Extracellular vesicle separating powder

Abstract

To provide an extracellular vesicle separating powder capable of selectively and efficiently separating and recovering exosome from a liquid containing exosome as extracellular vesicle.SOLUTION: An extracellular vesicle separating powder containing a plurality of inorganic particles is such that: in the inorganic particle, X / Y as a ratio of the atomic number X of boron to the total atomic number Y of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, measured by an X-ray photoelectron spectroscopy is 0.015 or more and 0.30 or less.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Exosomes are small extracellular vesicles known to efficiently mediate intercellular communication and signal transduction, including the transport of biologically active molecules, such as proteins, lipids, and nucleic acids, between cells and across biological barriers. Exosomes are not easily degraded and can transmit signals over long distances without dilution, making them promising candidates for drug delivery systems (DDS) and therapeutic drugs. In order to use exosomes for the above-mentioned medical applications, a separation method that can reduce the inclusion of unintended exosomes and impurities is required. Various methods for separating and recovering extracellular vesicles have been reported.

[0003] For example, Patent Document 1 discloses a method for isolating biological particles from a liquid sample, the method comprising the steps of: a) obtaining a liquid sample from a subject or a cell culture; b) contacting the liquid sample with a crystal / precipitation-inducing agent under conditions suitable for allowing crystal formation and / or precipitation, thereby producing 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 biological particles from the liquid sample. Patent Document 2 discloses a method for separating biologically derived micromaterials using size exclusion chromatography. According to such a separation method using size exclusion chromatography, biologically derived samples of various sizes repeatedly enter and exit the pores in the surface porous particles, thereby obtaining fractions of biologically derived components (for example, fractions of exosomes and impurity proteins). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2017 / 0252670 [Patent Document 2] International Publication No. 2019 / 202994 Summary of the Invention [Problem to be solved by the invention]

[0005] In order to use exosomes for medical purposes, it is necessary to establish a method for more selectively isolating and recovering exosomes. In addition, since commercial formulations are expected to be administered to a large number of patients, a method for efficiently isolating and recovering exosomes on a scale applicable to commercial formulations is required. In these respects, the methods described in Patent Documents 1 and 2 still have issues with respect to selective and efficient separation and recovery of exosomes.

[0006] The present disclosure has been made in consideration 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. [Means for solving the problem]

[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, The inorganic particles are powders for separating extracellular vesicles, in which the ratio X / Y, where X is the number of boron atoms and Y is the sum of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, as measured by X-ray photoelectron spectroscopy, is in the range of 0.015 or more and 0.30 or less. [2] The powder for isolating extracellular vesicles described in [1], wherein the inorganic particles are particles of silicon oxide containing boron. [3] The powder for separating extracellular vesicles described in [1] or [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. [4] The powder for separating extracellular vesicles described in any one of [1] to [3], wherein the inorganic particles have pores, and the average pore diameter of the pores, as measured by mercury intrusion porosimetry, is in the range of 50 nm or more and 1000 nm or less. Effect of the Invention

[0008] 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The powder for separating extracellular vesicles according to the present disclosure will be described in detail below. The explanation of the requirements described below may be based on the representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment, and can be modified as appropriate within the scope of the purpose of the present disclosure.

[0010] 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 limit and upper limit, respectively. In the numerical ranges described in the present disclosure, the upper or lower limit value described in a certain numerical range may be replaced with the upper or lower limit value of another numerical range described in the present disclosure. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit value described in a certain numerical range may be replaced with a value shown in the examples.

[0011] In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0012] 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.

[0013] Extracellular vesicles are vesicles that are derived from cells and exist outside the cells, and are classified into exosomes, microvesicles, and apoptotic bodies based on their size. Exosomes and microvesicles contain proteins, nucleic acids such as mRNA and miRNA, and apoptotic bodies contain fragmented nuclei and organelles. The difference between exosomes and microvesicles is not clear, and the exosomes described in the present disclosure may contain microvesicles.

[0014] [Powder for extracellular vesicle separation] The powder for separating extracellular vesicles according to the present disclosure is a powder for separating extracellular vesicles comprising a plurality of inorganic particles, in which the inorganic particles have a ratio X / Y, which is the ratio of the number of boron atoms X to the sum Y of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, as measured by X-ray photoelectron spectroscopy, in the range of 0.015 or more and 0.30 or less. According to the powder for separating extracellular vesicles according to the present disclosure, exosomes, which are extracellular vesicles, can be selectively and efficiently separated and recovered from a liquid containing exosomes. Although the reason why the powder for separating extracellular vesicles according to the present disclosure can exhibit such an effect is unclear, the present inventors speculate as follows. However, the following speculation is not intended to limit the interpretation of the powder for separating extracellular vesicles according to the present disclosure, but is merely an example.

[0015] The powder for separating extracellular vesicles according to the present disclosure was made based on the discovery that inorganic particles having boron in a specific atomic number range on their particle surfaces have high selective recovery properties for exosomes, which are extracellular vesicles, and that there is a correlation between the number of boron atoms on the particle surfaces of inorganic particles and the above-mentioned "X / Y" measured by X-ray photoelectron spectroscopy. The powder for separating extracellular vesicles according to the present disclosure includes a plurality of inorganic particles having boron in a specific atomic range on the surface. The boron present on the surface of the inorganic particles contributes to the adsorption of exosomes. For this reason, when the powder for separating extracellular vesicles according to the present disclosure is brought into contact with a liquid containing exosomes, it is considered that the exosomes are selectively and efficiently captured by the boron (specifically, a group containing boron) present on the surface of the inorganic particles, and the exosomes captured by the group containing boron are efficiently released from the group containing boron. An example of the group containing boron is a borate group.

[0016] In addition, when the inorganic particles are particles of silicon oxide containing boron, it is believed that the ratio of non-crosslinked sites (e.g., hydroxyl group terminals) to crosslinked sites forming the silicon oxide skeleton (non-crosslinked sites / crosslinked sites) is appropriately reduced by having a specific number of boron atoms or more on the surface of the inorganic particles. The inventors believe that hydroxyl groups contribute to the adsorption of exosomes, and it is presumed that the powder for separating extracellular vesicles according to the present disclosure can achieve the effect of selectively and efficiently separating and recovering exosomes, for example, by controlling the density of hydroxyl groups present on the surface of the inorganic particles with boron. From the above, it is presumed that the powder for separating extracellular vesicles according to the present disclosure makes it possible to selectively and efficiently separate and recover exosomes from a liquid containing exosomes, which are extracellular vesicles.

[0017] The boron present on the surface of inorganic particles is identified by X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopy used in the present disclosure is a quantitative and qualitative analysis method that can analyze the number of constituent elements and their electronic state in the surface region of a sample by irradiating a 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 rays and Mg-Kα rays, and in the present disclosure, when Al-Kα rays are used as the X-ray source, the surface region is the region where photoelectrons generated in the sample can escape outside the sample without losing energy. Although there are some differences depending on the energy of the generated photoelectrons, the depth of the surface region is about 5 nm.

[0018] <x y> The inorganic particles contained in the powder for separating extracellular vesicles according to the present disclosure have a ratio X / Y, which is the ratio of the number of boron atoms X to the sum Y of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, measured by X-ray photoelectron spectroscopy, in the range of 0.015 to 0.30, preferably in the range of 0.025 to 0.28, more preferably in the range of 0.050 to 0.25, even more preferably in the range of 0.075 to 0.23, and particularly preferably in the range of 0.10 to 0.20. When X / Y is 0.015 or more, exosomes can be appropriately adsorbed onto the surface of the inorganic particles, and the adsorbed exosomes tend to be efficiently detached from the surface of the inorganic particles. When X / Y is 0.30 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.

[0019] In the present disclosure, X / Y is determined by the following method. First, X-ray photoelectron spectroscopy is performed on the surface of inorganic particles contained in the powder for extracellular vesicles separation. An X-ray photoelectron spectroscopy analyzer is used as the analytical device, and X-rays are irradiated onto the powder for extracellular vesicles separation, which is filled on a dedicated substrate, while neutralizing the charge with electrons and Ar ions. Al-Kα rays are used as the X-ray source, the photoelectron takeoff angle is set to 45°, and the aperture diameter is set to 100 μm. The energy of the photoelectrons generated by the X-ray irradiation is measured to obtain a spectrum. The data analysis software of the X-ray photoelectron spectroscopy analyzer is used for the obtained spectrum, and charging correction is performed by setting the peak attributable to surface contaminating hydrocarbons in the 1s spectrum of carbon to 284.6 eV. Next, the detected peaks are identified as elemental species and peak fitting is performed. Next, the number of atoms is calculated by multiplying the area value of each peak obtained by peak fitting by the relative sensitivity coefficient of the device. From the number of atoms X determined from the peak area value of boron and the total number of atoms Y determined from the peak area values ​​of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, the ratio X / Y of the number of boron atoms X to the total number Y of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine is calculated. As the X-ray photoelectron spectrometer, for example, Quantera SXM (product name) manufactured by ULVAC-PHI, Inc. can be suitably used. 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.

[0020] -Composition of inorganic particles- The composition of the inorganic particles is not particularly limited as long as it contains boron. In other words, the inorganic particles may be particles of a compound containing boron. Compounds containing boron include, for example, oxides, nitrides, and carbides. The inorganic particles are preferably particles of an oxide containing boron. The composition of the boron-containing oxide is preferably a composition containing an element other than boron as a main component, from the viewpoint of being poorly soluble or insoluble in water. Here, the term "main component" refers to the component that has the largest content ratio on a mass basis. The boron-containing oxide preferably contains, as a main component, an element having a valence of 2 or more other than boron, more preferably at least one element selected from the group consisting of silicon, aluminum, titanium, zirconium, magnesium, calcium, zinc, and iron, and even more preferably silicon.

[0021] The inorganic particles preferably contain boron and silicon and are oxide particles whose main component is silicon, that is, are boron-containing silicon oxide particles. The inorganic particles may be particles of silicon oxide containing boron and sodium. When the inorganic particles are silicon oxide particles containing boron, exosomes, which are extracellular vesicles, tend to be able to be separated and recovered more selectively and efficiently from a liquid containing them. When the inorganic particles are particles of silicon oxide containing boron, the inorganic particles preferably have a ratio A1 / A2 of the number of boron atoms A1 to the number of silicon atoms A2, as measured by X-ray photoelectron spectroscopy, in the range of 0.015 or more and 0.50 or less, more preferably in the range of 0.025 or more and 0.40 or less, and even more preferably in the range of 0.050 or more and 0.30 or less. When A1 / A2 is 0.015 or more, exosomes can be more appropriately adsorbed onto the surface of the inorganic particles, and the adsorbed exosomes tend to be more efficiently detached from the surface of the inorganic particles. When A1 / A2 is 0.50 or less, exosomes can be more efficiently adsorbed onto the surface of inorganic particles, and more efficient detachment of the adsorbed exosomes from the particle surface tends not to be impaired.

[0022] -Physical properties of inorganic particles- The powder for separating extracellular vesicles according to the present disclosure contains a plurality of inorganic particles. The inorganic particles are preferably poorly soluble or insoluble in water. In this 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.

[0023] -Shape of inorganic particles- The shape of the inorganic particles is not particularly limited. Examples of the shape of the inorganic particles include spherical, elliptical, and irregular shapes.

[0024] -Structure of inorganic particles- The atomic arrangement structure of the inorganic particles is not particularly limited. The inorganic particles may be crystalline or amorphous.

[0025] 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 particle" in the present disclosure includes not only the outer surface of the inorganic particle but also the inner surface of the pores.

[0026] When the 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 or more and 1000 nm or less, from the viewpoint of efficiently capturing exosomes inside the pores. The average pore size of the pores in 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 in the inorganic particles is preferably 800 nm or less, more preferably 700 nm or less, and even more preferably 500 nm or less. In one embodiment, the average pore diameter of the pores of the inorganic particles may be in the range of 60 nm or more and 800 nm or less, 70 nm or more and 700 nm or less, 80 nm or more and 500 nm or less, or 80 nm or more and 400 nm or less.

[0027] The average pore size of the pores in the inorganic particles is measured by mercury intrusion porosimetry. For the measurement by the mercury intrusion method, a pore size distribution measuring device is used. Specifically, the 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 calculated from the pore distribution of pores of 1 μm or less in order to exclude the influence of the voids between the inorganic particles. The mercury parameters are set as follows: mercury contact angle 140°, mercury surface tension 480mN / m. 0.01g to 0.1g of sample is collected in a standard cell and measured under an initial pressure of 6.9kPa. The average pore size is calculated from the obtained pore distribution of 1μm or less. As the pore size distribution measuring device, for example, AutoPore V9620 manufactured by Micromeritics can be suitably used, although the pore size distribution measuring device is not limited thereto.

[0028] -Average particle size of inorganic particles- The average particle size of the inorganic particles is not particularly limited, but is preferably smaller from the viewpoint of efficiently capturing exosomes, which are extracellular vesicles contained in a liquid such as a sample solution. In addition, when the inorganic particles have pores, it is preferable that the average particle size is smaller from the viewpoint of efficiently capturing exosomes, which are extracellular vesicles contained in a liquid such as a sample solution, 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 or more and 150 μm or less, more preferably in the range of 1.0 μm or more and 150 μm or less, even more preferably in the range of 2.0 μm or more and 150 μm or less, particularly preferably in the range of 3.0 μm or more and 100 μm or less, and most preferably in the range of 5.0 μm or more and 50 μm or less.

[0029] In the present disclosure, the average particle size of inorganic particles means the volume average primary particle size. The average particle size of inorganic particles is determined as the particle size (D50) at which the cumulative amount from the small diameter side reaches 50% in the volume-based particle size distribution measured by a laser diffraction scattering type particle size distribution measuring device. As the laser diffraction scattering type particle size distribution measuring device, for example, Mastersizer 2000 manufactured by Malvern Panalytical can be suitably used. However, the laser diffraction scattering type particle size distribution measuring device is not limited to this.

[0030] <<Application>> Exosomes are small vesicles released by cells. They inherit the properties of the releasing cell (so-called donor cell). When applied to a receiving cell (so-called recipient cell), they can confer the properties of the donor cell to the recipient cell, 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, which are extracellular vesicles, from a liquid containing exosomes, 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.

[0031] [Manufacturing method of powder for extracellular vesicles isolation] The method for producing the powder for separating extracellular vesicles according to the present disclosure is not particularly limited. Examples of methods for producing the powder for separating extracellular vesicles 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. Hereinafter, as one suitable example of a method for producing a powder for separating extracellular vesicles according to the present disclosure, a method via a glass melting process will be described.

[0032] The manufacturing method of the powder for separating extracellular vesicles according to the present disclosure via a glass melting process (hereinafter also referred to as "manufacturing method X") includes, for example, a step A of firing and vitrifying the raw material, and a step B of crushing and granulating the glass.

[0033] Step A is a step of firing the raw material to vitrify it. According to step A, glass can be obtained. Raw materials include, for example, sodium carbonate (Na2CO3), sodium tetraborate (Na2B4O7), sodium metasilicate nonahydrate (Na2SiO3·9H2O), boron oxide (B2O3), and silicon dioxide (SiO2). As the raw materials, for example, a combination of Na2CO3, B2O3 and SiO2, a combination of Na2B4O7, B2O3 and SiO2, or a combination of Na2SiO3·9H2O, B2O3 and SiO2 is preferable because it is easy to control the surface boron concentration of the inorganic particles finally obtained.

[0034] When a combination of Na2CO3, B2O3, and SiO2 is selected as the raw materials, the compounding ratio of Na2CO3, B2O3, and SiO2 is preferably 5 parts by mass to 30 parts by mass of Na2CO3, 20 parts by mass to 60 parts by mass of B2O3, and 25 parts by mass to 65 parts by mass of SiO2, when the total of Na2CO3, B2O3, and SiO2 is 100 parts by mass, more preferably 10 parts by mass to 20 parts by mass of Na2CO3, 25 parts by mass to 40 parts by mass of B2O3, and 50 parts by mass to 65 parts by mass of SiO2, and even more preferably 13.5 parts by mass to 20 parts by mass of Na2CO3, 30 parts by mass to 40 parts by mass of B2O3, and 50 parts by mass to 58 parts by mass of SiO2. When a combination of Na2B4O7, B2O3, and SiO2 is selected as the raw materials, the compounding ratio of Na2B4O7, B2O3, and SiO2 is preferably 10 parts by mass to 50 parts by mass of Na2B4O7, 1 part by mass to 20 parts by mass of B2O3, and 30 parts by mass to 70 parts by mass of SiO2, when the total of Na2B4O7, B2O3, and SiO2 is 100 parts by mass, and more preferably 20 parts by mass to 40 parts by mass of Na2B4O7, 2 parts by mass to 15 parts by mass of B2O3, and 55 parts by mass to 70 parts by mass of SiO2. When a combination of Na2SiO3·9H2O, B2O3, and SiO2 is selected as the raw materials, the compounding ratio of Na2SiO3·9H2O, B2O3, and SiO2 is preferably 15 to 45 parts by mass of Na2SiO3·9H2O, 10 to 50 parts by mass of B2O3, and 15 to 55 parts by mass of SiO2, and more preferably 20 to 40 parts by mass of Na2SiO3·9H2O, 20 to 40 parts by mass of B2O3, and 30 to 55 parts by mass of SiO2, when the total of Na2SiO3·9H2O, B2O3, and SiO2 is 100 parts by mass.

[0035] The raw material may contain one or more different metal oxides other than Na2CO3, Na2B4O7, Na2SiO3·9H2O, B2O3, and SiO2. Examples of the different metal oxides include magnesium oxide (MgO), calcium oxide (CaO), aluminum oxide (Al2O3), zirconium oxide (ZrO2), and titanium oxide (TiO2). When a dissimilar metal oxide other than Na2CO3, Na2B4O7, Na2SiO3·9H2O, B2O3, and SiO2 is blended into the raw material, the blending amount of the dissimilar metal oxide may be, for example, 1 part by mass to 10 parts by mass per 100 parts by mass of the total of Na2CO3, Na2B4O7, Na2SiO3·9H2O, B2O3, and SiO2.

[0036] 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 more and 1600°C or less, and more preferably in the range of 1400°C or more and 1600°C or less. The rate of temperature rise when heating to the firing temperature is not particularly limited, but is preferably, for example, 10° C. / min to 50° C. / min. The firing may be carried out in an air atmosphere or in an inert atmosphere, but is preferably carried out in an air atmosphere. The firing time may be any time that allows the raw materials to be vitrified, and may be appropriately set depending on the firing temperature. The firing time may be, for example, 2 to 8 hours. After firing, it is preferable to cool the product to about room temperature. Examples of the cooling method include natural cooling and rapid cooling.

[0037] Before the step B, the glass obtained in the step A may be further subjected to a firing treatment. By carrying out the firing treatment, the glass can be made into a more stable structure. When the glass obtained in the 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 the product to about room temperature. Examples of the cooling method include natural cooling and rapid cooling.

[0038] Step B is a step of crushing the glass into particles. According to step B, glass particles can be obtained. The method for crushing the glass is not particularly limited. Examples of the crushing means include a crusher, a stamp mill, a ball mill, a bead mill, a jet mill, and a vibration mill.

[0039] The particle size distribution of the glass particles obtained by the crushing process is preferably narrowed by a classification process. The classification means may be, for example, a sieve.

[0040] The manufacturing method X may include a step other than the above-mentioned step A and step B. An example of a step other than step A and step B is step C in which glass particles are treated with an acid and then treated with a base. According to the step C, for example, inorganic particles having pores therein can be obtained. In addition, acid-soluble or base-soluble by-products, impurities, etc. generated during the production process of the glass particles can be removed.

[0041] The acid treatment can, for example, form a pore structure in the glass particles, and can also remove acid-soluble by-products, impurities, and the like that are generated during the manufacturing process of the glass particles. The acid treatment may be carried out, for example, by heating in an acidic solution. The acidic solution can be appropriately selected depending on the composition of the glass. The acidic solution is preferably a strong acid, and specifically, an aqueous solution of an inorganic acid such as an aqueous solution of hydrochloric acid, an aqueous solution of nitric acid, or an aqueous solution of perchloric acid is more preferable. In the acid treatment, two or more kinds of acid solutions may be used in combination. The heating temperature is not particularly limited, but is preferably from 60°C to 120°C, for example. The heating time is not particularly limited, but is preferably, for example, 2 to 24 hours.

[0042] The base treatment after the acid treatment can, for example, form pores in the glass particles, and can also remove base-soluble by-products, impurities, etc., generated during the manufacturing process of the glass particles. The base treatment may be carried out, for example, by mixing with a basic solution. The basic solution is preferably a strongly basic aqueous solution, and specifically, an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, or the like is more preferable. The treatment temperature is not particularly limited, but is preferably from 20°C to 60°C, for example. The treatment time is not particularly limited, but is preferably, for example, 2 to 72 hours.

[0043] According to the method via the above-mentioned glass melting process, the surface boron concentration of the finally obtained inorganic particles can be controlled, for example, by adjusting the compounding ratio of the raw materials, adjusting the firing temperature when the glass obtained in step A is further subjected to a firing treatment, and adjusting the firing time when the glass obtained in step A is further subjected to a firing treatment. For example, when a combination of Na2CO3, B2O3, and SiO2 is selected as the raw materials, increasing the compounding ratio of Na2CO3, decreasing 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, tends to increase the surface boron concentration of the inorganic particles, whereas decreasing the compounding ratio of Na2CO3, increasing the firing temperature when the glass obtained in step A is further fired, or lengthening the firing time when the glass obtained in step A is further fired, tends to decrease the surface boron concentration of the inorganic particles.

[0044] [How to use the powder for extracellular vesicles isolation] A method for using the powder for extracellular vesicle isolation according to the present disclosure includes step a of adsorbing exosomes to the powder for extracellular vesicle isolation by contacting a liquid containing exosomes, which are extracellular vesicles, with the powder, and step b of detaching the exosomes adsorbed to the powder for extracellular vesicle isolation. According to the method of using the powder for separating extracellular vesicles disclosed herein, exosomes can be selectively and efficiently separated and recovered from a liquid containing exosomes, which are extracellular vesicles.

[0045] Step a is a step of adsorbing exosomes to a powder for extracellular vesicle isolation by contacting a liquid containing exosomes, which are extracellular vesicles, with the powder for extracellular vesicle isolation. According to step a, for example, exosomes can be selectively separated from a liquid containing exosomes and contaminants. The liquid containing exosomes is not particularly limited as long as it is a dispersion liquid in which exosomes, which are extracellular vesicles, are dispersed in a liquid medium. The liquid containing exosomes may contain impurities. An example of the contaminants is a protein (so-called contaminant protein). Liquid media include, for example, serum, plasma, urine, saliva, lymph, and culture medium supernatants.

[0046] The method for contacting a liquid containing exosomes with a powder for isolating extracellular vesicles is not particularly limited. As a method for contacting a liquid containing exosomes with a powder for separating extracellular vesicles, for example, a method in which the liquid containing exosomes and the powder for separating extracellular vesicles are mixed by stirring is preferred, since this method allows effective contact between the exosomes in the liquid and the powder for separating extracellular vesicles. In addition, as a method for contacting a liquid containing exosomes with a powder for separating extracellular vesicles, for example, a method in which a liquid containing exosomes is passed through a column using the powder for separating extracellular vesicles as a carrier is also preferred.

[0047] Step b is a step of detaching exosomes adsorbed to the powder for extracellular vesicle isolation. According to step b, it is possible to recover exosomes separated by the powder for separating extracellular vesicles.

[0048] The method for detaching exosomes adsorbed to the powder for extracellular vesicle isolation is not particularly limited, and examples thereof include a method in which a detachment solution and the powder for extracellular vesicle isolation to which exosomes are adsorbed are mixed by stirring. The release liquid is not particularly limited as long as it can release exosomes from the powder for extracellular vesicle isolation. The release liquid is preferably an aqueous solution of an inorganic salt such as sodium hydrogen carbonate, sodium tetraborate, or sodium sulfate, and more preferably an aqueous solution of an inorganic salt such as sodium hydrogen carbonate or sodium sulfate. The aqueous solution of the inorganic salt preferably has a pH of 5 to 10, and more preferably has a pH of 7 to 9.

[0049] The method for using the powder for separating extracellular vesicles according to the present disclosure may include steps other than step a and step b (so-called other steps). Other steps include, for example, a step of recovering detached exosomes. For example, when exosomes are detached using a detachment solution, the detached exosomes are contained in the detachment solution. A preferred method for recovering the detachment solution containing exosomes is, for example, centrifugation. EXAMPLES

[0050] 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 exceed the gist of the present disclosure.

[0051] [Production of powder for extracellular vesicles isolation] <Production example A-1: ​​Powder A-1 for separating extracellular vesicles> 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 Co., Ltd.), and 2.12 g of sodium carbonate (manufactured by Fujifilm Wako Pure Chemical Co., 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, heated and 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, heated and 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 crushed particles of the second phase-separated glass obtained 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 obtained particles were decanted in distilled water to remove particles that did not naturally settle, and crushed and classified particles were obtained. The crushed and classified particles obtained were mixed with a 1N (1 mol / L) hydrochloric acid aqueous solution (Kishida Chemical Co., Ltd.) and heated at 90°C for 18 hours for acid treatment. The particles after the acid treatment were cooled to room temperature and then washed three times with distilled water. The washed particles were mixed with a 0.5N (0.5 mol / L) sodium hydroxide aqueous solution (prepared from sodium hydroxide powder manufactured by Fujifilm Wako Pure Chemical Co., Ltd.) and left to stand at room temperature for 4 hours for base treatment. The particles after the base treatment were washed three times with distilled water and then dried at 90°C to obtain powder A-1 for extracellular vesicle separation.

[0052] <Production example A-2: Powder A-2 for separating extracellular vesicles> A raw material mixture was obtained by mixing 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). The raw material mixture was placed in a platinum crucible, heated to 1500°C at a heating rate of 50°C / min, heated and 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, heated and 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. Next, the third phase-separated glass was crushed and granulated using a boron carbide mortar and pestle. The crushed particles of the obtained 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 obtained particles were decanted in distilled water to remove particles that did not naturally settle, and crushed and classified particles were obtained. The obtained crushed and classified particles were mixed with a 1N (1 mol / L) hydrochloric acid aqueous solution (manufactured by Kishida Chemical Co., Ltd.) and heated at 90°C for 18 hours to perform an acid treatment. The particles after the acid treatment were cooled to room temperature and then washed three times with distilled water. The washed particles were mixed with 0.5N (0.5mol / 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 particles after base treatment were washed three times with distilled water and then dried at 90°C to obtain powder A-2 for extracellular vesicle isolation.

[0053] <Production example A-3: Powder A-3 for extracellular vesicle separation> Powder A-3 for separating extracellular vesicles was obtained by the same procedure as in Production Example A-1 except that the raw material composition was changed. The differences from the procedure 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 together to obtain a raw material mixture."

[0054] <Production example A-4: Powder A-4 for separating extracellular vesicles> Powder A-4 for separating extracellular vesicles was obtained by the same procedure as in Production Example A-1 except that the raw material composition was changed. The differences from the procedure 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 together to obtain a raw material mixture."

[0055] <Production example A-5: Powder for extracellular vesicle separation A-5> Powder A-5 for separating extracellular vesicles was obtained by the same procedure as in Production Example A-1 except that the raw material composition was changed. The differences from the procedure 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 together to obtain a raw material mixture."

[0056] <Production example A-6: Powder A-6 for separating extracellular vesicles> Powder A-6 for separating extracellular vesicles was obtained by the same procedure as in Production Example A-1 except that the raw material composition was changed. The differences from the procedure 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 together to obtain a raw material mixture."

[0057] <Production example A-7: Powder A-7 for separating extracellular vesicles> Powder A-7 for separating extracellular vesicles was obtained by the same procedure as in Production Example A-1 except that the raw material composition was changed. The differences from the procedure in Production Example A-1 are as follows. "5.41 g of silicon oxide (manufactured by Kojundo Chemical Laboratory Co., Ltd.), 5.57 g of boron oxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 3.18 g of sodium carbonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed together to obtain a raw material mixture."

[0058] <Powder A-8 for extracellular vesicle separation> As powder A-8 for separating extracellular vesicles, diatomaceous earth (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared. The diatomaceous earth is a powder containing a plurality of silica particles that do not contain boron.

[0059] [X-ray photoelectron spectroscopy analysis] The particles contained in each of the powders A-1 to A-7 for extracellular vesicle isolation produced above and the prepared powder A-8 for extracellular vesicle isolation were subjected to X-ray photoelectron spectroscopy analysis of the surface. The analysis device used was an X-ray photoelectron spectroscopy analyzer (product name: Quantera SXM) manufactured by ULVAC-PHI, Inc. Specifically, the following operations were carried out using each powder as a sample. The sample was loaded onto a special substrate and irradiated with X-rays while neutralizing the charge with electrons and Ar ions. Al-Kα rays were used as the X-ray source, with a photoelectron take-off 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 spectrum was corrected for charge using the data analysis software "MultiPak" (ULVAC-PHI, Inc.) of the X-ray photoelectron spectrometer, with the peak attributable to surface contaminating hydrocarbons in the carbon 1s spectrum set at 284.6 eV. Then, for the powders A-1 to A-7 for separating extracellular vesicles, peak fitting was performed for the peaks detected in the sodium 1s spectrum region, the boron 1s spectrum region, and the silicon 2p spectrum region, and the area of ​​each peak was calculated. The area value of each peak was multiplied by the relative sensitivity coefficient of the device to calculate the number of atoms. Then, X / Y, which is the ratio of the number of boron atoms X to the total number Y of all detected elements except carbon, nitrogen, oxygen, fluorine, and chlorine, that is, sodium, boron, and silicon atoms, was calculated. In addition, A1 / A2, which is the ratio of the number of boron atoms A1 to the number of silicon atoms A2, was also calculated. For the powder A-8 for extracellular vesicles separation, in addition to sodium, boron, and silicon, peaks detected in the 2p spectrum region of aluminum, peaks detected in the 2p spectrum region of calcium, and peaks detected in the 2p spectrum region of iron were fitted, and the area of ​​each peak was calculated. The area value of each peak was multiplied by the relative sensitivity coefficient of the device to determine the number of atoms. Then, X / Y, which is the ratio of the number of boron atoms X to the total number Y of all detected elements except carbon, nitrogen, oxygen, fluorine, and chlorine, that is, sodium, boron, silicon, aluminum, calcium, and iron atoms, was calculated. In addition, A1 / A2, which is the ratio of the number of boron atoms A1 to the number of silicon atoms A2, was also calculated.

[0060] Table 1 shows the X / Y values ​​obtained by the above-mentioned X-ray photoelectron spectroscopy analysis for each of the powders A-1 to A-8 for isolating extracellular vesicles. Table 2 shows the A1 / A2 values ​​obtained by the above-mentioned X-ray photoelectron spectroscopy analysis for each of the extracellular vesicle isolation powders A-1 to A-8.

[0061] [Table 1]

[0062] From the results shown in Table 1, it was confirmed that, among the powders A-1 to A-8 for separating extracellular vesicles, powders A-1 to A-5 for separating extracellular vesicles correspond to the powders for separating extracellular vesicles according to the present disclosure.

[0063] [Table 2]

[0064] From the results shown in Table 2, it was confirmed that the ratio A1 / A2 of the number of boron atoms A1 to the number of silicon atoms A2 for all of the powders A-1 to A-5 for isolating extracellular vesicles was in the range of 0.015 or more and 0.50 or less.

[0065] [Preparation of sample solution] The sample liquid 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 centrifuged again (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 the sample liquid. Culture supernatant Z contains exosomes.

[0066] <Method for measuring exosome concentration> The concentration of exosomes in the sample solution was measured by sandwich ELISA as follows. 100μL of 1μg / mL mouse anti-human CD9 antibody (catalog number: 3C9-E12, manufactured by Cell Engineering Institute, Inc.) in PBS(-) was added to each well of a MaxiSorp 96 well plate (catalog number: 439454, manufactured by Thermo Fisher Scientific Co., Ltd.) and left to stand at room temperature for 1 hour. Next, each well was washed three times with 200μL of TBS solution containing 0.05% Tween 20 (50mM Tris and 0.14M NaCl, pH8) (hereinafter referred to as "washing solution T"), after which 200μL of washing solution T containing 0.01g / mL bovine serum albumin was added and left to stand at room temperature for 1 hour. Next, after removing washing solution T containing 0.01g / mL bovine serum albumin from each well, 100μL of exosome standard solution of each concentration was added and left to stand at 4℃ overnight. 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 left 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, Inc.) was added and shaken at room temperature for 20 minutes. Then, 50 μL of 2% sulfuric acid aqueous 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 above 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, which showed high linearity between the concentration of the exosome standard solution and the absorbance at a wavelength of 450 nm (R 2 =0.989).

[0067] <Method for measuring protein concentration> The protein concentration in the sample solution was measured using a BCA Protein Assay Kit (catalog number: 23227, Thermo Fisher Scientific) with bovine serum albumin (catalog number: A7906, Sigma-Aldrich) as the standard. A calibration curve for calculating the protein concentration in the sample solution was created using a BCA Protein Assay Kit (catalog number: 23227, Thermo Fisher Scientific) with bovine serum albumin as the protein standard. 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).

[0068] [evaluation] The following tests were performed using the extracellular vesicle isolation powders A-1 to A-7 produced above, and the extracellular vesicle isolation powder A-8 prepared above, to evaluate the efficiency of exosome separation and recovery using the extracellular vesicle isolation powders. The results are shown in Table 3. Hereinafter, powders for separating extracellular vesicles A-1, A-2, A-3, A-4 and A-5 will be referred to as Example 1, Example 2, Example 3, Example 4 and Example 5, respectively, and powders for separating extracellular vesicles A-6, A-7 and A-8 will be referred to as Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively.

[0069] 1.Separation test 500 μL of the sample solution prepared above and 30 mg of the powder for extracellular vesicles separation produced above were mixed in a microtube (ProteoSave (registered trademark) SS 1.5 mL microtube, manufactured by Sumitomo Bakelite Co., Ltd.), and then rotated and stirred at room temperature for 2 hours. Next, the microtube was centrifuged at a rotation speed of 8,000 rpm (revolutions per minute; the same applies below) and a rotation time of 5 seconds to separate the powder for extracellular vesicles separation from the supernatant, and the supernatant was removed. Next, after removing the supernatant, 500 μL of PBS(-) solution was added as a washing solution to the microtube, and the powder for extracellular vesicles separation was mixed with the PBS(-) solution, and then the powder for extracellular vesicles separation was washed by rotating and stirring at room temperature for 10 minutes. After that, the microtube was centrifuged at a rotation speed of 8,000 rpm and a rotation time of 5 seconds to separate the powder for extracellular vesicles separation from the PBS(-) solution, and the PBS(-) solution was removed. The above washing and removal operations were performed three times in succession.

[0070] 2. Recovery test The powder for extracellular vesicles separation after the above "1. Separation test" was mixed with 500 μL of 240 mM sodium bicarbonate aqueous solution (pH 9) as a release solution in a microtube, and then rotated and stirred at room temperature for 30 minutes to release the exosomes adsorbed on the powder for extracellular vesicles separation from the powder for extracellular vesicles separation. Next, the microtube was centrifuged at a rotation speed of 8,000 rpm and a rotation time of 5 seconds to separate the powder for extracellular vesicles separation from the release solution, and the release solution was collected. The above release treatment and collection operations were performed three times in succession, and the fractions of the collected release solution were named "E1 fraction", "E2 fraction", and "E3 fraction", respectively.

[0071] The exosome recovery rate (unit: %) was calculated using the following formula. Exosome recovery rate (unit: %) = [Sum of exosome concentrations (absorbance at 450 nm) in E1, E2, and E3 fractions / exosome concentration (absorbance at 450 nm) in sample solution] x 100

[0072] The protein removal rate (unit: %) was calculated according to the following formula. Protein removal rate (unit: %) = 100 - [the sum of protein concentrations (absorbance at 570 nm) in E1, E2, and E3 fractions / protein concentration (absorbance at 570 nm) in sample solution] x 100

[0073] [Table 3]

[0074] As shown in Table 3, it has been clarified that the powder for separating extracellular vesicles according to the present disclosure can selectively adsorb and separate exosomes from a sample liquid containing exosomes, which are extracellular vesicles, and impurity proteins, and that the adsorbed and separated exosomes can be selectively and efficiently detached and recovered.< / x>

Claims

1. A powder for separating extracellular vesicles comprising a plurality of inorganic particles, The inorganic particles are a powder for separating extracellular vesicles, in which the ratio X / Y, where X is the number of boron atoms and Y is the sum of the numbers of atoms of all elements excluding carbon, nitrogen, oxygen, fluorine and chlorine, as measured by X-ray photoelectron spectroscopy, is in the range of 0.015 or more and 0.30 or less.

2. The powder for isolating extracellular vesicles according to claim 1 , wherein the inorganic particles are particles of silicon oxide containing boron.

3. The powder for separating extracellular vesicles according to claim 1 or 2, wherein the inorganic particles have an average particle size in the range of 0.5 μm or more and 150 μm or less.

4. 3. The powder for separating extracellular vesicles according to claim 1 or 2, wherein the inorganic particles have pores, and the average pore diameter of the pores, as measured by mercury intrusion porosimetry, is in the range of 50 nm to 1000 nm.

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