COMPOSITION FOR PROMOTING EXTRACELLULAR VESICLE PRODUCTION CONTAINING PEPTIDE DERIVED FROM NOXA PROTEIN AND METHOD FOR PRODUCING EXTRACELLULAR VESICLES BY USING SAME

Abstract

The present invention relates to a method for producing extracellular vesicles (EV) in large quantities by using a peptide derived from Noxa protein that plays a key role in apoptosis and derivatives thereof, and the use of the peptide for promoting extracellular vesicle production and the method for producing extracellular vesicles by using the same of the present invention can efficiently and uniformly mass-produce extracellular vesicles, and can produce extracellular vesicles which load drugs passing poorly through cellular membranes as well as recombinant proteins or plasmid DNA.

Claims

1. A composition for promoting extracellular vesicle production, the composition comprising at least one peptide selected from the group consisting of peptides containing amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 21.

2. A medium for extracellular vesicle production, the medium comprising: at least one peptide selected from the group consisting of peptides containing amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 21; and a solution containing a sugar.

3. The medium of claim 2, wherein the sugar is at least one selected from the group consisting of glucose, sucrose, and sorbitol.

4. The medium of claim 2, wherein the solution further contains 3-(N-morpholino)propanesulfonic acid (MOPS).

5. The medium of claim 2, wherein the solution further contains at least one selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), sodium gluconate (Na-gluconate), potassium gluconate (K-gluconate), sodium phosphate (NaPO.sub.4), and potassium phosphate (KPO.sub.4).

6. A method for producing extracellular vesicles, the method comprising: a medium preparing step of preparing a medium containing: at least one peptide selected from the group consisting of peptides containing amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 21; and a solution containing a sugar; and a mixing step of mixing the medium and a cell line.

7. The method of claim 6, wherein the sugar is at least one selected from the group consisting of glucose, sucrose, and sorbitol.

8. The method of claim 6, wherein the solution further contains 3-(N-morpholino)propanesulfonic acid (MOPS).

9. The method of claim 6, wherein the solution further contains at least one selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), sodium gluconate (Na-gluconate), potassium gluconate (K-gluconate), sucrose, sodium phosphate (NaPO.sub.4), and potassium phosphate (KPO.sub.4).

10. The method of claim 6, wherein the cell line is any one selected from the group consisting of mouse colon carcinoma (CT26), human cervical cancer (HeLa), human renal epithelium (HEK293), mouse adipocytes (3T3- L1), and human mesenchymal stem cells (HMSC).

11. A method for loading an extracellular vesicle payload, the method comprising: a medium preparing step of preparing a medium containing: at least one peptide selected from the group consisting of peptides containing amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 21; and a solution containing a sugar; and a mixing step of mixing the medium, a payload, and a cell line.

12. The method of claim 11, wherein the payload is at least one selected from the group consisting of proteins, DNA, RNA, plasmid DNA, and an anticancer substance.

13. The method of claim 12, wherein the anticancer substance is at least one selected from the group consisting of an anticancer protein, a tumor suppressing gene, and an anticancer compound.

14. The method of claim 13, wherein the anticancer protein is at least one selected from the group consisting of asparaginase, Botulinum toxin, Tetanus toxin, Shiga toxin, Diphtheria toxin (DT), ricin, Pseudomonas exotoxin (PE), cytolysin A (ClyA), Y- Gelonin, Vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), transforming growth factor-β, TGF-βintegrin, vascular endothelial (VE)-cadherin, plasminogen activator (PA), ephrin, platelet-derived growth factor (PDGF), monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor (FGF), placenta growth factor (PIGF), von HippelLindau (VHL), adenomatous polyposis coli (APC), cluster of differentiation 95 (CD95), suppression of tumorigenicity 5 (ST5), Yippee like 3 (YPEL3), suppression of tumorigenicity 7 (ST7), and suppression of tumorigenicity 14 (ST14).

15. The method of claim 13, wherein the tumor suppressing gene is at least one selected from the group consisting of von HippelLindau (VHS), adenomatous polyposis coli (APC), cluster of differentiation 95 (CD95), suppression of tumorigenicity 5 (ST5), yippee like 3 (YPEL3), suppression of tumorigenicity 7 (ST7), and suppression of tumorigenicity 14 (ST14).

16. The method of claim 13, wherein the anticancer compound is at least one selected from the group consisting of methotrexate, 5-fluorouracil, gemcitabine, arabinosylcytosine, hydroxy urea, mercaptopurine, thioguanine, nitrogen Mustard, cyclosporamide, anthracycline, daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, actinomycin D, vincristine, Taxol, combretastatin A4, Fumagillin, herbimycin A, 2- methoxyestradiol, OGT 2115, TNP 470, tranilast, XRP44X, thalidomide, endostatin, salmosin, angiostatin, plasminogen, a kringle domain of apolipoprotein, oxalopatin, carboplatin, cisplatin, bortezomib, and radionuclides.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 shows microscopic observation images of extracellular vesicles generated and settled on the bottom of the plate when the HeLa cell line was treated with the eMTDA4 peptide in combination with a solution according to an example of the present invention.

[0064] FIG. 2 shows microscopic observation images of extracellular vesicles generated when the HeLa cell line was treated with the eMTDA4 peptide in combination with solutions according to examples of the present invention.

[0065] FIG. 3 shows microscopic observation images of extracellular vesicles generated when the HeLa cell line was treated with the eMTDA4 peptide in combination with a solution containing glucose, sorbitol, or sucrose.

[0066] FIG. 4 shows confocal microscopic observation images of extracellular vesicles formed after the transfection of the HeLa cell line with HSP90, an exosome indicator, with EGFP, a fluorescent protein, attached thereto.

[0067] FIG. 5 shows images, taken by using a confocal microscope every two seconds, of the generation of extracellular vesicles after the membrane proteins CD9 and CD81 to which mEmerald and mCherry were attached, respectively, were transfected into HeLa cell lines.

[0068] FIG. 6 shows images depicting the sizes of extracellular vesicles generated in the HeLa cell line, as measured by an atomic microscope.

[0069] FIG. 7 shows heat-maps depicting the quantities of extracellular vesicles produced by MTD, and eMTDA4 and derivatives thereof in the HEK-293 cell line, as quantified by Bradford solution.

[0070] FIG. 8 shows graphs depicting the sizes and quantities of extracellular vesicles produced by MTD, and eMTDA4 and derivatives thereof in the HEK-293 cell line, as analyzed by the Nanoparticle Tracking Analysis (NTA) system.

[0071] FIG. 9 shows the results of performing acrylamide gel electrophoresis after the TRAIL protein, which is a recombinant protein, was loaded in extracellular vesicles.

[0072] FIG. 10 shows the results of performing agarose gel electrophoresis after pUC19 and pEGFP-C1, which are plasmid DNA, were loaded in extracellular vesicles.

[0073] FIG. 11 is a schematic diagram of a procedure where plasmid DNA was loaded in extracellular vesicles and isolated therefrom.

[0074] FIG. 12 shows images confirming the results by a confocal microscope after doxorubicin, a drug favorably passing through cell membranes, was loaded in extracellular vesicles produced by the treatment of the HeLa cell line with eMTDΔ4.

[0075] FIG. 13 shows images confirming the results by a confocal microscope after propidium iodide (PI), a drug poorly passing through cell membranes, was loaded in extracellular vesicles produced by the treatment of the HeLa cell line with eMTDΔ4.

[0076] Mode for Carrying Out the Invention

[0077] Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

[0078] Preparative Example 1: Preparation of Noxa protein- derived peptide and derivatives thereof The mitochondrial targeting domain (MTD) of Noxa protein is shown as SEQ ID NO: 1 on Table 1. For the synthesis of a peptide derived from MTD, a manual Fmoc synthesis method in units of 0.25 mmol was basically used. Specifically, a resin was cleanly washed with dimethylformamide (DMF), and then 10 mL of a 20% piperidine/DMF solution was added to the resin. After stirring for 1 minute, 10 mL of a 20% piperidine/dimethylformamide solution was again added thereto and shaken for 30 minutes. After the resin was again washed with dimethylformamide, no piperidine remained was confirmed through the ninhydrin test (resin turned blue).

[0079] The following solutions were prepared for coupling: 1 mmol Fmoc-amino acid, 2.1 ml of 0.45 M 2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, hexafluorophosphate Benzotriazole Tetramethyl Uronium/Hydroxybenzotriazole (HBTU/HOBT, 1 mmol), 348 μL of N,N-diisopropylethylamine (DIEA, 2 mmol). The prepared solution was added to the resin, and shaken for 30 minutes. After the solution was drained from the resin, the resin was washed with dimethyl formamide. Then, for coupling of amino acids, the solution was again added, and then a coupling step was repeated to synthesize Noxa protein-derived eMTDΔ4 and derivative peptides thereof.

[0080] The peptides thus synthesized were analyzed by high-performance liquid chromatography, Instrument: Waters 2690 separations module, Flow rate: 1.0 ml/min, gradient: 0-20% B, 5 minutes; 20-50%, B 20 minutes; 50- 80%, B 5 minutes, A; 0.1% TFA water, B; 0.1% TFA acetonitrile, column: waters C18, 5 microns, Detection: 220 nm, purity: 95%) and a mass analyzer (HP 1100 series LC/MSD). The synthesized peptides are shown as SEQ ID NO: 2 to SEQ ID NO: 21 on Table 1.

TABLE-US-00001 TABLE 1 SEQ ID NO Name Sequence Note 1 MTD KLLNLISKLF 10aa 2 eMTDΔ4 KLNFRQKLLNLISKLF 16aa 3 TU17 RPARPARGGKLLNLISKLF 19aa 4 R(8):MTD RRRRRRRRKLLNLISKLF 18aa 5 TU103 KLLNLWSLLFGYTIK 15aa 6 TU104 MEWWYLLKLLNLISKLF 17aa 7 TU107 LRSLRDKLLNLISKLF 16aa 8 TU111 GLKSLRKLLNLISKLF 16aa 9 TU114 KWYAKLLNLISKLF 14aa 10 TU124 KLLNLWSLLKGYTIK 15aa 11 TU128 AEYRKLLNLISKLF 14aa 12 TU135 AEYSRKLLNLISKLF 15aa 13 TU146 YWLPLRKLLNLISKLF 16aa 14 TU149 AHFLRKLLNLISKLF 15aa 15 TU151 AFFLRKLLNLISKLF 15aa 16 TU155 QFAQYLRNLISKLF 14aa 17 TU156 KVSIFLKNLISKLF 14aa 18 TU166 KLNFAEFLRNLISKLF 16aa 19 TU168 KLNFRLGLRSLREKLF 16aa 20 TU171 KLNFRQKLARLLTKLF 16aa 21 TU172 LNFKWYSLLNLISKLF 16aa

[0081] Example 2: Preparation of solutions for extracellular vesicle production

[0082] For extracellular vesicle production using eMTDΔ4, derived from the mitochondrial targeting domain of Noxa protein, and the derivative peptides thereof, the solutions of Example 1 and Comparative Examples 1 to 18 containing the compositions and contents shown in Table 2 were prepared.

TABLE-US-00002 TABLE 2 Na- K- NaPO4 KPO4 (mM) Glucose MOPS NaCl KCl gluconate gluconate Sucrose (pH7.4) (pH7.4) Example 1 5 10 — — 250.00 — — Comparative 5 10 145 — — — — — — Example 1 Comparative 5 10 — 145 — — — — — Example 2 Comparative 5 10 — — 145 — — — — Example 3 Comparative 5 10 — — — 145 — — — Example 4 Comparative 5 10 50 — 95 — — — — Example 5 Comparative 5 10 — 50 — 95 — — — Example 6 Comparative 5 10 — — — — 241.37 10 — Example 7 Comparative 5 10 — — — — 232.37 20 — Example 8 Comparative 5 10 — — — — 200.00 58 — Example 9 Comparative 5 10 75 — — — 91.37 10 — Example 10 Comparative 5 10 75 — — — 82.75 20 — Example 11 Comparative 5 10 75 — — — 50.00 58 — Example 12 Comparative 5 10 — — — — 241.37 — 10 Example 13 Comparative 5 10 — — — — 232.37 — 20 Example 14 Comparative 5 10 — — — — 200.00 — 58 Example 15 Comparative 5 10 — 75 — — 91.37 — 10 Example 16 Comparative 5 10 — 75 — — 82.75 — 20 Example 17 Comparative 5 10 — 75 — — 50.00 — 58 Example 18

[0083] Experimental Example 1: Culture of cancer cell lines

[0084] 1-1. Preparation of cell lines and reagents

[0085] Cancer cell lines were cultured prior to testing the activity to induce extracellular vesicle production by the Noxa protein-derived peptide and the derivatives thereof in Preparative Example 1.

[0086] Dulbecco's modified eagle medium (DMEM), RPMI 1640, fatal bovine serum (FBS), trypsin- ethylenediaminetetraacetic acid (trypsin-EDTA), and Hank's balanced salt solution (HBSS) required for culture were purchased from Gibco, and Effectene, which is a transfection reagent, was purchased from Qiagen. HeLa and HEK293 cell lines were purchased from Korean Cell Line Bank (KCLB).

[0087] 1-2. Cell culture

[0088] HeLa and HEK293 cell lines were subcultured at least three times before use in experiments. The HeLa cell line was cultured using DMEM containing 10% fetal bovine serum (FBS) and the HEK-293 cell line was cultured using RPMI 1640 containing 10% fetal bovine serum, under a gas condition of 5% (v/v) CO.sub.2 and a temperature condition of 37° C.

[0089] Experimental Example 2: Production of extracellular vesicles

[0090] 2-1. Production of extracellular vesicles

[0091] After the HeLa cell line was cultured in a 6-well plate, the culture was removed, and then the HeLa cell line was treated with the solution in Example 1.

[0092] The control group was not treated with any peptide, and the experiment group was treated with eMTDA4 peptide of SEQ ID NO: 2 (final concentration: 20 μM). After 10 minutes, the generation of extracellular vesicles was observed by an optical microscope (bright field), and the observation results are shown in FIG. 1.

[0093] As can be confirmed in FIG. 1, the bottom of the plate was clean in the control group, but small extracellular vesicle particles were formed and settled on the bottom of the plate in the experiment group.

[0094] 2-2. Production of extracellular vesicles using various solutions

[0095] Extracellular vesicle production was carried out by the experimental method of Experimental Example 2-1 using the R(8):MTD peptide of SEQ ID NO: 4 and the solutions of Example 1 and Comparative Examples 1 to 18, and the results are shown in FIGS. 2A to 2S. As can be confirmed in FIG. 2A, many extracellular vesicle particles were generated and most of the particles were settled on the bottom of the plate.

[0096] However, as can be confirmed in FIGS. 2B to 2S, extracellular vesicle particles were not generated, and thus no extracellular vesicles settled on the bottom of the plate could be observed, and only the formation of particles that occurs at the time of apoptosis inside cells was observed.

[0097] 2-3. Production of extracellular vesicles using various sugars

[0098] Extracellular vesicle production was carried out by the experimental method of Example 2-1 using the R(8):MTD peptide of SEQ ID NO: 4 and the solutions of Examples 1 to 3 on Table 3, and the results are shown in FIGS. 3A to 3C.

TABLE-US-00003 TABLE 3 (mM) Glucose Sucrose Sorbitol MOPS Example 1 5 250 — 10 Example 2 255 — — 10 Example 3 5 — 250 10

[0099] As can be confirmed in FIGS. 3A to 3C, even when an excess of glucose or sorbitol, instead of sucrose, was added to the solution, extracellular vesicles were well formed.

[0100] Experimental Example 3: Identification of extracellular vesicle morphology

[0101] In order to transfect the HSP90 protein, an exosome indicator, into the HeLa cell line, the HeLa cell line was cultured overnight in a 6-well plate, and then treated with a plasmid into which Effectene and pEGFP- HSP90 were cloned, and after 4 hours, the culture was replaced.

[0102] The next day, the culture was removed from the HeLa cell line, and the solution of Example 1 was added. A control group was not treated with any peptide, and an experiment group was treated with the eMTDΔ4 peptide of SEQ ID NO: 2 (final concentration: 20 μM). The HSP90 protein was excited using an argon laser of 488 nm. After 10 minutes, the control group and the experiment group were observed using a confocal microscope (Leica TCS SP5 Microsystems), and the results are shown in FIG. 4.

[0103] As can be confirmed in FIG. 4, the cell morphology of the control group was not damaged and the HSP90 protein was located in the cytoplasm. However, in the experiment group (eMTDΔ4), the cell morphology was damaged and extracellular vesicles containing the HSP90 protein were settled on the bottom of the plate.

[0104] The reasoning was determined to be that the HSP90 protein of the cytoplasm was released out of the cell through the cell membrane defects that occurred during the production of extracellular vesicles.

[0105] Experimental Example 4: Investigation of production of extracellular vesicles

[0106] The HeLa cell line was cultured in a 12-well plate overnight, and in order to identify the CD9 protein, an extracellular vesicle indicator, the HeLa cell line was treated with a plasmid into which Effectene and mEmerald-CD9 were cloned, and after 4 hours, the culture was replaced.

[0107] The next day, the culture was removed from the HeLa cell line, and the HeLa cell line was treated with the solution of Example 1. After each HeLa cell line was treated with the eMTDΔ4 peptide (final concentration: 20 μM), a laser having a wavelength of 488 nm was used to excite mEmerald-CD9. Thereafter, the cells were observed for 10 minutes while images were taken every 2 seconds by using a confocal microscope, and the results are shown in FIG. 5A.

[0108] In order to identify the CD81 protein, an extracellular vesicle indicator, the same procedure as in the method for observing the CD9 protein was performed except that the HeLa cells were treated with a plasmid into which mCherry-CD81 instead of mEmerald-CD9 was cloned and mCherry-CD81 was excited using a laser having a wavelength of 561 nm, and the results are shown in FIG. 5B.

[0109] As can be confirmed in FIGS. 5A and 5B, both CD9 and CD81 proteins were sufficient to observe the formation of extracellular vesicles, and CD9 and CD81 proteins were released into the extracellular matrix. It could be verified that extracellular vesicles were released to the extracellular matrix within a few seconds after generation, although the release procedure could not be specifically identified.

[0110] Experimental Example 5: Identification of extracellular vesicle size

[0111] The HeLa cell line was cultured in a culture plate, treated with the eMTDΔ4 peptide (final concentration: 20 μM) in combination with the solution of Example 1 and, after 10 minutes, fixed with 4% (v/v) paraformaldehyde.

[0112] Thereafter, the cells were observed using an atomic microscope (Surface Imaging Systems, NANO Station II, with a cantilever with a pyramidal-shaped tip, a frequency of 146-236 kHz, a spring constant of 21-98

[0113] N/m, a length of 225 nm, and a resistance of 0.01- 0.02Ωcm), and the observation results are shown in FIGS. 6A to 6C.

[0114] As can be confirmed in FIG. 6A, extracellular vesicles with various sizes were generated, and the sizes thereof could be measured. The results of measuring the sizes of extracellular vesicles are shown as graphs in FIGS. 6B and 6C.

[0115] As can be confirmed in FIGS. 6B and 6C, the sizes of extracellular vesicles were measured to be about 200 nm. These results prove that the extracellular vesicles of the present invention have a size similar to the previously known extracellular vesicle sizes.

[0116] Experimental Example 6: Verification of extracellular vesicle output

[0117] The 293-HEK cell line was cultured in a 6-well plate at a rate of 2 ×10.sup.5 cells/mL, and the next day, the cell line was treated with the solution of Example 1 and a peptide of any one of SEQ ID NOs: 1 to 21. After minutes, the solution was removed, followed by centrifugation at an RCF of 12000, and the extracellular vesicles were quantified using the supernatant by Bradford solution. The results are shown in FIG. 7 and Table 4.

TABLE-US-00004 TABLE 4 Amount of proteins contained in extracellular vesicles (unit: μg/μl) Item Name 10 μM 20 μM 40 μM 1 Media 1.03 0.75 0.76 (Control) 2 MTD 0.80 1.17 2.87 3 eMTDΔ4 4.93 6.40 7.56 4 TU103 0.75 0.40 0.55 5 TU104 0.62 0.75 1.02 6 TU107 5.58 6.69 7.45 7 TU111 4.17 4.98 6.44 8 TU124 3.30 5.02 6.24 9 TU128 3.86 5.29 7.25 10 TU135 1.81 4.41 6.54 11 TU146 2.01 2.89 3.50 12 TU149 1.71 2.00 2.57 13 TU151 1.51 1.30 1.40 14 TU155 1.56 1.77 2.85 15 TU156 1.17 1.55 3.93 16 TU166 5.77 6.68 8.10 17 TU168 0.77 2.17 3.74 18 TU171 2.64 3.62 5.30 19 TU172 0.83 0.44 0.59

[0118] As can be confirmed in FIG. 7 and Table 4, most of the peptides shown in Table 4 well produced extracellular vesicles although there is a difference in output.

[0119] In addition, as the final peptide treatment concentration increased from 10 μM to 20 μM and 40 μM, the amount of proteins contained in the extracellular vesicles tended to increase.

[0120] Experimental Example 6: Identification of extracellular vesicle size and quantity

[0121] After the 293-HEK cell line was cultured to 90% confluency (about 2 x 10.sup.7 cells) in a 10-cm culture dish, the culture was removed, and the cells were treated with the eMTDΔ4 peptide (final concentration: 20 μm), which has been dissolved in 5 ml of the solution of Example 1. After 20 minutes, the solution was removed, followed by centrifugation at an RCF of 12000, and then the size and quantity of extracellular vesicles were measured using a

[0122] Nanoparticle Tracking Analysis (NTA) system (Nanosight LM10, Malvern Instruments). The results are shown in FIGS. 8A to 8D and Table 5.

TABLE-US-00005 TABLE 5 Quantity Stats: Merged data (nm) (/ml, 50-fold diluted) eMTDΔ4 Mean 184.5 7.44 × 10.sup.8 ± 6.51 × 10.sup.7 Mode 126.3 SD 87.8 D10 111.0 D50 152.6 D90 298.4 MTD Mean 181.1 8.01 × 10.sup.8 ± 1.75 × 10.sup.7 Mode 115.6 SD 75.2 D10 113.4 D50 154.9 D90 288.5 TU17 Mean 229.6 7.95 × 10.sup.8 ± 7.15 × 10.sup.7 Mode 150.9 SD 104.3 D10 131.6 D50 193.2 D90 407.9 TU114 Mean 231.1 8.75 × 10.sup.8 ± 9.27 × 10.sup.7 Mode 182.3 SD 110.2 D10 137.9 D50 191.3 D90 390.5

[0123] As can be confirmed in FIGS. 8A to 8D and Table 5, with respect to the size of extracellular vesicles, the mean was 184.5 nm, the mode was 126.3 nm, and the standard deviation (SD) was 87.8 nm when the eMTDΔpeptide was used.

[0124] The mean was 181.1 nm, the mode was 115.6 nm, and the standard deviation was 75.2 nm when the MTD peptide was used.

[0125] The mean was 229.6 nm, the mode was 150.9 nm, and the standard deviation was 104.3 nm when the TU17 peptide was used.

[0126] The mean was 231.1 nm, the mode was 182.3 nm, and the standard deviation was 110.2 nm when the TU114 peptide was used.

[0127] It could be therefore verified that when the peptide for promoting extracellular vesicle production of the present invention is used under a specific solution condition, extracellular vesicles with various sizes can be efficiently mass-produced.

[0128] Experimental Example 7: Production of extracellular vesicles loading recombinant protein

[0129] After the 293-HEK cell line was cultured to 90% confluency in a 6-well plate, the culture was removed, and the cell line was treated with the eMTDΔ4 peptide (final concentration: 25 μM) and the recombinant TRAIL, which have been dissolved to concentrations of 25 μM and 2 μg/ml in the solution of Example 1, respectively. The supernatant was collected, and PEG (final concentration:

[0130] 8%) was added, followed by centrifugation at an RCF of 12000 for 10 minutes, thereby settling extracellular vesicles, and the extracellular vesicles were loaded on the SDS-acrylamide gel to check the TRAIL protein. The results are shown in FIG. 9 and Table 6.

TABLE-US-00006 TABLE 6 Treated or not Cell − − − − + + + + Recombinant TRAIL − + − + − + − + Peptide − − + + − − + + Protein detected or not X X X X X X X ◯

[0131] As can be confirmed from FIG. 9 and Table 6, when the peptide for promoting extracellular vesicle production of the present invention was used under a specific solution condition, a recombinant protein externally added during the production of extracellular vesicles can be efficiently loaded in the extracellular vesicles.

[0132] Experimental Example 8: Production of extracellular vesicles loading plasmid DNA

[0133] After the 293-HEK cell line was cultured to 90% confluency in a 6-well plate, the culture was removed, and the cell line was treated with the eMTDΔ4 peptide (final concentration: 25 pM) and plasmid DNA (PUC19 3 μg/ml, EGFP-C1 6 μg/ml), which have been dissolved in the solution of Example 1.

[0134] 1.15 ml of the supernatant was collected and 0.35 ml of Qiagen buffer P3 was added, thereby making a total volume of 1.5 ml, of which 0.75 ml was then passed through the DNA miniprep column (Qiagen), and the passed solution was purified with phenol and precipitated with ethanol.

[0135] After 30 μl of DNA bound to the column and 30 μl of DNA precipitated with ethanol were dissolved in water, 10μl of DNA bound to the column and 10 μl of DNA precipitated with ethanol were subjected to agarose gel electrophoresis, and then stained with EtBr. The results are shown in FIG. 10.

[0136] As can be confirmed in FIG. 10, the plasmid DNA externally added during the production of extracellular vesicles can be loaded in the extracellular vesicles.

[0137] In addition, the plasmid DNA could be again isolated from the extracellular vesicles by the process shown in FIG. 11.

[0138] Therefore, when the peptide for promoting extracellular vesicle production of the present invention is used under a specific solution condition, plasmid DNA can be efficiently loaded in extracellular vesicles.

[0139] Experimental Example 9: Production of extracellular vesicles loading doxorubicin

[0140] Doxorubicin, which is a drug passing through cell membranes well, is used to treat cancers, such as breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, or acute lymphoblastic leukemia. First, the HeLa cell line was cultured in a 12-well plate overnight, then transfected with a plasmid expressing EGFP-HSP90 using effectene, and then the culture was replaced after 4 hours. The next day, the culture was removed from the HeLa cell line to prepare a cell line.

[0141] The control group was treated with only doxorubicin without any peptide, and the experiment group was treated with the eMTDΔ4 peptide (final concentration: 25 μM) and doxorubicin (final concentration: 100 μM), which have been dissolved in the solution of Example 1, and then after 10 minutes, the groups were observed by a confocal microscope. The results are shown in FIG. 12.

[0142] As can be confirmed in FIG. 12, the generated extracellular vesicles contained doxorubicin as well as the HSP90 protein, an exosome indicator.

[0143] Experimental Example 10: Production of extracellular vesicles loading propidium iodide (PI)

[0144] Propidium iodide (PI), which is a drug poorly passing through cell membranes, is a fluorescent substance capable of being used to stain cells. First, the HSP90 protein was transfected into the HeLa cell line to prepare a cell line.

[0145] The control group was treated with only PI without any peptide, and the experiment group was treated with the eMTDΔ4 peptide (final concentration: 25 μM) and 5 μg/ml PI, which have been in combination dissolved in the solution of Example 1, and then after 10 minutes, the groups were observed by a confocal microscope. The results are shown in FIG. 13.

[0146] As can be confirmed in FIG. 13, the generated extracellular vesicles contained PI as well as the HSP90 protein, an exosome indicator.

[0147] Therefore, proteins, DNA, various drugs that cannot pass through cell membranes, and the like can be loaded in the extracellular vesicles, which would be advantageously used as a new drug delivery system.