METHOD FOR MASS-PRODUCING PLANT EXOSOMES

Abstract

The present disclosure relates to a large-scale production method of plant exosomes. The method of the present disclosure can isolate high purity plant exosomes from a large amount of raw plants, using centrifugation and TFF, which can process a large amount of plant raw materials at once. This improves a conventional isolation process of plant exosomes stayed at the laboratory level, and thereby, suggests an easy process for large-scale production.

Claims

1. A method for isolating plant exosomes, comprising performing centrifugation; and performing tangential-flow filtration (TFF).

2. The method for isolating plant exosomes according to claim 1, wherein the centrifugation comprises ultracentrifugation.

3. The method for isolating plant exosomes according to claim 1, wherein the tangential-flow filtration is one or more selected from the group consisting of hollow fiber TFF and membrane TFF.

4. The method for isolating plant exosomes according to claim 1, wherein the tangential-flow filtration uses a TFF filter with a molecular weight cutoff (MWCO) of 100,000 Da to 500,000 Da.

5. The method for isolating plant exosomes according to claim 1, wherein the method further comprises crushing a raw plant before the centrifugation.

6. The method for isolating plant exosomes according to claim 1, wherein the raw plant comprises one or more selected from the group consisting of pulps, peels, seeds, stems, leaves, roots and flowers.

7. The method for isolating plant exosomes according to claim 1, wherein the plant exosomes have a diameter of 50 to 200 nm.

8. The method for isolating plant exosomes according to claim 1, wherein a high purity and high yield plant derived exosome extract is obtained by the method, and wherein the extract comprises plant exosomes at a concentration of 10.sup.7 to 10.sup.12 particles per 1 mL of the extract.

9. The method for isolating plant exosomes according to claim 1, wherein a high purity plant derived exosome extract is obtained by the method, and wherein the extract does not comprise vacuoles.

10. The method for isolating plant exosomes according to claim 1, wherein the method is for large-scale production of plant-derived exosomes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a schematic diagram of a process for a method of isolation of plant derived exosomes according to one embodiment of the present disclosure.

[0034] FIG. 2 is the result of form analysis (TEM) of aloe peel derived exosomes according to Example 1.

[0035] FIG. 3 is the result of characteristic analysis (NTA) of aloe peel derived exosomes according to Example 1.

[0036] FIG. 4 is a TEM photograph of comparing the results obtained when omitting the UC process and performing only TFF and the results obtained when combining UC and TFF.

[0037] FIG. 5 is the result of characteristic analysis (DLS) of aloe peel derived exosomes according to Example 1.

[0038] FIG. 6 is the result of form analysis (TEM) of garlic derived exosomes according to Example 2.

[0039] FIG. 7 is the result of characteristic analysis (NTA) of garlic derived exosomes according to Example 2.

[0040] FIG. 8 is the result of form analysis (TEM) of sea mustard peel derived exosomes according to Example 3.

[0041] FIG. 9 is the result of form analysis (NTA) of sea mustard peel derived exosomes according to Example 3.

MODE FOR INVENTION

[0042] Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for illustrating the present disclosure more specifically, and it will be apparent to those skilled in the art to which the present disclosure pertains that the scope of the present disclosure is not limited by these examples.

EXAMPLE

[0043] 1. Aloe Peel Derived Exosomes

[0044] (1) Isolation Method

Example 1 (UC+TFF)

[0045] Aloe peel derived exosomes were isolated using UC and TFF. Specifically, aloe peel was sufficiently grinded after putting in a mixer setting to 1:2 (w/w) with phosphate-buffered saline (PBS). Then, supernatant generated after centrifugation at 1,000×g for 10 minutes was centrifuged at 2000×g for 20 minutes, and then the supernatant was collected again. The supernatant was centrifuged at 3,000×g for 30 minutes and then centrifuged at 10,000×g for 60 minutes. The supernatant after 10,000×g was ultracentrifuged at 100,000×g for 70 minutes at 4° C. using UC.

[0046] The remaining pellets after UC were suspended and exosomes were isolated from the suspension through a tangential-flow filtration (TFF) system. Specifically, other impurity particles smaller than the pores of the filter were removed from the surface of the multi-filter having a cutoff value of 100 to 500 kDa, and the solution containing aloe peel derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at −70° C. or less until used in the experiment.

Comparative Example 1 (MF+TFF)

[0047] Aloe peel derived exosomes were isolated using microfiltration and TFF. Specifically, the aloe peel was sufficiently grinded after putting in a mixer setting to 1:2 (w/w) with phosphate-buffered saline (PBS). Then, to obtain exosomes with high purity in which the particle size distribution is uniform, 2% by weight of trehalose was added. After adding trehalose, this was filtered with a 0.22 μm filter, and impurities such as cell debris, wastes and large particles were removed.

[0048] After passing through the filtering process, through a tangential-flow filtration (TFF) system as ultrafiltration, exosomes were isolated. Specifically, other impurity particles smaller than the pores of the filter were removed from the surface of the multi-filter having a cutoff value of 100 to 500 kDa, and the solution containing aloe peel derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at −70° C. or less until used in the experiment.

[0049] (2) Evaluation of Characteristics of Exosomes

[0050] For aloe peel derived exosomes, evaluation of exosome characteristics was progressed. In order to confirm the shape of exosomes, a scanning electron microscope (TEM) was used, and in order to confirm the accurate microparticle size, a dynamic light scattering photometer (DLS) was used. The particle number of the isolated exosomes per unit volume was confirmed through nanoparticle tracking analysis (NTA).

[0051] As a result of the experiment, in Example 1, a mixture in which exosomes and vacuoles were present together was obtained after filtering solids with a relatively large particle size by microfiltration (pore size 0.1˜1.0 μm) of the supernatant after 10,000×g centrifugation, and it was confirmed that exosomes and vacuoles were mixed in the supernatant after 10,000×g centrifugation. In Example 1, it was confirmed that a large amount of impurities such as proteins and nucleic acids as well as exosomes were mixed in the pellet collected after performing 100,000×g ultracentrifugation. As a result of isolating exosomes by performing ultrafiltration, TFF in Example 1, 1×10.sup.9-11 high purity exosomes per unit volume of 1 mL were isolated. It was confirmed that the isolated aloe peel derived exosomes had a microstructure of 200 nm or less (FIG. 2), and the concentration of 1×10.sup.9-11 exosomes per unit volume of 1 mL was confirmed through nanoparticle tracking analysis (NTA) (FIG. 3).

[0052] On the other hand, it was observed that vacuoles similar to exosomes were mixed in the aloe peel derived extract isolated by the method of Comparative example 1, and thereby, it was confirmed that it was difficult to remove vacuoles contained in the plant extract by the MF and TFF methods. In other words, when performing TFF only without an ultracentrifugation (UC) process in the process of isolating exosomes from aloe peel, it was clearly confirmed that impurities including vacuoles (red arrow) were included in addition to exosomes, and these impurities cause a decrease in the efficacy of plant exosomes (See FIG. 4).

[0053] 2. Isolation of Garlic Derived Exosomes

[0054] (1) Isolation Method

Example 2 (UC+TFF)

[0055] Garlic derived exosomes were isolated using UC and TFF. Specifically, garlic from which peel was removed was sufficiently grinded after putting in a mixer setting to 1:2 (w/w) with phosphate-buffered saline (PBS). Then, supernatant generated after centrifugation at 1,000×g for 10 minutes was centrifuged at 2000×g for 20 minutes, and then the supernatant was collected again. The supernatant was centrifuged at 3,000×g for 30 minutes and then centrifuged at 10,000×g for 60 minutes. The supernatant after 10,000×g was ultracentrifuged at 100,000×g for 70 minutes at 4° C. using UC. By suspending the pellet remained after UC, exosomes were isolated through a tangential-flow filtration (TFF) system as ultrafiltration. Specifically, other impurity particles smaller than the pores of the filter were removed from the surface of the multi-filter having a cutoff value of 100 to 500 kDa, and the solution containing garlic derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at −70° C. or less until used in the experiment.

Comparative Example 2 (UC+S-DGUC)

[0056] Garlic derived exosomes were isolated using UC and Sucrose-Density Gradient Ultracentrifugation. Specifically, garlic from which peel was removed was sufficiently grinded after putting in a mixer setting to 1:2 (w/w) with phosphate-buffered saline (PBS). Then, supernatant generated after centrifugation at 1,000×g for 10 minutes was centrifuged at 2000×g for 20 minutes, and then the supernatant was collected again. The supernatant was centrifuged at 3,000×g for 30 minutes and then centrifuged at 10,000×g for 60 minutes. The supernatant after 10,000×g was ultracentrifuged at 100,000×g for 70 minutes at 4° C. using UC.

[0057] By suspending the pellet remained after UC, Sucrose-Density Gradient Ultracentrifugation was additionally performed. Specifically, a sucrose solution having a density gradient was added in a centrifugation tube for ultracentrifugation. These sucrose solutions were made into 90%, 80%, 70%, 60%, 50%, 40% and 30% solutions, respectively, and after sterilization at 124° C. for 15 minutes, the tube for ultracentrifugation was carefully placed to have a layer of 90%˜30%. After carefully adding the suspension containing the exosomes to the top of the sucrose gradient tube for ultracentrifugation prepared as above, using a centrifuge for ultracentrifugation, ultracentrifugation at 4° C., 200,000×g for 4 hours was performed. Then, fractions were obtained by 1 ml from the top of the tube, and the density was measured to obtain fractions corresponding to exosomes and washed to separate sucrose.

[0058] (2) Evaluation of Characteristics of Exosomes

[0059] As an experimental result, it was confirmed that the garlic derived exosomes isolated in Example 2 had a round microstructure of 200 nm or less through a scanning electron microscope (TEM) (FIG. 6), and as the result of confirming the particle number per unit volume of the isolated exosomes through nanoparticle tracking analysis (NTA), the concentration of 1×10.sup.10-12 exosomes per unit volume of 1 mL was confirmed (FIG. 7).

[0060] It was observed that the garlic derived exosomes isolated by the method of Comparative example 2 also had a round microstructure of 200 nm or less through a scanning electron microscope (TEM). However, in order to isolate high purity exosomes, an additional washing process for separating sucrose was required, and the exosome production per hour was less than 1/100 of the production of Example 2. Therefore, it was confirmed that the method of Comparative example 2 was difficult to utilize in a mass production process for commercialization of exosomes.

[0061] 3. Isolation of Sea Mustard Derived Exosomes

[0062] (1) Isolation Method

Example 3 (UC+TFF)

[0063] Sea mustard derived exosomes were isolated using UC and TFF. Specifically, sea mustard from which moisture was removed was sufficiently grinded after putting in a mixer setting to 1:2 (w/w) with phosphate-buffered saline (PBS). Then, supernatant generated after centrifugation at 1,000×g for 10 minutes was centrifuged at 2000×g for 20 minutes, and then the supernatant was collected again. The supernatant was centrifuged at 3,000×g for 30 minutes and then centrifuged at 10,000×g for 60 minutes. The supernatant after 10,000×g was ultracentrifuged at 100,000×g for 70 minutes at 4° C. using UC.

[0064] By suspending the pellet remained after UC, exosomes were isolated through a tangential-flow filtration (TFF) system as ultrafiltration. Specifically, other impurity particles smaller than the pores of the filter were removed from the surface of the multi-filter having a cutoff value of 100 to 500 kDa in the TFF system, and the solution containing sea mustard derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at −70° C. or less until used in the experiment.

Comparative Example 3 (SEC+TFF)

[0065] Size Exclusion Chromatography was known as a useful method capable of separating exosomes without exosome damage or change in characteristics. Accordingly, sea mustard derived exosomes were isolated using SEC and TFF. Specifically, separation of exosomes by SEC was performed by the method disclosed in Boing et al (Single-step isolation of extracellular vesicles by size-exclusion chromatography, J Extracell Vesicles, 2014:1-11). 12 mL of Sepharose CL-2B (Sigma Aldrich, St. Louis, Mo., USA) was stacked in a 20 mL syringe (BD Plasticpakc™, San Jose, Calif.), washed with PBS and equilibrated. 2 mL of sample was loaded onto a column, and fractions were collected using PBS as an elution buffer.

[0066] Exosomes were isolated from the collected fractions through a tangential-flow filtration (TFF) system. Specifically, other impurity particles smaller than the pores of the filter were removed from the surface of the multi-filter having a cutoff value of 100 to 500 kDa in the TFF system, and the solution containing sea mustard derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at −70° C. or less until used in the experiment.

[0067] (2) Evaluation of Characteristics of Exosomes

[0068] It was confirmed that the sea mustard derived exosomes isolated in Example 3 had a round microstructure of 200 nm or less through a scanning electron microscope (TEM) (FIG. 8), and as the result of confirming the particle number per unit volume of the isolated exosomes through nanoparticle tracking analysis (NTA), the concentration of 1×10.sup.9-11 exosomes per unit volume of 1 mL was confirmed (FIG. 9).

[0069] On the other hand, it was observed that vacuoles similar to exosomes were mixed in the sea mustard derived extract isolated by the method of Comparative example 3, and therefore, it was confirmed that it was difficult to remove vacuoles contained in the plant extract by the SEC and TFF methods.