KR-20260063904-A - Method for seperationg extracellular vesicles comprising diatomaceous earth surface modified with amine group, crosslinker and filter

Abstract

One aspect relates to a method for isolating extracellular vesicles (EVs) from a biological sample and a kit for isolation. According to one aspect, it was confirmed that extracellular vesicles selectively bind using amine-modified diatomite and a crosslinking agent. Additionally, it was confirmed that extracellular vesicles can be isolated from the biological sample by using the diatomite, as it does not pass through a filter. Therefore, the isolation method enables the isolation of extracellular vesicles from a biological sample in a short time and at low cost, and thus can be applied to various analytical methods using the isolated extracellular vesicles.

Inventors

• 신용
• 이효주
• 김남헌

Assignees

• 연세대학교 산학협력단
• 주식회사 인퓨전텍

Dates

Publication Date

20260507

Application Date

20241031

Claims (19)

1. A method for isolating extracellular vesicles (EVs) from a biological sample, comprising the following steps: (a) A step of modifying diatomite with amine groups; (b) a step of adding amine-modified diatomite and a crosslinking agent represented by the following chemical formula 1 to a biological sample; [Chemical Formula 1] In the above equation, X is (CH 2 ) p -SS-(CH 2 ) q , and p or q are integers from 1 to 3, respectively. (c) a step of separating the diatomite from a biological sample using a filter; and (d) A step of separating extracellular vesicles from the separated diatomite.
2. A method according to claim 1, wherein the modification in step (a) is characterized by treating the diatomite with a silane compound.
3. In claim 2, the silane compound is (3-aminopropyl)triethoxysilane (APTES), (3-aminopropyl)trimethoxysilane, (1-aminomethyl)triethoxysilane, (2-aminoethyl)triethoxysilane, (4-aminobutyl)triethoxysilane, (5-aminopentyl)triethoxysilane, (6-aminohexyl)triethoxysilane, 3-aminopropyl(diethoxy)methylsilane (APDM). A method characterized by comprising one or more selected from the group consisting of N-[3-(trimethoxysilyl)propyl]ethylenediamine, N-[3-(trimethoxysilyl)propyl]diethylenetriamine, [3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMS), and 3-[(trimethoxysilyl)propyl]diethylenetriamine (TMPTA).
4. The method of claim 1, characterized in that the crosslinking agent is added at a concentration of 1-40 mg/ml per 1 mL of biological sample.
5. The method of claim 1, wherein the crosslinking agent is one or more selected from the group consisting of dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), and dimethyl 3,3'-dithiobispropionimidate (DTBP).
6. A method according to claim 1, characterized in that the amine-modified diatomite is added at a concentration of 1-10 mg/ml per 1 mL of biological sample.
7. A method according to claim 1, wherein in step (d), the separation of extracellular vesicles is performed using a high pH solution containing NaHCO₃ or a RIPA (Radio-Immunoprecipitation Assay) solution as the elution buffer.
8. A method according to claim 1, characterized by additionally including a step of incubating for 5-20 minutes in step (b).
9. A method according to claim 1, further comprising the step of washing the diatomite concentrated on the filter surface with phosphate-buffered saline (PBS) after step (c).
10. The method according to claim 1, wherein the material of the filter is one or more selected from the group consisting of nylon (NY), cellulose acetate (CA), glass fiber (GF), regenerated cellulose (RC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polyethylene (PE), polytetrafluoroethylene (PTFE, including hydrophilic PTFE-W and hydrophobic PTFE-O), polypropylene (PP), and mixed cellulose ester (MCE).
11. The method of claim 1, wherein the filter has a pore size of 0.22 to 3 μm.
12. A method according to claim 1, wherein the biological sample is one or more selected from the group consisting of blood, serum, plasma, saliva, urine, cerebrospinal fluid, gastric secretion, ascites, nasal secretion, sputum, and pharyngeal exudate.
13. A kit for separating extracellular vesicles (EVs) from a biological sample, comprising amine-modified diatomite (ADE), a crosslinking agent represented by the following chemical formula 1, and a syringe filter. [Chemical Formula 1] In the above equation, X is (CH2) p -SS-(CH2) q , and p or q are integers from 1 to 3, respectively.
14. A kit according to claim 13, wherein the crosslinking agent is one or more selected from the group consisting of dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), and dimethyl 3,3'-dithiobispropionimidate (DTBP).
15. A kit according to claim 13, characterized in that it further comprises a high pH solution containing NaHCO₃ or a RIPA solution as an elution buffer.
16. A kit according to claim 13, wherein the material of the syringe filter is one or more selected from the group consisting of nylon (NY), cellulose acetate (CA), glass fiber (GF), regenerated cellulose (RC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polyethylene (PE), polytetrafluoroethylene (PTFE, including hydrophilic PTFE-W and hydrophobic PTFE-O), polypropylene (PP), and mixed cellulose ester (MCE).
17. A kit according to claim 13, wherein the filter is characterized by having a pore size of 0.22 to 3 μm.
18. A kit according to claim 13, further comprising an antibody or ligand that binds to a surface marker protein of an extracellular vesicle.
19. A kit according to claim 13, characterized in that the surface marker is CD9 or CD81.

Description

Method for separating extracellular vesicles comprising diatomaceous earth modified with an amine group, a crosslinker, and a filter One example of the present invention relates to a method for isolating extracellular vesicles (EVs) from a biological sample and a kit for isolation. Extracellular vesicles are known to play a crucial role in various biological processes, such as intercellular communication, disease diagnosis, and treatment. Therefore, the effective isolation and analysis of extracellular vesicles are recognized as a critical task in life science research and the medical field. Currently used EV separation technologies include ultracentrifugation, density gradient centrifugation, size exclusion chromatography, immunoaffinity separation, and polymer-based precipitation. Each of these methods has its own advantages and disadvantages, and they are used selectively depending on the research objective. However, existing EV separation technologies have several limitations. Ultracentrifugation is time-consuming and requires expensive equipment, while density gradient centrifugation is time-consuming and labor-intensive due to its complex process. Size exclusion chromatography suffers from the problem of EV dilution, and immunoaffinity separation is expensive and limited to separating only specific subgroups. Due to the limitations of existing technologies, there is a continuous demand in EV research and application fields for the development of new technologies capable of separating EVs more efficiently, economically, and with higher purity. In particular, polymer-based separation technologies are attracting attention due to their advantages of simple and rapid separation; however, existing polymer-based technologies still require room for improvement. Figures 1a and 1b are schematic diagrams illustrating the ADE principle and a method for separating extracellular vesicles using ADE. Figure 1a shows the process in which diatomite is treated with APDMS to expose amine groups on its surface, and DMS acts as a crosslinking agent to capture negatively charged intracellular vesicles through electrostatic bonding. Figure 1b shows the process of adding ADE and DMS together to a sample, removing the supernatant using a syringe filter, and then using a separation solution on the remaining ADE to break the electrostatic bonds between the diatomite and extracellular vesicles to perform separation. Figures 2a to 2d show the optimized results of the extracellular vesicle separation process using ADE. Figure 2a shows the protein concentration analysis results, Western blotting band intensity values, and miRNA RT-PCR results according to the concentration of DMS, i.e., the crosslinking agent. Figure 2b shows the protein concentration analysis results, Western blotting band intensity values, and miRNA RT-PCR results according to the concentration of the ADE used. Figure 2c shows the protein concentration analysis results, Western blotting band intensity values, and miRNA RT-PCR results according to the material type of the syringe filter. Figure 2d shows the protein concentration analysis results, Western blotting band intensity values, and miRNA RT-PCR results according to the incubation time after addition to the sample. Figures 3a and 3b show the results of evaluating the separation efficiency of extracellular vesicles under various conditions. Figure 3a measured the separation efficiency in samples of various pH values. Figure 3b measured the separation efficiency of the same amount of extracellular vesicles in various volumes using protein concentration. Figures 4a to 4c show the results of a comparative evaluation of separation performance for extracellular vesicles using conventional methods. Figure 4a shows the results using nanoparticle tracking analysis. Figure 4b shows the results regarding protein concentrations in extracellular vesicles separated by various techniques. Figure 4c shows the results of Western blotting. The present invention will be described in detail below. To achieve the above objective, a method for isolating extracellular vesicles (EVs) from a biological sample is provided, comprising the following steps: (a) A step of modifying diatomite with amine groups; (b) a step of adding amine-modified diatomite and a crosslinking agent represented by the following chemical formula 1 to a biological sample; [Chemical Formula 1] In the above equation, X is (CH 2 ) p -SS-(CH 2 ) q , and p or q are integers from 1 to 3, respectively. (c) a step of separating the diatomite from a biological sample using a filter; and (d) A step of separating extracellular vesicles from the separated diatomite. The term "diatomite modified with amine groups" as used herein refers to diatomite modified with amine groups ( -NH2 ) that impart a positive charge to the surface of the diatomite, and is used interchangeably with the English abbreviation "ADE" in this document. The term "diatomeous earth" used herein refers