JP7160294B1 - Method for collecting microparticles or extracellular vesicles covered with lipid bilayer membrane and collection kit for collecting microparticles or extracellular vesicles covered with lipid bilayer membrane - Google Patents

Abstract

An object of the present invention is to provide a method for recovering microparticles or extracellular vesicles covered with a lipid bilayer membrane that can sufficiently separate extracellular vesicles. A method for collecting microparticles or extracellular vesicles covered with lipid bilayer membranes, comprising: By contacting the substance containing the amino group with a carrier capable of supporting the substance containing the amino group, the microparticles or extracellular vesicles covered with the lipid bilayer membrane and the amino group supported by the carrier bind to each other. a complex forming step of forming a complex formed by the above, and the complex obtained by the complex forming step is brought into contact with a dissociation buffer solution containing a metal cation, and the lipid bilayer is separated from the complex and a dissociation step of dissociating the membrane-covered microparticles or extracellular vesicles. [Selection drawing] Fig. 1

Description

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

Exosomes, which are microparticles or extracellular vesicles covered with a lipid bilayer membrane, are membrane-covered particles with a diameter of 20-150 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, exosomes have been suggested to have an intercellular communication function through the substances contained therein, and are attracting attention as biomarkers for various diseases and life phenomena.

Several methods have been reported as techniques for collecting exosomes, for example, (1) ultracentrifugation (Patent Document 1), (2) pellet down by centrifugation, (3) depending on particle size Fractionation, (4) immunoprecipitation, and the like are known.

JP 2017-038566 A Drug Delivery System 17-4 2002

However, the above 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 exosomes-antibody complexes is strong, in order to recover exosomes that do not impair the structure and function of exosomes by dissociating them from exosomes and antibodies, , must be dissociated under harsh conditions that involve protein denaturation. Therefore, it is difficult to recover exosomes without damaging the structure and function of exosomes.
Therefore, an object of the embodiments is to provide an exosome recovery method and an exosome recovery kit that can separate exosomes efficiently without impairing the structure and function of the exosomes.

In order to solve the above problems, a lipid bilayer-coated microparticle or extracellular vesicle is collected from a sample containing the lipid bilayer-coated microparticle or extracellular vesicle. A method for collecting dispersed microparticles or extracellular vesicles,
The sample, a substance containing an amino group, and a carrier capable of supporting the substance containing the amino group are brought into contact, or the sample is brought into contact with the substance containing the amino group supported on the carrier. a complex formation step of forming a complex in which the microparticles or extracellular vesicles covered with the lipid bilayer membrane and the amino groups supported on the carrier are bonded to each other;
The complex obtained by the complex formation step is brought into contact with a dissociation buffer solution containing metal cations to dissociate the microparticles or extracellular vesicles covered with the lipid bilayer membrane from the complex. and a dissociation step.

In addition, an exosome collection kit for performing the exosome collection method, which is characterized by including the peptide, the carrier, and the dissociation buffer, is used. Furthermore, an exosome collection kit for collecting exosomes from a sample containing exosomes, which contains a peptide containing polyornithine and a carrier carrying the peptide, is used.
Further, a collection kit for microparticles or extracellular vesicles covered with a lipid bilayer membrane for performing the method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane,
A collection kit for microparticles or extracellular vesicles covered with a lipid bilayer membrane is used, which contains the substance containing an amino group, the carrier, and the dissociation buffer. Furthermore, a lipid bilayer-covered microparticle or extracellular vesicle is recovered from a sample containing the lipid bilayer-covered microparticle or extracellular vesicle. A vesicle recovery kit is used for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane containing a peptide containing polyornithine and a carrier carrying the peptide.

The exosome recovery method and exosome recovery kit of the present application can sufficiently separate exosomes.

1 is a perspective view of a column for separating fine particles or vesicles according to an embodiment; FIG. 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)
An 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 modifications are possible within the scope of the claims. Embodiments also include embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples, respectively, within the technical scope of the embodiments.

[1. Exosome collection method]
A method for collecting exosomes according to one embodiment of the embodiment is a method for collecting exosomes, which collects exosomes from a sample containing exosomes, and includes a sample, a peptide containing ornithine, and a carrier capable of carrying the peptide. Or, by contacting the sample and the peptide supported on the carrier, the exosomes and the peptide supported on the carrier bind to form a complex that forms a complex Formation step, the complex obtained by the complex formation step, and a dissociation step of dissociating the exosomes from the complex by contacting a dissociation buffer containing metal cations.
Exosomes recovery method according to one embodiment of the embodiment has the above configuration, the following effects (1) to (3).

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

(2) Since ornithine binds to the membrane of exosomes, exosomes can be widely captured and collected regardless of the expression state of antigen molecules in exosomes.

(3) Dissociate exosomes from the complex under mild (mild) conditions using a dissociation buffer containing metal cations, which does not affect 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, the structure and function of the exosomes are not impaired, or the structure and functions of the exosomes are almost intact).

By recovering exosomes that do not impair the structure and function of exosomes (or that the structure and function of exosomes are almost unimpaired), for example, (1) functional analysis such as physiological effects of exosomes, and (2) biomarkers (proteins derived from exosomes, nucleic acids such as miRNA) and the like.

In addition, exosomes are expected to be applied to the treatment of a wide range of diseases such as cancer, Alzheimer's disease, myocardial infarction, cerebral infarction, neurodegenerative diseases, knee osteoarthritis and infectious diseases. Therefore, by recovering exosomes in a state in which the structure and function of exosomes are not impaired (or in a state in which the structure and function of exosomes are not nearly impaired), the recovered exosomes themselves are used as (3) therapeutic drugs, and (4) drug delivery. Can be used as a carrier for the system. In particular, the above (3) use of exosomes as a therapeutic agent includes (A) supplementary use in regenerative medicine, (B) use for immunoregulation, and (C) use as a vaccine. It is

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

(sample)
In the recovery method of the embodiment, the "exosome-containing sample" (also referred to herein simply as a sample) is not particularly limited as long as it is a mixture containing exosomes, for example, containing exosomes Biological samples are included.

As used herein, the term "biological sample" means a specimen collected from the body of a living organism, an extract from the living organism, a culture solution, or a microbial component. Examples of biological samples include blood, plasma, serum, saliva, urine, tears, sweat, breast milk, amniotic fluid, cerebrospinal fluid (cerebrospinal fluid), bone marrow fluid, pleural fluid, ascites, synovial fluid, aqueous humor, Examples include, but are not limited to, vitreous body, extracts from the living body itself or some tissues, culture fluids, and components derived from microorganisms. Selection of a biological sample can be appropriately set by a person skilled in the art according to the purpose. For example, from the viewpoint of ease of collection, blood, plasma, serum, saliva, urine, etc. are preferably used. In addition, extracts of each organ, culture solutions after animal/plant cell culture, microorganisms or microorganism-derived components, and the like are included.

In one embodiment, the origin of the biological sample is not particularly limited as long as it is derived from a species that possesses exosomes. Mammals, for example, are used as biological species from which biological samples are derived. Examples of mammals include mice (Musmusculus), rats (Rattus norvegicus), cats (Felis catus), dogs (Canis lupus familiaris), pigs (Sus scrofa domesticus), sheep (Ovis aries), cows (Bosaurus), green monkeys ( Cercopithecus aethiops), humans (Homosapiens), and the like. As the culture solution, a culture solution obtained by culturing stem cells, cancer cells, oral mucosa-derived cells, or the like can be used.
In addition, extracts from leaves, fruits, and fruit juice components of plants are used. Examples of plants include grapes, grapefruits, acerola, dragon fruit, ginger, carrots, citrus fruits such as lemons, broccoli, and apples. Microorganisms include Aspergillus oryzae, lactic acid bacteria, yeast and the like. Food products (miso, soy sauce, sake, yogurt, milk, and other fermented foods) are also included.

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

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

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

(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. A carrier of embodiments can bind to a peptide of embodiments bound to an exosome to form a complex that binds in the order exosome-peptide of embodiments-carrier of embodiments.

In the recovery method of the embodiment, by using a carrier, it is possible to efficiently and simply recover a complex containing exosomes, thereby enabling highly efficient recovery of exosomes.

Other configurations of the carrier are not particularly limited as long as the carrier is a structure capable of directly or indirectly supporting (holding) 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 bound to the carrier, such as glass, nylon membrane, semiconductor wafer, latex particles, cellulose particles, μ beads, silica Beads, magnetic particles, monolithic carriers and the like are included. As the carrier of the embodiment, a monolithic carrier is particularly preferable in that the collection of complexes containing exosomes and the collection of exosomes can be easily performed. Monolith 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 and the carrier of the embodiment)
As described above, by binding the peptide of the embodiment to the carrier, the peptide of the embodiment can be supported by the carrier of the embodiment.

In one embodiment, the method for binding the peptide to the carrier is not particularly limited, and a well-known method can be used as appropriate. Also, 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 are particularly preferable as the carrier. Embodiments are a monolith body in which the surface of the monolith body is coated with a polymer and the polymer is modified with an ion-exchange group, and a column for separating microparticles or vesicles comprising such a monolith body. Also, the ion exchange group is not particularly limited, but an anion exchange group can be used.

Microparticles or vesicles are particles with a particle diameter of 1 nm to several 300 nm. may or may not contain substances such as proteins and nucleic acids. Microparticles or vesicles include those made by organisms and those produced industrially. Microparticles or vesicles include, but are not limited to, drug-loaded nanomedicine, including liposome formulations, protein-conjugated formulations, exosomes, and viruses. The surface of many nanomedicine is negatively charged to enable stable circulation within blood vessels. Drugs included in nanomedicine include, but are not limited to, low-molecular-weight compounds, high-molecular-weight antibodies, oligonucleic acids, peptides, proteins, DNA, messenger RNA, and μRNA (miRNA). Also, the matrix of the microparticles or vesicles can be composed of, but not limited to, gold, silver, carbon, and the like.

Separation of microparticles or vesicles includes separation for analysis, purification, etc. of microparticles or vesicles.
A monolithic body has a three-dimensional net-like skeleton structure and is formed by the skeleton and is continuous from one end to the other end of the skeleton, in other words, it penetrates from the upper end to the lower end of the skeleton and communicates with each other. It is a porous body that is a single structure with macropores, which are through-holes, and is an integrated porous body. In addition, the monolith body has a structure with mesopores, which are pores smaller than the through-holes in the skeleton in the through-holes (macropores) and communicates or does not communicate between the through-holes, and a structure without mesopores. exists. The monolith body preferably has through-holes forming a continuous and regular three-dimensional network structure, but is not limited to this. Moreover, it is preferable that the through-hole has a circular or nearly circular cross-section in a direction perpendicular to the axis of the monolith body.

Macropores are cultured cells, proteins contained in blood, etc., fine particles or vesicles, low-molecular substances released or released from fine particles or vesicles, nanomedicine, and drugs contained in nanomedicine. , act as passages for released drugs and free drugs from nanomedicine, and mesopores are small molecules released or released from microparticles or vesicles, drugs contained in nanomedicine, and released drugs from nanomedicine. and the function of separating free drugs. In addition, by selecting the size of the mesopores according to the size of the microparticles or vesicles and the size of the substances released or released from the microparticles or vesicles, it is possible to It is possible to separate fine particles or vesicles from fine particles or vesicles and released or free substances from small molecules to macromolecular substances such as antibodies and nucleic acids.

The size of the mesopores is believed to alter their binding strength to microparticles or vesicles, and the varying surface area results in the mesopores with the highest recovery. That is, if the mesopores are smaller than the target microparticles or vesicles, they will not be able to trap inside the mesopores, whereas if the mesopores are made large enough, the surface area will decrease, thus allowing the microparticles to trap. Or the number of vesicles becomes smaller. As a condition for maximizing the recovery rate, it is preferable to set the mesopore size to 0.005 to 200 nm.

On the other hand, it has been found that if the mesopores are made sufficiently small, it becomes difficult to collect large microparticles or vesicles. This is probably because large particles or vesicles are partly trapped in the mesopores, making the cations in the dissociation solution inaccessible to the anionic groups in the particles or vesicles and mesopores. be done. Being able to selectively obtain small microparticles or vesicles is effective when using the microparticles 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 the macropores through which the liquid containing the sample flows is not particularly limited, but is preferably 1 to 50 μm. Basically, the smaller the diameter of the macropores, the more the number of contact times, but the resistance to the flow of the liquid containing the sample also increases. The diameter of the pores is preferably 1-20 μm.

As the monolith body, a ceramic monolith body including a silica monolith body and a glass monolith body and a polymer monolith body can be used. The raw material of the monolithic body is not particularly limited, but inorganic materials such as ceramics and glass are preferable. Furthermore, in the case of a biological sample component, adsorption of non-objects may occur depending on the raw material for forming the monolith, and a silica monolith 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 produced by sol-gel synthesis carried out singly or after mixing these raw materials is preferred.

The production of the silica monolith body is not particularly limited, but can be carried out as follows using a generally known method such as the sol-gel method. First, a water-soluble polymer is dissolved in an aqueous solution of acetic acid, the above-mentioned raw materials are added to the solution, a hydrolysis reaction is performed, and the resulting solution is solidified after stirring. Then, the solidified gel is immersed in an aqueous ammonia solution, dried and heated to obtain a porous silica monolith made of amorphous silica. The higher the temperature of the ammonia solution immersion, 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. Also, the macropores can be controlled by the ratio of raw materials and the temperature during solidification.

As a specific example, when the sol-gel method is used, 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 having an inner diameter of 8 mm and a length of 50 cm, for example, and the pipe is sealed and incubated for 20 hours. In this manner, a cylindrical monolithic sol, for example, a monolithic sol having 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 aqueous ammonia solution, incubation is performed to form mesopores, and the gel is washed with methanol to obtain a silica monolith.

By changing the PEO concentration from 1% to 20%, the macropore diameter of the monolith porous body can be changed from 0.1 μm to 200 μm. Further, by changing the concentration of the aqueous ammonia solution from 0.01% to 1%, the diameter of the mesopores of the monolithic porous material 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. is produced (formation of sol), further hydrolyzed to promote polymerization and cross-linking, and synthesized by making a three-dimensional network skeleton structure (formation of gel). Finally, it is baked to completely gel and harden. If the baking temperature is 300° C. or higher, the gelation is completed. By setting the firing temperature to a high temperature of about 1100° C., the silica monolith can be made non-porous. If the gelation is completed, it will not be denatured even by heat such as fusion bonding.

The glass monolith body and the ceramic monolith body are also porous bodies having the same structure as the silica monolith body, although the raw materials and manufacturing methods are different.

Ceramic monolithic bodies made from ceramics are not particularly limited, but may be silica, titanium, zirconium, hafnium or other group 4 element compounds, alumina silicate A (sintered hard magnetic particles), silica sand, and alumina. , alumina silitic B (sintered chamotte particles), porous mullite, diatomite, and the like.

In this case, the production method is not limited to this, but for example, ceramic particles (hard magnetic powder, silica, alumina, chamotte, etc.) having a certain particle size, a pore-forming material such as crystalline cellulose, and a suitable dispersion It can be produced by mixing with a solvent, molding, and sintering. More specifically, but not limited to, for example, silica sand, boric _acid , soda ash, and various metal oxides such as _{Al2O3 ,} ZrO2, _ZnO2 , _TiO2 , SnO2, and _{MgO2 are} mixed. , 1200 to 1400° C., a ceramic monolith having a composition of metal oxide—Na ₂ O—B ₂ O ₃ —SiO ₂ —CaO can be produced.

The glass monolith body made from glass is not particularly limited, but examples thereof include NaO--B ₂ O ₃ --SiO ₂ --CaO-based compositions such as Al ₂ O ₃ , ZrO ₂ , ZnO ₂ , TiO ₂ , In some cases, glass to which various oxides such as SnO ₂ and MgO ₂ are added is used. In this case, the production method is not limited to this, but for example silica sand, boric acid, soda ash and alumina are mixed and melted at 1200 to 1400° C. to make a lump of glass. The composition becomes Na ₂ O—B ₂ O ₃ —SiO ₂ —CaO and becomes a porous glass body.

After molding this at 800 to 110° C., an unsplit phase borosilicate glass is obtained, which is separated into a SiO ₂ phase and a B ₂ O ₃ --NaO--CaO phase by heat treatment, and a glass with an SiO ₂ skeleton remaining by acid treatment. A monolithic body can be manufactured. When the raw material of the monolithic body is inorganic, a monolithic body having a uniform pore distribution can be produced by changing the conditions during the heat treatment.

The ceramic monolith body and the glass monolith body can control the diameter of macropores by changing the composition and phase separation conditions, and can form mesopores at the same time as macropores.

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

The material of the polymer monolith is not particularly limited, but specific examples include copolymers such as styrene-divinylbenzene, methacrylic acid ester, and acrylamide. The monolithic body may be constructed by introducing an anion exchange group into a polymer that is a copolymer of these. The monomer constituting the polymer monolith is not particularly limited as long as it has copolymerizability, and a monomer suitable for the purpose may be selected. Moreover, the hydrophobicity can be controlled by adjusting the mixing ratio of the monomers. Further, the uniformity of the macropore diameter and mesopore diameter of the polymer monolith is controlled by the mixing ratio of the porogen that dissolves the monomer, the type of solvent, the concentration of the monomer, the ratio of the cross-linking agent and the polymerization initiator, the polymerization temperature, and the like. can do

As an example of manufacturing a polymer monolith, an anion-exchange polymer monolith using a ring-opening reaction of epoxy groups will be described. 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 prepare a GMA-based polymer monolith. can be done.

The surface of the monolithic body is coated with a polymer, and the surface of the polymer is modified with ion exchange groups. Ion exchange groups are exposed on the surface of the polymer coated on the surface of the monolith, in other words, on the surfaces of the macropores and mesopores of the monolith. Ion-exchange chromatography, in which embodiments are utilized, is a technique that utilizes the charge state of molecules in microparticles or vesicles to reversibly bind and cleave oppositely charged carriers. In addition to reversible interactions with ion-exchange groups, binding between molecules of microparticles or vesicles and carriers may involve, for example, Van der Waals forces and hydrophobic interactions, but generally these is a very weak bond compared to ionic bonds and can be neglected by controlling the surface charge of microparticles or vesicle molecules.

However, when using a monolithic silica matrix as a carrier, not only ionic bonds but also fine particles or vesicles are strongly adsorbed to the silanol groups remaining after surface treatment of the monolithic body, and are eluted only by breaking the ionic bonds. I can't Therefore, in order to eliminate the influence of the residual silanol groups, the surface of the monolith is coated with a polymer to cover the silanol groups with the polymer, thereby eliminating the influence of the silanol groups and enabling the elution of fine particles or vesicles. A polymer monolith can be used as the monolith. With such a structure, there is no influence of silanol, and since the surface of the monolith body is previously coated with a polymer, it is unnecessary to separately coat the monolith body with a polymer.

The polymer that coats the surface of the monolith may be configured by directly bonding a polymer material to the surface of the monolith, or may be configured by bonding to the surface of the monolith via a linker. When a linker is interposed, a preconfigured polymer may be bound to the linker, or a monomer may be bound to the linker, and a polymer formed by graft polymerization of the monomer may be bound.

A linker 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.

Moreover, the coating of the polymer on the surface of the monolith body may be configured such that the polymer itself having anion exchange ability is coated. Although such a configuration is not particularly limited, it can be configured as follows. The surface of the silica monolith is modified with a linker with a silane coupling agent having an epoxy group, isocyanate, isothiocyanate, acid anhydride, etc., which is reactive with amino groups. DEAE, polyallylamine, aminoethylated acrylic polymers, acrylamide-diallyldimethylammonium copolymers, poly(diallyldimethylammonium) polymers, poly(trimethylaminoethyl methacrylate) polymers can be coated. The polymer having anion-exchange ability may have an anion-exchange group in itself, or may have anion-exchange groups introduced into the polymer in advance.

Linkers targeting silanol groups on silica monolith include 3-glycidoxypropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane, 3-trimethoxysilylpropylsuccinic anhydride, and trimethylsilylisothiocinate. A silane coupling agent such as silane coupling agent can be used.

Further, the silanol group is N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane having an amino group, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxy An amino group-introduced silica monolith can be produced by modification with a silane coupling agent such as silane or 3-aminopropyltriethoxysilane. Also, a carboxyl group-introduced silica monolith can be produced by introducing 3-trimethoxysilylpropylsuccinic anhydride into silanol groups and then subjecting the succinic acid to a ring-opening reaction.

Homobifunctional cross-linking agents and hetero-bifunctional cross-linking agents having functional groups such as isothiocyanate, isocyanate, acylazide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, and carbodiimide, which are functional groups that form chemical bonds with the above amino groups and carboxyl groups. Agents can also be used as crosslinkers. As homobipolarity, for example, DSS (disuccinimidylsuberate), DMP (dimethylpimelimidate), etc. can be used, and immobilization via an amino group is possible. 3-(Dimethylaminopropyl)carbodiimide and the like can be used, and cross-linking is possible through carboxyl groups and amino groups.

The anion exchange group used 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. Further, as the anion exchange group, quaternary ammonium (Q), which is a strong anion exchange group, diethylaminoethyl (DEAE), diethylaminopropyl (ANX), etc., which are weak anion functional groups, can be used. It can be appropriately selected according to the pH range to be used and the fine particles or vesicles to be separated. Furthermore, as the anion exchange group, a strong anion exchange functional group having a quaternary amine such as trimethylammonium, a polyamine having primary and secondary amines, a basic polyamino acid such as polylysine, poly Arginine, polyhistidine, polyornithine, polydiaminobutyric acid are preferred.

The microparticles or vesicles containing nanomedicine and the released material containing the released drug from the microparticles or vesicles and the free material containing the free drug, which are contained in the mobile phase and sent to the monolithic body, pass through the macropores. At this time, the fine particles or vesicles are bound to the ion-exchange groups including the anion-exchange groups exposed on the sides of the macropores excluding the mesopores. and is released from the monolith body. On the other hand, released substances and free substances from microparticles or vesicles pass through macropores while entering mesopores, and are released from the monolith body.

In one embodiment, the monolithic body is coated with a polymer having ion-exchange groups according to the charge characteristics of the particles or vesicles to be separated, thereby eliminating adsorption of the particles or vesicles to silanol and interacting with the ion-exchange groups. Microparticles or vesicles are retained by action and eluted by ionic strength. On the other hand, by passing through the mesopores, the released substances and educts from the microparticles or vesicles undergo ion-exchange groups in the mesopores and ion-exchange groups in the macropores and the microparticles according to the charge characteristics of the educts and educts. Alternatively, they can be separated by interacting with a different strength than the interaction with the vesicles.

Surface coating of the monolith body with a polymer and modification of the surface of the polymer with ion-exchange groups can be carried out as follows. Covering the surface of the monolith body includes cases where the surface is not completely covered and partially exposed. As a specific example, when the silica monolith is coated with acrylamide, the method is not limited to this method, but a linker is bound to the silica surface of the silica monolith, and the silica monolith is coated with a polymer by graft polymerization. can be done.

Specifically, the N-(3 -triethoxysilylpropyl)methacrylamide to prepare a silane as a linker (spacer), 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 linker-modified silica monolith was treated 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 relative to the weight of the reaction solution) and peroxodiacyl chloride. Ammonium sulfate-containing methanol aqueous solution is reacted at 70°C for 15 hours to coat the surface of the monolith with a polymer and introduce ion-exchange groups, followed by washing with water and acetone to coat the monolith with a polymer and modify the anion-exchange groups. A silica monolith was obtained.

Thus, as a method of coating the surface of the monolith with a polymer, there is a method of attaching a linker to the surface of the monolith and graft-polymerizing the monomer to the linker. can also In addition, as a method of modifying a polymer with ion exchange groups, in addition to a method of modifying a polymer with ion exchange groups, a method of polymerizing a monomer modified with ion exchange groups to form a polymer and introduce ion exchange groups. can be adopted. Methods for directly bonding the polymer material to the surface of the monolith body include the following methods, but are not limited to these methods.

As a method of coating the silica monolith with polylysine, a silane agent (5 wt %) having an epoxy group, such as 3-glycidoxypropyltrimethoxysilane, was mixed and then purged with nitrogen. 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 4000 to 15000 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 up to about 500,000 can be used. By the same method, other basic polyamino acids and polymers in which basic amino acids and other amino acids are combined can be introduced into the silica monolith.

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

The surface of the 3 mm diameter monolith obtained as described above is coated with a polymer and the polymer is modified with ion-exchange groups. Covering the sides of the body and then inserting it into a stainless steel tube (6 mm OD, 1.2 mm thick, 50 mm long), a second polymer such as epoxy resin is placed between the monolith body and the stainless steel tube, which is coated on the sides with a first polymer layer. A polymer layer was packed to obtain a column for separating microparticles or vesicles.

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 a low liquid feeding pressure can be produced with sufficient performance. The end of the stainless steel tube can be cut off and used as a cartridge-type microparticle or vesicle separation HPLC column (3 mm ID×50 mm). The cartridge type column can be used as a column by inserting it into a special holder, which is also a stainless steel pipe, and tightening it with an end nut connectable to a 1/16 inch pipe.

The monolithic bodies of the embodiments can also be used for solid-phase extraction columns that enable easy separation and purification. For example, as a solid-phase extraction column, as shown in FIG. 1, a form (recovery kit) such as a spin column 1 configured by installing a monolithic body 3 in an empty column 2 regardless of the material and diameter thereof is used. can take
The solid-phase extraction column here refers to a monolithic cartridge that replaces conventional methods such as column chromatography packed with large particles and thin-layer chromatography, which are complicated and require special equipment and techniques. It was established by the method of packing the column. In addition to suction by a centrifuge, vacuum pump, aspirator, or the like, pressurization can be used to pass the liquid through, making it possible 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 using devices such as pumps and purification devices featuring low-pressure pumps can also be used. is.

As a conventional technique 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 exosome elution time span is about 4 to 5 minutes, and due to size exclusion chromatography, column equilibration before sample application requires careful attention, so it is not easy for anyone to do it. It is not a method that can be used in time. On the other hand, using the spin column 1 as a column for separating particles or vesicles of the embodiment, as shown in FIG. It has become possible.

(dissociation buffer)
In the recovery method of the embodiment, in the dissociation step to be described later, the complex and the metal cation are contained in a pH-adjusted dissociation buffer (hereinafter, appropriately referred to as the "dissociation buffer of the embodiment"). dissociates the exosomes from the complex and recovers the exosomes. The dissociation buffer of the embodiment does not contain a surfactant and / or protein denaturant, etc., or contains it at a very low concentration, and exosomes can be dissociated from the complex under mild (mild) conditions , Exosomes can be recovered in a state where the structure and function of exosomes are not impaired (a state in which the structure and function of exosomes are almost not impaired). In addition, the effect that exosomes can be easily recovered in a state in which the structure and function of exosomes are not impaired (a state in which the structure and functions of exosomes are almost unimpaired) is a remarkable and advantageous effect of the embodiment.

The metal cation contained in the dissociation buffer of the embodiment is not particularly limited. divalent metals such as ions, calcium ions, zinc ions, nickel ions, barium ions, copper(II) ions, iron(II) ions, tin(II) ions, cobalt(II) ions, and lead(II) ions cations, and trivalent metal cations such as aluminum ions and iron (III) ions, and the like. 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 contains sodium ions, potassium ions, or magnesium ions among the metal cations described above, alone or in combination. preferable. Various buffers 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, etc., phosphate buffers, carbonate buffers, citrate buffers, Various buffers such as can be used. Moreover, a reagent such as a bactericide can be added to the dissociation solution according to the intended use.

The purpose of the embodiments is to collect exosomes simply and without impairing the structure and function of the exosomes (or almost without impairing the structure and function of the exosomes). Therefore, in the recovery 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 without damaging exosomes (almost without) means that the exosomes in the sample are separated from substances other than exosomes in the sample (eg, proteins, etc.) and recovered. In other words, in the recovery method of the embodiment, "recovering exosomes without impairing the structure and function of exosomes (or in a state where the structure and function of exosomes are almost not impaired)" means the membrane structure of exosomes (specifically Specifically, the exosomes in the sample are exosomes in the It means separating and recovering from substances other than exosomes.

The dissociation buffer may contain substances other than metal cations to the extent that it does 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 is possible to 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 concentration of cations can vary depending on the binding strength between the peptide and exosomes of the embodiment used and the type (valence) of the metal cation, so after considering the optimal concentration as appropriate, You just have to decide. Although the concentration of the metal cation in the dissociation buffer of the embodiment is not particularly limited, it is preferably, for example, 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 salt concentration is low, it can be expected to obtain more exosomes, but there is a concern that contaminants such as proteins may be mixed. It is possible to appropriately select the salt concentration according to the intended use.

Also, the pH of the dissociation buffer can be set appropriately. 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. If the pH of the dissociation buffer is within the above range, it is preferable because it does not affect the membrane structure of exosomes and the structure of proteins present in the membrane.
[1-2. process]

(Complex formation step)
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) is a sample containing exosomes, the peptide of the embodiment, and the carrier of the embodiment. Or, by contacting the sample with the peptide of the embodiment supported on the carrier of the embodiment, the exosome and the peptide supported on the carrier are combined. It is a complex formation step of forming a body (complex in which exosomes-peptides of embodiments-carriers of embodiments are bound in this order, hereinafter referred to as "complexes of embodiments"). That is, in the complex formation step of the embodiment, exosomes after contacting the sample with the peptide of the embodiment and the carrier of the embodiment in a state in which the peptide of the embodiment is not supported on the carrier of the embodiment - The peptide of the embodiment - the carrier of the embodiment may be bound in this order to form a complex, or the peptide of the embodiment is already supported on the carrier of the embodiment, and then the peptide and the sample are combined. may be brought into contact to form the composite of the embodiment. However, it can be said that the latter mode is more preferable from the viewpoint of forming the composite of the embodiment more efficiently.

As a method of contacting a sample containing exosomes with the peptide of the embodiment and the carrier of the embodiment, the exosome in the sample and the peptide of the embodiment and the carrier of the embodiment are bound, respectively, Other methods are not limited as long as they are carried out under conditions capable of forming a complex of. For example, a method of adding the peptide of the embodiment and the carrier of the embodiment to a sample and mixing them can be mentioned. The order of addition and mixing when the sample, the peptide of the embodiment, and the carrier of the embodiment are brought into contact is not particularly limited. For example, (1) the peptide of the embodiment and the carrier of the embodiment may be added to the sample at the same time and mixed, (2) the peptide of the embodiment is added to the sample and mixed, and then the The carrier of the embodiment may be added and mixed, or (3) the carrier of the embodiment may be added to the sample and mixed, and then the peptide of the embodiment may be added and mixed. However, the peptide of the embodiment binds to exosomes, and the peptide of the embodiment and the carrier of the embodiment bind to the procedure of (2) above may be preferable. Contacting the sample, the peptide, and the carrier by the methods described above can form a complex of the embodiments comprising the exosome, the peptide, and the carrier.

Further, the sample, as a method of contacting the peptide of the embodiment supported on the carrier of the embodiment, exosomes in the sample and the peptide of the embodiment supported on the carrier of the embodiment bind Other methods are not limited as long as they are carried out under conditions that allow formation of the complexes of the embodiments. For example, a method of adding the peptide of the embodiment carried on the carrier of the embodiment to the sample and mixing the peptide can be used.
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 forms the complex of the embodiment. It is not limited as long as it is a sufficient time for the process, and it can be determined after examining the optimum conditions as appropriate.

In addition, in the contact between the sample containing exosomes, the peptides of the embodiments, and the carriers of the embodiments, for example, a columnar or cylindrical container (column) prepared by filling the carriers of the embodiments, including the carrier A column (referred to herein as a "carrier column") can also be used. As a method of obtaining a complex of the embodiment by passing a solution containing a sample and a solution containing a peptide through a carrier column, the exosomes in the sample and the peptide of the embodiment and the carrier of the embodiment bind to each other. However, other methods are not limited as long as they are carried out under conditions capable of forming the composite 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 the carrier column, and the solution is passed through the carrier of the embodiment, thereby performing After binding the peptide in the form of, 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 previously applied to the sample By adding and mixing, etc., the sample and the peptide of the embodiment are brought into contact to form a complex (exosome-peptide complex) in which exosomes and peptides in the sample are bound, and then (3 ) A solution containing the sample and a solution containing the peptide of the embodiment are added to the carrier column at the same time, and the solutions are passed through to form the complex of the embodiment.
In the above (1) to (3), the contact time between the carrier column and the peptide and / or sample, or the sample containing the exosome-peptide complex is determined by exosomes, peptides and carriers in the sample. It is preferred that the time be sufficient to form the complex of

The above time is the flow rate of the sample solution (sample containing exosomes and / or a solution containing the peptide of the embodiment, or a solution containing the exosome-peptide complex) to the carrier column, or the addition of the sample solution It can be appropriately set by adjusting the time, etc., from the start of feeding to the start of passing the solution.

In addition, in the contact with the peptide of the embodiment supported on the carrier of the embodiment and the sample containing exosomes, for example, the peptide of the embodiment is supported in a columnar or cylindrical container (column) A column prepared by packing a carrier (referred to herein as a "peptide column") can also be used. By passing a solution containing the 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 with each other to obtain the complex of the embodiment. The contact time between the peptide column and the sample is preferably sufficient time for the exosomes in the sample and the peptides of the embodiments supported on the carriers of the embodiments to form the complexes of the embodiments. . The time can be appropriately set by adjusting the flow rate of the solution containing the sample or the time from the addition of the solution to the start of passage.

A conventionally known method can be used as a physical method for passing the sample solution through the carrier column or the peptide column. For example, the following methods (1) to (3) can be mentioned.

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

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

(3) After adding a sample solution to one of the carrier columns (or peptide columns), the carrier columns (or peptide columns) are loaded into a centrifuge tube, and the centrifuge tube is centrifuged to obtain a carrier column (or This is a method in which the sample solution is passed through a peptide column). 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 in order to prevent drying.

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

As a method for contacting the complex of the embodiment with the dissociation buffer of the embodiment, other methods are not limited as long as it is performed under conditions that dissociate exosomes from the complex of the embodiment. . For example, there is a method of adding the dissociation buffer of the embodiment to a container containing the complex of the embodiment and mixing. 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 a sufficient time for exosomes to dissociate from the complex. By contacting the complexes of the embodiments with the dissociation buffer of the embodiments, the exosomes are dissociated from the complexes of the embodiments and migrate to the dissociation buffer. Then, 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. Moreover, the peptide of the embodiment may be contained in the dissociation buffer recovered after the separation. 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 separation, in the recovered dissociation buffer Preferably, the peptides of the embodiments are not included.

Further, when a column is used in the complex formation step, the dissociation buffer of the embodiment is passed through the column in which the complex of the embodiment is formed in the complex formation step, thereby obtaining the dissociation buffer of the embodiment. can be brought into contact with the dissociation buffer of the embodiment. Physical methods for passing the dissociation buffer of the embodiment through the column include the methods (1) to (3) described in the section (complex formation step). When the column and the dissociation buffer of the embodiment are contacted, the exosomes are dissociated from the complex and move 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 complex formation step and the dissociation step described above. Other steps include, for example, a washing step and a recovery step.

(Recovery 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. For the recovery step, conventionally known methods can be suitably employed depending on the complex formation step, the carrier, and the like.

The carrier is [1-1. In the case of the structure described in (Carrier) section of Material], the carrier containing the composite of the embodiment can be recovered by centrifugation. For example, by centrifuging, it is possible to collect the carrier containing the complex of the embodiment.

Further, when the carrier is a magnetic particle, the carrier can be easily recovered by applying a magnetic force from the outside using a conventionally known magnetic body such as a 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 centrifugal method: (1) the carrier which is a magnetic particle is Because it is attracted to the magnetic material, it is possible to remove the supernatant almost completely, and the purity of the exosomes obtained is high; (2) when removing the supernatant, the magnetic particles are removed together. The risk is reduced and the loss of the resulting exosomes can be prevented (the exosome recovery rate can be improved).

When a carrier column or a peptide column is used in the complex forming step of the embodiment, the carrier containing the complex of the embodiment remains in the column. Therefore, the recovery process described above is required.

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

Washing solutions used in the washing step include, for example, physiological saline or phosphate buffered saline (PBS).

When an embodiment of the embodiment includes a recovery step, the cleaning step includes methods such as the following. (1) By mixing the recovered carrier containing the complex of the embodiment with the washing liquid, the carrier is resuspended in the washing liquid. (2) Again, the carrier containing the complex of the embodiment is recovered (that is, the carrier containing the complex of the embodiment and the washing solution are separated).

In addition, when a carrier column or a peptide column is used in the complex forming step of the embodiment, an appropriate amount of the washing liquid may be passed through the column after the complex of the embodiment has been formed.

[2. Exosome recovery kit]
An exosome recovery kit according to one embodiment of the embodiment (hereinafter referred to as "kit of the embodiment" as appropriate) is a kit for performing the recovery method of the above-described embodiment, and the above-described embodiment , a carrier of embodiments, and a dissociation buffer of embodiments. Therefore, for the description of the configuration of the kit, see [1. exosome collection method] can be used.

The peptide of the embodiment and the carrier of the embodiment included 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 previously supported on the carrier of the embodiment. Alternatively, each may be included separately.

In addition, the kit of the embodiment may contain magnetic particles, polyornithine-immobilized magnetic particles, a magnetic substance such as a magnetic stand, biotin, and the like.
Moreover, the kit of the embodiment may include a container (for example, bottle, plate, tube, dish, column, etc.) containing a specific material in addition to the above configuration. Kits of embodiments may include containers containing diluents, solvents, wash solutions, or other reagents. The term "comprises" used in the description of the kit of the embodiment can mean the state of inclusion in any of the individual containers that make up 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 that collects exosomes from a sample containing exosomes, and includes a peptide containing polyornithine and a carrier carrying the peptide. This recovery 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, which recover the microparticles or extracellular vesicles covered with the lipid bilayer membrane from a sample containing the microparticles or extracellular vesicles covered with the lipid bilayer membrane A method for collecting outer vesicles, comprising:
The sample, a substance containing an amino group, and a carrier capable of supporting the substance containing the amino group are brought into contact, or the sample is brought into contact with the substance containing the amino group supported on the carrier. a complex forming step of forming a complex in which the microparticles or extracellular vesicles covered with the lipid bilayer membrane and the amino group supported on the carrier are bound;
The complex obtained in the complex forming step is brought into contact with a dissociation buffer solution containing metal cations to dissociate the microparticles or extracellular vesicles covered with the lipid bilayer membrane from the complex. and a dissociation step.
[2] The substance containing an amino group is an ornithine-containing peptide.
[3] The particle size of the collected microparticles or extracellular vesicles covered with the lipid bilayer membrane 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, and 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 microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of [1] to [4], wherein the dissociation buffer solution is a solution containing magnesium as divalent metal ions. Use the collection method of
[6] The concentration of the dissociation buffer solution is 0.1 to 1.5M. Use collection methods.
[7] Microparticles or cells covered with a lipid bilayer membrane for performing the method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to any one of [1] to [6] An outer vesicle recovery kit,
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane is used, which contains the substance containing an amino group, the carrier, and the dissociation buffer.
[8] Microparticles or cells covered with a lipid bilayer membrane, which recover the microparticles or extracellular vesicles covered with the lipid bilayer membrane from a sample containing the microparticles or extracellular vesicles covered with the lipid bilayer membrane An outer vesicle recovery kit,
A collection kit for microparticles or extracellular vesicles covered with a lipid bilayer membrane containing a peptide containing polyornithine and a carrier supporting the peptide is used.

An embodiment will be described below in more detail with reference to examples, but the embodiments are not limited only to these examples.
<Example A>
A peptide-supported column (modifier-immobilized column) was prepared. A silica monolith was used as a carrier.

A silica monolith body (3 mm ID×50 mm length) was used with 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 a positive charge to the surface of the silica monolith body. In order to incorporate a trimethylammonium group into the surface of the silica monolith, a column using a silica monolith modified with the same functional group (trimethylammonium group) by two different functional group modification methods of acrylamide and siloxane (hereinafter referred to as "trimethylammonium group (also referred to as "modified column") was used.

The surface modification of the silica monolith body was performed as follows. As an example, the surface of a silica monolith is coated with acrylamide by polymerizing an acrylamide monomer having a trimethylammonium group, and a column using a polymer-coated silica monolith in which anion exchange groups are introduced (hereinafter "acrylamide coating column”) was used. N-(3-triethoxysilylpropyl prepared by adding a solution of methacryloyl chloride dropwise under toluene and stirring 3-aminopropyltriethoxysilane and triethylamine in toluene at 0° C. for 0.5 hours or more. ) Preparing silane with methacrylamide, reacting the silica monolith with a 1:1:1 mixture of silane, pyridine and toluene at 80° C. for 12 hours under a nitrogen atmosphere to obtain a linker-modified silica monolith. The body was washed with toluene and methanol. Next, the linker-modified silica monolith is reacted with each solution of polyornithine (Example A4), polylysine (Example A2), aminosilane (Example A1), and polyallylamine (Example A3) to obtain polyornithine. , polylysine, aminosilane, and polyallylamine modified silica monoliths were 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 under a 37° C., 5% CO ₂ atmosphere, and the culture supernatant was collected when 50% or more confluent. The collected culture supernatant was centrifuged at 10000×g for 30 minutes at 4° C. using a centrifuge (CF16RN, manufactured by Koki Holdings), and the supernatant was collected. Then, 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 culture supernatant (0.5 mL) was passed through the column

(2) Exosomes were bound to the modified product in the column. That is, in the compounding step, a complex containing exosomes, modified products (peptide) and a column was formed

(3) The complex was washed: 0.5 mL of ultrapure water was passed through the column

(4) The operation (washing) of (3) above was performed twice more, and the exosomes in the culture supernatant were collected as a complex containing polyornithine-immobilized columns.

(5) The dissociation buffer was run through the column to separate the exosomes from the complex. _The dissociation buffer used was MgCl2.

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

(7) Using CD9/CD63 Exosome ELISA Kit (Cosmo Bio, EXH0102EL) and Sunrise Thermo RC-R (TECAN, 551-40081), the amount of exosomes was measured according to the attached standard protocol <Results>
The results are shown in Table 1 and FIG. Polyornithine of Example A4 was able to recover exosomes 4 times or more unexpectedly compared to other Examples A1 to A3. Amino acid polymers such as polylysine and polyornithine increase the positive charge on the column carrier and can adhere lipid bilayers and the like. Polyornithine has a lower molecular weight and is less hydrophobic than polylysine. Therefore, polyornithine is more likely to be charged and has lower hydrophobicity, so it is thought that the conditions for easier adsorption were derived.

Silica monoliths are preferred as supports. 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 particle size and amount of the collected exosomes were measured by changing the mesopore diameter of the silica monolith. Matters not explained are the same as those of Examples A and A4.
For measuring the particle size of exosomes, a particle size distribution analyzer (Lightsizer: manufactured by Anton Paar, laser diffraction/scattering method, average value based on volume) or a transmission electron microscope (JEM1230R: manufactured by JEOL Ltd., 1% uranyl acetate (Negative staining of samples with ) was used.
The mesopore diameter was measured using a pore size distribution analyzer (mercury intrusion method or nitrogen adsorption method).
Conditions and results are shown in Table 2, FIGS. It can be seen that the size and amount of exosomes that can be recovered differ depending on the mesopore size.
From Figure 3, mesopores can be set to obtain exosomes of the required diameter. For example, to obtain exosomes with a diameter of 140 nm, use a mesopore size (A region) of around 10 nm. To obtain exosomes with a diameter of 105 nm, use a mesopore size (C region) around 300 nm. To obtain exosomes with a diameter of 170-180 nm, a mesopore size of 20-200 nm (B region) is used.
It is possible to control the particle size of exosomes collected by limiting the mesopore size of the carrier.
Exosomes of the desired particle size obtained thereby can be effectively used in a drug delivery system (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.
Further, 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 constant.

<Example C>
Under the conditions of Example A4, the washing solution was NaCl, the concentration was changed, and the particle size of the recovered exosomes was measured. Matters not explained are the same as those of Examples A and A4.
Conditions and results are shown in Table 3 and FIG.
The concentration of the washing liquid is preferably 0.1-1.5M. 1.6M to 16M is also good. 0.1 to 0.9M is preferred. Although NaCl was used as the cleaning liquid, other cleaning liquids may be used with similar concentrations.

<Example D>
Under the conditions of Example A4, the dissociation buffer solution was MgSO ₄ , Na ₂ SO ₄ and the amount of recovered exosomes was measured. Matters not explained are the same as in Example A4.
Conditions and results are shown in Table 4 and FIG.
MgSO ₄ containing divalent metal ions could recover nearly twice as much exosomes as Na ₂ SO ₄ containing monovalent metal ions. This is probably because multivalent 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 columns may be beads or porous bodies other than silica monoliths.
The microparticles or extracellular vesicles covered with a lipid bilayer membrane are particles surrounded by a lipid bilayer membrane that do not have a nucleus (cannot be replicated) and are released from cells. Microparticles or extracellular vesicles covered with a lipid bilayer membrane are classified into (1) exosomes, (2) μ vesicles, and (3) apoptotic vesicles, depending on the production mechanism. The above applies 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, it is possible to collect exosomes in a state where the structure and function of exosomes are not impaired (state where the structure and function of exosomes are not substantially impaired). Therefore, according to the embodiments, it is possible to easily and in detail analyze substances contained inside exosomes. Accordingly, the embodiments are useful in various technical fields, especially in the pharmaceutical and medical fields.

1 spin column 2 column 3 monolith body

Claims (7)

Microparticles or extracellular vesicles covered with a lipid bilayer membrane for recovering the microparticles or extracellular vesicles covered with a lipid bilayer membrane from a sample containing the microparticles or extracellular vesicles covered with a lipid bilayer membrane A method of collecting the
Fine particles or extracellular vesicles covered with the lipid bilayer membrane and the amino group supported by the carrier are passed through the sample through the carrier holding the substance containing the amino group. A complex forming step of forming a complex formed by binding with
The complex obtained in the complex forming step is brought into contact with a dissociation buffer solution containing metal cations to dissociate the microparticles 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,
A method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, wherein the substance containing an amino group is a peptide containing polyornithine . The recovery method according to claim 1, wherein the particle size of the recovered microparticles or extracellular vesicles covered with the lipid bilayer membrane is controlled by limiting the pore size of the mesopores of the carrier. 3. The method for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane according to claim 1 or 2 , wherein the carrier is a silica monolith. The method for recovering microparticles or extracellular vesicles covered with lipid bilayer membranes according to any one of claims 1 to 3 , wherein the dissociation buffer solution is a solution containing magnesium as divalent metal ions. The method for collecting microparticles or extracellular vesicles covered with lipid bilayer membranes according to any one of claims 1 to 4 , wherein the dissociation buffer solution has a concentration of 0.1 to 1.5M. Collection of microparticles or extracellular vesicles covered with lipid bilayer membranes for performing the method for collecting microparticles or extracellular vesicles covered with lipid bilayer membranes according to any one of claims 1 to 5 is a kit,
A collection kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane, comprising the carrier on which the substance containing the amino group is supported , and the dissociation buffer. Microparticles or extracellular vesicles covered with a lipid bilayer membrane for recovering the microparticles or extracellular vesicles covered with a lipid bilayer membrane from a sample containing the microparticles or extracellular vesicles covered with a lipid bilayer membrane a collection kit for
A kit for collecting microparticles or extracellular vesicles covered with a lipid bilayer membrane containing a peptide containing polyornithine and a carrier carrying the peptide,
By passing the sample through the carrier holding the peptide containing polyornithine, the microparticles or extracellular vesicles covered with the lipid bilayer membrane bind to the amino groups carried on the carrier. The resulting complex is brought into contact with a dissociation buffer solution containing metal cations, and the microparticles or extracellular vesicles covered with the lipid bilayer membrane are separated from the complex. A collection kit for collecting dissociated microparticles or extracellular vesicles covered with a lipid bilayer membrane.

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