Extracellular vesicle loaded with neuropeptide and use thereof

Abstract

The present invention relates to an extracellular vesicle loaded with a neuropeptide and, more specifically, to the use of an extracellular vesicle loaded with a neuropeptide for preventing, alleviating, or treating nervous system damage or nervous system diseases. The extracellular vesicle loaded with a neuropeptide according to the present invention was experimentally verified to have excellent effects in increasing brain-derived neurotrophic factor (BDNF) expression, promoting neuronal cell proliferation, and promoting neuronal cell differentiation. This means that the extracellular vesicle loaded with a neuropeptide of the present invention exhibits excellent neuroprotective and neuroregenerative effects and can thus be variously used in the field of treating nervous system damage or nervous system diseases.

Description

Extracellular vesicles loaded with neuropeptides and uses thereof

[0001] The present invention relates to extracellular vesicles loaded with neuropeptides, and more particularly, to the use of extracellular vesicles loaded with neuropeptides for preventing, improving or treating nervous system damage or nervous system diseases.

[0002] Extracellular vesicles (EVs) are tiny spheres, each smaller than a cell, enclosed by a phospholipid bilayer and originating from a cell. They are present in all body fluids. EVs are a key vehicle for intercellular communication, containing key cellular components like nucleic acids and proteins. Recently, they have attracted attention for their potential as a novel drug delivery method. Significant advantages over other drug delivery methods include their immunity to the individual immune system, their tolerance to natural barriers like the cell membrane, and their ability to target specific cells.

[0003] Extracellular vesicles have a wide variety of names, depending on their cellular origin and method of production. Depending on how they are produced within the cell, they are classified as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. Among these extracellular vesicles, exosomes and microvesicles are attracting attention as novel drug delivery vehicles.

[0004] Meanwhile, neurological diseases or nerve damage occur when nerve cells in the brain or peripheral nervous system degenerate, decrease, die, become injured, or are eliminated due to environmental, genetic, or other factors. Therefore, treating these neurological diseases requires replenishing neurotransmitters lost due to nerve cell damage or regenerating nerve cells. Methods for regenerating nerve cells include undifferentiated neural stem cells and embryonic stem cells, which can differentiate into various cell types.

[0005] Numerous studies have been conducted on nerve regeneration of damaged nerves. Clinical surgical treatment for traumatic peripheral nerve injuries typically involves a complex series of steps, starting with the removal of damaged tissue near the site of injury and providing an environment conducive to peripheral nerve regeneration. Surgical procedures typically involve directly connecting or fusing the proximal and distal portions of the injured area. Additional surgical techniques, such as peripheral nerve grafting, are also used, along with more cautious outpatient treatments, such as maintaining electrical stimulation to generate muscle contractions that help suppress degeneration of the muscle-nerve junction while awaiting spontaneous nerve regeneration. Furthermore, most of these procedures still involve extensive physical therapy, including long-term, controlled exercise therapy, to prevent muscle weakness and contractures and promote nerve sprouting.

[0006] Accordingly, the inventors of the present invention developed extracellular vesicles loaded with neuropeptides and confirmed their excellent neuroprotective and nerve regeneration effects, thereby completing the present invention.

[0007] Accordingly, the purpose of the present invention is to provide an extracellular vesicle loaded with a peptide; and a composition comprising the same.

[0008] Another object of the present invention is to provide a method for treating nervous system damage or nervous system disease, comprising the step of administering the extracellular vesicle to a subject in need thereof.

[0009] To achieve the above purpose, the present invention provides an extracellular vesicle loaded with a peptide comprising an amino acid sequence represented by the following structural formula 1:

[0010]

[0011] [Structural formula 1]

[0012] N'-(X1)α-(X2)-(X3)β-C'

[0013]

[0014] In the above structural formula 1,

[0015] N' is the N-terminus of the peptide and C' is the C-terminus of the peptide;

[0016] α is 0 or 1, and if α is 1, X1 is R or M;

[0017] X2 is D or V;

[0018] β is 0 or 1, and if β is 1, then X3 is G,

[0019] α and β do not become 0 at the same time.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating nervous system damage or nervous system disease, which comprises the extracellular vesicles.

[0021] Additionally, the present invention provides an in vitro composition for promoting nerve regeneration comprising the extracellular vesicles.

[0022] The present invention also provides a method for treating nervous system damage or nervous system disease, comprising the step of administering the extracellular vesicle to a subject in need thereof.

[0023] It was experimentally confirmed that the neuropeptide-loaded extracellular vesicles of the present invention exhibit excellent effects in increasing BDNF (Brain-derived neurotrophic factor) expression, promoting neuronal proliferation, and promoting neuronal differentiation. This indicates that the neuropeptide-loaded extracellular vesicles of the present invention exhibit excellent neuroprotective and neuroregenerative effects, and thus can be utilized in various fields of treatment for nervous system injuries or diseases.

[0024] Figure 1a is a diagram showing the results of evaluating the effect of neuropeptides on BDNF (Brain-derived neurotrophic factor) expression (p<0.0001; ****).

[0025] Figure 1b is a diagram showing the results of evaluating the effects of neuropeptides on neuroproliferation and differentiation (p<0.05; *, p<0.01; **, p<0.001; ***).

[0026] Figure 2 is a diagram showing the results of evaluating the loading efficiency of extracellular vesicles loaded with neuropeptides according to the present invention.

[0027] Figure 3a is a diagram showing the results of analyzing the size distribution of extracellular vesicles loaded with neuropeptides according to the origin of the extracellular vesicles.

[0028] Figure 3b is a diagram showing the results of observing extracellular vesicles loaded with neuropeptides according to the present invention using TEM (upper image) and cryo-TEM (lower image).

[0029] Figure 3c is a diagram showing the results of analyzing the peptide loading amount and loading efficiency of extracellular vesicles loaded with neuropeptides according to the origin of the extracellular vesicles.

[0030] Figure 3d shows the results of analyzing the topology of extracellular vesicles loaded with neuropeptides according to their origin using a 3D-STORM microscope (top) and measuring the size of the cell vesicles and loaded peptides using CellMask dye (middle and bottom).

[0031] Figure 3e is a diagram showing the results of analyzing the Zeta-Potential of peptide loading of extracellular vesicles loaded with neuropeptides according to the origin of the extracellular vesicles.

[0032] Figure 4a is a diagram showing the results of evaluating the effects of extracellular vesicles loaded with various neuropeptides and NP8 peptides of the present invention on the expression of BDNF, TrkB, and p-CREB in SY5Y cells (p<0.05; *, p<0.01; **, p<0.001; ***).

[0033] Figure 4b is a diagram showing the results comparing the effects of extracellular vesicles loaded with the NP8 neuropeptide of the present invention on p-CREB expression in SY5Y cells (p<0.05; *, p<0.01; **, p<0.01; ##).

[0034] Figure 5a is a diagram showing the results of evaluating the effects of extracellular vesicles loaded with a neuropeptide according to the present invention on neuronal cell proliferation and nerve regeneration in SH-SY5Y neurons using fluorescence and optical microscopy.

[0035] Figure 5b is a diagram showing the results of measuring neurite length per cell, neurite tip number per cell, and total cell number based on the microscopic observation results of Figure 5a (p<0.05; *, p<0.01; **, p<0.001; ***).

[0036] Figure 5c is a diagram showing the results of evaluating the effect of extracellular vesicles loaded with neuropeptides according to the present invention on the death of nerve cells (p<0.01; **, p<0.0001; ****).

[0037] Figure 6 is a diagram showing the results of analyzing the delivery and uptake of extracellular vesicles loaded with a neuropeptide according to the present invention into SH-SY5Y neurons.

[0038] Figure 7a is a diagram showing the process of constructing a model for passage of an extracellular vesicle loaded with a neuropeptide according to the present invention through the blood-brain barrier in an in vitro BBB model.

[0039] Figure 7b is a diagram showing the results of analyzing the transepithelial electrical resistance (TEER) and the permeability of the BBB according to the treatment of extracellular vesicles loaded with the neuropeptide of the present invention in an in vitro BBB model.

[0040] Figure 7c is a diagram showing the results of measuring fluorescence intensity according to treatment of extracellular vesicles loaded with the neuropeptide of the present invention in an in vitro BBB model (p<0.001; ***).

[0041] Figure 7d is a diagram showing the results of immunofluorescence staining according to treatment of extracellular vesicles loaded with the neuropeptide of the present invention in an in vitro BBB model.

[0042] Figure 8a is a diagram showing the results of fluorescence distribution analysis of extracellular vesicles loaded with neuropeptides according to the present invention in each mouse brain tissue (olfactory bulb, cortex, hippocampus).

[0043] Figure 8b is a diagram showing the quantitative results of the fluorescence distribution of the extracellular vesicles loaded with the neuropeptide according to the present invention in the whole brain tissue of a mouse (p<0.01; **, p<0.001; ***).

[0044] Figure 8c is a diagram showing the results of investigating the effect of extracellular vesicles loaded with neuropeptides according to the present invention on BDNF expression in each mouse brain tissue (olfactory bulb, cortex, hippocampus) (p<0.05; *, p<0.01; **, p<0.001; ***, p<0.0001;****).

[0045] Figure 8d is a diagram showing the results of measuring the distribution of extracellular vesicles loaded with neuropeptides according to the present invention using IVIS equipment.

[0046] Figure 9 is a diagram showing the results of analyzing the expression of CD63 in extracellular vesicles loaded with a neuropeptide according to the present invention (p<0.001; ***).

[0047] Figure 10 is a diagram showing the results of analyzing the expression of five major miRNAs in extracellular vesicles loaded with neuropeptides according to the present invention (p<0.01; **).

[0048] Figure 11a is a diagram showing an experimental design for evaluating the efficacy of extracellular vesicles loaded with neuropeptides according to the present invention in a stroke mouse model.

[0049] Figure 11b is a diagram showing the results of quantitative analysis of mRNA expression of CREB transcription factor in RNA extracted from brain tissue of stroke mice administered with extracellular vesicles loaded with a neuropeptide according to the present invention (p<0.001; ***).

[0050] Figure 11c is a diagram showing the results of an MRI examination of brain damage in a stroke mouse administered with an extracellular vesicle loaded with a neuropeptide according to the present invention.

[0051] Figure 11d is a diagram showing the results of quantifying the volume of brain damage lesion areas in stroke mice following treatment with extracellular vesicles loaded with a neuropeptide according to the present invention (p<0.01; **, p<0.0001; ****).

[0052] Figure 11e is a diagram showing the results of Western blotting analysis of the expression of BDNF in the brain tissue of stroke mice administered with extracellular vesicles loaded with a neuropeptide according to the present invention.

[0053] Figure 11f is a diagram showing the results of quantifying the expression level of BDNF in proteins extracted from brain tissue of stroke mice administered with extracellular vesicles loaded with neuropeptides according to the present invention (p<0.05; *, p<0.01; **, p<0.05; #).

[0054] Hereinafter, the present invention will be described in detail.

[0055] According to an aspect of the present invention, the present invention provides an extracellular vesicle loaded with a peptide. The peptide may comprise an amino acid sequence represented by the following structural formula 1:

[0056]

[0057] [Structural formula 1]

[0058] N'-(X1)α-(X2)-(X3)β-C'

[0059] In the above structural formula 1,

[0060] N' is the N-terminus of the peptide and C' is the C-terminus of the peptide;

[0061] α is 0 or 1, and if α is 1, X1 is R or M;

[0062] X2 is D or V;

[0063] β is 0 or 1, and if β is 1, then X3 is G,

[0064] α and β do not become 0 at the same time.

[0065]

[0066] In a specific embodiment of the present invention, the peptide may be loaded with a peptide comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4, and may include a peptide having 1 to 5 amino acids added to the N- or C-terminus of the peptide.

[0067] The N- or C-terminus of the peptide of the present invention may be linked to additional components, such as an active ingredient or a labeling substance, to achieve the purpose of drug delivery. In addition, the active ingredient or labeling substance may be included within the extracellular vesicle loaded with the peptide of the present invention.

[0068] The above-mentioned active ingredient may be a drug for the prevention, improvement, or treatment of a specific disease, and its type is not limited. For example, the drug may be a substance for the treatment of a neurological disease.

[0069] The above-mentioned labeling material may be a fluorescent material, and the fluorescent material may be a fluorescent protein, a molecule that emits light in an excited state, a metal ion, a complex compound, an organic dye, a conductor, a semiconductor, an insulator, a quantum dot, a quantum wire, etc., but the type thereof is not limited.

[0070] Examples of the fluorescent proteins include enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP), and red fluorescent protein (RFP).

[0071] In addition, examples of the fluorescent material include pyrene or derivatives thereof, cyanine (Cy) series, Alexa Fluor series, BODIPY series, DY series, rhodamine or derivatives thereof, fluorescein or derivatives thereof, coumarin or derivatives thereof, acridine homodimer or derivatives thereof, acridine orange or derivatives thereof, 7-aminoactinomycin D (7-AAD) or derivatives thereof, actinomycin D or derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) or derivatives thereof, DAPI or derivatives thereof, Dihydroethidium or a derivative thereof, Ethidium bromide or a derivative thereof, Ethidium homodimer-1 (EthD-1) or a derivative thereof, Ethidium homodimer-2 (EthD-2) or a derivative thereof, Ethidium monoazide or a derivative thereof, Hexidium iodide or a derivative thereof, Bisbenzimide (Hoechst 33258) or a derivative thereof, Hoechst 33342 or a derivative thereof, Hoechst 34580 or a derivative thereof, Hydroxystilbamidine or a derivative thereof, LDS 751 751) or a derivative thereof, Propidium Iodide (PI) or a derivative thereof, Calcein or a derivative thereof, Oregon Green or a derivative thereof,Magnesium Green or a derivative thereof, Calcium Green or a derivative thereof, JOE or a derivative thereof, Tetramethylrhodamine or a derivative thereof, TRITC or a derivative thereof, N,N,N',N'-tetrametyl-6-carboxyrhodamine (TAMRA) or a derivative thereof, Pyronin Y or a derivative thereof, Lissamine or a derivative thereof, ROX or a derivative thereof, Calcium Crimson or a derivative thereof, Texas Red or a derivative thereof, Nile Red or a derivative thereof, Thiadicarbocyanine or a derivative thereof, dansylamide or a derivative thereof, cascade blue, DAPI (4',6-diamidino-2-phenylindole), etc. there is.,

[0072] The extracellular vesicle of the present invention may be incorporated into a cell and internalized when treated with a cell, and when incorporated into a cell and internalized, it may effectively deliver a clinically significant substance highly expressed in the extracellular vesicle to the cell, so that it is highly expressed in the cell, and it may effectively deliver the peptide of the present invention to promote nerve regeneration.

[0073] In a specific embodiment of the present invention, the extracellular vesicles contain peptides in an amount of 1 to 50 nmol / 5x10 9 It is preferable to load particles at a concentration of 10 to 25 nmol / 5x10 9 particles concentration, more preferably 15 to 20 nmol / 5x10 9 It can be loaded with particles concentration.

[0074] In a specific example of the present invention, the extracellular vesicle has at least one effect selected from the group consisting of increasing expression of BDNF (Brain-derived neurotrophic factor), promoting neuronal proliferation, and promoting neuronal differentiation.

[0075] In a specific example of the present invention, the extracellular vesicle may be an extracellular vesicle derived from at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells, preferably stem cells, more preferably mesenchymal stem cells, but the present invention is not limited thereto.

[0076] In a specific embodiment of the present invention, the mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, Wharton's jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, teeth, hair root cells, or placenta, and preferably derived from Wharton's jelly, but the present invention is not limited thereto.

[0077] The present inventors have confirmed the excellent neuroprotective and neuroregenerative effects of extracellular vesicles loaded with the peptide of the present invention. In particular, we experimentally confirmed that mesenchymal stem cell-derived extracellular vesicles loaded with the neuropeptide NP-8 (SEQ ID NO: 3) exhibited more pronounced neuroprotective and neuroregenerative effects than other experimental and control groups. Therefore, the extracellular vesicles loaded with the neuropeptide of the present invention can be utilized in various fields of treatment for nervous system diseases or injuries.

[0078]

[0079] According to another aspect of the present invention, a composition comprising an extracellular vesicle loaded with the peptide is provided. The composition may be a pharmaceutical composition for preventing or treating nervous system damage or nervous system disease; or an in vitro composition for promoting nerve regeneration.

[0080] In a specific embodiment of the present invention, the nervous system damage or nervous system disease may be at least one selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic spinal lateral sclerosis, motor nerve damage, peripheral nerve damage due to trauma, nerve damage due to ischemic brain damage, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury.

[0081] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0082] The composition of the present invention may contain at least one known effective ingredient having a preventive or therapeutic effect on nervous system damage or nervous system disease together with extracellular vesicles loaded with peptides.

[0083] The composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0084] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. It is preferable to use suitable formulations known in the art as disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

[0085] The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0086] The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0087] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the desired treatment.

[0088] The dosage of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 0.05 to 5 mg / kg, more preferably 0.1 to 0.4 mg / kg, even more preferably 0.2 to 0.35 mg / kg, and even more preferably 0.25 mg / kg, but the dosage does not limit the scope of the present invention in any way.

[0089] The route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0090] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0091] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of nervous system damage or nervous system disease.

[0092] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0093] The in vitro composition for promoting nerve regeneration of the present invention can be used for experimental purposes, and can be a composition for the purpose of treating isolated cells or tissues requiring nerve regeneration due to nerve damage.

[0094] The in vitro composition for promoting nerve regeneration of the present invention may be a medium composition, and the medium may include, without limitation, a medium known to those skilled in the art, for example, a medium containing serum (e.g., fetal bovine serum, horse serum, and human serum). The medium that can be used in the present invention may include, for example, RPMI series, EMEM, MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, a mixture of DMEM and F12, Way-mo, McCoy's 5A, or a medium known in the art suitable for culturing cells requiring nerve regeneration. The nerve regeneration may be used to mean all of inducing nerve regeneration, promoting BDNF expression, inducing neural differentiation, proliferation of neural cells, or inducing neural circuit recovery.

[0095]

[0096] According to another aspect of the present invention, the present invention provides a method for treating nervous system damage or nervous system disease, comprising the step of administering the extracellular vesicle to a subject in need thereof.

[0097] In a specific embodiment of the present invention, the subject may be an subject expected to develop a nervous system damage or a nervous system disease; an individual who has developed the disease; or an individual who has been judged to have been cured; but the scope of the present invention is not limited thereto.

[0098] In addition, the treatment method of the present invention can simultaneously or simultaneously administer a therapeutic agent for nervous system damage or nervous system disease known in the art in addition to the extracellular vesicles.

[0099] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0100]

[0101] Information on the neuropeptides used in the examples described below is shown in Table 1.

[0102] Sequence number Peptide molecular weight (Da) Sequence Note 1 NP-4330.4 RVG-2 NP-5321.36 MDG-3 NP-8248.35 MV-4 NP-9174.21 VG--NP-8-TRITC804 MV NP-8 with TRITC, a fluorescent substance, conjugated to the C-terminus -NP-8-FITC832 MV NP-8 with FITC, a fluorescent substance, conjugated to the C-terminus

[0103]

[0104] Example 1. Evaluation of the effect of neuropeptides on BDNF (Brain-derived neurotrophic factor) expression.

[0105] Human dopaminergic neuron SH-SY5Y cell line, differentiated for more than 7 days with retinoic acid, was seeded in a 6-well plate at a density of 2 × 10 per well. 5Cells were seeded. The seeded cells were cultured in a CO2 incubator for more than 18 h, then treated with neuropeptide NP-4, 5, 8, or 9 at various concentrations (10 pM or 1 μM) and cultured for an additional 12 h. Cells were harvested by adding 1×RIPA buffer containing protease inhibitor (SIGMA) to each well. The harvested cells were transferred to a 1.5 ml tube and centrifuged (Centrifuge, MiCRO 17TR, Hnil Science Industrial Co., Ltd.) to obtain cell pellets and supernatants.

[0106] After loading 20 μg of sample onto a 15% acrylamide gel, the gel was run using an electrophoresis device (electrophoresis power supply, EPS 601, Amersham) at 100 V for 2 hours and 30 minutes. The gel was then bio-transferred using an electrophoresis device (electrophoresis power supply, EPS 301, GE healthcare) at 400 mA for 2 hours and 30 minutes using PVDF (polyvinylidene difluoride, Millipore). The PVDF was blocked with 5% skim milk (Difco Skim milk, BD) for 1 hour, and the primary reaction was performed with mouse anti-human BDNF antibody (mouse anti-human BDNF monoclonal antibody, MAB248, R&D systems) at a ratio of 1:500. After washing three times for 5 minutes each with 0.05% TBST (Tris-Buffered Saline Tween 20), the membrane was incubated with goat anti-mouse IgG:HRP (Goat anti-mouse IgG:Horseradish Peroxidase Conjugate, SC-2005, Santa Cruz Biotechnology) at a ratio of 1:1000 for 1 hour. Subsequently, the membrane was washed four times for 10 minutes each with 0.05% TBST buffer, and detection was performed with Amersham ECL Prime Western Blotting Detection Reagent. The Western blotting results were quantified, and the results are shown in Fig. 1a.

[0107] As shown in Figure 1a, the neuropeptide-treated group showed increased expression of BDNF compared to the control group. In particular, the NP-8-treated group showed a higher expression level of BDNF compared to the other neuropeptide-treated groups.

[0108]

[0109] Example 2. Evaluation of the effects of neuropeptides on neuroproliferation and differentiation.

[0110] To induce differentiation into neural cells, SH-SY5Y cells were cultured for more than 7 days in a medium containing 0.5 μM retinoic acid. The differentiated SH-SY5Y cell line was seeded in 6-well plates at a density of 2 × 10 per well. 5 Cells were seeded one by one. The seeded cells were cultured in a CO2 incubator for more than 18 hours, then treated with neuropeptides at a concentration of 1 μM and cultured for an additional 48 hours. Using an optical microscope, the increase in neurite length, total cell number, and the number of branches extending from one end of the neurite were measured. The results of calculating neurite length per cell and neurite tip number per cell are shown in Figure 1b.

[0111] As shown in Figure 1b, the neuropeptide-treated group showed increased neuronal length and number of branches per cell compared to the control group. In particular, the NP-8-treated group showed significantly higher neuronal length and number of branches compared to the other neuropeptide-treated groups.

[0112]

[0113] Example 3. Preparation of extracellular vesicles loaded with neuropeptides

[0114] Based on the results of Examples 1 and 2, in this Example, extracellular vesicles (EVs) loaded with neuropeptide NP-8 were produced.

[0115]

[0116] 3-1. Manufacturing of extracellular vesicles loaded with neuropeptides

[0117] A diffusion-based method was adopted for loading peptide drugs, and neuropeptides were loaded into Wharton's jelly MSC-derived EVs through passive incubation. Specifically, EVs (2 x 10 9 The neuropeptides were loaded into EVs by incubating with 20 nmol of peptide (particle number) for more than 12 hours.

[0118] Ultrafiltration was used to remove unloaded substances smaller than EVs. In addition, 5kDa Amicon ® (Centrifugal Filter Units Merck Millipore) The EV loaded with neuropeptides was separated by centrifugation at 3500 rpm for 30 minutes using a filter.

[0119] After centrifugation, MV-loaded EVs remained in the supernatant of Amicon at approximately 50 to 100 μl, and all EVs attached to the Amicon filter membrane were recovered.

[0120] In the examples described below, extracellular vesicles loaded with the neuropeptide NP-8 are referred to as 'MSC-3D EV-NP8'. In addition, Wharton jelly mesenchymal stem cell-derived extracellular vesicles are referred to as 'MSC-3D EV', and HEK293 cell-derived EVs are referred to as 'Hek293-EV'.

[0121]

[0122] 3-2. Evaluation of the loading efficiency of EVs loaded with neuropeptides

[0123] In this example, the loading efficiency of EVs (MSC-3D EV-NP8) loaded with neuropeptide NP-8 was confirmed. Specifically, NP-8-TRITC, which is a fluorescent substance conjugated to the C-terminus of NP-8, was reacted with EVs, and then EVs were separated and fluorescence was measured to measure the loading efficiency. The quantification of the unloaded drug and the EV-loaded drug was quantified by measuring the fluorescence value using a fluorescence Microplater reader. 1 X 10 9 The quantification of the drug loaded in the particle EV was performed using fluorescence equipment to derive the quantitative value of the fluorescent peptide, and the results are shown in Fig. 2.

[0124] As shown in Figure 2, 1 X 10 9 5-40 nmol of neuropeptides could be loaded into particle EVs, with a total of 5 X 10 9 The peptide loaded inside the particle EV was confirmed to be 17.4 nmol. In other words, the loading efficiency (encapsulation efficiency, EE) was confirmed to be over 86.9%.

[0125]

[0126] Example 4. Characteristics of EVs loaded with neuropeptides according to EV type

[0127] In this example, neuropeptides were loaded onto mesenchymal stem cell-derived EVs (MSC-3D EVs) and HEK293 cell-derived EVs (HEK293 EVs), and their characteristics were analyzed.

[0128]

[0129] 4-1. Analysis of neuropeptide loading efficiency

[0130] Using the TRITC fluorescent labeling method, the loading amount of MSC-3D EVs or HEK293 EVs loaded with the neuropeptide NP-8 was analyzed. The fluorescently labeled NP-8 peptide was diluted to a concentration of 0.011 pmol to 25,000 pmol and loaded onto MSC-3D EVs or HEK293 EVs according to the method described in 3-1. After removing the unloaded material, the TRITC value of the EVs (i.e., EVs loaded with the fluorescently labeled neuropeptide) was read with a micro reader, and the loading amount relative to the initial input amount (i.e., loading efficiency) was calculated.

[0131]

[0132] EE(%) = (Amount of peptide loaded in EV / Total amount of peptide injected) X 100(%)

[0133]

[0134] 4-2. Nanoparticle tracking analysis (NTA)

[0135] The characteristics of EVs loaded with neuropeptides were analyzed using NTA. NTA tracks particle trajectories by adjusting a camera according to the intensity of light scattered from particles when irradiated with a laser, allowing analysis of particle size (size), concentration (concentration, particle number / ml), particle size distribution, and concentration (number).

[0136]

[0137] 4-3. Cryo-TEM

[0138] The EVs loaded with neuropeptides and the NP-8 peptide-loaded samples were imaged at high resolution using an ultra-low temperature 300 kV field emission transmission electron microscope platform to determine the double lipid membrane structure and circular structure of the EVs and whether they were denatured after internal and external loading.

[0139]

[0140] 4-4. 3D-STORM

[0141] EVs loaded with neuropeptides were stained with CellMak Deep Red (C10046; Thermo Fisher Scientific) and analyzed by 3D-STROM microscopy using an electron-multiplying charge-coupled device (EMCCD) camera (iXon Ultra 888, Andor).

[0142]

[0143] 4-5. Zeta-Potential

[0144] The zeta potential of EVs loaded with neuropeptides was measured using a Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). The ZP of EVs was measured three times under conditions of sensitivity 85, shutter speed 70, frame rate 30 frames / s, and temperature 25°C. ZetaView software was used to collect and analyze the measured data.

[0145]

[0146] 4-6. Characteristic Analysis Results

[0147] The results of analyzing the characteristics of EVs loaded with neuropeptides using the methods of Examples 4-1 to 4-3 are shown in Figures 3a to 3e. In addition, the experimental results under the 20 nmol condition are shown in Table 2.

[0148] EVEV size average (nm)EV size mode (nm)EV recovered after ultrafiltration (numbers / ml)EV recovery after ultrafiltration (%)Loading efficiency (EE %)NP-8 TRITC mole (nmol / 5x10) loaded on EV 9 particles)Naive-MSC 3D EV(MSC-3D EV)138.9112.98.6 x 10 9 86--NP-8-TRITC-equipped MSC 3D EV (MSC-3D EV-NP8)148.1117.68.3 x 10 9838717.4NP-8-TRITC equipped HEK 293 EV (Hek293- EV-NP8)138.5118.38.2 x 10 9 828617.2

[0149]

[0150] As shown in Table 2 and Figures 3a to e, the stability of EVs after drug loading was tested by examining the EV size, number, distribution, and external shape deformation using NTA and Cryo-TEM. As a result, EVs loaded with neuropeptides showed a similar distribution pattern to unloaded EVs. The EV recovery rate measured by NTA was 82–86%, resulting in a final volume of 8.2–8.6 X 10 9 EVs could be recovered at 1000 particles / ml (Table 2, Fig. 3a). In addition, EVs loaded with neuropeptides showed no apparent changes in their appearance even after drug loading (Fig. 3b). The loading efficiency of neuropeptides was over 86.9% at 20 nmol, and the loading amount was 17.4 nmol / 5x10 9 It was confirmed that they were particles (Table 2, Fig. 3c).

[0151] 3D-STORM analysis using a fluorescently tagged peptide (TAMRA peptide) confirmed that the EVs loaded with the neuropeptide contained the peptide inside the EVs. Furthermore, measurement of the EV size using CellMask dye confirmed that the EVs loaded with the neuropeptide (CellMASK labeled) were 100 nm in size, while the loaded peptide (TAMRA labeled) was smaller, measuring 84 nm (Fig. 3d).

[0152] Mesenchymal stem cell-derived EVs were confirmed to have zeta potential values ​​of 43 to 48 mV. Furthermore, EVs loaded with neuropeptides showed no significant change in zeta potential value despite being loaded with neuropeptides (Fig. 3e).

[0153] The above results show that MSC-3D EVs and HEK293 EVs loaded with neuropeptides have the same particle number (5 X 10 9 This means that the loading efficiency and loading amount are similar in particle EV / ml.

[0154]

[0155] Example 5. Evaluation of the efficacy of EVs loaded with neuropeptides in SY5Y cells.

[0156] 5-1. Evaluation of the effects of EVs loaded with neuropeptides on the expression of BDNF, TrkB, and pCREB in SY5Y cells.

[0157] SY5Y cells were prepared under the same conditions as in Example 1. The experimental and control groups of this experiment are as follows.

[0158] - MOCK: control group, PBS treatment

[0159] - MSC 3D-EV: Negative control, MSC-3D EV treatment

[0160] - NP4: Neuropeptide NP4 treatment

[0161] - NP8: Neuropeptide NP8 treatment

[0162] - NP9: Neuropeptide NP9 treatment

[0163] Prepared SY5Y cells were treated with MSC-3D EVs loaded with neuropeptides. The expression of BDNF, TrkB, and pCREB was then analyzed using the same method as in Example 1, and the results are shown in Figure 4a.

[0164] As shown in Figure 4a, all neuropeptide-treated groups showed higher expression of BDNF, TrkB, and pCREB than the PBS-treated group. In particular, the NP-8-treated group showed significantly higher expression of BDNF, TrkB, and pCREB, and BNDF expression was more than twice as high as that of the NP-4-treated group.

[0165]

[0166] Additionally, the effects of NP-8 peptide and NP-8-loaded MSC-3D EVs on CREB expression were evaluated. Specifically, prepared SY5Y cells were treated with various concentrations (1X, 2X) of NP-8 peptide or NP-8-loaded MSC-3D EVs, and then CREB expression was analyzed. The results of the CREB expression analysis are shown in Fig. 4b.

[0167] As shown in Fig. 4b, the NP-8-loaded MSC-3D EV treatment group showed higher CREB expression than the NP-8 peptide treatment group. In addition, it was confirmed that the NP-8-loaded MSC-3D EV increased CREB expression in a concentration-dependent manner. The above results indicate that the NP-8-loaded MSC-3D EV increases CREB expression in a concentration-dependent manner.

[0168]

[0169] The above results indicate that when NP-8 peptide is treated alone, it is easily decomposed due to the inherent instability of the peptide, resulting in reduced target drug reachability, whereas when loaded onto a drug delivery vehicle based on MSC-3D EV, the stability of the peptide's efficacy is increased and the drug delivery period is extended, thereby increasing drug delivery efficiency.

[0170]

[0171] 5-2. Evaluation of the Effects of Neuropeptide-Loaded EVs on Neuronal Proliferation and Nerve Regeneration

[0172] SY5Y cells were prepared under the same conditions as in Example 2, and the prepared SY5Y cells were treated with MSC-3D EVs (MSC-3D EV-NP8) and naive EVs loaded with the neuropeptide NP-8. Thereafter, the effects on neuronal proliferation and nerve regeneration were evaluated using the same method as in Example 2. After the evaluation, the morphology of differentiated neurons was analyzed by immunostaining using Tuj1 antibody on SY5Y cells using an immunohistochemical method. The control group in this experiment was treated with PBS, NGF (nerve growth factor), BDNF, or naive EVs. The results of evaluating the effects of EVs loaded with neuropeptides on neuronal proliferation and nerve regeneration are shown in Figures 5a and b.

[0173] As shown in Fig. 5a and b, the MSC-3D EV-treated group loaded with NP-8 had longer neurite length per cell, higher neurite tip number per cell, and higher total cell number than the control and naive EV-treated groups. Since the MSC-3D EV-loaded with NP-8 increased neurite proliferation and regeneration in all aspects compared to the naive EV-treated group, it was confirmed that the delivery of the NP-8 peptide can effectively increase the neural regeneration capacity and induction of neural differentiation.

[0174]

[0175] The effects of neuropeptide-loaded EVs (MSC-3D EV-NP8) on neuronal cell death were evaluated using fluorescence microscopy. Specifically, to determine whether neuropeptide-loaded EVs exhibit neuroprotective properties, SH-SY5Y neurons were cultured under hypoxic conditions, and neuronal survival and apoptosis following treatment with neuropeptide-loaded EVs were examined. The results confirming the neuroprotective properties of neuropeptide-loaded EVs are shown in Figure 5c.

[0176] As shown in Figure 5c, EVs loaded with neuropeptides showed a significantly higher number of surviving neurons compared to the MSC 3D EV treatment group and the NP8 peptide treatment group. This suggests that EVs loaded with neuropeptides exhibit excellent neuroprotective properties.

[0177]

[0178] Example 6. Analysis of delivery and uptake of extracellular vesicles loaded with neuropeptides into SH-SY5Y neurons.

[0179] SH-SY5Y neuronal cells were cultured in 24-well plates. The cultured cells were treated with EVs loaded with fluorescently labeled neuropeptides (MSC-3D EV-NP8), incubated for 6 hours, and the medium was replaced for analysis. The EVs loaded with neuropeptides delivered to the cells were then observed under a fluorescence microscope, and the results are shown in Figure 6.

[0180] As shown in Figure 6, fluorescence signals were detected from EV labeling dyes PKH67 and PKH26. This indicates that EVs loaded with neuropeptides are delivered and absorbed into neurons.

[0181]

[0182] Example 7. Analysis of the blood-brain barrier passage of neuropeptide-loaded extracellular vesicles in an in vitro BBB model.

[0183] As shown in Fig. 7a, an in vitro BBB model was created. Specifically, HBEC (cerebrovascular endothelial cells) and C6 (astrocytic cells) were cultured on both sides of the Trans-well, and the formation of the blood-brain barrier was measured using a transepithelial electrical resistance (TEER) measuring device. On the 6th day of model creation, EVs loaded with neuropeptides were treated in the upper chamber of the Trans-well, and the neuropeptide-loaded EVs that had migrated to the lower chamber were measured using a fluorescence reader. The results of confirming TEER and BBB permeability are shown in Fig. 7b, and the results of measuring the fluorescence intensity according to the treatment of EVs loaded with neuropeptides in the in vitro BBB model are shown in Fig. 7c. In addition, the results of immunofluorescence staining according to the treatment of EVs loaded with neuropeptides in the in vitro BBB model are shown in Fig. 7d.

[0184] As shown in Figures 7b to d, the peptide-only group rapidly crossed the BBB due to the very small size of the peptide, but exhibited permeability at quantitatively low concentrations. In contrast, the EV-treated group and the neuropeptide-loaded EV-treated group showed a gradual increase in permeability after 1 hour, and exhibited higher permeability than the peptide-only group. This suggests that the neuropeptide-loaded EVs maintained their EV characteristics (i.e., BBB permeability) despite being loaded with the neuropeptide.

[0185]

[0186] Example 8. Evaluation of the efficacy of EVs loaded with neuropeptides in experimental animals.

[0187] 8-1. Nasal administration method for experimental animals

[0188] - Animal: C57bl / 6 mouse (5-week-old male mouse)

[0189] - Administered drugs: Fluorescently labeled MSC-3D EV, MSC-3D EV-NP8, NP8 neuropeptide.

[0190] - Capacity: 6 x 10 8 EVs / 10 μL / mouse

[0191] - Anesthesia: Usually, inhalation anesthesia is used, but since the amount of inhalation is not active during sleep breathing, the patient is induced to inhale the drug during active breathing without anesthesia.

[0192] - Administration method: Using the thinnest and finest fine tip, 5 μl was inhaled into each nostril of the mouse by aligning the tip with the nostril. The mouse was held upright for 15-20 seconds to ensure complete inhalation and delivery to the brain (administration to the left side 10 minutes after administration to the right side). After 24 hours, tissues were prepared differently for each experimental purpose (ELISA analysis, fluorescence distribution analysis).

[0193] - Mouse preparation method: For ELISA analysis, brains were dissected without perfusion, and proteins were dissolved in each brain region. ELISA was performed with the lysates, and total protein was quantified through BCA quantitation, followed by protein analysis.

[0194] For fluorescence distribution analysis, the brains were removed after perfusion and fixed in 4% PFA for 2 hours. After dehydration in a 15% sucrose solution until they sank, they were transferred to a 30% sucrose solution for further dehydration. The dehydrated brains were embedded in OCT compound and cryosectioned (sectioned at 40–60 μm).

[0195]

[0196] 8-2. Analysis of the distribution of EVs loaded with neuropeptides in mouse brain tissue and investigation of their effects on BDNF expression.

[0197] Naive MSC-3D EV and MSC-3D EV loaded with NP-8 (MSC-3D EV-NP8) were prepared by fluorescent labeling (total 5 X 10 9 ). The prepared fluorescently labeled EVs were administered intranasally to adult mice (Example 6-1). 24 hours after administration, brain tissues (olfactory bulb, frontal cortex, clausetrocortex, hippocampus) were sectioned and the fluorescence distribution in the mouse brain tissues was analyzed using a fluorescence microscope. The results of analyzing the fluorescence distribution in the brain tissues of the sacrificed mice are shown in Figures 8a and b, and the results of analyzing BNDF expression in each region are shown in Figure 8c.

[0198] As shown in Figures 8a and b, the group administered NP-8-loaded MSC-3D EVs via nasal administration confirmed that neuropeptides were directly delivered to each region of the brain cells, namely the olfactory bulb, cortex, and hippocampus.

[0199] As shown in Fig. 8c, the group administered NP-8-loaded MSC-3D EVs via nasal administration showed a significant increase in BNDF expression in each region of the brain compared to the control groups (PBS, NP8 peptide only treatment, MSC-3D EV only treatment).

[0200]

[0201] The brain delivery rate of EVs loaded with neuropeptides following intranasal administration was measured using the IVIS imaging system, a preclinical tomography optical and fluorescence imaging device. Specifically, peptide-loaded EVs were injected into the nasal region of mice at a density of 6 × 10 9 After a single administration of particles, IVIS analysis was performed, and the results are shown in Fig. 8d.

[0202] As shown in Fig. 8d, as a result, fluorescent signals (i.e., EVs loaded with neuropeptides) were observed in the brain 30 minutes after administration. This indicates that EVs loaded with neuropeptides were effectively delivered and distributed to the brain.

[0203]

[0204] The above results imply that while the single NP-8 peptide has a long half-life in the body and is difficult to act as a therapeutic agent and promote the expression of BDNF, MSC-3D EV (MSC-3D EV-NP8) loaded with NP-8 can overcome the shortcomings of this peptide and induce direct access to brain cells and increased expression of BDNF in functional aspects, thereby demonstrating the efficacy of neural regeneration and differentiation.

[0205]

[0206] Example 9. Quality control (QC) analysis of EVs loaded with neuropeptides

[0207] After loading the neuropeptide NP8 peptide into MSC-3D EVs, a quality control analysis was performed to measure the changes in the contents of the EV marker CD63 and five major miRNAs (hsa-miR27a-3p, hsa-miR92a-3p, hsa-miR132a-3p, hsa-miR181a-5p, hsa-miR210a-3p). Specifically, the NP8 peptide was loaded onto each MSC-3D EV and then purified. To confirm the unique characteristic denaturation of EVs, the expression of CD63 in the purified MSC-3D EV-NP8 was measured by ELISA. In addition, the expression of five major miRNAs in the purified MSC-3D EV-NP8 was analyzed by ddPCR. The results of analyzing the expression of CD63 and five major miRNAs are shown in Figures 9 and 10, respectively.

[0208] As shown in Figures 9 and 10, it was confirmed that the content of EV-specific embedded miRNA was preserved unchanged even after peptide loading in MSC-3D EVs loaded with NP-8, and that there was no effect on the expression of CD63, an EV marker.

[0209]

[0210] Example 10. Efficacy test of extracellular vesicles loaded with neuropeptides in stroke mice.

[0211] As shown in Figure 11a, the efficacy of neuropeptide-loaded EVs (MSC-3D EV-NP8) was evaluated in stroke mice. Specifically, EVs loaded with neuropeptides were administered intranasally three times to a stroke-induced mouse model. qPCR analysis was performed on samples taken on day 1. MRI and protein expression analysis (ELISA, western blot) were also performed on samples taken on day 3. The qPCR results are shown in Figure 11b, and the MRI analysis results are shown in Figures 11c and d. Furthermore, the protein expression analysis results are shown in Figures 11e and f.

[0212] As shown in Figures 11b to f, the EV-treated group loaded with neuropeptides significantly increased the expression of CREB, a transcriptional regulator in the nucleus (Figure 11b). MRI analysis results showed that the EV-treated group loaded with neuropeptides showed a reduction in the brain lesion area in the stroke model (Figures 11c and d). Furthermore, protein expression analysis results showed that the EV-treated group loaded with neuropeptides showed a significant increase in the protein expression of BDNF in brain tissue (Figures 11e and f). These results suggest that EVs loaded with neuropeptides exhibit neuroprotective properties by activating CREB and increasing BNDF expression.

[0213]

[0214] In summary, the present inventors have developed extracellular vesicles loaded with neuropeptides and confirmed their superior neuroprotective and neuroregenerative effects. In particular, mesenchymal stem cell-derived extracellular vesicles loaded with the neuropeptide NP-8 (SEQ ID NO: 3) demonstrated more pronounced neuroprotective and neuroregenerative effects than other experimental and control groups. Therefore, the neuropeptide-loaded extracellular vesicles of the present invention can be utilized in various fields of treatment for nervous system diseases or injuries.

[0215]

[0216] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An extracellular vesicle loaded with a peptide comprising an amino acid sequence represented by the following structural formula 1: [Structural formula 1] N'-(X1)α-(X2)-(X3)β-C' In the above structural formula 1, N' is the N-terminus of the peptide and C' is the C-terminus of the peptide; α is 0 or 1, and if α is 1, X1 is R or M; X2 is D or V; β is 0 or 1, and if β is 1, X3 is G, α and β do not become 0 at the same time.

2. In the first paragraph, the peptide is an extracellular vesicle loaded with a peptide having at least one amino acid sequence selected from the group consisting of sequence numbers 1 to 4.

3. In the first paragraph, the extracellular vesicle contains 1 to 50 nmol / 5x10 peptide. 9 Extracellular vesicles loaded with particles at a concentration of 100 μm.

4. In the first paragraph, the extracellular vesicle has at least one effect selected from the group consisting of increasing the expression of BDNF (Brain-derived neurotrophic factor), promoting neuronal proliferation, and promoting neuronal differentiation.

5. In the first paragraph, the extracellular vesicle is derived from at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells.

6. In paragraph 5, the mesenchymal stem cell is an extracellular vesicle derived from umbilical cord, cord blood, Wharton jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, tooth, hair root cell, or placenta.

7. A pharmaceutical composition for preventing or treating nervous system damage or nervous system disease, comprising an extracellular vesicle according to any one of claims 1 to 6.

8. A composition according to claim 7, wherein the nervous system damage or nervous system disease is at least one selected from the group consisting of spinal cord damage, Parkinson's disease, stroke, amyotrophic spinal lateral sclerosis, motor nerve damage, peripheral nerve damage due to trauma, nerve damage due to ischemic brain damage, neonatal hypoxic brain damage, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury.

9. An in vitro composition for promoting nerve regeneration comprising the extracellular vesicle of any one of claims 1 to 6.

10. A method for treating nervous system damage or nervous system disease, comprising the step of administering an extracellular vesicle of any one of claims 1 to 6 to a subject in need thereof.

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