissociation step included in the recovery method of the embodiment (hereinafter referred to as "dissociation step of the embodiment" as appropriate) includes the complex of the embodiment obtained by the above complex formation step and a metal cation. This is a step of dissociating exosomes from the complex of the embodiment by contacting the dissociation buffer of the embodiment.

The method of bringing the complex of the embodiment into contact with the dissociation buffer of the embodiment is not limited to any other method as long as it is carried out under conditions that dissociate the exosome from the complex of the embodiment. . For example, a method may be mentioned in which the dissociation buffer of the embodiment is added to a container containing the complex of the embodiment and mixed. The contact time between the complex of the embodiment and the dissociation buffer of the embodiment is not particularly limited as long as it is sufficient time for exosomes to dissociate from the complex. By contacting the complex of the embodiment with the dissociation buffer of the embodiment, the exosomes are dissociated from the complex of the embodiment and migrate to the dissociation buffer. Then, the exosomes can be recovered by separating the dissociation buffer and the carrier of the embodiment and recovering the dissociation buffer.

In the dissociation step of the embodiment, the peptide of the embodiment may remain bound to the carrier of the embodiment or may be dissociated from the carrier of the embodiment. Furthermore, after the above separation, the peptide of the embodiment may be contained in the dissociation buffer recovered. From the viewpoint of recovering exosomes with higher purity, it is preferable that the peptide of the embodiment remains bound to the carrier of the embodiment in the dissociation step of the embodiment, and after the above separation, the peptide of the embodiment is added to the dissociation buffer recovered. Preferably, the peptide of the embodiment is not included.

Further, when a column is used in the complex formation step, the dissociation buffer of the embodiment can be passed through the column on which the complex of the embodiment has been formed in the complex formation step. It is possible to contact the complex with the dissociation buffer of the embodiment. Physical methods for passing the dissociation buffer of the embodiment through the column include methods (1) to (3) described in the section (complex formation step). When the column and the dissociation buffer of the embodiment are brought into contact, the exosomes are dissociated from the complex and migrate to the dissociation buffer. Therefore, exosomes can be recovered by recovering the dissociation buffer after passing through the column.

The recovery method of the embodiment may include steps other than the above-described complex formation step and dissociation step. Other steps include, for example, a washing step and a recovery step.

(Collection process)
The recovery step is provided between the complex formation step and the dissociation step, and is a step of recovering the complex of the embodiment obtained by the complex formation step. In the recovery step, a conventionally known method may be appropriately adopted depending on the complex formation step, carrier, and the like.

The carrier is [1-1. In the case of a structure as explained in the section of (Carrier) in [Materials], the carrier containing the composite of the embodiment can be recovered by centrifugation. For example, by performing centrifugation, it is possible to recover the carrier containing the complex of the embodiment.

Further, when the carrier is a magnetic particle, the carrier can be easily recovered by applying magnetic force from the outside using a magnetic material such as a conventionally known magnetic stand. When the carrier is a magnetic particle and is recovered using a magnetic material, it has the following advantages (1) and (2) compared to the centrifugation method: (1) The carrier is a magnetic particle, Because they are attracted to the magnetic material, it is possible to remove the supernatant almost completely, increasing the purity of the resulting exosomes; (2) When removing the supernatant, magnetic particles are also removed. It is possible to reduce the risk and prevent the loss of the obtained exosomes (the recovery rate of exosomes can be improved).

Note that when a carrier column or a peptide column is used in the complex formation step of the embodiment, the carrier containing the complex of the embodiment remains in the column. For this reason, the above-mentioned recovery step is required.

(Washing process)
Preferably, the recovery method of the embodiment further includes a washing step before the dissociation step. The washing step is a step of washing the recovered complex of the embodiment or the complex of the embodiment in the column an appropriate number of times using an appropriate washing solution. As long as the washing step is carried out under conditions that do not dissociate the complex, the type of washing liquid used, the number of washings, etc. are not particularly limited.

Examples of the washing solution used in the washing step include physiological saline or phosphate buffered saline (PBS).

When one embodiment of the embodiment includes a recovery step, examples of the washing step include the following methods. (1) By mixing the recovered carrier containing the complex of the embodiment with the above-mentioned washing solution, the carrier is resuspended in the washing solution. (2) Collect the carrier containing the complex of the embodiment again (that is, separate the carrier containing the complex of the embodiment from the washing liquid).

Further, in the complex forming step of the embodiment, when a carrier column or a peptide column is used, an appropriate amount of the above-mentioned washing liquid may be passed through the column after the complex of the embodiment is formed.

[2. Exosome recovery kit]
An exosome collection kit according to one embodiment (hereinafter referred to as "kit of embodiment" as appropriate) is a kit for carrying out the collection method of the embodiment described above, and is a kit for performing the collection method of the embodiment described above. , a carrier according to an embodiment, and a dissociation buffer according to an embodiment. Therefore, regarding the explanation of the configuration of the kit, please refer to [1. Exosome collection method] can be referred to.

The peptide of the embodiment and the carrier of the embodiment contained in the kit of the embodiment may be included in the kit of the embodiment in a state in which the peptide of the embodiment is pre-supported on the carrier of the embodiment. Alternatively, each may be included separately.

Further, the kit of the embodiment may include magnetic particles, magnetic particles on which polyornithine is immobilized, magnetic substances such as a magnetic stand, biotin, and the like.
In addition to the above configuration, the kit of the embodiment may also include a container (for example, a bottle, plate, tube, dish, column, etc.) containing a specific material. Kits of embodiments may include containers containing diluents, solvents, wash solutions, or other reagents. The term "comprising" used in the description of the kit of the embodiment may mean that the kit is contained in any of the individual containers constituting the kit.

Also. The kit of the embodiment may include instructions for carrying out the recovery method of the embodiment. Furthermore, it may be an exosome collection kit for collecting exosomes from a sample containing exosomes, which includes a peptide containing polyornithine and a carrier on which the peptide is supported. This collection kit, sample, and dissociation buffer may be used.
One embodiment of the embodiment has the following configuration.
[1] Microparticles or cells covered with a lipid bilayer membrane to collect microparticles or extracellular vesicles covered with a lipid bilayer membrane from a sample containing microparticles or extracellular vesicles covered with a lipid bilayer membrane. A method for collecting external vesicles, the method comprising:
Bringing the sample into contact with a substance containing an amino group and a carrier capable of supporting the substance containing an amino group, or bringing the sample into contact with the substance containing the amino group supported on the carrier. a complex forming step in which a complex is formed by bonding the fine particles or extracellular vesicles covered with the lipid bilayer membrane to the amino group supported on the carrier;
The complex obtained by the complex formation step is brought into contact with a dissociation buffer solution containing metal cations to dissociate fine particles or extracellular vesicles covered with the lipid bilayer membrane from the complex. A method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane includes a dissociation step.
[2] The substance containing an amino group is a peptide containing ornithine.The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to [1] is used.
[3] The particle size of the microparticles or extracellular vesicles covered with the lipid bilayer membrane to be collected is controlled by limiting the pore size of the mesopores of the carrier [1] or [2]. Use collection methods.
[4] The carrier is a silica monolith.The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of [1] to [3] is used.
[5] The dissociation buffer solution is a solution containing magnesium as a divalent metal ion, and the microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of [1] to [4]. Use the following collection method.
[6] The concentration of the dissociation buffer solution is 0.1 to 1.5 M. [1] to [5] above, in which microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of [1] to [5] are Use collection methods.
[7] Microparticles or cells covered with a lipid bilayer membrane for carrying out the method for recovering microparticles covered with a lipid bilayer membrane or extracellular vesicles according to any one of [1] to [6]. A kit for collecting external vesicles,
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane is used, which includes the substance containing the amino group, the carrier, and the dissociation buffer.
[8] Microparticles or cells covered with a lipid bilayer membrane to collect microparticles or extracellular vesicles covered with a lipid bilayer membrane from a sample containing microparticles or extracellular vesicles covered with a lipid bilayer membrane. A kit for collecting external vesicles,
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane containing a peptide containing polyornithine and a carrier on which the peptide is supported is used.

Hereinafter, one embodiment of the embodiment will be described in more detail with reference to Examples, but the embodiments are not limited to these Examples.
<Example A>
A peptide-supported column (modifier-immobilized column) was prepared. Silica monolith was used as the carrier.

The silica monolith body (3 mm ID x 50 mm length) used had a macropore size of 1 μm, a mesopore size of 11 nm, and a surface area of 340 m ² /g. Trimethylammonium groups were chosen as the source to add positive charges to the surface of the silica monolith. In order to incorporate trimethylammonium groups onto the surface of the silica monolith, we used a column (hereinafter referred to as "trimethylammonium group") using a silica monolith modified with the same functional group (trimethylammonium group) using two different functional group modification methods: acrylamide and siloxane. (Also referred to as "modified column.") was used.

Surface modification of the silica monolith body was performed as follows. As an example, a column using a polymer-coated silica monolith (hereinafter referred to as "acrylamide-coated") in which the surface of the silica monolith was coated with acrylamide by polymerizing an acrylamide monomer having a trimethylammonium group and an anion exchange group was introduced. ) was used. N-(3-triethoxysilylpropyl) prepared by dropwise addition of a solution of methacryloyl chloride in toluene and stirring 3-aminopropyltriethoxysilane and triethylamine in toluene at 0 °C for over 0.5 h. ) Preparing silane with methacrylamide, reacting the silica monolith with a 1:1:1 mixture of silane, pyridine, and toluene for 12 hours at 80°C under a nitrogen atmosphere to obtain a silica monolith modified with a linker. The body was washed with toluene and methanol. Next, the linker-modified silica monolith body was reacted with solutions of polyornithine (Example A4), polylysine (Example A2), aminosilane (Example A1), and polyallylamine (Example A3) to form polyornithine. A silica monolith modified with each of polylysine, aminosilane, and polyallylamine was obtained.

<Sample>
HADSC-adipose derived stem cells (Lonza) were seeded in Corning CellBIND surface hyper flask M (Corning), and KBM ADSC-4 (Kohjin Bio) was used as the medium. The cells were cultured in an incubator at 37° C. under a 5% CO ₂ atmosphere, and the culture supernatant was collected when the cells reached 50% confluence or more. The collected culture supernatant was centrifuged using a centrifuge (Koki Holdings, CF16RN) at 4° C. and 10,000×g for 30 minutes, and the supernatant was collected. Thereafter, the culture supernatant was obtained by passing through Millex GP 0.22 μm PES (Merck Millipore, SLGP033RS). Exosomes in the culture supernatant (0.5 mL) were collected by the following method.
<Method>

(1) The above culture supernatant (0.5 mL) was passed through the above column.

(2) Exosomes were bound to the modified product in the column. That is, in the compositing process, a complex containing an exosome, a modified product (peptide), and a column was formed.

(3) Washed the complex: 0.5 mL of ultrapure water was passed through the column.

(4) The operation (washing) in (3) above was performed two more times to recover the polyornithine-immobilized column containing exosomes in the culture supernatant as a complex.

(5) A dissociation buffer was passed through the column to separate exosomes from the complex. _MgCl2 was used as the dissociation buffer.

(6) The amount of exosomes in the dissociation buffer was measured by ELISA

(7) The amount of exosomes was measured using the CD9/CD63 Exosome ELISA Kit (Cosmo Bio, EXH0102EL) and Sunrise Thermo RC-R (TECAN, 551-40081) according to the attached standard protocol <Results>
The results are shown in Table 1 and Figure 2. Using the polyornithine of Example A4, it was unexpectedly possible to recover four times more exosomes than in other Examples A1 to A3. Amino acid polymers such as polylysine and polyornithine increase the positive charge on the column carrier, allowing lipid bilayer membranes to adhere. Polyornithine has a lower molecular weight and lower hydrophobicity than polylysine. Therefore, since polyornithine is more easily charged and has lower hydrophobicity, it is thought that conditions were derived that make it easier to adsorb.

Silica monolith is preferred as carrier. This is because the polymer can be easily fixed.

＜実施例Ｂ＞
実施例Ａ４の条件で、シリカモノリスのメソ穴径を変えて、回収されたエクソソームの粒子径、量を測定した。説明しない事項は、実施例Ａ、Ａ４と同様である。
エクソソームの粒子径の測定には、粒子径分布測定機（Ｌｉｇｈｔｓｉｚｅｒ：アントンパール製、レーザー回析・散乱法、体積基準で平均値）もしくは透過型電子顕微鏡（ＪＥＭ１２３０Ｒ：日本電子製、１％酢酸ウラニルで試料をネガティブ染色）を用いた。
メソ穴径の測定は、細孔分布測定装置（水銀圧入法あるいは窒素吸着法）を用いた。
条件と結果を表２、図３、４に示す。メソ孔径により、回収できるエクソソームの径、量が異なることがわかる。
図３より、必要な径のエクソソームを得るために、メソ孔を設定できる。たとえば、１４０ｎｍ径のエクソソームを得るには、１０ｎｍ前後のメソ孔径（Ａ領域）を使用する。１０５nｍ径のエクソソームを得るには、３００ｎｍ前後のメソ孔径（C領域）を使用する。１７０～１８０nｍ径のエクソソームを得るには、２０～２００ｎｍのメソ孔径（B領域）を使用する。
担体のメソ孔径を制限することで回収されるエクソソームの粒子径を制御することができる。
これにより得られる所望の粒子径のエクソソームは、ドラッグデリバリーシステム（Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ Ｓｙｓｔｅｍ：ＤＤＳ）で有効に使用され得る。
図４より、エクソソームをより多く回収するには、メソ孔径が、３０～２００ｎｍのシリカモノリスの担体を使用するのが好ましい。
また、図３，４より、メソ孔径３０～２００ｎｍの担体を用いると、エクソソームの回収率が高く、エクソソームの粒子径も一定のものが得られる。

＜実施例Ｃ＞
実施例Ａ４の条件で、洗浄液をＮａＣｌとして、その濃度を変えて、回収されたエクソソームの粒子径を測定した。説明しない事項は、実施例Ａ、Ａ４と同様である。
条件と結果を表３、図５に示す。
洗浄液の濃度は、０．１～１．５Ｍがよい。１．６Ｍ～１６Ｍもよい。０．１～０．９Ｍが好ましい。洗浄液としてＮａＣｌを用いたが、他の洗浄液でも同様の濃度がよい。

＜実施例Ｄ＞
実施例Ａ４の条件で、解離バッファー液をＭｇＳＯ_４、Ｎａ_２ＳＯ_４として、回収されたエクソソームの量を測定した。説明しない事項は、実施例Ａ４と同様である。
条件と結果を表４、図６に示す。
２価の金属イオンを含むＭｇＳＯ_４は、１価金属イオンを含むＮａ_２ＳＯ_４より、２倍近い量のエクソソームを回収できた。これは、多価の陽イオンのほうがカラムに修飾されたイオン交換基に対する親和性が強いためと思われる。２価の金属イオンを含む酸性溶液なら同様に効果がある。

（全体として）
カラムは、シリカモノリス以外のビーズや多孔質体でもよい。
なお、脂質二重膜に覆われた微粒子又は細胞外小胞は、細胞から放出される核を持たない（複製できない）脂質二重膜で囲まれた粒子である。脂質二重膜に覆われた微粒子又は細胞外小胞は産生機構の違いから（１）エクソソーム、（２）μベシクル、（３）アポトーシス小胞に分類されている。上記では、エクソソームだけでなく、（２）～（３）に同様に適用できる。つまり、脂質二重膜に覆われた微粒子又は細胞外小胞に適用できる。

<Example B>
Under the conditions of Example A4, the mesopore diameter of the silica monolith was changed and the particle diameter and amount of recovered exosomes were measured. Items not explained are the same as in Examples A and A4.
To measure the particle size of exosomes, use a particle size distribution analyzer (Lightsizer: made by Anton Paar, laser diffraction/scattering method, average value on a volume basis) or a transmission electron microscope (JEM1230R: made by JEOL, 1% uranyl acetate). Negative staining of the sample) was used.
The mesopore diameter was measured using a pore distribution measuring device (mercury intrusion method or nitrogen adsorption method).
The conditions and results are shown in Table 2 and Figures 3 and 4. It can be seen that the diameter and amount of exosomes that can be recovered differ depending on the mesopore diameter.
From FIG. 3, mesopores can be set in order to obtain exosomes with a required diameter. For example, to obtain exosomes with a diameter of 140 nm, a mesopore diameter (region A) of around 10 nm is used. To obtain exosomes with a diameter of 105 nm, use a mesopore diameter (region C) of around 300 nm. To obtain exosomes with a diameter of 170-180 nm, use a mesopore size of 20-200 nm (region B).
By limiting the mesopore size of the carrier, the particle size of the recovered exosomes can be controlled.
Exosomes with a desired particle size obtained thereby can be effectively used in a drug delivery system (DDS).
From FIG. 4, in order to recover more exosomes, it is preferable to use a silica monolith carrier with a mesopore diameter of 30 to 200 nm.
Furthermore, from FIGS. 3 and 4, when a carrier with a mesopore diameter of 30 to 200 nm is used, the recovery rate of exosomes is high and the particle size of exosomes is also constant.

<Example C>
The particle size of the recovered exosomes was measured under the conditions of Example A4, using NaCl as the washing solution and varying its concentration. Items not explained are the same as in Examples A and A4.
The conditions and results are shown in Table 3 and FIG. 5.
The concentration of the cleaning solution is preferably 0.1 to 1.5M. 1.6M to 16M is also good. 0.1 to 0.9M is preferred. Although NaCl was used as the cleaning solution, other cleaning solutions with similar concentrations are also suitable.

<Example D>
The amount of recovered exosomes was measured under the conditions of Example A4, using MgSO ₄ and Na ₂ SO ₄ as the dissociation buffer. Items not explained are the same as in Example A4.
The conditions and results are shown in Table 4 and FIG. 6.
MgSO ₄ containing divalent metal ions was able to recover nearly twice as many exosomes as Na ₂ SO ₄ containing monovalent metal ions. This seems to be because polyvalent cations have a stronger affinity for the ion exchange groups modified on the column. Acidic solutions containing divalent metal ions are similarly effective.

(as a whole)
The column may be beads or porous materials other than silica monolith.
Note that a microparticle or extracellular vesicle covered with a lipid bilayer membrane is a particle surrounded by a lipid bilayer membrane that does not have a nucleus (cannot replicate) and is released from a cell. Microparticles or extracellular vesicles covered with a lipid bilayer membrane are classified into (1) exosomes, (2) μ vesicles, and (3) apoptotic vesicles based on different production mechanisms. The above can be applied not only to exosomes but also to (2) to (3). That is, it can be applied to microparticles or extracellular vesicles covered with a lipid bilayer membrane.

According to the embodiment, exosomes can be recovered easily and in a state where the structure and function of the exosome are not impaired (state where the structure and function of the exosome are substantially unchanged). Therefore, according to the embodiment, it becomes possible to easily and precisely analyze substances contained inside exosomes. Therefore, the embodiments are useful in various technical fields, particularly in the pharmaceutical and medical fields.

1 Spin column 2 Column 3 Monolith body

Claims (8)

Microparticles or extracellular vesicles covered with a lipid bilayer are recovered from a sample containing microparticles or extracellular vesicles covered with a lipid bilayer. A collection method,
Bringing the sample into contact with a substance containing an amino group and a carrier capable of supporting the substance containing an amino group, or bringing the sample into contact with the substance containing the amino group supported on the carrier. a complex forming step in which a complex is formed by bonding the fine particles or extracellular vesicles covered with the lipid bilayer membrane to the amino group supported on the carrier;
The complex obtained by the complex formation step is brought into contact with a dissociation buffer solution containing metal cations to dissociate fine particles or extracellular vesicles covered with the lipid bilayer membrane from the complex. A method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, comprising: a dissociation step. 2. The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to claim 1, wherein the substance containing an amino group is a peptide containing ornithine. 3. The recovery method according to claim 1, wherein the particle size of the microparticles or extracellular vesicles covered with the lipid bilayer membrane to be recovered is controlled by limiting the pore size of the mesopores of the carrier. The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of claims 1 to 3, wherein the carrier is a silica monolith. The method for collecting fine particles or extracellular vesicles covered with a lipid bilayer membrane according to any one of claims 1 to 4, wherein the dissociation buffer solution is a solution containing magnesium as a divalent metal ion. The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of claims 1 to 5, wherein the concentration of the dissociation buffer solution is 0.1 to 1.5M. Recovery of microparticles or extracellular vesicles covered with a lipid bilayer membrane for carrying out the method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of claims 1 to 6. It is a kit,
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, comprising the substance containing the amino group, the carrier, and the dissociation buffer. Microparticles or extracellular vesicles covered with a lipid bilayer are recovered from a sample containing microparticles or extracellular vesicles covered with a lipid bilayer. A collection kit for
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, which includes a peptide containing polyornithine and a carrier on which the peptide is supported.