Wound treatment composition comprising exosomes of mast cells

Abstract

The present invention relates to a composition for preventing, alleviating or treating skin wounds, comprising exosomes of mast cells. The composition according to the present invention enhances the growth and activity of fibroblasts, and thus has the effects of significantly reducing the size of wounds and shortening an initial wound treatment period.

Description

Composition for wound treatment comprising exosomes from mast cells

[0001] The present invention relates to a composition for preventing, improving or treating skin wounds, comprising exosomes of mast cells.

[0002] Exosomes are vesicles composed of a lipid bilayer and are components of substances secreted by cells into the extracellular space. Exosomes are known to transport intracellular biomolecules such as proteins, bioactive lipids, and RNA (miRNA) to perform functional roles in mediating cell-to-cell communication and cellular immunity. These exosomes are also being studied as biomarkers for neurological diseases such as Alzheimer's disease, and due to their high selective permeability, capable of penetrating the blood-brain barrier (BBB), which separates cerebrospinal fluid and blood, they are also being utilized in the development of drug delivery systems such as nanocarriers for specific drugs.

[0003] Mast cells are cells involved in inflammatory responses. When IgE antibodies are activated and crosslinked by external antigens or allergens, various signaling pathways trigger the release of inflammatory substances such as histamine, β-hexosaminidase, and leukotrienes that were trapped in the granules of mast cells to the plasma membrane. These released inflammatory substances activate receptors and cytokines that induce inflammation, thereby further aggravating the inflammation. In particular, the release of these substances from mast cells is closely related to allergic diseases, and allergic diseases are generally known to be caused by sensitization through contact and exposure to antigens, which are genetically / immunologically related to IgE antibodies and acquired factors such as environmental and psychological factors. Among these, immunological factors are currently being targeted for treatment, and it can be said that they can be applied as a treatment for allergic diseases by controlling the release of these allergenic substances into the cell outer membrane from an immunological perspective.

[0004] However, to date, it has not been known whether exosomes released from mast cells are effective in treating skin wounds.

[0005] Accordingly, the inventors of the present invention completed the present invention by confirming that a therapeutic effect was observed in a wound mouse model when exosomes from mast cells were treated.

[0006] One object of the present invention is to provide a pharmaceutical composition for preventing or treating skin wounds, comprising exosomes of mast cells.

[0007] Another object of the present invention is to provide a composition for external application to the skin containing exosomes of mast cells.

[0008] Another object of the present invention is to provide a food composition for preventing or improving skin wounds, which comprises exosomes of mast cells.

[0009] To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0010] Hereinafter, the present invention will be described in detail.

[0011] The present invention provides a pharmaceutical composition for preventing or treating skin wounds, comprising exosomes of mast cells.

[0012] In the present invention, the term "mast cell" refers to a cell that is widely distributed in organs throughout the body, such as the skin, respiratory tract, gastrointestinal mucosa, lymphatic vessels, blood vessels, and brain, and secretes cytokines related to inflammatory responses.

[0013] The term "exosome" in the present invention refers to a membrane vesicle with a lipid bilayer membrane structure secreted by a cell or present within a cell, and refers to a substance present in the body fluids of almost all eukaryotic organisms. It is well known that exosomes play a role in transporting intracellular biomolecules such as proteins, bioactive lipids, and RNA (miRNA) to perform functional roles in mediating coagulation, cell-to-cell communication, and cellular immunity. In the present invention, the exosome is a concept encompassing microvesicles.

[0014] In the present invention, “mast cell exosome” means an exosome isolated from a culture medium obtained by culturing mast cells, and can be used interchangeably with “mast cell-derived exosome.”

[0015] In the present invention, the term “prevention” refers to any act of inhibiting or delaying the onset of skin wounds by administering mast cell exosomes according to the present invention.

[0016] In the present invention, the term “treatment” refers to any act of improving or beneficially changing the symptoms of a skin wound by administering mast cell exosomes according to the present invention.

[0017] In the present invention, the term “improvement” means any act of improving a bad condition of a skin wound by administering or ingesting the composition of the present invention to a subject.

[0018] In the present invention, the term "wound" refers to a damaged state of a living body, encompassing a pathological state in which tissues forming the internal or external surface of a living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organ, or vascular tissue, are divided or destroyed, and can be used interchangeably with "injury."

[0019] In the present invention, "skin wound" may be specifically any one selected from the group consisting of temperature damage such as burns, thermal burns, thermal burn ulcers, and frostbite; trauma such as lacerations, abrasions, incisions, cuts, acne, and strangulation; blood vessel and lymphatic vessel damage such as Buerger's disease, lymphedema, and leg ulcers; post-surgical wounds such as skin scraping and suture wounds; stomas, bedsores, pressure ulcers, diabetic ulcers / exfoliation, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, chemical damage, and other skin wounds.

[0020] According to one embodiment of the present invention, the mast cell may be a human-derived mast cell.

[0021] The exosomes of the mast cells of the present invention can promote the growth and activity of skin cells, preferably fibroblasts.

[0022] In the present invention, the exosomes of mast cells include exosomes of cells isolated from an individual, cultured, and manufactured through special manipulation, and can be used as a medicine for the purposes of treatment, diagnosis, and prevention, and can be used on skin wounds.

[0023] The above mast cell culture medium may be a culture medium, culture supernatant, a concentrate thereof, or a lyophilized product thereof obtained after culturing mast cells and removing the cells.

[0024] In the present invention, the mast cell culture solution can be obtained by subculturing mast cells in a serum medium and then culturing them in a serum-free medium.

[0025] The above mast cell culture medium can be obtained by subculturing mast cells in a serum-free medium.

[0026] The above mast cells can be conventionally cultured using cell culture media. Mast cell culture fluid is obtained by subculturing mast cells in serum-free media, followed by subculturing in serum-free media. This can be used as is, or the supernatant obtained after removing mast cells and macromolecules by centrifugation or filtration using a filter can be used. In addition, the obtained supernatant can be used as is, or the resulting concentrate can be used after concentration.

[0027] Culture media and culture conditions for culturing mast cells are well known in the technical field to which the present invention pertains, and can be appropriately selected or modified and used by a person skilled in the art.

[0028] The above serum medium is a medium suitable for maintaining and storing the same cell type as mast cells, and may be an IMDM medium supplemented with serum. The serum may be, but is not limited to, fetal bovine serum (FBS), and may be 1 to 20 wt% based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents, and mycoplasma inhibitors may be included, and these may be 1 to 5 wt% based on the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include, but are not limited to, gentamicin, ciprofloxacin, tylosin, and the like.

[0029] Culturing in the above serum-free medium can be performed after removing the culture medium from the serum medium and washing the cells with a phosphate buffer solution.

[0030] Preferably, the serum medium may be IMDM medium supplemented with 10% FBS and 1% antibiotics.

[0031] Preferably, the serum-free medium may be IMDM medium.

[0032] According to one embodiment of the present invention, the mast cell culture solution can be obtained by a step of removing cells by centrifuging or filtering the mast cell culture solution.

[0033] According to one embodiment of the present invention, the exosomes of mast cells may be obtained from a cell pellet obtained by centrifuging the mast cell culture solution.

[0034] Additionally, the exosome may be characterized by a particle diameter of 50 to 300 nm, preferably 100 to 160 nm.

[0035] Additionally, the exosomes express exosomal marker proteins, preferably CD63 and CD9. Furthermore, the exosomes may include SEMA7A, a protein known to be involved in the normal migration of fibroblasts and the assembly of the extracellular matrix, essential for the wound healing process. For example, the exosomes may be characterized by high levels of SEMA7A expression.

[0036] The above exosomes can be obtained using an exosome extraction method known in the art, and are not limited thereto, but can be obtained by an extraction method including, for example, the following steps:

[0037] 1) Step of culturing mast cells;

[0038] 2) A step of recovering the mast cell culture supernatant;

[0039] 3) A step of centrifuging the recovered cell culture supernatant to remove cell residue; and

[0040] 4) A step of obtaining exosomes separated and purified from the cell culture supernatant from which the cell debris has been removed using one selected from the group consisting of tangential flow filtration (TFF), ultracentrifugation, size exclusion chromatography, and an exosome isolation kit.

[0041] The pharmaceutical composition of the present invention can be formulated into various dosage forms, such as a solution, suspension, emulsion, lotion, ointment, and lyophilisate, according to conventional methods.

[0042] The pharmaceutical composition of the present invention can be formulated into a unit dosage form suitable for intravenous administration to a patient according to conventional methods in the pharmaceutical field, and administered. The formulation contains an effective dosage amount for single or multiple administrations. Preferred formulations for this purpose include parenteral administration preparations such as injections and infusions. In addition, the pharmaceutical composition for preventing or treating skin wounds may include conventional inert pharmaceutically acceptable carriers and diluents. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and bulking agents such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with mast cell exosomes and recipients to be transplanted therewith. Diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media. In addition, for example, in the case of injectables, preservatives, analgesics, solubilizers, or stabilizers may be additionally included, and in the case of topical formulations, bases, excipients, lubricants, or preservatives may be additionally included. The compositions of the present invention may be used unfrozen or frozen for later use. If frozen, standard cryopreservatives (e.g., DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) may be added to the cell population prior to freezing.

[0043] In addition, it can be transplanted and / or administered using a method of administration commonly used in the art, and preferably, direct engraftment or transplantation is possible at the disease site of a patient requiring treatment, but is not limited thereto. In addition, the administration can be carried out using various methods such as oral administration, non-surgical administration using a catheter, and injection or transplantation after incision of the disease site. The dosage may vary depending on the degree of concentration, but can be administered once or in several divided doses at 10 μl / kg to 1 ml / kg.

[0044] However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage does not limit the scope of the present invention in any way.

[0045] The present invention also provides a cosmetic composition for promoting skin regeneration comprising exosomes of mast cells.

[0046] The cosmetic composition according to the present invention can be manufactured in any formulation commonly manufactured in the art, and examples thereof include creams, emulsions, toners, packs, foundations, etc. Specifically, the cosmetic composition of the present invention includes formulations of skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisture lotions, nutritional lotions, massage creams, nutritional creams, moisture creams, hand creams, foundations, essences, nutritional essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, and body cleansers.

[0047] The present invention also provides a composition for external application to the skin comprising exosomes of mast cells.

[0048] The external skin composition of the present invention is not particularly limited as long as it is a formulation that can directly administer an active ingredient to the local surface of the skin. For example, it can be used as a preparation such as an ointment, an ointment patch, a liquid (suspension, emulsion, lotion, etc.), a cataplasm, a tape, an external powder, and an aerosol. Any of the mixing ingredients used in a typical external skin composition can be used as the mixing ingredients used in the external skin composition of the present invention.

[0049] In the case of ointments, creams, gels, and lotions, bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin, and squalene; solvents and solubilizers such as oleic acid, isopropyl myristate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylated hydroxyanisole; preservatives such as p-hydroxybenzoate; moisturizers such as glycerin, propylene glycol, and sodium hyaluronate; polyoxyethylene derivatives, and glycerol esters of fatty acids; Surfactants such as sucrose esters of fatty acids, sorbitan esters of fatty acids, propylene glycol esters of fatty acids, lecithin, etc.; thickeners such as carboxy vinyl polymer, xanthan gum, carboxymethyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc.; propellants such as liquefied petroleum gas, liquefied carbon dioxide, dimethyl ether, nitrogen, kerosene, carbon dioxide, etc.; stabilizers, preservatives, absorption promoters, etc.; and other appropriate additives can be blended.

[0050] In the case of wet compresses, adhesives such as polyacrylic acid and polyacrylic acid copolymers, crosslinking agents such as aluminum sulfate, potassium aluminum sulfate, aluminum chloride, magnesium aluminometasilicate, and dihydroxy aluminum acetate, thickeners such as sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose, polyhydric alcohols such as glycerin, polyethylene glycol (macrogol), propylene glycol, and 1,3-butanediol, surfactants such as polyoxyethylene derivatives, fragrances such as 1-menthol, preservatives such as p-hydroxybenzoate, purified water, and other appropriate additives can be blended.

[0051] In the case of tapes, adhesives such as styrene-isoprene-styrene block copolymers and acrylic resins, tackifiers such as alicyclic saturated-hydrocarbon resins, rosin resins, and terpene resins, softeners such as liquid rubber and liquid paraffin, antioxidants such as dibutylhydroxytoluene, polyhydric alcohols such as propylene glycol, absorption promoters such as oleic acid, surfactants such as polyoxyethylene derivatives, and other suitable additives can be blended. In addition, a polymer that can contain water, such as sodium polyacrylate or polyvinyl alcohol, and a small amount of purified water can be added to produce an aqueous tape.

[0052] In the case of external powder, excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, and other appropriate additives may be mixed.

[0053] In the case of aerosols, bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin, and squalene used in ointments, creams, gels, suspensions, emulsions, solutions, lotions, and external powders; solvents and solubilizers such as oleic acid, isopropyl myristate, diisopropyl adipate, diisopropyl sebacate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylated hydroxyanisole; preservatives such as p-hydroxy benzoate; glycerin, propylene glycol; Humectants such as sodium hyaluronate, polyoxyethylene derivatives, glycerol esters of fatty acids, sucrose esters of fatty acids, sorbitan esters of fatty acids, propylene glycol of fatty acids, surfactants such as lecithin, carboxyvinyl polymers, xanthan gum, carboxymethyl cellulose, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, thickeners such as excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, propellants such as liquefied petroleum gas, liquefied carbon dioxide, dimethyl ether, nitrogen, kerosene, carbon dioxide, buffers, correctives, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, or other appropriate additives may be blended.

[0054] Meanwhile, the external skin composition of the present invention may be formulated for topical application to the skin by containing a cosmetically or dermatologically acceptable medium or base. This may be any formulation suitable for topical application, for example, a solution, gel, solid, anhydrous paste, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule, or an ionic (liposome) or non-ionic vesicle dispersion, or a cream, toner, lotion, powder, ointment, spray, or concealer stick. It may also be used in the form of a foam or an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

[0055] In addition, the external skin composition of the present invention may contain adjuvants commonly used in the field of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. The adjuvants are introduced in amounts commonly used in the field of cosmetology or dermatology.

[0056] The present invention also provides a food composition for preventing or improving skin wounds, which comprises exosomes of mast cells.

[0057] The present invention can be generally used as a commonly used food.

[0058] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body, as defined by the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological functions.

[0059] The food composition of the present invention may include conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0060] Items listed in the above "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0061] The food composition of the present invention may contain exosomes of mast cells in an amount of 0.01 to 95 wt%, preferably 5 to 90 wt%, based on the total weight of the composition.

[0062] In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and / or improving skin wounds.

[0063] For example, among health functional foods in capsule form, hard capsules can be manufactured by filling a conventional hard capsule with a mixture of mast cell exosomes according to the present invention and additives such as excipients, and soft capsules can be manufactured by filling a capsule base such as gelatin with a mixture of the food composition according to the present invention and additives such as excipients. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.

[0064] The definitions of terms for the above excipients, binders, disintegrants, lubricants, maturing agents, flavoring agents, etc. are described in documents known in the art and include those with the same or similar functions.

[0065] There are no special restrictions on the types of the above foods, and all health functional foods in the conventional sense are included.

[0066] The present invention provides a method for treating a skin wound, comprising administering exosomes of mast cells to a subject.

[0067] In the present invention, the terms “mast cell exosome,” “skin wound,” “administration,” etc. are the same as those described above.

[0068] The subject refers to an animal, typically a mammal that can exhibit beneficial effects from treatment using mast cell exosomes of the present invention. Preferred examples of such subjects include primates such as humans. Furthermore, such subjects may include any subject with skin wound symptoms or at risk of developing such symptoms.

[0069] The present invention also provides the use of exosomes from mast cells in the manufacture of a medicament for the treatment of skin wounds.

[0070] The present invention also provides a composition comprising exosomes of mast cells for use in the treatment of skin wounds.

[0071] The present invention also provides the use of exosomes from mast cells for the treatment of skin wounds.

[0072] The composition according to the present invention has the effect of promoting the growth and activity of fibroblasts, significantly reducing the size of a wound, and shortening the initial wound healing period.

[0073] Figure 1 is a diagram confirming the characteristics of mast cell-derived exosomes.

[0074] (a) This is a diagram showing the results of confirming the size distribution profile of mast cell-derived exosomes through Nanosight particle tracking analysis.

[0075] (b) The expression of exosome marker proteins (CD63, CD9, CD81) and endoplasmic reticulum (ER) membrane protein Calnexin was confirmed through Western blot.

[0076] (c to e) The size and concentration (number of particles) of exosomes were confirmed and compared with exosomes derived from tonsil stem cells (T-MSCs).

[0077] Figure 2 is a diagram confirming the effect of mast cell-derived exosomes on promoting fibroblast growth and activity.

[0078] (a) This is a diagram showing the results of measuring the survival rate (% of cell viability) of fibroblasts when treated with mast cell-derived exosomes using the MTT assay (* P <0.1, ns (not significant)).

[0079] (b) This figure shows changes in the expression of fibroblast activation markers and extracellular matrix proteins due to mast cell-derived exosomes through Western blot.

[0080] Figure 3 is a schematic diagram of a mouse model experiment to confirm the wound healing effect of mast cell-derived exosomes.

[0081] Figures 4a and 4b show a group of genetically modified mice deficient in mast cells (B6.Cg-KitW-sh ) and wild type mouse groups (C57BL / 6J WT) were treated with PBS or mast cell-derived exosomes to determine the change in wound size.

[0082] Figure 5 shows a group of genetically modified mice deficient in mast cells (B6.Cg-Kit W-sh ) and wild type mouse groups (C57BL / 6J WT) after applying PBS or mast cell-derived exosomes.

[0083] Figure 6 is a diagram confirming the epithelial regeneration and tissue remodeling effects of mast cell-derived exosomes using H&E staining.

[0084] Figure 7 is a diagram showing the results of proteomics and functional abundance analysis of mast cell-derived exosome proteins.

[0085] (a) This is a diagram showing the results of analyzing the KEGG pathway of mast cell-derived exosome proteins.

[0086] (b) This is a diagram confirming the Cellular Component through Gene Ontology.

[0087] (c) This is a diagram that confirms the Biological Process through Gene Ontology.

[0088] (d) This is a diagram that confirms the molecular function through Gene Ontology.

[0089] Figure 8 is a diagram showing the results of Western blot performed to confirm SEMA7A protein expression and function.

[0090] (a) High expression of SEMA7A in mast cell-derived exosomes was confirmed and compared with exosomes derived from tonsillar stem cells (T-MSCs).

[0091] (b) This figure confirms the decrease in SEMA7A expression in mast cell-derived exosomes following SEMA7A knockdown treatment.

[0092] (c) This figure confirms the effect of SEMA7A knockdown exosomes on the expression of ECM proteins (Collagen I, Fibronectin) and α-SMA in dermal fibroblasts.

[0093] Figures 9a and 9b show a group of genetically modified mice deficient in mast cells (B6.Cg-Kit W-sh ) and wild type mouse groups (C57BL / 6J WT) were treated with PBS, mast cell-derived exosomes, and SEMA7A KD exosomes to determine the change in wound size.

[0094] Figures 10a and 10b show a group of genetically modified mice deficient in mast cells (B6.Cg-Kit W-sh ) and wild type mouse groups (C57BL / 6J WT) were treated with PBS, mast cell-derived exosomes, and SEMA7A KD exosomes, and the degree of tissue regeneration was compared through H&E staining.

[0095] Figures 11a and 11b show a group of genetically modified mice deficient in mast cells (B6.Cg-Kit W-sh ) and wild type mouse groups (C57BL / 6J WT) were treated with PBS, mast cell-derived exosomes, and SEMA7A KD exosomes. The degree of collagen deposition was compared and quantitatively represented using Masson's trichrome staining.

[0096] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0097] Experimental Example 1. Cell Culture

[0098] Human mast cell line (HMC-1) was donated by Radiation Health Institute, Korea Hydro & Nuclear Power Co., Ltd. and cultured in IMDM medium (GIBCO) containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin. Tonsil-derived mesenchymal stem cells (T-MSC) were purchased from Cellatoz Therapeutics Inc. and cultured in low-glucose DMEM (Welgene) containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin. Additionally, human dermal fibroblasts were purchased from the American Type Culture Collection (ATCC) and cultured in high-glucose DMEM (Welgene) medium containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin.

[0099] Experimental Example 2. Preparation of exosomes derived from mast cells or tonsil-derived mesenchymal stem cells.

[0100] According to Experimental Example 1 above, human mast cell line (HMC-1) or tonsil-derived mesenchymal stem cells (T-MSC) were cultured, and when the cells reached 80-90% confluency in a 100 mm tissue culture plate, they were washed with PBS and then cultured in serum-free IMDM medium for 48 hours. Afterwards, the culture supernatant was passed through a 0.2-μm filter (Pall Corporation) and centrifuged at 4°C and 5000 Xg for 1 hour (Sorvall LYNX4000, Thermo Fisher Scientific). After that, 1 / 5 volume of ExoQuick-TC reagent (System Biosciences) was added to the supernatant, mixed thoroughly by pipetting, and stored at 4°C overnight before centrifuging at 1500 Xg for 35 minutes. After centrifugation, the pellet containing exosomes was completely dissolved in PBS, and the concentration was measured using a BCA protein assay kit, and Nanosight particle tracking analysis (ZetaView, Particle Metrix Inc.) was performed.

[0101] Experimental Example 3. Western blot

[0102] Western blot analysis of HMC-1 and T-MSC-derived exosomes was performed to evaluate the expression of CD63, CD9, CD81, calnexin, and SEMA7A, and all exosomes were quantified using a BCA protein assay kit (Thermo Fisher Scientific). A total of 15 μg of exosomes were loaded onto an SDS-polyacrylamide gel for Western blotting. The separated proteins were electrotransferred to membranes and incubated overnight with primary antibodies against CD9 (sc-13118, Santa Cruz Biotechnology), CD63 (sc-59284, Santa Cruz Biotechnology), CD81 (sc-166029, Santa Cruz Biotechnology), and SEMA7A (sc-376149, Santa Cruz Biotechnology).

[0103] Meanwhile, the expression of fibroblast activation markers was determined using antibodies against type I collagen (sc-293182, Santa Cruz Biotechnology), fibronectin (sc-8422, Santa Cruz Biotechnology), and α-SMA (sc-32251, Santa Cruz Biotechnology). The expression of β-actin was measured using anti-β-actin (sc-47778, Santa Cruz Biotechnology) antibody as an internal control.

[0104] After thorough washing, the membrane was reacted with HRP-conjugated secondary antibody (HRP anti-mouse IgG, Sigma-Aldrich).

[0105] Afterwards, the expression signal of each marker protein was detected using SuperSignal West Femto Substrate (Thermo Fisher Scientific) and scanned using ImageQuant LAS 3000 equipment (GE Healthcare, Little Chalfont, UK).

[0106] Experimental Example 4. MTT Assay

[0107] To assess the viability of dermal fibroblasts in the presence of HMC-1 exosomes, an MTT (Sigma-Aldrich) assay was performed. Specifically, fibroblasts in 96-well plates were treated with various concentrations of HMC-1 exosomes (0.2, 0.5, 1, 2, 5, and 10 μg / mL). After 24 h, MTT and dimethyl sulfoxide were sequentially added to all wells to dissolve the dark blue formazan crystals.

[0108] Experimental Example 5. Transfection of HMC-1 cells to suppress SEMA7A expression.

[0109] To reduce the expression of endogenous SEMA7A, HMC-1 cells were transfected with SEMA7A-specific siRNA oligonucleotide (sc-63010, Santa Cruz Biotechnology) using siRNA transduction reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions.

[0110] Non-targeting siRNA oligonucleotides (Santa Cruz Biotechnology) were used as negative controls, and conditioned media were harvested for exosome isolation 48 h after transduction.

[0111] Experimental Example 6. Mouse Model Experiment with Wound Induction

[0112] To evaluate the wound healing efficacy, a full-thickness excisional wound mouse model was established.

[0113] Specifically, 10-week-old male C57BL / 6 mice (OrientBio, Gyeonggi-do, Korea) and B6.Cg-KitW-sh mice (# 030764, The Jackson Laboratory) were anesthetized, and the dorsal hair was removed. A full-thickness excisional wound was then induced on the mid-dorsal skin using a sterile 8-mm biopsy punch. Immediately after wound induction, the mice were randomly divided into two groups. The control group received PBS, and the treatment group received HMC-1-derived exosomes (20 μg / mouse) directly applied to the wound site. The wounds were left uncovered, and the wound size was monitored and photographed at designated time points for analysis. All mice were sacrificed 8–9 days after wound induction, and skin tissues from the wound site were collected for subsequent analysis.

[0114] This experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University College of Medicine (Approval No. EWHA MEDIACUC 24-048-t).

[0115] Experimental Example 7. Histological Analysis

[0116] Skin tissue samples from the wound site were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness. The tissue sections were then stained with hematoxylin-eosin (H&E) to evaluate the overall histological characteristics and tissue morphology.

[0117] Additionally, tissue sections were stained with Masson's trichrome to assess the degree of collagen deposition. Masson's trichrome-stained sections were then analyzed using ImageJ software (NIH, USA) to quantify collagen deposition.

[0118] Experimental Example 8. Proteomics Analysis of HMC-1 Exosomes

[0119] Protein samples were concentrated by ultrafiltration and separated by SDS-PAGE. Gel plugs were reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide, and digested with trypsin overnight.

[0120] Digested peptides were extracted, dried, and analyzed using a nano LC-MS / MS analyzer (Dionex Ultimate 3000 and Eclipse, Thermo Fisher Scientific) using data-independent acquisition (DIA). Peptides were separated using a linear gradient of acetonitrile on a C18 column, and mass spectrometry (MS) spectra were collected at high resolution in the range of 400–1200 m / z. Data were searched using DIA-NN (v1.8.1) in a predicted-library and library-free manner. Functional annotation was performed through KEGG pathway analysis using DAVID (2021), and the top 500 protein-coding genes were identified.

[0121] Experimental Example 9. Statistical Analysis

[0122] Data are expressed as mean ± standard error of the mean (SEM). Statistical significance was analyzed using one-way and two-way ANOVA using GraphPad Prism 10 software (GraphPad Software Inc., San Diego, CA, USA). A P value less than 0.05 was considered statistically significant.

[0123] Example 1. Characterization of mast cell-derived exosomes

[0124] The size distribution profile of mast cell-derived exosomes was confirmed through Nanosight particle tracking analysis, and it was found to contain nanoparticles with an average size of 130 nm (Fig. 1a).

[0125] Meanwhile, Western blotting was performed to confirm marker proteins expressed in exosomes derived from mast cells or tonsil-derived mesenchymal stem cells. As a result, typical exosome marker proteins (CD63, CD9, CD81) were detected, and Calnexin, an endoplasmic reticulum (ER) membrane protein, was not detected, confirming that it was a pure exosome preparation (Fig. 1b).

[0126] In addition, there was no significant difference in the average size of exosomes (Size) between exosomes derived from tonsil stem cells (T-MSCs) and exosomes derived from mast cells, but it was confirmed that the particle number contained a significantly larger number of nanoparticles in exosomes derived from mast cells (Figures 1c to 1e).

[0127] Example 2. Confirmation of the effect of mast cell-derived exosomes on promoting fibroblast growth and activity.

[0128] According to the MTT assay, mast cell-derived exosomes were found to significantly promote the growth of fibroblasts, and in particular, when the exosomes were treated at a concentration of 1 μg / mL, the proliferation (% of cell viability) of fibroblasts was the highest (Fig. 2a).

[0129] Based on the above experimental results, fibroblasts were treated with mast cell-derived exosomes at a concentration of 1 μg / mL and cultured for 24 hours. Changes in marker expression were then confirmed through Western blotting. As a result, compared to the control group, the expression of fibroblast activation markers (α-SMA) and extracellular matrix proteins (type I collagen, fibronectin) increased (Fig. 2b).

[0130] That is, it was confirmed that the growth and activity of fibroblasts were enhanced by mast cell-derived exosomes (especially, at a concentration of 1 μg / mL).

[0131] Example 3. Confirmation of the wound healing effect of mast cell-derived exosomes.

[0132] B6.Cg-Kit, a genetically modified mouse lacking mast cells W-sh After inducing wounds of 8 mm in diameter in the C57BL / 6J WT group, which is a group and wild type mice, PBS or mast cell-derived exosomes were applied and observed for a total of 8 days (Fig. 3). It was confirmed that there was a difference in the degree of wound healing between each group (Figs. 4a and 4b).

[0133] When PBS was applied to the wound site to induce natural healing, B6.Cg-Kit was compared with the C57BL / 6J WT group (WT-PBS). W-sh Group (Kit W-sh -PBS) showed little reduction in wound size. In contrast, B6.Cg-Kit coated with mast cell-derived exosomes W-sh Group (Kit W-sh -Exosome) reduced the wound size to a degree comparable to the natural healing power of the C57BL / 6J WT group (WT-Exosome) (Fig. 5). In addition, in both the mast cell-deficient and wild type mouse groups, the wound size was statistically significantly reduced when mast cell-derived exosomes were applied compared to when PBS was applied (***P = 0.0002, **** P <0.0001). In particular, when mast cell-derived exosomes were applied to the wounds of the wild type mouse group (WT-Exosome), the time it took for the wound size to be reduced by 50% was 5 days faster than that of the control group (WT-PBS) (Fig. 5).

[0134] Next, the dorsal skin tissue was stained with H&E 8 days after wound induction, and the results showed that B6.Cg-Kit applied with mast cell-derived exosomes W-sh Both the group and the C57BL / 6J WT group showed a decrease in the number of adipocytes and an increase in tissue density at the wound base, indicating enhanced tissue remodeling (Fig. 6).

[0135] Therefore, we confirmed that mast cell-derived exosomes significantly reduce wound size and shorten the initial wound healing period.

[0136] Example 4. Characterization and Proteomics Analysis of Mast Cell-Derived Exosomes

[0137] To identify components contributing to the wound healing effect of mast cell-derived exosomes, proteomic profiling and functional enrichment analysis were performed.

[0138] First, we performed KEGG pathway analysis on the top 500 proteins. As a result, we confirmed that exosomal proteins were significantly distributed in pathways related to ribosome function, neurodegeneration, and cytoskeletal regulation (Fig. 7a).

[0139] Furthermore, analysis of cellular components in Gene Ontology revealed that these proteins were primarily associated with the cytoplasm, extracellular exosomes, and cytoplasmic compartments (Fig. 7b). This suggests that these proteins originate from exosomes and have extracellular functions.

[0140] Furthermore, biological process analysis revealed significant correlations with various processes, including translation, mRNA splicing, apoptosis inhibition, and positive regulation of cell proliferation (Fig. 7c), suggesting their potential contribution to tissue regeneration.

[0141] Furthermore, molecular function analysis revealed significant activity related to protein binding, RNA binding, and ATP binding (Fig. 7d), suggesting an active involvement in cell signaling and structural remodeling during wound healing.

[0142] Through the above experiments, it was confirmed that the above proteins were derived from exosomes, and that mast cell-derived exosomes play a key role in protein synthesis and processing and tissue regeneration, and can contribute to cell signaling and structural remodeling during wound healing.

[0143] Example 5. ECM synthesis and wound healing effects of SEMA7A in mast cell-derived exosomes.

[0144] Example 5-1. Evaluation of the ECM synthesis regulation function of SEMA7A

[0145] Western blot was performed to confirm the expression of SEMA7A (Semaphorin 7A), a key regulator of in vivo tissue regeneration, in mast cell-derived exosomes.

[0146] As a result, SEMA7A, known to be related to the normal migration of fibroblasts and the assembly function of the extracellular matrix, was highly expressed compared to exosomes derived from tonsillar stem cells (T-MSCs), which was the control group (Fig. 8a), confirming that mast cell-derived exosomes contain marker proteins that play an important role in wound healing.

[0147] Next, siRNA knockdown was performed to determine the effect of SEMA7A knockdown HMC-1 exosome treatment on ECM proteins (Collagen Type I, Fibronectin) and fibroblast activation marker (α-SMA) in dermal fibroblasts (Dermal FBs).

[0148] As a result, it was confirmed that the expression level of SEMA7A was significantly reduced in the knockdown (KD) group compared to the control group, confirming that the expression of SEMA7A was effectively suppressed through siRNA (Fig. 8b).

[0149] Next, we confirmed that the expression levels of ECM proteins (Collagen Type I, Fibronectin) were significantly reduced in the SEMA7A knockdown HMC-1 exosome-treated group (KD Exo) compared to the normal control group (Cont Exo). Meanwhile, we confirmed that there was no significant effect on the expression of the fibroblast activation marker (α-SMA) (Fig. 8c).

[0150] That is, the above experiment confirmed that SEMA7A is not essential for inducing fibroblast activity, but plays a role in selectively regulating ECM protein synthesis.

[0151] Example 5-2. Evaluation of the wound healing efficacy of SEMA7A using a mast cell-deficient mouse model.

[0152] B6.Cg-Kit coated with SEMA7A KD exosomes W-sh Both the group and the C57BL / 6J WT group showed significantly lower wound healing efficacy compared to the control exosomes. Specifically, on days 3 and 5, B6.Cg-Kit treated with mast cell-derived exosomes W-sh Both the group (***P < 0.001) and the C57BL / 6J WT group (**P < 0.01) showed significantly improved wound healing effects compared to the PBS treatment group. In particular, the B6.Cg-Kit W-sh We confirmed that there was a significant difference (*P < 0.05) between the group treated with mast cell-derived exosomes and the group treated with SEMA7A KD exosomes (Figures 9a and 9b).

[0153] This means that mast cell-derived SEMA7A plays a key role in an environment where endogenous mast cells are deficient.

[0154] Example 6. Confirmation of the tissue regeneration and ECM remodeling effects of SEMA7A.

[0155] Example 6-1. H&E stained tissue observation

[0156] To confirm the tissue regeneration and ECM remodeling effects of SEMA7A in mast cell-derived exosomes, histological analysis was performed based on H&E staining.

[0157] As a result, B6.Cg-Kit treated with mast cell-derived exosomes W-sh Both the group and the C57BL / 6J WT group showed significantly enhanced epithelial regeneration and dermal tissue regeneration compared to the PBS-treated group. In particular, wounds treated with mast cell-derived exosomes showed a thicker and more continuous epidermal layer and dense granulation tissue. In contrast, both mouse groups treated with SEMA7A KD exosomes still formed scabs, and the dermal structure remained irregular, showing similar patterns to the PBS-treated group (Figs. 10a and 10b).

[0158] Example 6-2. Observation of Masson's trichrome stained tissue

[0159] To determine the effect of SEMA7A in mast cell-derived exosomes on collagen deposition and ECM remodeling, histological analysis using Masson's trichrome staining was performed.

[0160] As a result, B6.Cg-Kit treated with mast cell-derived exosomes W-sh Both the group and the C57BL / 6J WT group showed significantly higher collagen deposition compared to the PBS treatment group, confirming that ECM remodeling was promoted (Fig. 11a, collagen deposition area is indicated in blue).

[0161] On the other hand, both groups of mice treated with SEMA7A KD exosomes showed a significantly reduced collagen production effect compared to the mast cell-derived exosome-treated group, and showed a somewhat improved level compared to the PBS-treated group (Fig. 11b).

[0162] In other words, these results suggest that SEMA7A in mast cell-derived exosomes is a key factor regulating collagen synthesis and ECM remodeling during wound healing.

Claims

1. A pharmaceutical composition for preventing or treating skin wounds containing exosomes from mast cells.

2. A pharmaceutical composition according to claim 1, wherein the mast cells are human-derived mast cells.

3. A pharmaceutical composition according to claim 1, characterized in that the exosomes of the mast cells have a diameter of 100 to 160 nm.

4. A pharmaceutical composition according to claim 1, characterized in that the exosome of the mast cell contains SEMA7A.

5. A pharmaceutical composition according to claim 1, wherein the exosomes of the mast cells promote the growth and activity of fibroblasts.

6. A pharmaceutical composition according to claim 1, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wound, stomatitis, bedsore, pressure ulcer, diabetic ulcer / exfoliation, post-herpetic ulcer, drug-induced ulcer, skin ulcer, damage due to dermatitis, radiation damage, and chemical damage.

7. A composition for external application to the skin, comprising exosomes from mast cells.

8. A composition for external application to the skin, wherein the mast cells in paragraph 7 are human-derived mast cells.

9. A food composition for preventing or improving skin wounds containing exosomes of mast cells.

10. A food composition according to claim 9, wherein the mast cells are human-derived mast cells.

11. A cosmetic composition for skin regeneration, comprising exosomes from mast cells.

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