Powder for extracellular vesicle separation

Abstract

A powder for extracellular vesicle separation containing a plurality of inorganic particles, wherein the composition of the surface of the inorganic particles is represented by the formula MalphaObeta(OZ)gammaYtheta, where M represents at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, Z represents at least one element selected from the group consisting of hydrogen, alkali metals, and alkaline earth metals, Y represents at least one element selected from the group consisting of carbon, nitrogen, and halogens, alpha, beta, gamma, and theta represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, as measured by X-ray photoelectron spectroscopy, and gamma / (alpha + gamma), which is the ratio of gamma to the total of alpha and gamma, is in the range of 0.01-0.08.

Description

Powder for extracellular vesicle separation

[0001] The present disclosure relates to a powder for isolating extracellular vesicles.

[0002] Extracellular vesicles called exosomes are known to efficiently mediate intercellular communication and signal transduction, transporting bioactive molecules such as proteins, lipids, and nucleic acids between cells and across biological barriers. Because exosomes are resistant to degradation and capable of long-distance signal transduction without dilution, they are considered promising drug delivery systems (DDS) and therapeutic agents. To use exosomes for the above-mentioned medical applications, a separation method that can reduce the contamination of unintended exosomes and impurities is required. Various methods for the separation and recovery of extracellular vesicles have been reported.

[0003] For example, Patent Document 1 discloses a method for isolating biological particles from a liquid sample, comprising the following steps: a) obtaining a liquid sample from a subject or cell culture; b) contacting the liquid sample with a crystal / precipitation inducer under conditions suitable for crystal formation and / or precipitation, thereby forming a mixture; c) incubating the mixture for a time sufficient to allow crystal formation and / or precipitation; and d) separating the mixture to obtain a particle fraction containing biological particles, thereby isolating the biological particles from the liquid sample. Patent Document 2 also discloses a method for separating biologically derived micromaterials using size exclusion chromatography. According to this separation method using size exclusion chromatography, biologically derived samples of various sizes repeatedly enter and exit the pores of superficially porous particles, thereby obtaining a fraction of biologically derived components (e.g., a fraction of exosomes and contaminating proteins).

[0004] US Patent Application Publication No. 2017 / 0252670 International Publication No. 2019 / 202994

[0005] In order to use exosomes for medical purposes, it is necessary to establish a method that can more selectively isolate and recover exosomes. Furthermore, since commercially available formulations are expected to be administered to a large number of patients, a method that can efficiently isolate and recover exosomes on a scale that can be applied to commercial formulations is required. In this regard, the methods described in Patent Documents 1 and 2 both still have issues with the selective and efficient isolation and recovery of exosomes.

[0006] The present disclosure has been made in view of the above circumstances. An object of one embodiment of the present disclosure is to provide a powder for separating extracellular vesicles that can selectively and efficiently separate and recover exosomes, which are extracellular vesicles, from a liquid containing exosomes.

[0007] Specific means for solving the above problems include the following aspects: [1] A powder for separating extracellular vesicles, comprising a plurality of inorganic particles, wherein the surface composition of the inorganic particles is represented by the formula M α O β (OZ) γ Y θ wherein M represents at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron; Z represents at least one element selected from the group consisting of hydrogen, an alkali metal, and an alkaline earth metal; Y represents at least one element selected from the group consisting of carbon, nitrogen, and a halogen; α, β, γ, and θ represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, as measured by X-ray photoelectron spectroscopy; and γ / (α+γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 or more and 0.08 or less.

[0008] [2] A powder for separating extracellular vesicles, comprising a plurality of inorganic particles, wherein the inorganic particles have at least one OZ group, and Z represents a hydrogen atom, an alkali metal atom, or an alkaline earth metal atom, and the inorganic particles are subjected to a sodium ion adsorption treatment under the following condition A. The ratio X / Y of the number of sodium atoms X to the total number Y of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens, as measured by X-ray photoelectron spectroscopy, is in the range of 0.01 to 0.08.

[0009] (Condition A) 1. In a sealed container, 10 mg of dried inorganic particles are impregnated with 0.25 mL of 0.5 mol / L aqueous sodium hydroxide solution and allowed to stand for 4 hours in an environment with an ambient temperature of 25°C. 2. After removing the supernatant, 0.3 mL of distilled water is added to the residue, and the mixture is allowed to stand for 24 hours in an environment with an ambient temperature of 25°C. 3. The above operation "2." is repeated three times. 4. After removing the supernatant, the residue is dried at 90°C.

[0010] [3] The powder for separating extracellular vesicles according to [1] or [2], wherein the inorganic particles contain silicon, and the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms, as measured by X-ray photoelectron spectroscopy, is in the range of 50% to 98.5%. [4] The powder for separating extracellular vesicles according to [3], wherein the inorganic particles further contain boron. [5] The powder for separating extracellular vesicles according to any one of [1] to [4], wherein the inorganic particles have an average particle size in the range of 0.5 μm to 150 μm. [6] The powder for separating extracellular vesicles according to any one of [1] to [5], wherein the inorganic particles have pores, and the average pore size of the pores, as measured by mercury intrusion porosimetry, is in the range of 50 nm to 1,000 nm.

[0011] According to one embodiment of the present disclosure, there is provided a powder for separating extracellular vesicles that can selectively and efficiently separate and recover exosomes, which are extracellular vesicles, from a liquid containing exosomes.

[0012] The powder for separating extracellular vesicles according to the present disclosure will be described in detail below. The following description of the requirements may be based on a representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment and can be implemented with appropriate modifications within the scope of the purpose of the present disclosure.

[0013] In the present disclosure, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits, respectively. In the numerical ranges described in stages in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in the numerical ranges described in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with a value shown in the examples.

[0014] In the present disclosure, a combination of two or more preferred aspects is a more preferred aspect.

[0015] In the present disclosure, the term "process" includes not only an independent process but also a process that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved.

[0016] Extracellular vesicles are vesicles that exist outside cells and are classified into exosomes, microvesicles, and apoptotic bodies based on their size. Exosomes and microvesicles contain proteins and nucleic acids such as mRNA and miRNA, while apoptotic bodies contain fragmented nuclei and organelles. The difference between exosomes and microvesicles is unclear, and the exosomes described in the present disclosure may also include microvesicles.

[0017] [Powder for Separating Extracellular Vesicles] One embodiment of the powder for separating extracellular vesicles according to the present disclosure is a powder for separating extracellular vesicles containing a plurality of inorganic particles, wherein the surface composition of the inorganic particles is represented by the formula M α O β (OZ) γ Y θwherein M represents at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, Z represents at least one element selected from the group consisting of hydrogen, alkali metals, and alkaline earth metals, Y represents at least one element selected from the group consisting of carbon, nitrogen, and halogens, and α, β, γ, and θ represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, as measured by X-ray photoelectron spectroscopy, and γ / (α+γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 to 0.08. The powder for extracellular vesicle isolation according to this embodiment will hereinafter also be referred to as "powder (a) for extracellular vesicle isolation."

[0018] Furthermore, one embodiment of the powder for separating extracellular vesicles according to the present disclosure is a powder for separating extracellular vesicles comprising a plurality of inorganic particles, the inorganic particles having at least one OZ group, wherein Z represents a hydrogen atom, an alkali metal atom, or an alkaline earth metal atom, and the inorganic particles after sodium ion adsorption treatment under condition A have a ratio X / Y of the number of sodium atoms X to the total number of atoms Y of elements excluding carbon, nitrogen, oxygen, and halogens, measured by X-ray photoelectron spectroscopy, in the range of 0.01 to 0.08. The powder for separating extracellular vesicles according to this embodiment will hereinafter also be referred to as "powder for separating extracellular vesicles (b)".

[0019] The powder for separating extracellular vesicles according to the present disclosure can selectively and efficiently separate and recover exosomes from a liquid containing exosomes, which are extracellular vesicles. The reason why the powder for separating extracellular vesicles according to the present disclosure can achieve such an effect is unclear, but the present inventors speculate as follows. However, the following speculation is not intended to limit the scope of the powder for separating extracellular vesicles according to the present disclosure, but is provided as an example.

[0020] The powder for separating extracellular vesicles according to the present disclosure was developed based on the discovery that inorganic particles having OZ groups on their surface within a specific density range have a high selective recovery ability for exosomes, which are extracellular vesicles, and that there is a correlation between the density of OZ groups on the particle surface of inorganic particles (hereinafter also referred to as "surface OZ groups") and the above "γ / (α+γ)" and "X / Y" measured by X-ray photoelectron spectroscopy. The powder for separating extracellular vesicles according to the present disclosure contains a plurality of inorganic particles, and these inorganic particles have OZ groups on their surfaces within a specific density range that contribute to the adsorption of exosomes. Therefore, when the powder is brought into contact with a liquid containing exosomes, it is thought that exosomes are selectively and efficiently captured by the OZ groups on the surface of the inorganic particles, and that the exosomes captured by the OZ groups are efficiently detached from the OZ groups. From the above, it is presumed that the powder for separating extracellular vesicles according to the present disclosure will enable selective and efficient separation and recovery of exosomes, which are extracellular vesicles, from a liquid containing them.

[0021] X-ray photoelectron spectroscopy, as used in the present disclosure, is a quantitative and qualitative analysis technique that can analyze the number of constituent elements and their electronic states in the surface region of a sample by irradiating the sample with X-rays of a specific energy and measuring the number and energy of photoelectrons generated by the photoelectric effect. Examples of X-ray sources in X-ray photoelectron spectroscopy include Al-Kα radiation and Mg-Kα radiation. In the present disclosure, when Al-Kα radiation is used as the X-ray source, the surface region is defined as the region where photoelectrons generated in the sample can escape to the outside of the sample without losing energy. The depth of the surface region is approximately 5 nm, although this varies slightly depending on the energy of the generated photoelectrons.

[0022] [Powder (a) for separating extracellular vesicles] The powder (a) for separating extracellular vesicles according to the present disclosure comprises a plurality of inorganic particles, and the surface composition of the inorganic particles is represented by the formula M α O β (OZ) γ Y θIn the formula, M represents at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, Z represents at least one element selected from the group consisting of hydrogen, alkali metals, and alkaline earth metals, Y represents at least one element selected from the group consisting of carbon, nitrogen, and halogens, α, β, γ, and θ represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, measured by X-ray photoelectron spectroscopy, and γ / (α+γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 or more and 0.08 or less.

[0023] The surface composition of the inorganic particles contained in the powder (a) for separating extracellular vesicles according to the present disclosure is represented by the formula M α O β (OZ) γ Y θ Here, "formula M α O β (OZ) γ Y θ " is composed of M, O, OZ, and Y, and indicates that the proportions of M, O, OZ, and Y are α, β, γ, and θ, respectively, and is different from the composition formula, which is the bulk composition.

[0024] In the formula, M represents at least one element selected from the group consisting of silicon (Si), boron (B), aluminum (Al), titanium (Ti), zirconium (Zr), magnesium (Mg), calcium (Ca), zinc (Zn), and iron (Fe). For example, from the viewpoint of controlling the density of surface OZ groups, it is preferably at least one element selected from the group consisting of silicon, boron, and aluminum, and more preferably contains silicon. When M contains silicon, the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms measured by X-ray photoelectron spectroscopy is preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more, from the viewpoint of controlling the density of surface OZ groups. The upper limit is preferably 98.5% or less, more preferably 97.5% or less, and even more preferably 95.0% or less. In an embodiment, the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms, as measured by X-ray photoelectron spectroscopy, may be in the range of 50% to 98.5%, or in the range of 60% to 97.5%, or in the range of 70% to 98.5%.

[0025] In the formula, Z represents at least one element selected from the group consisting of hydrogen, alkali metals, and alkaline earth metals, and is preferably at least one element selected from the group consisting of hydrogen (H), lithium (Li), sodium (Na), potassium (K), and calcium (Ca).

[0026] In the formula, Y represents at least one element selected from the group consisting of carbon, nitrogen, and halogen, and is preferably at least one element selected from the group consisting of carbon (C), nitrogen (N), fluorine (F), and chlorine (Cl).

[0027] In the formula, α, β, γ, and θ represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, as measured by X-ray photoelectron spectroscopy. α, β, γ, and θ are not particularly limited as long as γ / (α + γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 to 0.08. γ / (α + γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 to 0.08, preferably in the range of 0.01 to 0.07, more preferably in the range of 0.016 to 0.07, even more preferably in the range of 0.02 to 0.06, even more preferably in the range of 0.02 to 0.05, and particularly preferably in the range of 0.03 to 0.05. γ / (α + γ) being 0.01 or more means that the inorganic particles have OZ groups on their surface that contribute to the adsorption of exosomes. When γ / (α+γ) is 0.08 or less, exosomes can be efficiently adsorbed onto the surface of inorganic particles, and efficient detachment of the adsorbed exosomes from the particle surface tends not to be impaired.

[0028] In the present disclosure, γ / (α+γ) is determined by the following method. First, X-ray photoelectron spectroscopy is performed on the surfaces of inorganic particles contained in the powder for extracellular vesicle isolation. Using an X-ray photoelectron spectroscopy analyzer as the analytical device, the powder for extracellular vesicle isolation packed on a dedicated substrate is irradiated with X-rays while charge neutralization is performed using electrons and Ar ions. Al-Kα rays are used as the X-ray source, with a photoelectron takeoff angle of 45° and an aperture diameter of 100 μm. The energy of photoelectrons generated by X-ray irradiation is measured to obtain a spectrum. Using the data analysis software of the X-ray photoelectron spectroscopy analyzer, charge correction is performed on the obtained spectrum, setting the peak attributable to surface contaminating hydrocarbons in the carbon 1s spectrum to 284.6 eV. Next, elemental species identification and peak fitting are performed on the detected peaks. The area value of each peak determined by peak fitting is then multiplied by the relative sensitivity coefficient of the instrument to determine the number of atoms. γ / (α+γ) is calculated from the number of atoms determined from the peak area value of the element corresponding to M and the number of atoms determined from the peak area value of the element corresponding to Z. As the X-ray photoelectron spectrometer, for example, Quantera SXM (product name) manufactured by ULVAC-PHI, Inc. can be suitably used. Furthermore, as the data analysis software, for example, MultiPak (product name) manufactured by ULVAC-PHI, Inc. can be suitably used. However, the X-ray photoelectron spectrometer and the data analysis software are not limited to the above products.

[0029] Since X-ray photoelectron spectroscopy cannot detect hydrogen, when Z in the formula contains hydrogen, prior to the X-ray photoelectron spectroscopy analysis, the Z atom portion of the OZ group on the surface of the inorganic particle is substituted with sodium by ion exchange (so-called sodium ion adsorption treatment), and the substituted sodium is then detected by X-ray photoelectron spectroscopy. The conditions for the sodium ion adsorption treatment are the same as "Condition (A)" described in the description of the powder (b) for separating extracellular vesicles according to the present disclosure below, and therefore will not be described here.

[0030] [Powder (b) for separating extracellular vesicles] The powder (b) for separating extracellular vesicles according to the present disclosure is a powder for separating extracellular vesicles comprising a plurality of inorganic particles, wherein the inorganic particles have at least one OZ group, wherein Z represents a hydrogen atom, an alkali metal atom, or an alkaline earth metal atom, and the inorganic particles after sodium ion adsorption treatment under condition A have a ratio X / Y of the number of sodium atoms X to the sum Y of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens, measured by X-ray photoelectron spectroscopy, in the range of 0.01 or more and 0.08 or less.

[0031] The inorganic particles contained in the powder (b) for separating extracellular vesicles according to the present disclosure have at least one OZ group, where Z represents a hydrogen atom, an alkali metal atom, or an alkaline earth metal atom, and is preferably at least one element selected from the group consisting of hydrogen (H), lithium (Li), sodium (Na), potassium (K), magnesium (Mg), and calcium (Ca).

[0032] In the powder (b) for separating extracellular vesicles according to the present disclosure, the ratio X / Y, which is the ratio of the number of sodium atoms X to the sum Y of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens, measured by X-ray photoelectron spectroscopy after sodium ion adsorption treatment of inorganic particles under condition A, is in the range of 0.01 to 0.08, preferably 0.01 to 0.07, more preferably 0.02 to 0.06, even more preferably 0.02 to 0.05, and particularly preferably 0.03 to 0.05. An X / Y ratio of 0.01 or greater means that the inorganic particles have OZ groups on their surface that contribute to the adsorption of exosomes. When X / Y is 0.08 or less, exosomes can be efficiently adsorbed onto the surface of the inorganic particles, and efficient detachment of the adsorbed exosomes from the particle surface tends to be less impaired.

[0033] The number of sodium atoms X and the sum Y of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens of the inorganic particles after the sodium ion adsorption treatment under condition A, as measured by X-ray photoelectron spectroscopy, are not particularly limited as long as X / Y is in the range of 0.01 or more and 0.08 or less. However, the number of sodium atoms X is, for example, preferably in the range of 0.01 or more and 0.07 or less, more preferably in the range of 0.016 or more and 0.07 or less, even more preferably in the range of 0.02 or more and 0.06 or less, still more preferably in the range of 0.02 or more and 0.05 or less, and particularly preferably in the range of 0.03 or more and 0.05 or less.

[0034] In the present disclosure, X / Y is determined by the following method: First, inorganic particles contained in a powder for separating extracellular vesicles are subjected to a sodium ion adsorption treatment under the following condition A, thereby substituting the Z atom portion of the OZ group on the surface of the inorganic particles with sodium.

[0035] (Condition A) 1. In a sealed container, 10 mg of dried inorganic particles are impregnated with 0.25 mL of 0.5 mol / L aqueous sodium hydroxide solution and allowed to stand for 4 hours in an environment with an ambient temperature of 25°C. 2. After removing the supernatant, 0.3 mL of distilled water is added to the residue, and the mixture is allowed to stand for 24 hours in an environment with an ambient temperature of 25°C. 3. The above operation "2." is repeated three times. 4. After removing the supernatant, the residue is dried at 90°C.

[0036] Next, X-ray photoelectron spectroscopy is performed on the surface of the inorganic particles after the above treatment. An X-ray photoelectron spectroscopy analyzer is used as the analytical device, and the inorganic particles after the above treatment, which are packed on a dedicated substrate, are irradiated with X-rays while charge neutralization is performed with electrons and Ar ions. Al-Kα rays are used as the X-ray source, with a photoelectron take-off angle of 45° and an aperture diameter of 100 μm. The energy of photoelectrons generated by X-ray irradiation is measured, and a spectrum is acquired. Using the data analysis software of the X-ray photoelectron spectroscopy analyzer, charge correction is performed on the acquired spectrum, setting the peak attributable to surface contaminating hydrocarbons in the carbon 1s spectrum to 284.6 eV. Next, elemental species identification and peak fitting are performed on the detected peaks. The area value of each peak determined by peak fitting is then multiplied by the relative sensitivity coefficient of the instrument to determine the number of atoms. From the number of atoms X determined from the peak area value of sodium and the total number of atoms Y determined from the peak area values ​​of all elements excluding carbon, nitrogen, oxygen, and halogens, X / Y, which is the ratio of the number of atoms X of sodium to the total number Y of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens, is calculated.

[0037] -Composition of Inorganic Particles- The composition of the inorganic particles contained in the powder for extracellular vesicle separation according to the present disclosure is not particularly limited, as long as the surface composition of the inorganic particles satisfies the requirements described for powder for extracellular vesicle separation (a) according to the present disclosure and powder for extracellular vesicle separation (b) according to the present disclosure. The inorganic particles may be, for example, oxide particles, nitride particles, or carbide particles, but are preferably oxide particles. The inorganic particles preferably contain at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, more preferably silicon and / or boron, and even more preferably silicon. The inorganic particles may be, for example, oxide particles containing silicon and having silicon as the main component, i.e., silicon oxide particles, or silicon oxide particles containing boron. Here, "main component" refers to the component with the largest content by mass. The inorganic particles may also be silicon oxide particles containing boron and sodium.

[0038] The inorganic particles preferably contain silicon, and the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms measured by X-ray photoelectron spectroscopy is in the range of 50% to 98.5%, and more preferably contain silicon and boron, and the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms measured by X-ray photoelectron spectroscopy is in the range of 50% to 98.5%. When the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms measured by X-ray photoelectron spectroscopy is 50% or more, exosomes can be more efficiently adsorbed to the surface of the inorganic particles, and more efficient detachment of adsorbed exosomes from the particle surface tends to be less impaired. When the ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms, as measured by X-ray photoelectron spectroscopy, is 98.5% or less, exosomes can be more appropriately adsorbed to the surface of inorganic particles, and the adsorbed exosomes tend to be more efficiently detached from the surface of inorganic particles. The ratio of the number of silicon atoms to the total number of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron atoms, as measured by X-ray photoelectron spectroscopy, is more preferably in the range of 60% to 97.5%, and even more preferably in the range of 70% to 95%.

[0039] The inorganic particles may contain, for example, at least one element selected from the group consisting of carbon, nitrogen, oxygen, and halogen.

[0040] -Physical Properties of Inorganic Particles- The inorganic particles are preferably poorly soluble or insoluble in water. In the present disclosure, "poorly soluble in water" means that the solubility in water at 25°C is 0.01% by mass to 1% by mass, and "insoluble in water" means that the solubility in water at 25°C is less than 0.01% by mass.

[0041] -Shape of inorganic particles- The shape of the inorganic particles is not particularly limited, and examples of the shape of the inorganic particles include spherical, elliptical, and irregular shapes.

[0042] -Structure of Inorganic Particles- There are no particular limitations on the atomic arrangement structure of inorganic particles, and inorganic particles may be crystalline or amorphous.

[0043] The inorganic particles may have a porous structure or a non-porous structure. When the inorganic particles have a porous structure, the "surface of the inorganic particles" in the present disclosure includes not only the outer surfaces of the inorganic particles but also the inner surfaces of the pores.

[0044] When inorganic particles have a porous structure, the average pore size of the pores of the inorganic particles is not particularly limited, but is preferably in the range of 50 nm to 1000 nm, from the viewpoint of efficiently capturing exosomes inside the pores. The average pore size of the pores of the inorganic particles is preferably 60 nm or more, more preferably 70 nm or more, and even more preferably 80 nm or more. The average pore size of the pores of the inorganic particles is preferably 800 nm or less, more preferably 700 nm or less, and even more preferably 500 nm or less. In some embodiments, the average pore size of the pores of the inorganic particles may be in the range of 60 nm to 800 nm, 70 nm to 700 nm, 80 nm to 500 nm, or 80 nm to 400 nm.

[0045] The average pore size of the pores in the inorganic particles is measured by mercury intrusion porosimetry. A pore size distribution measurement device is used for measurement by mercury intrusion porosimetry. Specifically, measurement is performed under the following conditions and procedures. Here, when calculating the average pore size of the pores in the inorganic particles, the average pore size is determined from the distribution of pores of 1 μm or less to exclude the influence of voids between the inorganic particles. Mercury parameters are set to a mercury contact angle of 140° and a mercury surface tension of 480 mN / m. 0.01 g to 0.1 g of sample is collected in a standard cell, and measurement is performed under an initial pressure of 6.9 kPa. The average pore size is determined from the resulting distribution of pores of 1 μm or less. An AutoPore V9620 manufactured by Micromeritics, Inc., for example, can be suitably used as the pore size distribution measurement device. However, the pore size distribution measurement device is not limited to this.

[0046] -Average particle size of inorganic particles- The average particle size of inorganic particles is not particularly limited, but is preferably smaller, for example, from the viewpoint of efficiently capturing exosomes, which are extracellular vesicles contained in liquids such as sample solutions. Furthermore, when the inorganic particles have pores, it is preferable that the average particle size be smaller, for example, from the viewpoint of efficiently capturing exosomes, which are extracellular vesicles contained in liquids such as sample solutions, inside the pores of the inorganic particles and efficiently releasing them. From this viewpoint, the average particle size of the inorganic particles is preferably in the range of 0.5 μm to 150 μm, more preferably in the range of 1.0 μm to 150 μm, even more preferably in the range of 2.0 μm to 150 μm, particularly preferably in the range of 3.0 μm to 100 μm, and most preferably in the range of 5.0 μm to 50 μm.

[0047] In the present disclosure, the average particle size of inorganic particles refers to the volume-average primary particle size. The average particle size of inorganic particles is determined as the particle size (D50) at which the cumulative total from the smallest diameter side reaches 50% in a volume-based particle size distribution measured using a laser diffraction scattering particle size distribution analyzer. A suitable example of a laser diffraction scattering particle size distribution analyzer is the Mastersizer 2000 manufactured by Malvern Panalytical. However, the laser diffraction scattering particle size distribution analyzer is not limited to this.

[0048] <<Applications>> Exosomes are vesicles released by cells that inherit the properties of the releasing cells (so-called donor cells) and can be applied to receiving cells (so-called recipient cells) to impart properties derived from the donor cells to the recipient cells, making them useful as materials for drug delivery systems, therapeutic drugs, etc. The powder for separating extracellular vesicles according to the present disclosure can selectively and efficiently separate and recover exosomes from a liquid containing exosomes, which are extracellular vesicles, and can therefore be preferably used for separating and recovering exosomes as materials for drug delivery systems and therapeutic drugs, where ensuring purity and quantity is required.

[0049] [Method for producing powder for extracellular vesicle separation] The method for producing the powder for extracellular vesicle separation according to the present disclosure is not particularly limited. Examples of methods for producing the powder for extracellular vesicle separation according to the present disclosure include a method via a glass melting process, a method via a sol-gel method, and a method via a solid-phase reaction. Below, a method via a glass melting process will be described as one suitable example of the method for producing the powder for extracellular vesicle separation according to the present disclosure.

[0050] The method for producing a powder for separating extracellular vesicles according to the present disclosure via a glass melting process (hereinafter also referred to as "production method X") includes, for example, step A of firing and vitrifying raw materials, and step B of crushing the glass to form particles.

[0051] Step A is a step of firing raw materials to vitrify them. According to Step A, glass can be obtained. The raw materials include, for example, sodium carbonate (Na ２ CO ３ ), sodium tetraborate (Na ２ B ４ O ７ ), sodium metasilicate nonahydrate (Na ２ SiO ３ ・9H ２ O), boron oxide (B ２ O ３ ), and silicon dioxide (SiO ２As the raw material, for example, Na is preferred in that it is easy to control the density of the surface OZ group of the inorganic particles to be finally obtained. ２ CO ３ , B ２ O ３ and SiO ２ Combination of Na ２ B ４ O ７ , B ２ O ３ and SiO ２ or a combination of Na ２ SiO ３ ・9H ２ O, B ２ O ３ and SiO ２ The combination is preferred.

[0052] As a raw material, Na ２ CO ３ , B ２ O ３ and SiO ２ When selecting a combination of ２ CO ３ and B ２ O ３ and SiO ２ The compounding ratio is Na ２ CO ３ , B ２ O ３ and SiO ２ When the total of ２ CO ３ is 5 parts by mass to 30 parts by mass, and B ２ O ３ is 20 parts by mass to 60 parts by mass, and SiO ２ is preferably 25 to 65 parts by mass, and Na ２ CO ３ is 10 parts by mass to 20 parts by mass, and B ２ O ３ is 25 parts by mass to 40 parts by mass, and SiO ２ is more preferably 50 parts by mass to 65 parts by mass, and Na ２ CO ３ is 13.5 parts by mass to 20 parts by mass, B ２ O ３ is 30 parts by mass to 40 parts by mass, and SiO２ It is more preferable that the amount of Na is 50 parts by mass to 58 parts by mass. ２ B ４ O ７ , B ２ O ３ and SiO ２ When selecting a combination of ２ B ４ O ７ and B ２ O ３ and SiO ２ The compounding ratio is Na ２ B ４ O ７ , B ２ O ３ and SiO ２ When the total of ２ B ４ O ７ is 10 parts by mass to 50 parts by mass, and B ２ O ３ is 1 part by mass to 20 parts by mass, and SiO ２ is preferably 30 parts by mass to 70 parts by mass, and Na ２ B ４ O ７ is 20 parts by mass to 40 parts by mass, and B ２ O ３ is 2 parts by mass to 15 parts by mass, and SiO ２ It is more preferable that the amount of Na is 55 parts by mass to 70 parts by mass. ２ SiO ３ ・9H ２ O, B ２ O ３ and SiO ２ When selecting a combination of ２ SiO ３ ・9H ２ O and B ２ O ３ and SiO ２ The compounding ratio is Na ２ SiO ３ ・9H ２ O, B ２ O ３ and SiO ２ When the total of ２ SiO ３ ・9H２ O is 15 parts by mass to 45 parts by mass, and B ２ O ３ is 10 parts by mass to 50 parts by mass, and SiO ２ is preferably 15 parts by mass to 55 parts by mass, and Na ２ SiO ３ ・9H ２ O is 20 parts by mass to 40 parts by mass, and B ２ O ３ is 20 parts by mass to 40 parts by mass, and SiO ２ is more preferably 30 parts by mass to 55 parts by mass.

[0053] The raw materials include Na ２ CO ３ , Na ２ B ４ O ７ , Na ２ SiO ３ ・9H ２ O, B ２ O ３ and SiO ２ The different metal oxides may be blended with one or more kinds of different metal oxides other than magnesium oxide (MgO), calcium oxide (CaO), aluminum oxide (Al ２ O ３ ), zirconium oxide (ZrO ２ ), zinc oxide (ZnO ２ ), and titanium oxide (TiO ２ ) Na ２ CO ３ , Na ２ B ４ O ７ , Na ２ SiO ３ ・9H ２ O, B ２ O ３ and SiO ２ When a different metal oxide other than Na is blended in the raw material, the blending amount of the different metal oxide is, for example, ２ CO ３ , Na ２ B ４ O ７ , Na ２ SiO ３ ・9H ２ O, B ２O ３ and SiO ２ The amount may be 1 part by mass to 10 parts by mass relative to 100 parts by mass of the total of the above.

[0054] The firing temperature is not particularly limited as long as it is a temperature at which the raw materials are vitrified. The firing temperature is, for example, preferably in the range of 1200°C or higher and 1600°C or lower, and more preferably in the range of 1400°C or higher and 1600°C or lower. The temperature rise rate when heating to the firing temperature is not particularly limited, but is preferably, for example, 10°C / min to 50°C / min. Firing may be performed in an air atmosphere or an inert atmosphere, but is preferably performed in an air atmosphere. The firing time may be a time at which the raw materials are vitrified, and can be set appropriately depending on the firing temperature. The firing time can be, for example, 2 hours to 8 hours. After firing, it is preferable to cool to near room temperature. Cooling methods include, for example, natural cooling and rapid cooling.

[0055] Before step B, the glass obtained in step A may be further subjected to a firing treatment. By performing the firing treatment, the glass can be made into a more stable structure. When the glass obtained in step A is further subjected to a firing treatment, the firing temperature is preferably, for example, 400°C to 700°C, and the firing time is preferably, for example, 2 hours to 100 hours. The temperature rise rate when heating to the firing temperature can be, for example, 5°C / min to 20°C / min. After firing, it is preferable to cool to around room temperature. Examples of cooling methods include natural cooling and rapid cooling.

[0056] Step B is a step of crushing the glass to form particles. According to Step B, glass particles can be obtained. The method of crushing the glass is not particularly limited. Examples of crushing means include a crusher, a stamp mill, a ball mill, a bead mill, a jet mill, and a vibration mill.

[0057] The particle size distribution of the glass particles obtained by the crushing treatment is preferably narrowed by classification treatment. Examples of classification methods include a sieve.

[0058] Manufacturing method X may include steps other than the above-described steps A and B. Examples of steps other than steps A and B include step C, in which glass particles are treated with an acid and then with a base. Step C can produce inorganic particles having pores therein. In addition, acid-soluble or base-soluble by-products, impurities, and the like generated during the manufacturing process of the glass particles can be removed.

[0059] Acid treatment can, for example, form a pore structure in the glass particles. Furthermore, acid treatment can, for example, remove acid-soluble by-products, impurities, and the like generated during the manufacturing process of the glass particles. Examples of methods for acid treatment include a method of heating in an acidic solution. The acidic solution can be appropriately selected depending on the composition of the glass. As the acidic solution, a strong acid is preferred, and specifically, an inorganic acid aqueous solution such as an aqueous hydrochloric acid solution, an aqueous nitric acid solution, or an aqueous perchloric acid solution is more preferred. Two or more types of acidic solutions may be used in combination for the acid treatment. The heating temperature is not particularly limited, but is preferably, for example, 60°C to 120°C. The heating time is not particularly limited, but is preferably, for example, 2 hours to 24 hours.

[0060] By performing a base treatment after an acid treatment, for example, pores can be formed within the glass particles. Furthermore, by performing a base treatment, for example, base-soluble by-products, impurities, etc. generated during the manufacturing process of the glass particles can be removed. An example of a method for the base treatment is a method of mixing with a basic solution. As the basic solution, a strongly basic aqueous solution is preferred, and specifically, an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, etc. are more preferred. The treatment temperature is not particularly limited, but is preferably, for example, 20°C to 60°C. The treatment time is not particularly limited, but is preferably, for example, 2 hours to 72 hours.

[0061] According to the method using the glass melting process, the density of the surface OZ groups of the finally obtained inorganic particles can be controlled by, for example, adjusting the compounding ratio of the raw materials, adjusting the firing temperature when the glass obtained in step A is further subjected to firing treatment, and adjusting the firing time when the glass obtained in step A is further subjected to firing treatment. ２ CO ３ , B ２ O ３ and SiO ２ When selecting a combination of ２ CO ３ Increasing the compounding ratio of Na, lowering the firing temperature when the glass obtained in step A is further fired, or shortening the firing time when the glass obtained in step A is further fired, the density of the OZ group on the surface of the inorganic particles decreases, and ２ CO ３ When the compounding ratio of is lowered, when the firing temperature when the glass obtained in step A is further subjected to firing treatment is increased, or when the firing time when the glass obtained in step A is further subjected to firing treatment is extended, the density of the OZ groups on the surface of the inorganic particles tends to increase.

[0062] [Method of Using Powder for Isolating Extracellular Vesicles] The method of using the powder for isolating extracellular vesicles according to the present disclosure includes step a of contacting a liquid containing exosomes, which are extracellular vesicles, with the powder for isolating extracellular vesicles to adsorb exosomes to the powder for isolating extracellular vesicles, and step b of detaching the exosomes adsorbed to the powder for isolating extracellular vesicles. According to the method of using the powder for isolating extracellular vesicles according to the present disclosure, exosomes can be selectively and efficiently separated and recovered from a liquid containing exosomes, which are extracellular vesicles.

[0063] Step a is a step of adsorbing exosomes to the powder for extracellular vesicle isolation by contacting a liquid containing exosomes, which are extracellular vesicles, with the powder for extracellular vesicle isolation. Step a allows, for example, selective separation of exosomes from a liquid containing exosomes and impurities. The liquid containing exosomes is not particularly limited as long as it is a dispersion containing exosomes, which are extracellular vesicles, dispersed in a liquid medium. The liquid containing exosomes may also contain impurities. Examples of impurities include proteins (so-called impurity proteins). Examples of liquid media include serum, plasma, urine, saliva, lymph, and culture medium supernatant.

[0064] The method for contacting a liquid containing exosomes with a powder for separating extracellular vesicles is not particularly limited. A preferred method for contacting a liquid containing exosomes with a powder for separating extracellular vesicles is, for example, mixing the liquid containing exosomes with the powder for separating extracellular vesicles by stirring, as this allows for effective contact between the exosomes in the liquid and the powder for separating extracellular vesicles. Another preferred method for contacting a liquid containing exosomes with a powder for separating extracellular vesicles is to pass the liquid containing exosomes through a column using the powder for separating extracellular vesicles as a carrier.

[0065] Step b is a step of detaching exosomes adsorbed to the powder for extracellular vesicle isolation. Step b enables recovery of exosomes separated by the powder for extracellular vesicle isolation.

[0066] The method for releasing exosomes adsorbed to the extracellular vesicle isolation powder is not particularly limited, and examples include a method in which the release liquid and the exosome-adsorbed extracellular vesicle isolation powder are mixed by stirring. The release liquid is not particularly limited as long as it can release exosomes from the extracellular vesicle isolation powder. The release liquid is preferably an aqueous solution of an inorganic salt such as sodium bicarbonate, sodium tetraborate, or sodium sulfate, and more preferably an aqueous solution of an inorganic salt such as sodium bicarbonate or sodium sulfate. The aqueous solution of the inorganic salt preferably has a pH of 5 to 10, and more preferably a pH of 7 to 9.

[0067] The method of using the powder for separating extracellular vesicles according to the present disclosure may include steps other than steps a and b (so-called other steps). Examples of other steps include a step of recovering released exosomes. For example, when exosomes are released using a release solution, the released exosomes are contained in the release solution. Centrifugation, for example, is a preferred method for recovering the release solution containing exosomes.

[0068] The powder for separating extracellular vesicles according to the present disclosure will be described in more detail below with reference to examples. The powder for separating extracellular vesicles according to the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the present disclosure.

[0069] [Production of Powder for Extracellular Vesicle Separation] <Production Example A-1: ​​Powder for Extracellular Vesicle Separation A-1> 7.00 g of silicon oxide (manufactured by Kojundo Chemical Laboratory Co., Ltd.), 4.53 g of boron oxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 2.12 g of sodium carbonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to obtain a raw material mixture. The raw material mixture was placed in a platinum crucible, heated to 1500°C at a heating rate of 50°C / min, fired at 1500°C for 4 hours, and then cooled to room temperature (25°C; the same applies below) over 12 hours to obtain a first phase-separated glass. Next, the first phase-separated glass was heated to 700°C at a heating rate of 10°C / min, fired at 700°C for 24 hours, and then cooled to room temperature over 7 hours to obtain a second phase-separated glass. Next, the second phase-separated glass was crushed and granulated using a boron carbide mortar and pestle. The resulting crushed particles of the second phase-separated glass were classified using a stainless steel sieve with a mesh size of 20 μm to obtain particles with a maximum particle size of 20 μm or less. After classification using this sieve, the resulting particles were decanted in distilled water to remove particles that did not settle naturally, obtaining crushed and classified particles. The resulting crushed and classified particles were mixed with a 1 N (1 mol / L) hydrochloric acid aqueous solution (manufactured by Kishida Chemical Co., Ltd.) and heated at 90°C for 18 hours for acid treatment. After cooling to room temperature, the acid-treated particles were washed three times with distilled water. The washed particles were mixed with a 0.5 N (0.5 mol / L) sodium hydroxide aqueous solution (prepared from sodium hydroxide powder manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and allowed to stand at room temperature for 4 hours for base treatment. The base-treated particles were washed three times with distilled water and then dried at 90°C to obtain powder A-1 for separating extracellular vesicles.

[0070] <Production Example A-2: Powder A-2 for Extracellular Vesicle Separation> 10.27 g of silicon oxide (manufactured by Kojundo Chemical Laboratory Co., Ltd.), 5.64 g of boron oxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 6.82 g of sodium metasilicate nonahydrate (manufactured by Sigma-Aldrich Co. LLC) were mixed to obtain a raw material mixture. The raw material mixture was placed in a platinum crucible, heated to 1500°C at a heating rate of 50°C / min, fired at 1500°C for 4 hours, and then cooled to room temperature over 12 hours to obtain a first phase-separated glass. Next, the first phase-separated glass was heated to 700°C at a heating rate of 10°C / min, fired at 700°C for 24 hours, and then cooled to room temperature over 7 hours to obtain a second phase-separated glass. Next, the second phase-separated glass was heated to 400°C at a heating rate of 13°C / min, heated at 400°C for 30 minutes, and then quenched with a large amount of water to obtain a third phase-separated glass. The third phase-separated glass was then crushed and granulated using a boron carbide mortar and pestle. The resulting crushed particles of the third phase-separated glass were classified using a stainless steel sieve with a mesh size of 20 μm to obtain particles with a maximum particle size of 20 μm or less. After classification using this sieve, the resulting particles were decanted in distilled water to remove particles that did not settle naturally, thereby obtaining crushed and classified particles. The resulting crushed and classified particles were mixed with a 1 N (1 mol / L) hydrochloric acid aqueous solution (manufactured by Kishida Chemical Co., Ltd.) and heated at 90°C for 18 hours for acid treatment. The acid-treated particles were cooled to room temperature and then washed three times with distilled water. The washed particles were mixed with 0.5 N (0.5 mol / L) aqueous sodium hydroxide solution (prepared from sodium hydroxide powder manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and allowed to stand at room temperature for 25 hours for base treatment. The base-treated particles were washed three times with distilled water and then dried at 90°C to obtain powder A-2 for extracellular vesicle isolation.

[0071] <Production Example A-3: Powder A-3 for separating extracellular vesicles> Powder A-3 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "7.02 g of silicon oxide [manufactured by Kojundo Chemical Laboratory Co., Ltd.], 4.64 g of boron oxide [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.], and 2.12 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0072] <Production Example A-4: Powder A-4 for separating extracellular vesicles> Powder A-4 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "7.41 g of silicon oxide [manufactured by Kojundo Chemical Laboratory Co., Ltd.], 4.14 g of boron oxide [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.], and 1.91 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0073] <Production Example A-5: Powder A-5 for separating extracellular vesicles> Powder A-5 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "7.20 g of silicon oxide [manufactured by Kojundo Chemical Laboratory Co., Ltd.], 4.34 g of boron oxide [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.], and 2.01 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0074] <Production Example A-6: Powder A-6 for separating extracellular vesicles> Powder A-6 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "8.80 g of silicon oxide [manufactured by Kanto Chemical Co., Inc.], 4.60 g of boron oxide [manufactured by Merck & Co.], and 1.68 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0075] <Production Example A-7: Powder A-7 for separating extracellular vesicles> Powder A-7 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "9.80 g of silicon oxide [manufactured by Kanto Chemical Co., Inc.], 4.51 g of boron oxide [manufactured by Merck & Co.], and 2.65 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0076] <Production Example A-8: Powder A-8 for separating extracellular vesicles> Powder A-8 for separating extracellular vesicles was obtained by performing the same operation as in Production Example A-1, except that the raw material composition was changed. The differences from the operation in Production Example A-1 are as follows: "7.01 g of silicon oxide [manufactured by Kojundo Chemical Laboratory Co., Ltd.], 4.22 g of boron oxide [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.], and 1.71 g of sodium carbonate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] were mixed to obtain a raw material mixture."

[0077] <Powder A-9 for Isolating Extracellular Vesicles> Diatomaceous earth (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared as powder A-9 for isolating extracellular vesicles. The diatomaceous earth is a powder containing a plurality of silica particles having hydroxyl groups on the surface.

[0078] [Analysis by X-ray photoelectron spectroscopy] The particles contained in each of the extracellular vesicle isolation powders A-1 to A-8 produced above and the prepared extracellular vesicle isolation powder A-9 were subjected to X-ray photoelectron spectroscopy analysis of the surface. The analytical device used was an X-ray photoelectron spectroscopy analyzer (product name: Quantera SXM) manufactured by ULVAC-PHI, Inc. In order to quantify the OZ group density on the particle surface, a treatment was performed prior to analysis in which the Z atom moiety of the OZ group on the particle surface was ion-exchanged with sodium ions (so-called sodium ion adsorption treatment). Specifically, the treatment was performed under the following condition A.

[0079] (Condition A) 1. In a sealed container, 10 mg of dried particles are impregnated with 0.25 mL of 0.5 mol / L aqueous sodium hydroxide solution and allowed to stand for 4 hours in an environment with an ambient temperature of 25°C. 2. After removing the supernatant, 0.3 mL of distilled water is added to the residue, and the mixture is allowed to stand for 24 hours in an environment with an ambient temperature of 25°C. 3. The above operation "2." is repeated three times. 4. After removing the supernatant, the residue is dried at 90°C.

[0080] The particles after the above treatment were packed into a dedicated substrate and irradiated with X-rays while charge neutralization was performed using electrons and Ar ions. Al-Kα radiation was used as the X-ray source, with a photoelectron takeoff angle of 45° and an aperture diameter of 100 μm. The energy of the photoelectrons generated by X-ray irradiation was measured to obtain a spectrum. The acquired spectrum was subjected to charge correction using the data analysis software "MultiPak" (manufactured by ULVAC-PHI, Inc.) for the X-ray photoelectron spectrometer, with the peak attributable to surface contaminating hydrocarbons in the carbon 1s spectrum set at 284.6 eV. Subsequently, for extracellular vesicle separation powders A-1 to A-8, peak fitting was performed on the peaks detected in the sodium 1s spectrum region, the silicon 2p spectrum region, the boron 1s spectrum region, and the carbon 1s spectrum region, and the area of ​​each peak was calculated. The number of atoms was determined by multiplying the area value of each peak by the relative sensitivity coefficient of the instrument. Then, the ratio of the OZ percentage γ to the sum of the M percentage α and the OZ percentage γ was calculated from the number of atoms determined from the peak area values ​​of silicon and boron, which are elements corresponding to M, and the number of atoms determined from the peak area value of sodium, which is the element corresponding to Z, γ / (α + γ). Furthermore, X / Y, the ratio of the number of sodium atoms X to the sum Y of the numbers of all elements excluding carbon, nitrogen, oxygen, and halogens, i.e., sodium, boron, and silicon, was calculated. For powder A-9 for extracellular vesicle isolation, peak fitting was performed for each of the peaks detected in the fluorine 1s spectrum region, the carbon 1s spectrum region, the aluminum 2p spectrum region, the calcium 2p spectrum region, and the iron 2p spectrum region, in addition to sodium, boron, and silicon, to determine the area of ​​each peak. The area value of each peak was multiplied by the relative sensitivity coefficient of the instrument to determine the number of atoms.Then, γ / (α+γ), which is the ratio of the OZ proportion γ to the sum of the M proportion α and the OZ proportion γ, was calculated from the number of atoms calculated from the peak area values ​​of silicon, boron, aluminum, and calcium, which are elements corresponding to M, and the number of atoms calculated from the peak area value of sodium, which is an element corresponding to Z. In addition, X / Y, which is the ratio of the number of sodium atoms X to the total sum Y of the numbers of all elements excluding carbon, nitrogen, oxygen, and halogens, that is, sodium, boron, silicon, aluminum, calcium, and iron, was calculated.

[0081] The values ​​of α / (α+γ) and the particle surface composition of each of the powders A-1 to A-9 for separating extracellular vesicles are shown in Table 1, and the values ​​of X / Y are shown in Table 2.

[0082]

[0083] From the results shown in Table 1, it was confirmed that, among the powders A-1 to A-9 for separating extracellular vesicles, powders A-1 to A-7 for separating extracellular vesicles correspond to the powder (a) for separating extracellular vesicles according to the present disclosure.

[0084]

[0085] From the results shown in Table 2, it was confirmed that, among the powders A-1 to A-9 for separating extracellular vesicles, powders A-1 to A-7 for separating extracellular vesicles correspond to the powder (b) for separating extracellular vesicles according to the present disclosure.

[0086] [Preparation of Sample Solution] The sample solution used was the culture supernatant of human pancreatic cancer cell line BxPC-3 cells (cell registration number: JCRB1448, JCRB Cell Bank). Specifically, the sample solution was prepared as follows. Human pancreatic cancer cell line BxPC-3 cells were cultured in bovine serum-free RPMI-1640 medium (catalog number: 189-02025, Fujifilm Wako Pure Chemical Industries, Ltd.) for 3 days. The resulting culture solution was then centrifuged (centrifugal force: 300 × g, rotation time: 5 minutes, temperature: 4°C) to recover culture supernatant X. The recovered culture supernatant X was again centrifuged (centrifugal force: 1,200 × g, rotation time: 20 minutes, temperature: 4°C) to recover culture supernatant Y. The recovered culture supernatant Y was centrifuged again (centrifugal force: 10,000 × g, rotation time: 30 minutes, temperature: 4°C) to recover culture supernatant Z. This recovered culture supernatant Z was used as a sample solution. Culture supernatant Z contains exosomes.

[0087] <Method for measuring exosome concentration> The concentration of exosomes contained in the sample solution was measured by sandwich ELISA. Specifically, the measurement was performed as follows. 100 μL of a 1 μg / mL mouse anti-human CD9 antibody (catalog number: 3C9-E12, manufactured by Cell Engineering Institute, Inc.) PBS(-) solution was added to each well of a MaxiSorp 96-well plate (catalog number: 439454, manufactured by Thermo Fisher Scientific) and allowed to stand at room temperature for 1 hour. Next, each well was washed three times with 200 μL of TBS solution containing 0.05% by mass Tween 20 [50 mM Tris and 0.14 M NaCl, pH 8] (hereinafter referred to as "washing solution T"). After that, 200 μL of washing solution T containing 0.01 g / mL bovine serum albumin was added and allowed to stand at room temperature for 1 hour. Next, after removing the washing solution T containing 0.01 g / mL bovine serum albumin from each well, 100 μL of exosome standard solution of each concentration was added and allowed to stand overnight at 4 ° C. Next, each well was washed five times with 200 μL of washing solution T, and then 100 μL of HRP-labeled rat anti-human CD63 antibody [catalog number: 1C8-2B11, manufactured by Cell Engineering Institute, Inc.] was added and allowed to stand at room temperature for 1 hour. Next, each well was washed five times with 200 μL of washing solution T, and then 100 μL of HRP substrate solution (catalog number: 5120-0053, manufactured by SeraCare Life Sciences) was added, followed by shaking at room temperature for 20 minutes. Thereafter, 50 μL of a 2% by weight aqueous sulfuric acid solution was added to stop the reaction, and the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader. Here, the culture supernatant Z was ultracentrifuged (centrifugal force: 100,000×g, rotation time: 1 hour, temperature: 4°C), the resulting pellet was suspended in 1 mL of PBS(-) solution (phosphate buffered saline), the resulting suspension was ultracentrifuged again (centrifugal force: 100,000×g, rotation time: 1 hour, temperature: 4°C), and the resulting pellet was resuspended in 1 mL of PBS(-) solution. Using the recovered exosomes as a standard, a calibration curve was created, showing high linearity between the concentration of the exosome standard solution and the absorbance at a wavelength of 450 nm (R ２ = 0.989).

[0088] <Method for measuring protein concentration> The protein concentration contained in the sample solution was measured using a BCA Protein Assay Kit (catalog number: 23227, manufactured by Thermo Fisher Scientific) with bovine serum albumin (catalog number: A7906, manufactured by Sigma-Aldrich) as the standard. A calibration curve for calculating the protein concentration contained in the sample solution was created using a BCA Protein Assay Kit (catalog number: 23227, manufactured by Thermo Fisher Scientific) with bovine serum albumin as the protein standard, and a high linearity was observed between the concentration of the protein standard solution and the absorbance at a wavelength of 570 nm (R ２ = 0.995).

[0089] [Evaluation] The following tests were conducted using the extracellular vesicle isolation powders A-1 to A-8 produced above and the prepared extracellular vesicle isolation powder A-9 to evaluate the efficiency of exosome separation and recovery using the extracellular vesicle isolation powders. The results are shown in Table 3. Hereinafter, extracellular vesicle isolation powders A-1, A-2, A-3, A-4, A-5, A-6, and A-7 will be referred to as Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, and Example 7, respectively, and extracellular vesicle isolation powders A-8 and A-9 will be referred to as Comparative Example 1 and Comparative Example 2, respectively.

[0090] 1. Separation Test: 500 μL of the sample solution prepared above and 30 mg of the extracellular vesicle separation powder prepared above were mixed in a microtube (ProteoSave® SS 1.5 mL microtube, manufactured by Sumitomo Bakelite Co., Ltd.) and then rotated and stirred at room temperature for 2 hours. The microtube was then centrifuged at 8,000 rpm (revolution per minute; hereinafter the same) for 5 seconds to separate the extracellular vesicle separation powder and the supernatant, which was then removed. Next, after the supernatant was removed, 500 μL of PBS(-) solution was added to the microtube as a washing solution. The extracellular vesicle separation powder and the PBS(-) solution were mixed, and then rotated and stirred at room temperature for 10 minutes to wash the extracellular vesicle separation powder. The microtube was then centrifuged at a rotation speed of 8,000 rpm for 5 seconds to separate the powder for extracellular vesicle isolation from the PBS(-) solution, and the PBS(-) solution was then removed. The above washing and removal procedures were repeated three times in succession.

[0091] 2. Recovery Test The extracellular vesicle separation powder obtained after the above "1. Separation Test" was mixed with 500 μL of 240 mM sodium bicarbonate aqueous solution (pH 9) as the release solution in a microtube, and then rotated and stirred at room temperature for 30 minutes to release the exosomes adsorbed to the extracellular vesicle separation powder from the powder. The microtube was then centrifuged at 8,000 rpm for 5 seconds to separate the extracellular vesicle separation powder and the release solution, which was then recovered. The above release treatment and recovery procedure was performed three times in succession, and the fractions of the recovered release solution were designated "E1 fraction," "E2 fraction," and "E3 fraction," respectively.

[0092] The exosome recovery rate (unit: %) was calculated using the following formula: Exosome recovery rate (unit: %) = [Sum of exosome concentrations (absorbance at a wavelength of 450 nm) contained in E1 fraction, E2 fraction, and E3 fraction / Exosome concentration (absorbance at a wavelength of 450 nm) contained in the sample solution] × 100

[0093] The protein removal rate (unit: %) was calculated using the following formula: Protein removal rate (unit: %) = 100 - [total of protein concentrations (absorbance at a wavelength of 570 nm) contained in fractions E1, E2, and E3 / protein concentration (absorbance at a wavelength of 570 nm) contained in sample solution] × 100

[0094]

[0095] As shown in Table 3, it has been revealed that the powder for separating extracellular vesicles according to the present disclosure can selectively adsorb and separate exosomes from a sample solution containing exosomes, which are extracellular vesicles, and impurity proteins, and that the adsorbed and separated exosomes can be selectively and efficiently detached and recovered.

Claims

1. A powder for separating extracellular vesicles comprising a plurality of inorganic particles, wherein the surface composition of the inorganic particles is represented by the formula M α O β (OZ) γ Y θ wherein in the formula, M represents at least one element selected from the group consisting of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, Z represents at least one element selected from the group consisting of hydrogen, an alkali metal, and an alkaline earth metal, Y represents at least one element selected from the group consisting of carbon, nitrogen, and a halogen, and α, β, γ, and θ represent the proportion of M, the proportion of O, the proportion of OZ, and the proportion of Y, respectively, as measured by X-ray photoelectron spectroscopy, and γ / (α+γ), which is the ratio of γ to the sum of α and γ, is in the range of 0.01 or more and 0.08 or less.

2. A powder for separating extracellular vesicles, comprising a plurality of inorganic particles, wherein the inorganic particles have at least one type of OZ group, the Z representing a hydrogen atom, an alkali metal atom, or an alkaline earth metal atom, and the ratio X / Y of the number of sodium atoms X to the total number Y of atoms of all elements excluding carbon, nitrogen, oxygen, and halogens, measured by X-ray photoelectron spectroscopy of the inorganic particles after sodium ion adsorption treatment under the following condition A, is in the range of 0.01 to 0.

08. (Condition A) 1. In a sealed container, 10 mg of dried inorganic particles are impregnated with 0.25 mL of 0.5 mol / L aqueous sodium hydroxide solution, and allowed to stand for 4 hours in an environment at an atmospheric temperature of 25°C.

2. After removing the supernatant, add 0.3 mL of distilled water to the residue and leave it to stand for 24 hours in an environment with an ambient temperature of 25° C.

3. Repeat the above operation of “2.” three times.

4. After removing the supernatant, dry the residue at 90° C.

3. A powder for isolating extracellular vesicles as described in claim 1 or claim 2, wherein the inorganic particles contain silicon, and the ratio of the number of silicon atoms to the total number of atoms of silicon, boron, aluminum, titanium, zirconium, magnesium, calcium, zinc and iron, as measured by X-ray photoelectron spectroscopy, is in the range of 50% or more and 98.5% or less.

4. The powder for isolating extracellular vesicles according to claim 3, wherein the inorganic particles further contain boron.

5. A powder for separating extracellular vesicles as described in claim 1 or claim 2, wherein the average particle size of the inorganic particles is in the range of 0.5 μm or more and 150 μm or less.

6. A powder for separating extracellular vesicles as described in claim 1 or claim 2, wherein the inorganic particles have pores, and the average pore diameter of the pores, measured by mercury intrusion porosimetry, is in the range of 50 nm or more and 1000 nm or less.

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