Zirconia-containing composition for isolating extracellular vesicles or nucleic acids therefrom

Abstract

The present invention relates to a zirconia-containing composition for isolating extracellular vesicles and / or extracellular vesicle-derived nucleic acids, and a method for isolating extracellular vesicles and / or extracellular vesicle-derived nucleic acids by using the composition.

Description

Extracellular vesicles containing zirconia and composition for isolating nucleic acids thereof

[0001] The present invention relates to a composition for isolating extracellular vesicles and / or extracellular vesicle-derived nucleic acids comprising zirconia, and a method for isolating extracellular vesicles and / or extracellular vesicle-derived nucleic acids using the composition.

[0002] Extracellular vesicles (EVs) are lipid bilayer compartments involved in intercellular signaling and material transport, containing proteins, lipids, and nucleic acids (miRNA, mRNA, cfDNA, etc.). The composition of EVs varies depending on cell type, physiological state, and isolation method. These components hold significant potential as biomarkers for disease diagnosis and therapeutics utilizing their physiological and pathological functions. Unlike living cells, EVs do not divide, thus reducing the risk of tumorigenesis. They also have low immunogenicity and possess a membrane topology identical to that of cells, enabling drug delivery to target cells through the cell membrane.

[0003] However, the quantity of naturally secreted extracellular vesicles is extremely limited. Therefore, separation technologies capable of obtaining high yields and purity of extracellular vesicles are essential for clinical and industrial applications. Currently, the widely used ultracentrifugation (UC) method requires long, high-speed rotation, which limits the amount of treatment and repetitive use. Furthermore, the high g-force can damage the extracellular vesicle membrane and cause protein and lipid aggregates to separate together.

[0004] In the case of the reagent precipitation method, the yield varies greatly depending on changes in the ionic strength or pH of the sample, and the purity is low due to co-precipitated proteins, etc., which increases noise in subsequent analyses.

[0005] In particular, in order to extract nucleic acids from extracellular vesicles, steps of destruction of extracellular vesicles, isolation and purification of nucleic acids must be performed. However, protein or lipid contaminants remaining in this process reduce the specificity and sensitivity of nucleic acid analysis.

[0006] Accordingly, there is a need for research and development of a technology that efficiently separates extracellular vesicles and extracellular vesicle-derived nucleic acids from samples while improving purity.

[0007] The purpose of the present invention is to provide a composition for separating extracellular vesicles containing zirconia and a method for separating extracellular vesicles from a sample using the same.

[0008] Another object of the present invention is to provide a composition for isolating extracellular vesicle-derived nucleic acid comprising zirconia and a method for isolating extracellular vesicle-derived nucleic acid from a sample using the same.

[0009] One aspect of the present invention for achieving the above-described purpose relates to a composition for separating extracellular vesicles, comprising zirconia.

[0010] In the present invention, "extracellular vesicles" are nano-sized vesicles secreted by all cells into the external environment for intercellular information exchange, containing various biologically active substances such as proteins, lipids, nucleic acids, and metabolites. Since the quantity of these extracellular vesicles is limited, the present invention aims to increase the separation efficiency of extracellular vesicles while ensuring their stable separation.

[0011] In the present invention, 'zirconia (ZrO2)' refers to zirconium dioxide, which is an amphoteric metal oxide that has a stable monoclinic structure at room temperature, but can exist in a tetragonal or cubic structure when stabilized with alumina, yttria (Y2O3), potassium (K2O), etc. Such crystal phases can also be applied to the composition of the present invention. Zirconia has high density and high hardness characteristics, so it is resistant to mechanical wear, and has excellent heat resistance and chemical resistance, so there is almost no structural change even in ultraviolet rays, high-temperature sterilization, strong acid, and strong base processes. It is recognized as a biocompatible material in ISO 10993 and FDA guidelines, and is used for dental implants, bone substitutes, chromatography fillers, etc., and has low cytotoxicity and pyrogenicity, so there is no great risk of contamination or toxicity.

[0012] The above zirconia can have any geometric shape, such as nanoparticles (diameter 10 to 200 nm), bead particles (diameter 0.1 to 5 mm), porous scaffolds, core-shell structures, etc.

[0013] Specifically, the zirconia may be particles having a diameter of 10 nm to 1.0 mm. The diameter is an average particle diameter (D) determined by a laser diffraction particle size analyzer or a dynamic light scattering device. 50 ) and can be adjusted to have a particle size distribution of ±10% or less. More specifically, the diameter of the zirconia may be 50 nm to 1.0 mm, and most specifically, 0.1 mm to 0.5 mm, but is not limited thereto, and the diameter can be appropriately selected and adjusted according to process conditions and intended use, such as target recovery rate, processing capacity, density, design of separation device, mixing intensity, type and viscosity of sample.

[0014] Also specifically, the extracellular vesicles can be isolated from any sample that actually contains or is expected to contain extracellular vesicles, including not only biological samples but also environmental samples. Biological samples may include human and / or animal-derived body fluids such as whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, synovial fluid, amniotic fluid, breast milk, tears, sweat, urine, and feces; tissue and / or cell-derived samples such as cut or biopsied tissues, tissue washes, tissue homogenates, cultured cells or cell culture supernatants, interstitial fluids, exudates, pus, bioprinted tissue compositions; fermentation and / or production process samples such as cell cultures, fermentation products, by-products, precipitates, purification process intermediates, feeds, and feed additives. Environmental samples may include aquatic samples such as groundwater, surface water (rivers, lakes, reservoirs), seawater, sewage, domestic and industrial wastewater, freshwater and marine sediments, and sludge; and soil and / or compost samples such as soil, compost, composted slurry, waste, and waste rock.

[0015] More specifically, the extracellular vesicles of the present invention may be isolated from one or more samples selected from the group consisting of tissue, cell, whole blood, plasma, serum, blood, saliva, sputum, interstitial fluid, lymph, urine, feces, culture fluid, exudate, homogenate, tissue washing fluid, fermentation product, by-product, sediment, feed, soil, sludge, sediment, groundwater, surface water, and sewage, but are not limited thereto.

[0016] The present invention can be applied not only to diagnosis, treatment, and monitoring using extracellular vesicles, but also to environmental toxicity assessments, microbial community analysis, monitoring of biocommunity genetic diversity, food or feed safety testing, and quality control of industrial processes. Therefore, any sample containing or expected to contain extracellular vesicles is within the scope of the present invention, and the type of sample, pretreatment method, and separation and / or purification process can be appropriately modified and / or combined depending on the intended purpose.

[0017] In the present invention, 'extracellular vesicle (EV)' is a vesicle secreted by cells into the external environment for information exchange between cells, and has been called by various names such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles based on its origin, secretion mechanism, size, etc., and the Ministry of Food and Drug Safety's guidelines (extracellular vesicle therapeutic product quality, nonclinical and clinical evaluation guidelines) use the name 'extracellular vesicle'.

[0018] Specifically, in the present invention, the extracellular vesicle may be a nanovesicle, a microvesicle, an exosome, an exosome-like vesicle, or an ectosome, but is not limited thereto and also includes an artificially synthesized form. More specifically, the extracellular vesicle of the present invention may be an exosome.

[0019] Specifically, the diameter of the extracellular vesicles may be, but is not limited to, 10 nm to 1,000 nm, more specifically 20 nm to 800 nm, and most specifically 50 nm to 800 nm. The average diameter of the extracellular vesicles of the present invention may vary depending on the separation method applied as needed.

[0020] Another aspect of the present invention relates to an extracellular vesicle isolation kit comprising the composition for extracellular vesicle isolation. The kit may include a composition containing zirconia, a buffer, a container, a manual or instructions for use, and may additionally include some or all of the necessary reagents. Furthermore, the kit may include a control sample and reagents used with the control sample.

[0021] Another aspect of the present invention relates to a composition for isolating nucleic acids from extracellular vesicles comprising zirconia.

[0022] In the present invention, “zirconia” is as described above.

[0023] In the present invention, “extracellular vesicle-derived nucleic acid” encompasses all nucleic acids extracted, separated, and / or purified from extracellular vesicles. It may include all natural and artificial nucleic acids (including naturally occurring sequences, chemically or biologically modified sequences, and complementary sequences thereof) directly isolated from extracellular vesicles or obtained by fragmentation or extraction of extracellular vesicles. Specifically, it may be a nucleic acid present within an extracellular vesicle, but is not limited thereto.

[0024] Also, specifically, the nucleic acid may be single-stranded, double-stranded, or hybrid, and may include chemical modifications such as modified oligonucleotides and LNA-introduced sequences, as needed. More specifically, the nucleic acid may be RNA, but is not limited thereto, and may include genomic DNA fragments, etc. The RNA may include mRNA, small RNA, lncRNA, and circRNA, and there is no limitation on the RNA form or length.

[0025] Specifically, the zirconia may be particles having a diameter of 10 nm to 1.0 mm. The diameter is an average particle diameter (D) determined by a laser diffraction particle size analyzer or a dynamic light scattering device. 50) and can be adjusted to have a particle size distribution of ±10% or less. More specifically, the diameter of the zirconia may be 50 nm to 1.0 mm, and most specifically, 0.1 mm to 0.5 mm, but is not limited thereto, and the diameter can be appropriately selected and adjusted according to process conditions and intended use, such as target recovery rate, processing capacity, density, design of separation device, mixing intensity, type and viscosity of sample.

[0026] In addition, specifically, the extracellular vesicles may be isolated from one or more samples selected from the group consisting of tissues, cells, whole blood, plasma, serum, blood, saliva, sputum, interstitial fluid, lymph, urine, feces, cultures, exudates, homogenates, tissue washings, fermented products, by-products, sediments, feed, soil, sludge, sediments, groundwater, surface water, and sewage, but are not limited thereto. Any sample that contains or is expected to contain extracellular vesicles may be included. The description of the sample from which the extracellular vesicles of the present invention can be isolated is as described above.

[0027] Also specifically, the extracellular vesicle may be a nanovesicle, a microvesicle, an exosome, an exosome-like vesicle, or an ectosome, but is not limited thereto, and also includes an artificially synthesized form. More specifically, the extracellular vesicle of the present invention may be an exosome.

[0028] Another aspect of the present invention relates to a kit for isolating nucleic acids from extracellular vesicles, comprising the composition for isolating nucleic acids from extracellular vesicles. The kit may include a composition containing zirconia, a buffer, a container, a manual or instructions for use, and may additionally include some or all of the necessary reagents. Furthermore, the kit may include a control sample and reagents for use with the control sample.

[0029] Another aspect of the present invention relates to a method for isolating extracellular vesicles, comprising a step of mixing the composition for isolating extracellular vesicles with a sample.

[0030] The process of isolating extracellular vesicles from the above sample can be performed using a known process, and the composition of the present invention can be added to the known process, or the composition of the present invention can be applied alone. The form in which the composition of the present invention is applied can be changed as needed. Specifically, the composition of the present invention can be specifically applied to the process for isolating extracellular vesicles. In addition, the size and content of zirconia included in the composition for isolating extracellular vesicles, the concentration of the composition, the stirring speed, the reaction time, etc. can be changed or adjusted depending on the type of sample, the purpose of separation, and the production scale, and the present invention includes all such modifications.

[0031] Another aspect of the present invention relates to a method for isolating extracellular vesicle-derived nucleic acid, comprising a step of mixing the composition for isolating extracellular vesicle-derived nucleic acid with a sample.

[0032] By appropriately combining nonionic or anionic surfactants, chaotropic salts, eluting agents such as metal chelators, nucleic acid stabilizers including RNase / DNase inhibitors, buffers, precipitants, etc., nucleic acids isolated from extracellular vesicles can be concentrated and purified without damage. The composition for isolating nucleic acids from extracellular vesicles of the present invention can be added as an additive to a known extracellular vesicle isolation process to improve the recovery rate and purity of nucleic acids from extracellular vesicles, or the process of extracting and purifying nucleic acids from extracellular vesicles can be performed alone.

[0033] The size and content of zirconia included in the composition for isolating nucleic acids from extracellular vesicles, the concentration of the composition, the stirring speed, the reaction time, etc. can be changed or adjusted depending on the type of sample, the purpose of separation, and the production scale, and the present invention includes all such modifications.

[0034] The extracellular vesicle-derived nucleic acids obtained by the method for isolating such extracellular vesicle-derived nucleic acids can be applied to various fields such as biomarkers for disease diagnosis and / or prognosis monitoring, therapeutic agents, environmental analysis, and food and / or feed safety evaluation.

[0035] The zirconia (ZrO2)-based separation composition of the present invention increases the efficiency of extracellular vesicle separation, and significantly improves the nucleic acid derived from extracellular vesicles that are extracted and purified, and greatly improves the purity, thereby enabling extracellular vesicles and / or extracellular vesicle-derived nucleic acids to be stably separated.

[0036] In particular, when using the composition of the present invention compared to existing technologies, extracellular vesicles can be obtained with high efficiency even from a small amount of sample due to the high efficiency of extracellular vesicle separation.

[0037] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0038] Figure 1 shows the results of confirming the separation efficiency of extracellular vesicles and / or extracellular vesicle-derived nucleic acids depending on the presence or absence of zirconia treatment.

[0039] Figure 2 shows the results of confirming the separation efficiency of extracellular vesicles and / or extracellular vesicle-derived nucleic acids according to the content of 0.1 mm diameter zirconia.

[0040] Figure 3 shows the results of confirming the separation efficiency of extracellular vesicles and / or extracellular vesicle-derived nucleic acids according to the content of 0.5 mm diameter zirconia.

[0041] Figure 4 shows the results of a cross-comparison of the separation efficiency of extracellular vesicles and / or extracellular vesicle-derived nucleic acids according to the zirconia particle size and content.

[0042] Figure 5 shows the results of confirming the separation efficiency of extracellular vesicles and / or extracellular vesicle-derived nucleic acids when zirconia particles with diameters of 0.1 mm and 0.5 mm were processed singly and mixed.

[0043] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0044] Example 1. Separation of extracellular vesicles using zirconia

[0045] Fecal samples were suspended in 1X PBS buffer at a concentration of 100 mg / ml, and 1 ml of the suspension was used for the isolation of extracellular vesicles. Samples with and without 1 g of 0.1 mm diameter zirconia particles were dissolved by vortexing for 3 minutes, respectively. After centrifugation at 4°C and 4500 g for 10 minutes, 600 μl of the supernatant was transferred to a new tube. After removing the supernatant, 600 μl of 1X PBS buffer was added to the remaining pellet layer, vortexed for 3 minutes to dissolve, and centrifuged at 4°C and 4500 g for 10 minutes, and all supernatants were transferred to a new tube. All supernatants transferred to new tubes were combined and vortexed to mix, then 500 μl was dispensed into 1.5 ml tubes and centrifuged at 4 ℃, 20,000 g, for 15 minutes, and the supernatant was obtained again. The obtained supernatant was transferred to a new tube, mixed well, and filtered through a 0.2 μm filter. After filtering, the obtained sample was mixed well, 800 μl was dispensed into 1.5 ml tubes and 400 μl of XENO-EVIMEDI (Extracellular Vesicle Isolation Kit, Xenohelix Co., Ltd., cat. 9366-EVI-M) was added and mixed. After 2 hours of reaction at room temperature, the mixture was centrifuged at 4 ℃, 12,300 g, for 15 minutes. After discarding the supernatant, centrifugation was performed at 4°C, 12300 g for 2 minutes, and then the supernatant was discarded to obtain a pellet to obtain extracellular vesicles.

[0046] Afterwards, NTA (Nanoparticle Tracking Analysis, Zetaview) ® ) was used for analysis. RNA was purified from the isolated extracellular vesicles using the XENO-EVARI kit (Small RNA Purification in Exosome, Xenohelix Co., Ltd., cat. 9366-EVI-A), and the purified RNA was quantified using NanoDrop One (ThermoFisher Scientific) and loaded onto a 2% agarose gel for confirmation.

[0047] As a result, as shown in Fig. 1, when zirconia was used, it was confirmed that the RNA extracted and purified from each extracellular vesicle significantly increased by about 5 times compared to when zirconia was not used. In addition, the A260 / A280 value indicating the purity of the extracted RNA was also improved, confirming that contamination by proteins, lipids, and other suspended solids was reduced and purity was improved. In other words, it can be seen that the quality of the extracellular vesicles obtained through zirconia treatment is improved, and accordingly, the separation efficiency and purity of nucleic acids within the extracellular vesicles are significantly increased.

[0048] Example 2. Confirmation of extracellular vesicle separation efficiency according to zirconia size and content.

[0049] 2-1. Separation of extracellular vesicles using 0.1 mm diameter zirconia particles

[0050] Extracellular vesicles were separated using the same method as in Example 1, but samples containing or not containing 0.5, 1, and 2 g of zirconia particles with a diameter of 0.1 mm were dissolved by vortexing for 3 minutes. After centrifugation at 4°C and 4500 g for 10 minutes, 600 μl of the supernatant was transferred to a new tube. After removing the supernatant, 600 μl of 1X PBS buffer was added to the remaining pellet layer, vortexed for 3 minutes to dissolve, centrifuged at 4°C and 4500 g for 10 minutes, and all supernatants were transferred to new tubes. All supernatants transferred to new tubes were combined, vortexed to mix, and 500 μl was dispensed into 1.5 ml tubes, centrifuged at 4°C and 20000 g for 15 minutes, and the supernatants were obtained again. The obtained supernatant was transferred to a new tube, mixed well, and filtered through a 0.2 μm filter. After filtering, the obtained sample was mixed well, dispensed into 1.5 ml tubes (800 μl each), and 400 μl of XENO-EVIMEDI (Extracellular Vesicle Isolation Kit, Xenohelix Co., Ltd., cat. 9366-EVI-M) was added and mixed. After 2 hours of reaction at room temperature, centrifugation was performed at 4 °C, 12,300 g, and 15 minutes. After discarding the supernatant, centrifugation was performed again at 4 °C, 12,300 g, and 2 minutes, and then the supernatant was discarded and the pellet was obtained to obtain extracellular vesicles.

[0051] Afterwards, NTA (Nanoparticle Tracking Analysis, Zetaview) ® ) was used for analysis. RNA was purified from the isolated extracellular vesicles using the XENO-EVARI kit (Small RNA Purification in Exosome, Xenohelix Co., Ltd., cat. 9366-EVI-A), and the purified RNA was quantified using NanoDrop One (ThermoFisher Scientific) and loaded onto a 2% agarose gel for confirmation.

[0052] As a result, as shown in Fig. 2, when 0.1 mm zirconia particles were treated, the extracellular vesicles obtained in the 0.5, 1, and 2 g treatments all increased, and in particular, in the case of 1 g treatment, it was confirmed that approximately 2.2 times more extracellular vesicles were obtained compared to the control group without zirconia. In addition, in the case of 0.5 and 2 g treatments, the number increased by approximately 2 times or more, respectively, confirming that when a certain amount of zirconia particles is treated, an improvement in the yield of approximately 2 times can be seen.

[0053] RNA extracted and purified from extracellular vesicles also increased when treated with 0.5, 1, and 2 g, and in particular, when treated with 1 g, it increased by approximately 3 times compared to the control group without zirconia. When treated with 0.5 and 2 g, it increased by approximately 2.2 times and approximately 2.5 times, respectively, confirming that the isolation efficiency of nucleic acids within extracellular vesicles also increased significantly.

[0054] In addition, in terms of purity, the A260 / A280 values ​​were also improved, confirming that contamination by proteins, lipids, and other suspended solids was reduced and purity was improved.

[0055] In particular, the NTA analysis results showed that the peak diameter (approximately 110-120 nm) and distribution width were almost identical to those of the control group not treated with zirconia under all zirconia conditions, confirming that zirconia treatment selectively increased only extracellular vesicles without the influx of debris or microcontaminant particles.

[0056] 2-2. Separation of extracellular vesicles using 0.5 mm diameter zirconia particles

[0057] Extracellular vesicles were isolated using the same method as in Example 1. Fecal samples were suspended in 1X PBS buffer at a concentration of 100 mg / ml, and 0.5, 1, and 2 g of zirconia particles with a diameter of 0.5 mm were added to 1 ml of the suspension, respectively, and vortexed for 3 minutes to dissolve each sample. After centrifugation at 4°C and 4500 g for 10 minutes, 600 μl of the supernatant was transferred to a new tube. After removing the supernatant, 600 μl of 1X PBS buffer was added to the remaining pellet layer, vortexed for 3 minutes to dissolve, and centrifuged at 4°C and 4500 g for 10 minutes, and all supernatants were transferred to a new tube. All supernatants transferred to new tubes were combined and vortexed to mix, then 500 μl was dispensed into 1.5 ml tubes and centrifuged at 4 ℃, 20,000 g, for 15 minutes, and the supernatant was obtained again. The obtained supernatant was transferred to a new tube, mixed well, and filtered through a 0.2 μm filter. After filtering, the obtained sample was mixed well, 800 μl was dispensed into 1.5 ml tubes and 400 μl of XENO-EVIMEDI (Extracellular Vesicle Isolation Kit, Xenohelix Co., Ltd., cat. 9366-EVI-M) was added and mixed. After 2 hours of reaction at room temperature, the mixture was centrifuged at 4 ℃, 12,300 g, for 15 minutes. After discarding the supernatant, centrifugation was performed at 4°C, 12300 g for 2 minutes, and then the supernatant was discarded to obtain a pellet to obtain extracellular vesicles.

[0058] Afterwards, NTA (Nanoparticle Tracking Analysis, Zetaview) ®) was used for analysis. RNA was purified from the isolated extracellular vesicles using the XENO-EVARI kit (Small RNA Purification in Exosome, Xenohelix Co., Ltd., cat. 9366-EVI-A), and the purified RNA was quantified using NanoDrop One (ThermoFisher Scientific) and loaded onto a 2% agarose gel for confirmation.

[0059] As a result, as shown in Fig. 3, when 0.5 mm zirconia particles were treated, the yield of extracellular vesicles was improved by about 2 times or more when 0.5, 1, and 2 g were treated.

[0060] RNA extracted and purified from extracellular vesicles also increased when treated with 0.5, 1, and 2 g, and in particular, when treated with 2 g, it increased by approximately 0.5 times compared to the control group without zirconia. When treated with 0.5 and 1 g, it also increased by approximately 1.2 times or more, respectively, confirming that the isolation efficiency of nucleic acids within extracellular vesicles also increased significantly.

[0061] In addition, in terms of purity, the A260 / A280 values ​​were also improved, confirming that contamination by proteins, lipids, and other suspended solids was reduced and purity was improved.

[0062] In particular, the NTA analysis results showed that the peak positions and distribution widths under all zirconia conditions were almost identical to those of the control group that was not treated with zirconia, confirming that even when zirconia was treated, only extracellular vesicles were selectively increased without the influx of debris or micro-contaminants.

[0063] 2-3. Cross-comparison of extracellular vesicle separation efficiency according to zirconia particle size and content.

[0064] Extracellular vesicles were isolated using the same method as in Example 1. Fecal samples were suspended in 1X PBS buffer at a concentration of 100 mg / ml, and 1 ml of the suspension contained 1 g and 2 g of zirconia particles with diameters of 0.1 mm and 0.5 mm, respectively. The samples were dissolved by vortexing for 3 minutes. After centrifugation at 4°C and 4500 g for 10 minutes, 600 μl of the supernatant was transferred to a new tube. After removing the supernatant, 600 μl of 1X PBS buffer was added to the remaining pellet layer, vortexed for 3 minutes to dissolve, and centrifuged at 4°C and 4500 g for 10 minutes. All of the supernatant was transferred to a new tube. All supernatants transferred to new tubes were combined and vortexed to mix, then 500 μl was dispensed into 1.5 ml tubes and centrifuged at 4 ℃, 20,000 g, for 15 minutes, and the supernatant was obtained again. The obtained supernatant was transferred to a new tube, mixed well, and filtered through a 0.2 μm filter. After filtering, the obtained sample was mixed well, 800 μl was dispensed into 1.5 ml tubes and 400 μl of XENO-EVIMEDI (Extracellular Vesicle Isolation Kit, Xenohelix Co., Ltd., cat. 9366-EVI-M) was added and mixed. After 2 hours of reaction at room temperature, the mixture was centrifuged at 4 ℃, 12,300 g, for 15 minutes. After discarding the supernatant, centrifugation was performed at 4°C, 12300 g for 2 minutes, and then the supernatant was discarded to obtain a pellet to obtain extracellular vesicles.

[0065] Afterwards, NTA (Nanoparticle Tracking Analysis, Zetaview) ®) was used for analysis. RNA was purified from the isolated extracellular vesicles using the XENO-EVARI kit (Small RNA Purification in Exosome, Xenohelix Co., Ltd., cat. 9366-EVI-A), and the purified RNA was quantified using NanoDrop One (ThermoFisher Scientific) and loaded onto a 2% agarose gel for confirmation. This experiment was performed under the same conditions as Examples 2-1 and 2-2, and the relative comparison with the control group was applied to the control group without zirconia for 0.1 mm diameter zirconia particles, and the control group without zirconia for 0.5 mm diameter zirconia particles was analyzed by applying the control group in Example 2-2. The numerical values ​​for each zirconia treatment group are as shown in Fig. 4.

[0066] As a result, as shown in Fig. 4, when zirconia particles with a diameter of 0.1 mm were treated, the number of extracellular vesicles obtained increased compared to the control group without zirconia, and when 1 g was treated, it was confirmed that about 2.4 times more extracellular vesicles were obtained compared to the control group without zirconia, and when 2 g was treated, it was confirmed that about 2.5 times more extracellular vesicles were obtained compared to the control group without zirconia. RNA extracted and purified from extracellular vesicles also significantly increased compared to the control group without zirconia, and when 1 g was treated, it was confirmed that about 3 times more and when 2 g was treated, it was confirmed that about 3.6 times more, indicating that the separation efficiency of nucleic acids inside extracellular vesicles was also greatly increased.

[0067] Even when zirconia particles with a diameter of 0.5 mm were treated, the number of extracellular vesicles obtained increased compared to the control group without zirconia, and when 1 g was treated, it was confirmed that about 2 times more extracellular vesicles were obtained compared to the control group without zirconia, and when 2 g was treated, it was confirmed that about 2.2 times more extracellular vesicles were obtained compared to the control group without zirconia. RNA extracted and purified from extracellular vesicles also increased significantly compared to the control group without zirconia, and when 1 g was treated, it was confirmed that about 2 times more and when 2 g was treated, it was confirmed that about 2.1 times more, and the separation efficiency of nucleic acids inside extracellular vesicles was also greatly increased.

[0068] In addition, in terms of purity, the A260 / A280 values ​​were also significantly improved in both 0.1 mm diameter and 0.5 mm diameter zirconia treatments, confirming that contamination by proteins, lipids, and other suspended solids was reduced and purity was improved.

[0069] The NTA analysis results showed that the peak positions and distribution widths were almost constant under all zirconia conditions, reconfirming that even when zirconia was treated, only the original extracellular vesicles were selectively increased without the introduction of debris or micro-contaminants.

[0070] 2-4. Confirmation of extracellular vesicle separation efficiency according to zirconia particle mixing

[0071] The extracellular vesicle separation efficiency was compared when zirconia particles with diameters of 0.1 mm and 0.5 mm were processed singly and mixed.

[0072] Extracellular vesicles were separated using the same method as in Example 1, and fecal samples were suspended in 1X PBS buffer at a concentration of 100 mg / ml. Zirconia particles with diameters of 0.1 mm and 0.5 mm were added to 1 ml of the suspension, either singly or in combination, as shown in Table 1 below, and vortexed for 3 minutes to dissolve.

[0073] Zirconia particle diameter and content group 0.1 mm (g) 0.5 mm (g) 10 2 2 0.5 1.5 3 1 1 4 1.5 0.5 5 20

[0074] After centrifugation at 4℃, 4500g for 10 minutes, 600 μl of the supernatant was transferred to a new tube. After removing the supernatant, 600 μl of 1X PBS buffer was added to the remaining pellet layer, vortexed for 3 minutes to dissolve, and centrifuged at 4℃, 4500g for 10 minutes, and the supernatant was transferred to a new tube. All the supernatants transferred to the new tubes were combined, vortexed to mix, and 500 μl each was dispensed into 1.5 ml tubes, centrifuged at 4℃, 20000g for 15 minutes, and the supernatant was obtained again. The obtained supernatant was transferred to a new tube, mixed well, and filtered through a 0.2 μm filter. The sample obtained after filtering was well mixed and 800 μl was dispensed into 1.5 ml tubes, 400 μl of XENO-EVIMEDI (Extracellular Vesicle Isolation Kit, Xenohelix Co., Ltd., cat. 9366-EVI-M) was added, and mixed. After 2 hours of reaction at room temperature, centrifugation was performed at 4 °C, 12,300 g, and 15 minutes. After discarding the supernatant, centrifugation was performed again at 4 °C, 12,300 g, and 2 minutes, and then the supernatant was discarded and the pellet was obtained to obtain extracellular vesicles.

[0075] Afterwards, NTA (Nanoparticle Tracking Analysis, Zetaview) ®) was used for analysis. RNA was purified from the isolated extracellular vesicles using the XENO-EVARI kit (Small RNA Purification in Exosome, Xenohelix Co., Ltd., cat. 9366-EVI-A), and the purified RNA was quantified using NanoDrop One (ThermoFisher Scientific) and loaded onto a 2% agarose gel for confirmation. This experiment was performed under the same conditions as Examples 2-1 and 2-2, and the relative comparison with the control group was applied to the control group without zirconia for 0.1 mm diameter zirconia particles, and the control group without zirconia for 0.5 mm diameter zirconia particles was analyzed by applying the control group in Example 2-2. The numerical values ​​for each zirconia treatment group are as shown in Fig. 5.

[0076] As a result, as shown in Fig. 5, in both cases where zirconia particles with a diameter of 0.1 mm were treated alone and zirconia particles with a diameter of 0.5 mm were treated alone, it was confirmed that the amount of extracellular vesicles and extracellular vesicle-derived RNA obtained increased, and the A260 / A280 values ​​also improved, resulting in improved purity, similar to Examples 2-1 and 2-2.

[0077] When zirconia particles with a diameter of 0.1 mm and zirconia particles with a diameter of 0.5 mm were mixed and treated at a weight ratio of 1:3, extracellular vesicles increased by about 1.8 times and extracellular vesicle-derived RNA increased by about 1.2 times compared to the control group not treated with zirconia, and the purity was also greatly improved.

[0078] When zirconia particles with a diameter of 0.1 mm and zirconia particles with a diameter of 0.5 mm were mixed and treated at a weight ratio of 1:1, extracellular vesicles increased by about 2.4 times and extracellular vesicle-derived RNA increased by about 1.9 times compared to the control group not treated with zirconia, and the purity was also greatly improved.

[0079] When zirconia particles with a diameter of 0.1 mm and zirconia particles with a diameter of 0.5 mm were mixed and treated at a weight ratio of 3:1, extracellular vesicles increased by about 2.5 times and extracellular vesicle-derived RNA increased by about 3.6 times compared to the control group not treated with zirconia, and the purity was also greatly improved.

[0080] The above results show that even when 0.1 mm diameter and 0.5 mm diameter zirconia particles are mixed, extracellular vesicles and extracellular vesicle-derived nucleic acids can be separated with high efficiency, and even when mixed processing is performed, the NTA analysis results confirmed that no contaminating particles were introduced and extracellular vesicles were maintained at a constant level. Furthermore, in the case of mixed processing, extracellular vesicles increased by about 2.5 times and extracellular vesicle-derived RNA increased by about 3.6 times or more, showing a synergistic effect, so that it can be designed and applied in a customized manner according to the process and sample condition.

[0081] In the present invention, it was confirmed that when zirconia is treated, the yield of extracellular vesicles is significantly improved, and the nucleic acid derived from extracellular vesicles that is extracted and purified is also significantly improved, and the purity is greatly improved.

[0082] In addition, the NTA analysis results showed that the peak positions and distribution widths were almost constant under all zirconia conditions, indicating that even when zirconia was processed, only the original extracellular vesicles were selectively increased without the introduction of fragments or micro-contaminants, thereby confirming both an improvement in purity and maintenance of size distribution, suggesting that the technology of the present invention satisfies high efficiency, high quality, and reproducibility.

[0083] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0084] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0085] This invention is the result of research and development conducted with the support of the National Research and Development Program of the Republic of Korea.

[0086] Assignment ID: RS-2022-00414618

[0087] Ministry Name: All Ministry

[0088] Research Management Specialist Organization: Inter-Ministry Full-Cycle Medical Device Research and Development Project Group

[0089] Research Project Name: Development of Market-Friendly, Globally Competitive Products

[0090] Research Project Title: Commercialization of a Parkinson's Disease Diagnostic System Based on a Genosensor for Rapid Detection of MiRNA Biomarkers

[0091] Host organization: Genohelix Co., Ltd.

[0092] Research Period: April 1, 2022 - December 31, 2025

Claims

1. A composition for separating extracellular vesicles, comprising zirconia.

2. In paragraph 1, A composition for separating extracellular vesicles, wherein the above zirconia is a particle having a diameter of 10 nm to 1.0 mm.

3. In paragraph 1, A composition for isolating extracellular vesicles, wherein the extracellular vesicles are separated from at least one sample selected from the group consisting of tissue, cells, whole blood, plasma, serum, blood, saliva, sputum, intercellular fluid, lymph, urine, feces, culture fluid, exudate, homogenate, tissue washing fluid, fermentation product, by-product, sediment, feed, soil, sludge, sediment, groundwater, surface water, and sewage.

4. In paragraph 1, A composition for separating extracellular vesicles, wherein the extracellular vesicles are nanovesicles, microvesicles, exosomes, exosome-like vesicles or ectosomes.

5. In paragraph 1, A composition for separating extracellular vesicles, wherein the diameter of the extracellular vesicles is 20 to 1,000 nm.

6. A kit for separating extracellular vesicles comprising the composition of paragraph 1.

7. A composition for isolating nucleic acids derived from extracellular vesicles, comprising zirconia.

8. In paragraph 7, A composition for isolating nucleic acid derived from extracellular vesicles, wherein the nucleic acid is RNA.

9. In paragraph 7, A composition for separating nucleic acids from extracellular vesicles, wherein the above zirconia is a particle having a diameter of 10 nm to 1.0 mm.

10. In paragraph 7, A composition for isolating nucleic acid derived from extracellular vesicles, wherein the extracellular vesicles are isolated from at least one sample selected from the group consisting of tissue, cell, whole blood, plasma, serum, blood, saliva, sputum, intercellular fluid, lymph, urine, feces, culture fluid, exudate, homogenate, tissue washing fluid, fermentation product, by-product, sediment, feed, soil, sludge, sediment, groundwater, surface water, and sewage.

11. In paragraph 7, A composition for isolating nucleic acids from extracellular vesicles, wherein the extracellular vesicles are nanovesicles, microvesicles, exosomes, exosome-like vesicles, or ectosomes.

12. A kit for isolating nucleic acid derived from extracellular vesicles comprising the composition of Article 7.

13. A method for separating extracellular vesicles from a sample, comprising the step of mixing the composition of paragraph 1 with the sample.

14. A method for isolating extracellular vesicle-derived nucleic acid, comprising the step of mixing the composition of Article 7 with a sample.

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