Composition for promoting extracellular vesicle production containing peptide derived from Noxa protein, and method for producing extracellular vesicles using the same

Abstract

The present invention relates to a method for producing large amounts of extracellular vesicles (EVs) using a peptide derived from the Noxa protein, which plays an important role in apoptosis, and its derivatives. The peptide for promoting extracellular vesicle production of the present invention and the method for producing extracellular vesicles using the peptide enable efficient, uniform, and mass production of extracellular vesicles, and can produce extracellular vesicles loaded with not only recombinant proteins or plasmid DNA but also drugs that do not easily pass through the cell membrane.

Description

[Technical field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2020-0115145, filed with the Korean Intellectual Property Office on September 9, 2020, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for promoting extracellular vesicle production containing a peptide derived from a Noxa protein, and a method for producing extracellular vesicles (EVs) using the composition, particularly to a method for efficiently mass-producing extracellular vesicles using a composition containing a peptide derived from a Noxa protein that plays an important role in apoptosis, and a solution containing sugar. [Background technology]

[0003] Extracellular vesicles (EVs) are sacs that are smaller than cells and are enclosed by a phospholipid bilayer. They exist in all kinds of body fluids. They play a role in exchanging contents or information between cells, and in recent years have attracted attention because they can be used as a new drug delivery system. Compared to other drug delivery systems, EVs have great advantages, such as less resistance from the individual immune system, being largely unaffected by natural barriers such as cell membranes, and being selective for specific cells.

[0004] Extracellular vesicles are classified into three types according to their size and formation process: exosomes, apoptotic bodies, and microvesicles (ectosomes). The size of exosomes is 30-150 nm, and they are derived from intracellular endosomes. The size of apoptotic bodies is 100-5000 nm, and they are derived from the cytoskeleton that is destroyed in the early stage of apoptosis. The size of microvesicles is 50-1000 nm, and they are formed by the protrusion of the cell membrane due to certain drugs or physical stress. Among extracellular vesicles, exosomes and microvesicles have attracted attention as new drug delivery systems, and many studies using exosomes in particular have been conducted.

[0005] Exosomes are formed from the cell membrane that has entered the cell. When the cell membrane enters the inside of the cell by endocytosis, an early sorting endosome (ESE) is formed. The early endosome can bind to the endoplasmic reticulum (ER) or the trans-golgi network (TGN). After forming a late sorting endosome (LSE), the early endosome either incorporates other cytoplasmic substances into the endosome or releases endosomal substances into the cytoplasm to form multivesicular bodies (MVBs) containing intraluminal vesicles. The multivesicular bodies bind to autophagosomes and then either bind to lysosomes and are decomposed, or bind to the inside of the cell membrane and release the intraluminal vesicles outside the cell. The intraluminal vesicles released in this way are called exosomes. Indicators for isolating and purifying the exosomes include CD9, CD63, CD81, flotillin, HSP70, and HSP90.

[0006] Exosomes perform various functions by moving between cells. The exosomes interact with various organelles within the cells, and as a result, they contain cellular components such as proteins, DNA, or RNA, as well as metabolic products such as amino acids. Therefore, the exosomes play an important role in reproduction, development, immune response, and the occurrence of various diseases including cancer through cell-cell interactions.

[0007] The use of exosomes in diagnosis and treatment has been actively studied. Exosomes can reflect any substance present inside or outside a cell, and attempts have been made to diagnose cerebrovascular disease, central nervous system disease, etc. using exosomes, and in particular, much research has been conducted on cancer diagnosis. The possibility of cancer diagnosis by analyzing DNA, RNA, or proteins contained in exosomes has been presented very actively in recent years.

[0008] Exosomes are also attracting attention as a new drug delivery system. Not only do exosomes enter cells more easily than liposomes, but they also encounter little resistance from the immune system. Furthermore, because there are abundant ligands on the membrane surface of exosomes, they show the possibility of cell-specific signal transduction via receptors.

[0009] Microvesicles are formed when the cell membrane protrudes to the outside of the cell. Representative examples include microvesicles formed from platelets and red blood cells, which are formed when cells are activated, when shear stress is applied, or when apoptosis occurs. Since the microvesicles have a structure similar to that of the cell membrane of the cell from which they originate, they can easily enter the cell with little influence from the immune system, indicating the possibility of cell-specific signal transduction.

[0010] However, despite these advantages, it remains difficult to mass-produce extracellular vesicles efficiently and uniformly. Summary of the Invention [Problem to be solved by the invention]

[0011] In order to solve the above-mentioned problems, the inventors have developed a method for efficiently and uniformly mass-producing large amounts of extracellular vesicles (EVs) by treating cell lines with a peptide derived from the Noxa protein, which plays an important role in apoptosis, and its derivatives under specific conditions.

[0012] Furthermore, the present inventors have confirmed that substances that do not easily pass through cell membranes can be carried in extracellular vesicles.

[0013] Therefore, an object of the present invention is to provide a composition for promoting extracellular vesicle production, which contains a peptide derived from a Noxa protein.

[0014] Another object of the present invention is to provide a medium for producing extracellular vesicles, comprising: a peptide derived from a Noxa protein; and a solution containing sugar.

[0015] Yet another object of the present invention is to provide a method for producing extracellular vesicles using a medium for producing extracellular vesicles.

[0016] Yet another object of the present invention is to provide a method for loading an extracellular vesicle payload using a medium for producing extracellular vesicles. [Means for solving the problem]

[0017] The present invention relates to a composition for promoting extracellular vesicle production containing a peptide derived from a Noxa protein, and a method for producing extracellular vesicles (EVs) using the composition, particularly to a method for efficiently mass-producing extracellular vesicles using a composition containing a peptide derived from a Noxa protein that plays an important role in apoptosis, and a solution containing sugar.

[0018] The present invention will now be described in more detail.

[0019] One example of the present invention relates to a composition for promoting extracellular vesicle production, comprising a peptide derived from a Noxa protein, a derivative thereof, or a combination thereof.

[0020] In the present invention, it is believed that Noxa protein activates BAX and BAK proteins by binding to and inhibiting Mcl1 and Bcl2A1 via the Bcl-2 homology 3 (BH3) domain, and that the release of cytochrome C into the cytoplasm activates the caspase system, thereby inducing apoptosis.

[0021] As used herein, the term "peptide" refers to a linear molecule formed by the joining of amino acid residues together through peptide bonds.

[0022] The term "derivative" as used herein means, but is not limited to, a peptide in which the N-terminus, C-terminus, etc. of the peptide of the present invention has been chemically modified or altered by the addition of amino acids, and means a peptide that performs the same or similar functions as the peptide of the present invention.

[0023] In one embodiment of the present invention, the Noxa protein-derived peptide may be a peptide having a homology of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more to a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0024] In one specific example according to the present invention, a derivative of a peptide derived from a Noxa protein may be a peptide having a homology of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more to a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 2 to 21.

[0025] In the present invention, the peptide may be produced by a method such as direct chemical synthesis using solid phase peptide synthesis, synthesis using an automatic synthesizer, or expression by inserting a base sequence encoding the peptide into a vector, but is not limited thereto.

[0026] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell.

[0027] The vectors of the present invention may include, but are not limited to, for example, plasmid vectors, cosmid vectors, and bacteriophage vectors, as well as viral vectors such as adenovirus vectors, retrovirus vectors, and adeno-associated virus (AAV) vectors.

[0028] Another example of the present invention relates to a medium for producing extracellular vesicles, comprising: at least one peptide selected from the group of peptides comprising the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21; and a solution containing a sugar.

[0029] The concentration of the peptide contained in the solution of the present invention may be 10 to 80 μM, 10 to 70 μM, 10 to 60 μM, 10 to 50 μM, 10 to 40 μM, 15 to 80 μM, 15 to 70 μM, 15 to 60 μM, 15 to 50 μM, 15 to 40 μM, 20 to 80 μM, 20 to 70 μM, 20 to 60 μM, 20 to 50 μM, or 20 to 40 μM, for example, 20 to 40 μM, but is not limited to this.

[0030] The sugar in the present invention may be one or more selected from the group consisting of glucose, sorbitol, and sucrose, and may be, for example, sucrose, but is not limited thereto.

[0031] When the solution of the present invention contains a sugar other than the glucose of the present invention, the concentration of glucose contained in the solution may be 3 to 70 mM, 3 to 60 mM, 3 to 50 mM, 4 to 70 mM, 4 to 60 mM, 4 to 50 mM, 5 to 70 mM, 5 to 60 mM, or 5 to 50 mM, for example, 5 to 50 mM, but is not limited thereto.

[0032] In the present invention, when the solution does not contain any sugar other than the glucose of the present invention, the concentration of glucose contained in the solution may be 80 to 300 mM, 80 to 290 mM, 80 to 280 mM, 80 to 270 mM, 80 to 260 mM, 80 to 250 mM, 90 to 300 mM, 90 to 290 mM, 90 to 280 mM, 90 to 270 mM, 90 to 260 mM, 90 to 250 mM, 100 to 300 mM, 100 to 290 mM, 100 to 280 mM, 100 to 270 mM, 100 to 260 mM, or 100 to 250 mM, for example, but is not limited to this.

[0033] The concentration of sucrose contained in the solution of the present invention may be 80 to 300 mM, 80 to 290 mM, 80 to 280 mM, 80 to 270 mM, 80 to 260 mM, 80 to 250 mM, 90 to 300 mM, 90 to 290 mM, 90 to 280 mM, 90 to 270 mM, 90 to 260 mM, 90 to 250 mM, 100 to 300 mM, 100 to 290 mM, 100 to 280 mM, 100 to 270 mM, 100 to 260 mM, or 100 to 250 mM, for example, but is not limited to 100 mM to 250 mM.

[0034] The concentration of sorbitol contained in the solution of the present invention may be 100 to 350 mM, 100 to 340 mM, 100 to 330 mM, 100 to 320 mM, 100 to 310 mM, 100 to 300 mM, 125 to 350 mM, 125 to 340 mM, 125 to 330 mM, 125 to 320 mM, 125 to 310 mM, 125 to 300 mM, 150 to 350 mM, 150 to 340 mM, 150 to 330 mM, 150 to 320 mM, 150 to 310 mM, or 150 to 300 mM, for example, but is not limited to this.

[0035] The medium in the present invention may be one or more types selected from the group consisting of solid media and liquid media, and may be, for example, a liquid medium, but is not limited thereto.

[0036] The concentration of MOPS [3-(N-morpholino)propanesulfonic acid] contained in the solution of the present invention may be 7 to 13 mM, 7 to 12 mM, 7 to 11 mM, 8 to 13 mM, 8 to 12 mM, 8 to 11 mM, 9 to 13 mM, 9 to 12 mM, 9 to 11 mM, 10 to 13 mM, 10 to 12 mM, or 10 to 11 mM, for example, 10 mM, but is not limited to this.

[0037] The pH of the solution in the present invention may be 5-10, 5-9, 5-8, 6-10, 6-9, 6-8, 7-10, 7-9, or 7-8, for example, 7-8.

[0038] The pH of the solution in the present invention is important for producing extracellular vesicles, and if the pH is greater than 10 or less than 5, extracellular vesicles may not be produced.

[0039] The solution of the present invention may further contain one or more selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), sodium gluconate (Na-gluconate), potassium gluconate (K-gluconate), sodium phosphate (NaPO4), and potassium phosphate (KPO4).

[0040] The concentration of sodium chloride (NaCl) contained in the solution of the present invention may be 1 mM or less, or may be 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0041] The concentration of potassium chloride (KCl) contained in the solution of the present invention may be 1 mM or less, 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0042] The concentration of sodium gluconate (Na-gluconate) contained in the solution of the present invention may be 1 mM or less, or may be 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0043] The concentration of potassium gluconate (K-gluconate) contained in the solution of the present invention may be 1 mM or less, or may be 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0044] The concentration of sodium phosphate (NaPO4) contained in the solution of the present invention may be 1 mM or less, or may be 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0045] The concentration of potassium phosphate (KPO4) contained in the solution of the present invention may be 1 mM or less, or may be 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.4 mM, 0.1 to 1 mM, 0.1 to 0.8 mM, 0.1 to 0.6 mM, or 0.1 to 0.4 mM, for example, but is not limited to, 0.1 mM.

[0046] In one embodiment of the present invention, the solution may contain 5 mM glucose, 10 mM MOPS, and 250 mM sucrose.

[0047] In one embodiment of the present invention, the solution may contain 255 mM glucose and 10 mM MOPS.

[0048] In one embodiment of the present invention, the solution may contain 5 mM glucose, 10 mM MOPS, and 250 mM sorbitol.

[0049] Yet another example of the present invention relates to a method for producing extracellular vesicles, comprising the steps of: a medium preparation step of preparing a medium containing a solution containing at least one peptide selected from the group of peptides containing the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21 and sugar; and A mixing step in which the medium and the cell line are mixed.

[0050] In the present invention, the cell line may be one selected from the group consisting of CT26 (mouse colon carcinoma), HeLa (human cervical carcinoma), HEK293 (human kidney epithelium), 3T3-L1 (mouse adipocytes), and HMSC (human mesenchymal stem cell), for example, HeLa or HEK293, but is not limited thereto.

[0051] Yet another example of the present invention relates to a method for loading an extracellular vesicle payload, comprising the steps of: a medium preparation step of preparing a medium containing a solution containing at least one peptide selected from the group of peptides containing the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21 and sugar; and A mixing step in which media, payload, and cell line are mixed.

[0052] In the present invention, the payload may be one or more selected from the group consisting of protein, DNA, RNA, plasmid DNA, and anticancer substances, for example, plasmid DNA, but is not limited thereto.

[0053] In the present invention, loading refers to, but is not limited to, making extracellular vesicles carry a payload.

[0054] In the present invention, the anticancer substance may be one or more selected from the group consisting of anticancer proteins, tumor suppressor genes, and anticancer drugs, but is not limited thereto.

[0055] In the present invention, the anti-cancer protein is selected from the group consisting of asparaginase, botulinum toxin, tetanus toxin, Shiga toxin, diphtheria toxin (DT), ricin, Pseudomonas exotoxin (PE), cytolysin A (ClyA), γ-gelonin, vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), transforming growth factor-β, TGF-β integrin, vascular endothelial cadherin (VE-cadherin), plasminogen activator, and the like. The target protein may be one or more selected from the group consisting of, but is not limited to, phosphoinositide activator (PA), ephrin, platelet-derived growth factor (PDGF), monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor (FGF), placenta growth factor (PIGF), von Hippel Lindau (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).

[0056] In the present invention, the tumor suppressor gene may be one or more selected from the group consisting of VHL (von HippelLindau), APC (Adenomatous polyposis coli), CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 (Yippee like 3), ST7 (Suppression of tumorigenicity 7), and ST14 (Suppression of tumorigenicity 14), but is not limited thereto.

[0057] In the present invention, the anticancer drug is methotrexate, 5-fluorouracil, gemcitabine, arabinosylcytosine, hydroxyurea, mercaptopurine, thioguanine, nitrogen mustard, cyclosporamide, anthracycline, daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, actinomycin D, D), vincristine, taxol, combretastatin A4, fumagillin, herbimycin A, 2-methoxyestradiol, OGT 2115, TNP 470, tranilast, XRP44X, thalidomide, endostatin, salmosin, angiostatin, plasminogen, kringle domain of apolipoprotein The therapeutic agent may be one or more selected from the group consisting of, but is not limited to, oxaloplatin, carboplatin, cisplatin, bortezomib, and radionuclides. Effect of the Invention

[0058] The peptide for promoting extracellular vesicle production of the present invention and the method for producing extracellular vesicles using the same enable efficient and uniform mass production of extracellular vesicles, and also enable the efficient production of extracellular vesicles carrying not only recombinant proteins or plasmid DNA, but also drugs that do not easily pass through cell membranes. [Brief description of the drawings]

[0059] [Figure 1] Figure 1 is a microscopic photograph showing extracellular vesicles being produced and sinking to the bottom of a plate when a HeLa cell line was treated with eMTDΔ4 peptide in combination with a solution according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a microscopic photograph showing the production of extracellular vesicles when a HeLa cell line was treated with eMTDΔ4 peptide in combination with a solution according to one embodiment of the present invention. [Diagram 3] FIG. 3 shows microscopic photographs showing the production of extracellular vesicles when a HeLa cell line was treated with the eMTDΔ4 peptide in combination with a solution containing glucose, sorbitol, or sucrose. [Figure 4] Figure 4 shows a confocal micrograph of extracellular vesicles produced after transfection of a HeLa cell line with the fluorescent protein EGFP bound to HSP90, an indicator of exosomes. [Diagram 5] Figure 5 shows photographs taken at 2-second intervals using a confocal microscope showing the process of extracellular vesicle production after transfection into a HeLa cell line in which the membrane proteins CD9 and CD81 were linked with mEmerald and mCherry, respectively. [Figure 6] FIG. 6 shows photographs of the size of extracellular vesicles produced in a HeLa cell line, measured using an atomic force microscope. [Figure 7] FIG. 7 is a heat map showing the amount of extracellular vesicles produced by MTD, eMTDΔ4, and their derivatives in the HEK-293 cell line quantified using Bradford solution. [Figure 8]FIG. 8 is a graph showing the size and number of extracellular vesicles produced by MTD, eMTDΔ4, and their derivatives in a HEK-293 cell line, analyzed using an NTA (Nanoparticle Tracking Analysis) system. [Figure 9] FIG. 9 shows the results of acrylamide gel electrophoresis performed after the recombinant protein TRAIL was loaded onto extracellular vesicles. [Figure 10] FIG. 10 shows the results of agarose gel electrophoresis performed after plasmid DNA pUC19 and pEGFP-C1 were loaded onto extracellular vesicles. [Figure 11] FIG. 11 is a schematic diagram illustrating the process of loading plasmid DNA into extracellular vesicles and then isolating them. [Figure 12] Figure 12 shows a photograph of the results obtained by treating a HeLa cell line with eMTDΔ4 to produce extracellular vesicles, which were loaded with doxorubicin, a drug that easily passes through cell membranes, and then examined with a confocal microscope. [Figure 13] Figure 13 shows photographs of the results observed with a confocal microscope after extracellular vesicles produced by treating a HeLa cell line with eMTDΔ4 were loaded with PI (propidium iodide), a drug that does not easily pass through cell membranes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] 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 describing the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it will be apparent to those skilled in the art that these examples are not to be construed as limiting the scope of the present disclosure. EXAMPLES

[0061] Production Example 1. Production of Noxa protein-derived peptides and their derivatives The mitochondrial targeting domain (MTD) of the Noxa protein is shown in SEQ ID NO: 1 in Table 1. The synthesis of the peptide derived from the MTD was basically performed using the manual Fmoc synthesis method in 0.25 mmol units. Specifically, after washing the resin with dimethylformamide (DMF), 10 mL of 20% piperidine / dimethylformamide (piperidine / DMF) was added. After stirring for 1 minute, the resin was washed again with dimethylformamide, and the absence of remaining piperidine was confirmed by the ninhydrin test (the resin turned blue).

[0062] To carry out the coupling, the following solution was prepared: 1 mmol of Fmoc-amino acid, 2.1 ml of 0.45 M HBTU / HOBT [(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium / Hydroxybenzotriazole] (1 mmol), and 348 μL of DIEA (N,N-Diisopropylethylamine) (2 mmol).

[0063] The prepared solution was added to the resin and shaken for 30 minutes. After removing the solution from the resin, the resin was washed with dimethylformamide. Then, the solution was added again for coupling of amino acids, and the coupling process was repeated to synthesize eMTDΔ4 derived from Noxa protein and its derivative peptides.

[0064] The peptides thus synthesized were analyzed by high performance liquid chromatography (instrument: Waters 2690 separation module, flow rate: 1.0 ml / min, gradient: 0-20% B, 5 min; 20-50% B, 20 min; 50-80% B, 5 min, A; 0.1% TFA aqueous solution, B; 0.1% TFA acetonitrile, column: waters C18, 5 microns, detection: 220 nm, purity: 95%) and mass spectrometry (HP 1100 series LC / MSD). The synthesized peptides are shown as SEQ ID NOs: 2 to 21 in Table 1.

[0065] [Table 1]

[0066] Example 2. Preparation of solution for producing extracellular vesicles In order to produce extracellular vesicles using eMTDΔ4 derived from the mitochondrial targeting domain of Noxa protein and its derivative peptides, solutions of Example 1 and Comparative Examples 1 to 18 having the compositions and contents shown in Table 2 below were prepared.

[0067] [Table 2]

[0068] Experimental example 1. Cultivation of cancer cell lines 1-1. Preparation of cell lines and reagents Prior to testing the extracellular vesicle production-inducing activity of the Noxa protein-derived peptide and its derivatives of Preparation Example 1, a cancer cell line was cultured.

[0069] The culture media required for DMEM (Dulbecco's modified eagle medium), RPMI 1640, FBS (fatal bovine serum), trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid), and HBSS (Hank's balanced salt solution) were purchased from Gibco, and the transfection reagent Effectene was purchased from Qiagen. HeLa and HEK293 cell lines were purchased from the Korea Cell Line Bank (KCLB).

[0070] 1-2.Culture HeLa and HEK293 cell lines were subcultured three or more times for use in the experiment. HeLa cell line was cultured in DMEM containing 10% fetal bovine serum (FBS), and HEK-293 cell line was cultured in RPMI 1640 containing 10% fetal bovine serum, under conditions of 5% (v / v) CO2 and 37℃.

[0071] Experimental Example 2. Production of extracellular vesicles 2-1. Production of extracellular vesicles The HeLa cell line was cultured in a 6-well plate, and after removing the culture medium, the solution of Example 1 was applied to the HeLa cell line.

[0072] The control group was not treated with peptide, and the experimental group was treated with eMTDΔ4 peptide (final concentration: 20 μM) of SEQ ID NO: 2. After 10 minutes, the production of extracellular vesicles was observed using an optical microscope (bright field), and the results are shown in FIG.

[0073] As can be seen from FIG. 1, the bottom of the control plate was clean, whereas in the experimental group, small extracellular vesicle particles were formed and sunk to the bottom of the plate.

[0074] 2-2. Production of extracellular vesicles using various solutions The results of producing extracellular vesicles using the R(8):MTD peptide of SEQ ID NO:4 and the solutions of Example 1 and Comparative Examples 1 to 18 according to the experimental method of Experimental Example 2-1 are shown in Figures 2A to 2S.

[0075] As can be seen in Figure 2A, a large number of extracellular vesicles were produced, and most of the particles sank to the bottom of the plate. However, as can be seen from Figures 2B to 2S, no extracellular vesicles were produced, so no extracellular vesicles precipitated at the bottom of the plate could be observed, and only the formation of particles that occur during apoptosis within the cells was observed.

[0076] 2-3. Production of extracellular vesicles using various sugars The results of producing extracellular vesicles using the R(8):MTD peptide of SEQ ID NO:4 and the solutions of Examples 1 to 3 shown in Table 3 according to the experimental method of Experimental Example 2-1 are shown in Figures 3A to 3C.

[0077] [Table 3]

[0078] As can be seen from Figures 3A to 3C, extracellular vesicles were successfully formed even when glucose or sorbitol was added in excess instead of sucrose.

[0079] Experimental Example 3. Confirmation of the shape of extracellular vesicles To transfect the HSP90 protein, an indicator of exosomes, into HeLa cell lines, the HeLa cell lines were cultured overnight in 6-well plates, and then the cell lines were treated with a plasmid containing Effectene and pEGFP-HSP90 cloned therein, and the culture medium was replaced after 4 hours.

[0080] The next day, the culture medium was removed from the HeLa cell line, and the solution of Example 1 was added. The control group was not treated with peptide, and the experimental group was treated with eMTDΔ4 peptide of SEQ ID NO: 2 (final concentration: 20 μM). HSP90 protein was excited using a 488 nm argon laser. After 10 minutes, the control group and the experimental group were observed using a confocal microscope (Leica TCS SP5 Microsystems), and the results are shown in FIG. 4.

[0081] As can be seen from Figure 4, in the control group, the cell morphology was not disrupted and the HSP90 protein was located in the cytoplasm. However, in the experimental group (eMTDΔ4), the cell morphology was impaired and extracellular vesicles containing the HSP90 protein were confirmed to have precipitated at the bottom of the plate. The reason for this was believed to be that the cytoplasmic HSP90 protein was released outside the cell via defects in the cell membrane that occurred during the formation of extracellular vesicles.

[0082] Experimental Example 4. Confirmation of the extracellular vesicle production process HeLa cells were cultured overnight in a 12-well plate, and to confirm the CD9 protein, an indicator of extracellular vesicles, they were treated with Effectene and plasmids containing mEmerald-CD9. The culture medium was replaced after 4 hours.

[0083] The next day, the culture medium was removed from the HeLa cell line, and the solution of Example 1 was treated. After treating each HeLa cell line with eMTDΔ4 peptide (final concentration: 20 μM), a 488 nm wavelength laser was used to excite mEmerald-CD9. After that, the cells were observed for 10 minutes using a confocal microscope, taking pictures at intervals of about 2 seconds, and the results are shown in FIG. 5A.

[0084] In addition, to confirm CD81 protein, an indicator of extracellular vesicles, the same process as for observing CD9 protein was carried out, except that a plasmid into which mCherry-CD81 was cloned instead of mEmerald-CD9 was treated in a HeLa cell line, and mCherry-CD81 was excited using a 561 nm wavelength laser. The results are shown in Figure 5B.

[0085] As can be seen in Figures 5A and 5B, both CD9 protein and CD81 protein were sufficient to observe the formation of extracellular vesicles, and CD9 protein and CD81 protein were released into the extracellular matrix. Although the release procedure could not be specifically identified, it was confirmed that the extracellular vesicles were released into the extracellular matrix within a few seconds after production.

[0086] Experimental Example 5. Confirmation of the size of extracellular vesicles The HeLa cell line was cultured on a culture plate, and the cell line was treated with the eMTDΔ4 peptide (final concentration: 20 μM) together with the solution of Example 1, and after 10 minutes, the cells were fixed with 4% (v / v) paraformaldehyde.

[0087] The cantilevers were then observed using an atomic microscope (Surface Imaging Systems, NANO Station II) (cantilevers with pyramidal tips, frequencies of 146 to 236 kHz, spring constants of 21 to 98 N / m, lengths of 225 nm, and resistances of 0.01 to 0.02 Ω cm), and the results are shown in Figures 6A to 6C.

[0088] As can be seen from Figure 6A, extracellular vesicles of various sizes were generated and their sizes could be measured. The results of measuring the size of extracellular vesicles are shown in the graphs of Figures 6B and 6C. As can be seen from Figures 6B and 6C, the size of the extracellular vesicles was measured to be about 200 nm. Such results prove that the extracellular vesicles of the present invention have a size equivalent to that of conventionally known extracellular vesicles.

[0089] Experimental Example 6. Confirmation of the amount of extracellular vesicles produced 293-HEK cells were cultured in a 6-well plate at 2 × 10 5 The next day, the cell line was treated with the solution of Example 1 and any one of the peptides of SEQ ID NOs: 1 to 21. After 20 minutes, the solution was removed, and the mixture was centrifuged at 12,000 rcf. The supernatant was then collected and the extracellular vesicles were quantified using Bradford solution. The results are shown in FIG. 7 and Table 4.

[0090] [Table 4]

[0091] As can be seen from Figure 7 and Table 4, although there were differences in the production amounts, most of the peptides shown in Table 4 produced extracellular vesicles well. It was also confirmed that the amount of protein contained in extracellular vesicles tended to increase as the final concentration of peptide treatment increased from 10 μM to 20 μM and then to 40 μM.

[0092] Experimental Example 6. Confirmation of size and quantity of extracellular vesicles 293-HEK cells were cultured in a 10 cm culture dish at 90% confluency (approximately 2 × 10 7 After culturing in 1000 μg / ml of ...

[0093] [Table 5]

[0094] As can be seen from Figures 8A to 8D and Table 5, the size of extracellular vesicles using the eMTDΔ4 peptide showed a mean of 184.5 nm, a mode of 126.3 nm, and a standard deviation (SD) of 87.8 nm. Using the MTD peptide, a mean of 181.1 nm, a mode of 115.6 nm, and a standard deviation of 75.2 nm were observed. Using the TU17 peptide, a mean of 229.6 nm, a mode of 150.9 nm, and a standard deviation of 104.3 nm were observed. Using the TU114 peptide, a mean of 231.1 nm, a mode of 182.3 nm, and a standard deviation of 110.2 nm were observed.

[0095] Therefore, it was confirmed that by using the peptide for promoting extracellular vesicle production of the present invention under specific solution conditions, it is possible to efficiently mass-produce extracellular vesicles of various sizes.

[0096] Experimental Example 7. Production of extracellular vesicles carrying recombinant proteins After culturing 293-HEK cell lines at 90% confluency in a 6-well plate, the culture medium was removed, and eMTDΔ4 peptide (final concentration: 25 μM) and recombinant TRAIL were dissolved in the solution of Example 1 at concentrations of 25 μM and 2 μg / ml, respectively, and treated with the cell lines. The supernatant was collected, PEG (final concentration: 8%) was added, and the mixture was centrifuged at 12000 rcf for 10 minutes to precipitate extracellular vesicles, which were then subjected to SDS-acrylamide gel to confirm the TRAIL protein, and the results are shown in FIG. 9 and Table 6.

[0097] [Table 6]

[0098] As can be seen from Figure 9 and Table 6, when the peptide for promoting extracellular vesicle production of the present invention was used under specific solution conditions, the recombinant protein added externally could be efficiently loaded onto the extracellular vesicles during the extracellular vesicle production process.

[0099] Experimental Example 8. Production of extracellular vesicles carrying plasmid DNA 293-HEK cell lines were cultured in 6-well plates at 90% confluency, and the culture medium was then removed. The eMTDΔ4 peptide (final concentration: 25 μM) and plasmid DNA (PUC19 3 μg / ml, EGFP-C1 6 μg / ml) were dissolved in the solution of Example 1 and used to treat the cell lines.

[0100] 1.15 ml of the supernatant was collected and 0.35 ml of Qiagen buffer P3 was added to bring the total volume to 1.5 ml, of which 0.75 ml was passed through a DNA miniprep column (Qiagen), and the flow-through solution was purified with phenol and precipitated with ethanol.

[0101] 30 μl of the column-bound DNA and 30 μl of the ethanol-precipitated DNA were dissolved in water, and then 10 μl of the column-bound DNA and 10 μl of the ethanol-precipitated DNA were subjected to agarose gel electrophoresis and stained with EtBr. The results are shown in Figure 10.

[0102] As can be seen from FIG. 10, during the extracellular vesicle production process, the plasmid DNA added externally could be loaded into the extracellular vesicles.

[0103] Plasmid DNA can also be separated again from extracellular vesicles by the process shown in Figure 11.

[0104] Therefore, when the peptide for promoting extracellular vesicle production of the present invention is used under specific solution conditions, plasmid DNA can be efficiently loaded into extracellular vesicles.

[0105] Experimental Example 9. Production of extracellular vesicles carrying doxorubicin Doxorubicin is a drug that easily passes through cell membranes and is used to treat cancers such as breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. First, HeLa cell lines were cultured overnight in 12-well plates, and then treated with Effectene and a plasmid containing pEGFP-HSP90 cloned therein. After 4 hours, the culture medium was replaced. The next day, the culture medium was removed from the HeLa cell lines, and the cell lines were prepared.

[0106] The control group was not treated with any peptide, but was treated with doxorubicin only, while the experimental group was treated with eMTDΔ4 peptide (final concentration: 25 μM) and doxorubicin (final concentration: 100 μM) dissolved in the solution of Example 1, and then observed using a confocal microscope after 10 minutes. The results are shown in Figure 12.

[0107] As can be seen from Figure 12, the produced extracellular vesicles contained doxorubicin in addition to HSP90 protein, which is an exosome indicator.

[0108] Experimental Example 10. Production of extracellular vesicles carrying PI (propidium iodide) PI (propidium iodide) is a drug that does not easily pass through cell membranes and is a fluorescent substance that can be used to stain cells. First, HSP90 protein was transfected into a HeLa cell line to prepare a cell line.

[0109] The control group was not treated with peptide, but was treated with PI only, and the experimental group was treated with eMTDΔ4 peptide (final concentration: 25 μM) dissolved in the solution of Example 1 together with 5 μg / ml PI, and then observed using a confocal microscope after 10 minutes. The results are shown in Figure 13.

[0110] As can be seen from Figure 13, the generated extracellular vesicles contained PI in addition to HSP90 protein, which is an exosome indicator.

[0111] Therefore, proteins, DNA, and various drugs that cannot pass through the cell membrane can be loaded into extracellular vesicles, which may be useful as a new drug delivery system. [Industrial Applicability]

[0112] The present invention relates to a composition for promoting extracellular vesicle production containing a peptide derived from a Noxa protein and a method for producing extracellular vesicles (EVs) using the same, more specifically, to a composition containing a peptide derived from a Noxa protein that plays an important role in apoptosis, and a method for efficiently mass-producing extracellular vesicles using a solution containing sugar.

Claims

1. one or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; Sugar and MOPS [3-(N-morpholino)propanesulfonic acid], A composition for promoting extracellular vesicle production, comprising:

2. one or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; A solution containing sugar; MOPS [3-(N-morpholino)propanesulfonic acid], A medium for producing extracellular vesicles, comprising:

3. The medium for producing extracellular vesicles according to claim 2 , wherein the sugar is one or more selected from the group consisting of glucose, sucrose, and sorbitol.

4. The solution contains sodium chloride (NaCl), potassium chloride (KCl), sodium gluconate (Na-gluconate), potassium gluconate (K-gluconate), sodium phosphate (NaPO ４ ), and potassium phosphate (KPO ４ The medium for producing extracellular vesicles described in claim 2, further comprising one or more selected from the group consisting of:

5. a medium preparation step of preparing a medium containing one or more peptides having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, a solution containing a sugar, and MOPS [3-(N-morpholino)propanesulfonic acid]; A mixing step of mixing the medium and the cell line; A method for producing extracellular vesicles, comprising:

6. The method for producing extracellular vesicles according to claim 5 , wherein the sugar is one or more selected from the group consisting of glucose, sucrose, and sorbitol.

7. The solution contains sodium chloride (NaCl), potassium chloride (KCl), sodium gluconate (Na-gluconate), potassium gluconate (K-gluconate), sucrose, sodium phosphate (NaPO ４ ), and potassium phosphate (KPO ４ The method for producing extracellular vesicles described in claim 5, further comprising one or more selected from the group consisting of:

8. The method for producing extracellular vesicles according to claim 5, wherein the cell line is any one selected from the group consisting of CT26 (mouse colon cancer), HeLa (human cervical cancer), HEK293 (human kidney epithelium), 3T3-L1 (mouse adipocyte), and HMSC (human mesenchymal stem cell).

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