METHOD FOR ISOLATING EXTRACELLULAR VESICLE USING HYDROPHOBIC INTERACTION

Abstract

The present invention relates to a method for isolating extracellular vesicles and, more particularly, to a method for isolating extracellular vesicles using hydrophobicity of the extracellular vesicles, and extracellular vesicles isolated using the method. When used, the method for isolating extracellular vesicles according to the present invention allows for isolating, from various animal body fluids including blood and urine or various tissues including cancer tissues, extracellular vesicles free of contamination with lipoproteins that are difficult to eliminate using a conventional method, can solve conventional problems caused by lipoprotein contamination, and is expected to be an essential technology that has a great influence on various research using extracellular vesicles isolated from various animal body fluids or tissues, such as characterization and function research, multi-omics research, excavation of novel biomarkers, diagnosis and treatment, etc.

Claims

1.-36. (canceled)

37. A method for isolating extracellular vesicles, the method comprising the steps of: (a) immobilizing a hydrophobic functional group onto a stationary phase; (b) loading a sample containing extracellular vesicles to the stationary phase to bind the extracellular vesicles to the hydrophobic functional group; (c) washing out remaining sample residues not bound to the hydrophobic functional group on the stationary phase; and (d) eluting extracellular vesicles bound to the hydrophobic functional group from the stationary phase.

38. The method of claim 37, further comprising a step of washing the stationary phase having the hydrophobic functional group thereon with a solution containing salting-out ions, prior to step (b), wherein the salting-out ions have a concentration suitable for controlling the hydrophobicity of the stationary phase to enable the extracellular vesicles to bind to the stationary phase, and the concentration of the salting-out ions ranges from 0 M to 10 M.

39. The method of claim 37, further comprising a step of adding a solution containing salting-out ions to the sample to change the kind or concentration of salting-out ions contained in the sample, prior to step (b) of loading a sample containing extracellular vesicles to the stationary phase.

40. The method of claim 37, further comprising a step of controlling the kind or concentration of the salting-out ions to remove materials weaker in intensity of hydrophobic interaction than the extracellular vesicles, prior to step (d) of eluting the extracellular vesicles, wherein the concentration of the salting-out ions for removing materials weaker in intensity of hydrophobic interaction than the extracellular vesicles is controlled to be within a range of 0 M to 10 M.

41. The method of claim 37, wherein the salting-out ions are at least one selected from the group consisting of sulfate, phosphate, hydroxide, fluoride, formate, acetate, chloride, bromide, nitrate, iodide, thiocyanate, citrate, and tartrate; wherein the stationary phase is at least one selected from the group consisting of agarose beads, sepharose beads, sephadex beads, silica beads, magnetic beads, gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, a nylon membrane, nitrocellulose membrane, a PVDF membrane, paper, plastic, glass, and a metal sensor chip; and wherein the sample is at least one selected from the group consisting of a mammalian cell culture, a bacterial cell culture, a yeast culture, a tissue extract, a cancer tissue, serum, plasma, saliva, tear, nasal mucus, sweat, urine, feces, cerebrospinal fluid, ascite, amniotic fluid, bronchoalveolar lavage fluid, pleural effusion, semen, milk, dust, fresh water, seawater, soil, and fermented foods.

42. The method of claim 37, wherein the hydrophobic functional group is at least one selected from the group consisting of butyl, cyanobutyl, octyl, phenyl, and diphenyl.

43. The method of claim 37, further comprising a step of pre-treating the sample containing the extracellular vesicles, prior to step (b) of loading the sample to the stationary phase.

44. A method for isolating extracellular vesicles, the method comprising the steps of: (a) immobilizing a hydrophobic functional group onto a stationary phase; (b) loading a sample containing extracellular vesicles to the stationary phase; and (c) collecting a sample fraction containing extracellular vesicles not bound to the hydrophobic functional group from the stationary phase.

45. The method of claim 44, further comprising a step of washing the stationary phase having hydrophobic functional group thereon with a solution containing salting-out ions, prior to step (b), wherein the concentration of the salting-out ions ranges from 0 M to 10 M.

46. The method of claim 44, further comprising a step of adding a solution either containing or not containing salting-out ions to the sample to change kinds or concentrations of salting-out ions contained in the sample, prior to step (b) of loading a sample containing extracellular vesicles to the stationary phase.

47. The method of claim 44, wherein the salting-out ions are at least one selected from the group consisting of sulfate, phosphate, hydroxide, fluoride, formate, acetate, chloride, bromide, nitrate, iodide, thiocyanate, citrate, and tartrate; wherein the stationary phase is at least one selected from the group consisting of agarose beads, sepharose beads, sephadex beads, silica beads, magnetic beads, gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, a nylon membrane, nitrocellulose membrane, a PVDF membrane, paper, plastic, glass, and a metal sensor chip; wherein the sample is at least one selected from the group consisting of a mammalian cell culture, a bacterial cell culture, a yeast culture, a tissue extract, a cancer tissue, serum, plasma, saliva, tear, nasal mucus, sweat, urine, feces, cerebrospinal fluid, ascite, amniotic fluid, bronchoalveolar lavage fluid, pleural effusion, semen, milk, dust, fresh water, seawater, soil, and fermented foods.

48. The method of claim 44, wherein the hydrophobic functional group is at least one selected from the group consisting of butyl, cyanobutyl, octyl, phenyl, and diphenyl.

49. The method of claim 44, further comprising a step of pre-treating the sample containing the extracellular vesicles, prior to step (b) of loading the sample to the stationary phase, and/or a step of post-treating the extracellular vesicles in the sample fraction collected from step (c); wherein the pre-treatment step and/or the post-treating step is conducted by a technique selected from centrifugation, ultracentrifugation, filtration, ultrafiltration, dialysis, desalting column chromatography, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, precipitation, and an aqueous two-phase system.

50. A method for removing a material greater in intensity of hydrophobic interaction than extracellular vesicles from a sample containing the extracellular vehicles, the method comprising the steps of: (a) immobilizing a hydrophobic functional group onto a stationary phase; (b) loading a sample containing extracellular vesicles to the stationary phase having the hydrophobic functional group immobilized thereon; and (c) collecting a sample fraction containing extracellular vesicles not bound to the hydrophobic functional group from the stationary phase.

51. The method of claim 50, further comprising a step of washing the stationary phase having the hydrophobic functional group immobilized thereon with a solution containing salting-out ions, prior to step (b).

52. The method of claim 51, wherein the concentration of the salting-out ions ranges from 0 M to 10 M.

53. The method of claim 50, further comprising a step of adding a solution containing salting-out ions to the sample to change the kind or concentrations of salting-out ions contained in the sample, prior to step (b) of loading a sample containing extracellular vesicles to the stationary phase.

54. The method of claim 53, wherein the concentration of the salting-out ions ranges from 0 M to 10 M.

55. The method of claim 50, wherein the material greater in intensity of hydrophobic interaction than extracellular vesicles is a lipoprotein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 shows schematic views illustrating a method for isolation of extracellular vesicles according to an embodiment of the present invention.

[0064] FIG. 2 is a conceptual view illustrating an overall scheme for a method for isolation of reference extracellular vesicles from the colon cancer cell line SW480 according to an embodiment of the present invention.

[0065] FIG. 3 shows the identification of reference extracellular vesicles isolated according to an embodiment of the present invention, as analyzed by size exclusion chromatography (a), transmission electron microscopy (b), DLS analysis (c), and Western blotting (d).

[0066] FIG. 4 shows the binding behavior of reference extracellular vesicles to a stationary phase having a hydrophobic functional group immobilized thereonto according to an embodiment of the present invention, as identified by nanoparticle concentration analysis (a) and sandwich ELISA analysis (b).

[0067] FIG. 5 shows the binding behavior of reference extracellular vesicles to a stationary phase having a hydrophobic functional group immobilized thereonto in the environment of various concentrations of sodium chloride according to an embodiment of the present invention, as identified by nanoparticle concentration analysis (a) and Western blotting (b).

[0068] FIG. 6 shows the purification of human plasma-derived extracellular vesicles being removed of lipoproteins therefrom through an isolation method using hydrophobic interaction according to an embodiment, as identified by Western blotting (a), cholesterol assay (b), nanoparticle concentration analysis (c), and protein concentration analysis (d).

BEST MODE FOR INVENTION

Mode for Carrying Out the Invention

[0069] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Purification and Analysis of Reference Extracellular Vesicles

[0070] A fresh culture of the colon cancer cell line SW480 was centrifuged at 500g for 10 min (repeated twice in total). After removal of the cell debris and the precipitates, the supernatant was centrifuged again at 2,000g for 20 min (repeated twice in total) and the precipitate was removed.

[0071] The supernatant was added with a precipitation-inducing agent (8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH7.4) in order to primarily purify and precipitate extracellular vesicles existing therein. After being stored for 16 hours in a refrigerator, the supernatant was centrifuged at 12,000g for 30 min to harvest extracellular vesicles as a precipitate. The precipitate was dissolved in HEPES buffer (HEPES-buffered saline, 20 mM HEPES, 150 mM NaCl, pH 7.4).

[0072] In order to secondarily purify the extracellular vesicles by use of density and buoyancy, the sample was mixed with OptiPrep (final concentration 30%) and the mixture was loaded to the bottom of an ultracentrifugation vessel, followed by layering 20% OptiPrep and 5% OptiPrep thereon in the order. After buoyant density gradient ultracentrifugation at 200,000g for 2 hours (30%, 20%, and 5% OptiPrep triple layers), extracellular vesicles and a high-density (1.08-1.12 g/ml) area were harvested.

[0073] For tertiary purification of the purified extracellular vesicles, size exclusion chromatography was performed on a Sephacryl S500-filled column (10100 mm) into which the harvest was previously loaded with the aid of an HPLC apparatus. As a result, a fraction of finally purified extracellular vesicles was obtained. This isolation procedure of sample extracellular vesicles is illustrated in FIG. 2.

[0074] The extracellular vesicles finally purified, as described above, from the colon cancer cell line SW480 were identified using size exclusion chromatography, electron microscopy, and Dynamic Light Scattering (DLS). The results are depicted in FIG. 3. As shown in FIG. 3(a), the colon cancer cell line-derived extracellular vesicles were found to be highly pure. In addition, the colon cancer cell line-derived extracellular vesicles were morphologically observed under a transmission electron microscope as shown in FIG. 3(b) and were analyzed to have a diameter of about 164 nm as measured by DLS in FIG. 3(c).

[0075] Furthermore, as shown in FIG. 3(d), Western blot analysis indicated selectively high expression levels of Alix, CD63, and CD9 proteins, which are markers of extracellular vesicles, compared with the cell line, but did not detect Calnexin and histone H2B, which are cytosolic proteins, in the purified extracellular vesicles derived from the colon cancer cell line.

Example 2: Binding Behavior of Reference Extracellular Vesicles to Stationary Phase Having Hydrophobic Functional Group

[0076] Examination was made to see whether or not extracellular vesicles bind to a hydrophobic functional group. In this regard, an HPLC system was used to examine the binding behavior of the reference extracellular vesicles separated in Example 1.

[0077] First, a column (1 ml) filled with a stationary phase (Phenyl-Sepharose) having a hydrophobic functional group was washed with 20 column volume of a binding buffer (1 M NaCl, 20 mM HEPES, pH 7.2) at a pressure of 1 ml/min. The reference extracellular vesicles separated in Example 1 were dissolved in an amount of 410.sup.10 vesicles in the same binding buffer.

[0078] Using the same binding buffer, the reference extracellular vesicle solution was loaded to the washed stationary phase having a hydrophobic functional group for 5 min, followed by washing out unbound residues with the same buffer for 5 min. Then, 20 column volumes with a concentration gradient of 1 M-0 M NaCl were applied before washing with 10 column volumes of 0 M NaCl buffer.

[0079] Eluates from the extracellular vesicle binding, washing, NaCl concentration gradient, and final washing procedures were fractioned by 1 ml. All of the eluate fractions were subjected to nanoparticle concentration analysis and sandwich ELISA based on anti-extracellular vesicle marker antibodies (anti-CD9 and anti-SW480EV antibody) to assay binding behaviors of the extracellular vesicles.

[0080] Results are depicted in FIG. 4. As shown in FIG. 4, most of the reference extracellular vesicles bound to the stationary phase having a hydrophobic function group and thus was not detected in the eluates under the environment of 1 M NaCl buffer. When the concentration of NaCl in the mobile phase reached 0 M, the elution of the extracellular vesicles was detected. Therefore, it is understood that the extracellular vesicles bind to the hydrophobic function group in a high-concentration NaCl environment whereas being separated from the hydrophobic function group in a low-concentration NaCl environment. In addition, the results obtained above indicate that a stationary phase having a hydrophobic functional group allows the effective elimination of impurities present in the purified reference extracellular vesicles, or inactivated extracellular vesicles or large residual impurity complexes formed during storage.

Example 3: Binding Behavior of Reference Extracellular Vesicle to Stationary Phase Having Hydrophobic Functional Group According to Salt Concentration

[0081] In order to examine the binding behavior of the reference extracellular vesicles to a stationary phase having a hydrophobic functional group according to NaCl concentrations, the following experiment was performed.

[0082] The purified reference extracellular vesicles were precipitated using polyethylene glycol. The precipitated reference extracellular vesicles were added to buffers with various concentrations of NaCl (0 M, 50 mM, 150 mM, 500 mM NaCl in 20 mM HEPES, pH7.2).

[0083] An empty column was filled with 0.4 ml of a stationary phase (Phenyl-Sepharose) having a hydrophobic functional group and then washed three times with 0.5 ml of water by centrifugation (700g, 1 min) to prepare hydrophobic interaction chromatography column. Four of such hydrophobic interaction chromatography columns were washed three times with 0.5 ml of respective buffers (0 M, 50 mM, 150 mM, 500 mM NaCl in 20 mM HEPES, pH7.2) for each time. To each of the prepared columns was loaded 0.2 ml of the reference extracellular vesicles, followed by reaction for 10 min. Centrifugation at 700g for 1 min discharged unbound vesicles. The solutions thus harvested were measured for nanoparticle concentrations to quantitate the reference extracellular vesicles therein, and Western blot analysis against CD9, which is a marker of extracellular vesicles, was performed. The results are given in FIG. 5.

[0084] As shown in both the nanoparticle analysis result (a) and the Western blots (b) of FIG. 5, the highest nanoparticle concentration and the most intensive CD9 signal were detected in the buffer with 0 M NaCl, indicating that the extracellular vesicles bind to the hydrophobic functional group in the condition of a high concentration of NaCl, but escape out of the column without being bound to the hydrophobic functional group in the condition of a low concentration of NaCl.

Example 4: Isolation of Blood-Derived Extracellular Vesicles Free of Lipoprotein by Hydrophobic Interaction Chromatography

[0085] Using the method of the present invention, extracellular vesicles were isolated from human plasma and a lipoprotein elimination effect was analyzed as follows.

[0086] An empty column was filled with 2 ml of a stationary phase (Phenyl-Sepharose) having a hydrophobic functional group and washed three times with 2 ml of water by centrifugation (700g, 1 min). Subsequently, 1 ml of EDTA-treated human plasma sample was loaded into the stationary phase-filled column which was then centrifuged again at 700g for 1 min to harvest the plasma sample that were not bound to the column. Thereafter, the harvested plasma sample was loaded again to the same column and centrifuged in the same condition. This procedure was performed three times in total.

[0087] The resulting plasma sample passing three times through the stationary phase-filled, hydrophobic interaction chromatography column was diluted eight times in a HEPES buffer (150 mM NaCl, HEPES, pH7.2) while a plasma sample that had not passed through the hydrophobic interaction chromatography column was used as a control. Using polyethylene glycol, extracellular vesicles in the samples were precipitated and centrifuged centrifugation (12,000g, 10 min). The precipitates of extracellular vesicles were collected and dissolved in HEPES buffer, followed by additional purification through S500 size exclusion chromatography using an HPLC system.

[0088] The blood-derived extracellular vesicles purified above were analyzed for CD81, which is a marker protein of extracellular vesicles, and ApoA1 and ApoB, which are proteins specific for high- and low-density lipoproteins, respectively, by Western blotting. The results are given in FIG. 6(a). As is understood from the data, similar CD81 signals were detected in both the samples that passed and did not pass through the hydrophobic interaction chromatography column whereas no signals for ApoB and ApoA1, which are markers of lipoproteins, were detected from the sample that passed through the hydrophobic interaction chromatography column. Therefore, the results indicate that the use of a hydrophobic interaction chromatography column can selectively remove only lipoproteins while retaining the yield for the blood-derived extracellular vesicles.

[0089] In addition, the extracellular vesicle sample that passed through the hydrophobic interaction chromatography column was found to significantly decrease in cholesterol concentration as measured by an assay for cholesterol concentrations of the blood-derived extracellular vesicles purified above (FIG. 6(b)), as well as in G protein concentration as measured by a nanoparticle assay (FIG. 6(c) and a protein concentration assay (FIG. 6(d)), which accounts for the removal of lipoprotein nanoparticles.

[0090] Therefore, this Example demonstrates that the use of a hydrophobic interaction chromatography column can effectively prevent the contamination of blood-derived extracellular vesicles with lipoproteins, which are difficult to remove with conventional methods, while retaining the yield for the extracellular vesicles.