KR20210152151A - Composition comprising an exosome having enhanced effects of anti-inflammation, wound healing or accelerating wound healing as an active ingredient and manufacturing method thereof - Google Patents

Abstract

The present invention provides a method for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes, comprising a step of culturing animal-derived cells in a medium containing TNF-α and interferon-γ. The present invention can enhance the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes, and can economically and efficiently produce exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy in high yield, thereby being commercially and/or clinically useful. The composition comprising, as an active ingredient, an exosome having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention is excellent in anti-inflammatory effect, wound treatment or an effect of promoting wound treatment.

Description

Composition comprising an exosome having enhanced effects of anti-inflammation, wound healing or wound healing promotion as an active ingredient as an active ingredient, and a method for producing the same method

The present invention relates to a composition comprising, as an active ingredient, an exosome having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, and a method for producing the same.

In addition, the present invention relates to an anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound healing, quasi-drugs, external preparations for skin, or functional cosmetic composition comprising the composition.

Further, the present invention relates to a method for preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or accelerating wound healing by using the composition.

Inflammation is a defense reaction of the living body that appears in response to a pathological condition caused by physical or chemical trauma, infection by bacteria, fungi or viruses, and various allergens. The inflammatory response appears as part of the innate immune response. Various substances and physiological and chemical phenomena are involved in the inflammatory response, and recent studies have revealed that various inflammatory cytokines play an important role in the inflammatory response. Major cytokines involved in the inflammatory response include IL-1α, IL-1β, IL-27, TNF-α, IL-6, IL-8, IL-12, IFN-β, and their expression levels and Increased secretion and activation are related to a series of complex physiological responses such as secretion of inflammatory mediators, immune cell infiltration, cell migration and tissue destruction, and symptoms such as erythema, edema, fever, and pain.

In general, the inflammatory reaction is not a big problem if the source of infection is removed and the damaged tissue is regenerated, and the diseased area is restored to normal. lead to inflammatory diseases. Nonsteroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, neuropeptide antagonists, COX inhibitors, antihistamines, and immunosuppressive agents such as cyclosporin A are used for the relief or treatment of inflammatory reactions or inflammatory diseases resulting from skin atrophy, vasodilation, pigmentation There are problems causing side effects such as loss of room, hypersensitivity reaction, tolerance, and neutropenia. In addition, these drugs have limitations in helping to control symptoms to an appropriate level rather than fundamental treatment.

Recently, research on the development of inflammatory disease treatments or functional cosmetics using natural products is active. In the case of inflammatory disease treatment or functional cosmetics using these natural substances as raw materials, a large amount of use is required to obtain anti-inflammatory effects due to the low content of active ingredients in natural extracts, and most of these ingredients are natural substances. However, more scientific research is needed on the actual anti-inflammatory effect. In addition, functional cosmetics containing a plant extract as an active ingredient may cause a foreign body sensation in the process of evaporating the solution after application to the skin, and there is a problem that the effect duration is short.

On the other hand, wounds not only cause pain, bleeding, and dysfunction of tissue destruction, but also become defenseless against bacterial infection, which can lead to secondary infection or sepsis. Therefore, it is necessary for wounds to be healed as soon as possible while minimizing scarring and leaving no functional impairment in skin tissue.

Wound healing proceeds in four stages: homeostasis, inflammatory, proliferative, and remodeling. Each stage of wound healing involves clotting for hemostasis, replenishment of neutrophils to destroy bacterial and necrotic tissue, and replenishment of macrophages. During the proliferative phase, angiogenesis occurs, where endothelial cells migrate to the wound site and at the same time fibroblasts migrate to the wound site, helping to produce granulation tissue. Re-epithelialization occurs by the formation of granulation tissue. In the final stage, the remodeling phase, a delicate balance between collagen degradation and synthesis appears, and collagen remodeling and vascular maturation appear. Control of the inflammatory response is essential for skin regeneration, and if the inflammatory response is maintained continuously, it causes chronic wounds and creates excessive scarring. Therefore, it is important to control the excessive inflammatory response during wound healing.

In wound healing, cells such as keratinocytes, fibroblasts, endothelial cells, and macrophages cooperate, and processes such as migration, proliferation and differentiation of these cells are various growth factors. It is known to be regulated by either cytokines. For this reason, a wound treatment method using growth factors and cytokines is being developed.

However, the wound treatment method using growth factors and cytokines has a problem in that the treatment period is long and one specific growth factor or cytokine does not show a satisfactory therapeutic effect when considering the wound healing mechanism by the complex action of several proteins. In addition, a method of using a mesenchymal stem cell culture medium for wound healing has been proposed, focusing on the point that mesenchymal stem cells secrete growth factors and cytokines. However, in the case of non-activated mesenchymal stem cells, there is a problem in that the amount of cytokines and growth factors secreted from the cells is small. Since components such as derived serum are also included, there is a high possibility of exposure to various risks when used on a wound site.

Recently, it has been reported that cell secretome contains various bioactive factors that regulate cell behavior. Since it contains extracellular vesicles, research on its components and functions is being actively conducted.

Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs). The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.

Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made of a double phospholipid membrane identical to the structure of the cell membrane, and contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo inside. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cells from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behavior such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

That is, exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells. Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .

However, despite the usefulness of exosomes and various applications in medical and cosmetic fields, in order to apply exosomes commercially and/or clinically, limited productivity problems must be solved, and the physiological activity of exosomes, for example, There is a need to enhance anti-inflammatory efficacy, wound healing efficacy, and/or wound healing facilitating efficacy, and the like.

In this regard, although exosome production methods and efficacy enhancement methods have been proposed, continuous improvement can be made in the technical fields related to the production and efficacy enhancement of exosomes as in other technical fields.

On the other hand, it should be understood that the matters described as the above background art are only for improving understanding of the background of the present invention, and are not cited as an admission that they can be used as "prior art" of the present invention.

Republic of Korea Patent Publication No. 10-1985941 (2019.06.04)

It is an object of the present invention to provide a composition comprising as an active ingredient an exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, and a method for producing the same.

Another object of the present invention is to provide an anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment, quasi-drugs, external preparations for skin, or functional cosmetic composition comprising the composition.

Another object of the present invention is to provide a method for preventing, suppressing, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or accelerating wound healing by using the composition.

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby. Further, other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present inventors have been repeating intensive studies on exosomes with enhanced anti-inflammatory efficacy, wound healing efficacy, and / or wound healing efficacy, when producing exosomes using a combination of TNF-α and interferon-γ The present invention was completed by confirming that the anti-inflammatory efficacy of the produced exosomes, the efficacy of wound treatment, and/or the efficacy of promoting wound healing were improved.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used to mean Depending on the isolation environment, conditions and methods, etc., the extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may have meanings including nanovesicles that do not have the composition of exosomes.

As used herein, the term "exosomes" refers to endoplasmic reticulum with a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of the cell membrane (provided that the separation target is The particle size of exosomes may vary depending on the type of cell to be used, separation method and measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015)) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known

On the other hand, the term "exosome" used herein has a nano-sized vesicle structure that is secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -Similar vesicles) are included.

In the present invention, the type of animal-derived cells from which the exosomes are derived is not limited, but may be stem cells or immune cells as an example not limiting the present invention. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages.

As an example not limiting the present invention, the adult stem cells are one or more types of adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. It may be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly and placenta. Preferably, the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably may be adipose-derived stem cells. The type of the stem cell or the immune cell is not limited as long as there is no risk of infection by a pathogen and does not cause immune rejection, but preferably a human stem cell or human-derived immune cell.

However, as long as it does not cause adverse effects on the human body, of course, various animal cells that are used in the art or that can be used in the future can be used. For example, it is also possible to use HEK 293 cells or HEK 293T cells. Therefore, it should be understood that the adipose stem cells used in the examples to be described below are examples of animal cells that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "pre-treatment" refers to culturing animal-derived cells in a medium containing TNF-α and interferon-γ, and “pre-treatment exosomes” refers to animal-derived cells in a medium containing TNF-α and interferon-γ. It refers to exosomes obtained by culturing cells. "Untreated" means culturing animal-derived cells in a medium not containing TNF-α and interferon-γ, and "untreated exosomes or untreated control" refers to any stage of cell culture and exosome production. TNF-α and interferon-γ also refers to the obtained exosomes without treatment.

As used herein, the term "serum-free medium" or "SFM" refers to, for example, a culture medium in which serum such as Fetal Bovine Serum (FBS) is not added to a basal medium (optionally including antibiotics and/or antifungals) means, "EDM" is a platelet lysate, preferably a human platelet lysate, more preferably a human platelet lysate from which exosomes and extracellular vesicles derived from platelet lysate are added to the basal medium culture medium (optionally containing antibiotics and/or antifungals). As an example not to limit the present invention, the basal medium is α-MEM, DMEM, DMEM F12, 199 medium, RPMI-1640, L-15, Hum F-10, Hum F-12, DM-160, DM-170, etc. can

As used herein, the term "anti-inflammatory" means preventing, suppressing, alleviating, ameliorating or treating inflammation, and inflammatory diseases are examples that are not limited to the present invention, and include dermatitis, atopic dermatitis, eczema, bacterial infection, virus Inflammation due to infection or fungal infection, burns, inflammation due to burns, wounds, inflammation due to wounds, ulcerative colitis, arthritis, rheumatoid arthritis, hepatitis, nephritis, and the like.

As used herein, the term “wound” refers to a state in which a living body is damaged, and a tissue constituting the internal or external surface of the living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ or vascular tissue It encompasses segmented or disrupted pathological conditions. As an example not limiting the present invention, the wound is abrasion, laceration, cut, cut, avulsion, pressure sore, pit wound, tissue destruction by radiation, penetrated wound, gunshot wound ( gun shot wound), burns, frostbite, surgical wounds, sutures after plastic surgery, chemical wounds, etc., and may include damage to any part of an object.

As used herein, the term "iontophoresis" refers to a method of allowing an ionized active ingredient to penetrate the skin with an electrical repulsive force by flowing a microcurrent to the skin to which an active material is applied, giving a potential difference, and changing the electrical environment of the skin means Iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into an electrode patch on the skin and a microcurrent is introduced into the skin, and a battery is installed in the electrode patch itself to protect the skin. A method in which microcurrent is introduced, a method in which microcurrent is introduced into the skin through a patch equipped with a reversed electrodialysis means that generates an electric current through a difference in ion concentration between a high-concentration electrolyte solution and a low-concentration electrolyte solution, etc. can However, the present invention is not limited thereto, and it goes without saying that various types of iontophoresis may be used.

The present invention provides a method for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes, comprising the step of culturing animal-derived cells in a medium containing TNF-α and interferon-γ. For example, the method for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing TNF-α and interferon-γ; (b) washing the animal-derived cells, (c) culturing the washed animal-derived cells in a serum-free medium and collecting a culture solution, (d) separating the exosomes from the collected culture solution may include

In the method for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes of one embodiment of the present invention, platelet lysate may be added to the serum-free medium of step (c). The platelet lysate may be preferably a human platelet lysate, and more preferably a human platelet lysate from which exosomes and extracellular vesicles derived from platelet lysate are removed.

In the method for enhancing the anti-inflammatory, wound healing or wound healing promoting efficacy of exosomes of one embodiment of the present invention, the TNF-α and interferon-γ each have a concentration of about 1 ng/mL to 100 ng/mL in the medium, preferably may be included in a concentration of about 10 ng/mL to 30 ng/mL, for example, may be included in a concentration of about 20 ng/mL.

In the method for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of the exosome of one embodiment of the present invention, the culture may be performed for about 12 hours to 48 hours, for example, it may be performed for 24 hours.

The present invention provides a medium for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of animal cell-derived exosomes, comprising TNF-α and interferon-γ.

In the medium for enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of animal cell-derived exosomes of one embodiment of the present invention, the TNF-α and interferon-γ are each about 1 ng/mL to 100 ng/mL in the medium. It may be included at a concentration of, preferably, a concentration of about 10 ng/mL to 30 ng/mL, for example, it may be included at a concentration of about 20 ng/mL.

In addition, the present invention includes, as an active ingredient, an exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy obtained by culturing animal-derived cells in a medium containing TNF-α and interferon-γ, anti-inflammatory, wound treatment Or it provides a composition for promoting wound healing.

For example, the composition of one embodiment of the present invention comprises the steps of: (a) culturing animal-derived cells in a medium containing TNF-α and interferon-γ; (b) washing the animal-derived cells; (c) culturing the washed animal-derived cells in a serum-free medium and collecting a culture solution, and (d) separating the exosomes from the collected culture solution.

In the composition of one embodiment of the present invention, platelet lysate may be added to the serum-free medium of step (c). The platelet lysate may be preferably a human platelet lysate, and more preferably a human platelet lysate from which exosomes and extracellular vesicles derived from platelet lysate are removed.

In the composition of one embodiment of the present invention, the TNF-α and interferon-γ are each at a concentration of about 1 ng/mL to 100 ng/mL in the medium, preferably at a concentration of about 10 ng/mL to 30 ng/mL It may be included, for example, may be included at a concentration of about 20 ng / mL.

In the composition of one embodiment of the present invention, the culturing may be carried out for about 12 hours to 48 hours, for example, it may be carried out for 24 hours.

By way of example and not limitation of the present invention, the composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.

The composition of one embodiment of the present invention may be a pharmaceutical composition, a quasi-drug, an external preparation for skin, or a functional cosmetic composition. For example, the pharmaceutical composition may be in the form of an injection, injection, spray, liquid, or patch, and the quasi-drug or external preparation for skin may be a liquid, ointment, cream, spray, patch, gel, or aerosol. have. In addition, the functional cosmetic composition may be, for example, a lotion or a cream.

When the composition of one embodiment of the present invention is used as a pharmaceutical composition, it may include a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. .

Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance to the patient by any suitable method, and the administration route of the pharmaceutical composition is administered through any general route as long as the drug can reach the target tissue. can be For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration includes intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration. ) administration, intralesional and intracranial administration, transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intralymphatic administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, rectal administration, etc. can be mentioned However, it is not limited thereto and does not exclude various administration methods known in the art. In addition, the pharmaceutical composition of one embodiment of the present invention may be administered by any device capable of transporting an active substance to a target tissue or cell. In addition, the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect anti-inflammatory, wound healing, and/or wound healing promoting effects.

The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, or a suppository. The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be prepared as an injection. The injection of one embodiment of the present invention may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection used by dissolving or suspending, but is not limited thereto. The injection of one embodiment of the present invention is distilled water for injection, vegetable oil (eg, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth hyposalicylate, depending on the type , Arsenobenzol sodium, or at least one of streptomycin sulfate, and may optionally include a stabilizer or a preservative.

The blending ratio of the pharmaceutical composition of one embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional ingredients as described above. For example, based on the total amount of the injection, the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, a suitable dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted depending on the severity of the disease, the type of formulation, the formulation method, the patient's age, sex, weight, health status, diet, excretion rate, administration time and administration method. can For example, when administering the pharmaceutical composition of one embodiment of the present invention to an adult, it may be administered at a dose of 0.001 mg/kg to 100 mg/kg per day, divided into 1 to several times.

On the other hand, when the composition of one embodiment of the present invention is prepared as a quasi-drug, an external preparation for skin and/or a cosmetic composition, it is usually used as a quasi-drug, external preparation for skin and/or functional cosmetic composition within the range that does not impair the effects of the present invention. Ingredients such as moisturizing agents, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc. can be appropriately blended as needed.

In addition, the quasi-drugs, external preparations for skin and/or functional cosmetic composition of one embodiment of the present invention has an anti-inflammatory, wound treatment or wound treatment promoting effect, in addition to the enhanced exosome, its action (eg, anti-inflammatory, wound treatment or wound treatment promotion) etc.), conventionally used anti-inflammatory agents, anti-inflammatory agents, wound healing agents, and/or moisturizing agents may be mixed and used together. For example, the composition comprising, as an active ingredient, an exosome having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention is a hydrogel, hyaluronic acid, hyaluronic acid salt (eg sodium hyaluronate, etc.), or hyaluronic acid. It may be supported on or mixed with at least one kind of ronic acid gel. In the quasi-drugs, external preparations for skin and/or cosmetic composition of one embodiment of the present invention, the type of the hydrogel is not limited, but preferably a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. and the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin.

The quasi-drugs, external preparations for skin and/or functional cosmetic compositions of one embodiment of the present invention are, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, It can be applied to various forms such as spray, aerosol, mist, foundation, powder, and oily paper. For example, the quasi-drug, external preparation for skin and/or functional cosmetic composition may be applied or deposited on at least one surface of a patch, mask pack, or mask sheet.

The functional cosmetic composition of one embodiment of the present invention may be used for the purpose of anti-inflammatory, wound healing promotion, and/or wound improvement, etc., and the functional cosmetic formulation may be prepared in any formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, flexible lotion, nourishing lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , soap, shampoo, surfactant-containing cleansing, bathing agent, body lotion, body cream, body oil, body essence, body wash, hair dye, hair tonic, etc., but is not limited thereto.

The quasi-drugs, external preparations for skin and/or functional cosmetic composition of one embodiment of the present invention include components commonly used in quasi-drugs, external preparations for skin and/or functional cosmetic compositions, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and conventional adjuvants such as perfumes and carriers. In addition, in each formulation for quasi-drugs, external preparations for skin and/or functional cosmetic composition, other ingredients may be appropriately selected and formulated by those skilled in the art without difficulty depending on the type or purpose of use of the quasi-drug, external preparation for skin and/or functional cosmetic composition. have.

In addition, the present invention prevents, suppresses, alleviates, ameliorates or treats inflammatory diseases, or treats wounds, Or it provides a method for accelerating wound healing (accelerating wound healing).

The method of one embodiment of the present invention comprises administering a therapeutically effective amount of the composition to a mammal, or treating the composition to an inflamed site and/or a wound site of the mammal.

In the method of one embodiment of the present invention, the composition may be administered or treated to a mammal by injection, microneedling, iontophoresis, application, or a combination thereof.

In the method of one embodiment of the present invention, the composition is, for example, a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, It can be applied to various forms such as mist, foundation, powder, and oil paper. For example, the composition may be applied or deposited on at least one surface of the mask pack, mask sheet, or patch.

The method of one embodiment of the present invention may further include performing iontophoresis by flowing a microcurrent to an inflammatory site and/or a wound site of the mammal treated with the composition.

In the method of one embodiment of the present invention, performing the iontophoresis may be performed by contacting or attaching an iontophoresis device to an inflammatory site and/or a wound site of the mammal.

In the method of one embodiment of the present invention, the iontophoresis device comprises a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery. It may include at least one type of battery selected from.

In the method of one embodiment of the present invention, the mammal may be a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig.

The present invention can enhance the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes, and can economically and efficiently produce exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy in high yield, commercial and / or clinically useful. The composition comprising, as an active ingredient, an exosome having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention is excellent in anti-inflammatory effect and the effect of promoting wound treatment or wound treatment.

Therefore, the exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention may be usefully utilized as an active ingredient in a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion, quasi-drugs, external application for skin, or functional cosmetic composition. can

On the other hand, the scope of the present invention is not limited by the above-described effects.

1 is an untreated exosome obtained using ultrafiltration after culturing stem cells in a medium not containing TNF-α and interferon-γ, and a medium containing TNF-α and interferon-γ It is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on each of the pre-treated exosomes obtained using ultrafiltration after culturing stem cells in
Figure 2 is a comparative graph showing that when TNF-α and interferon-γ are pretreated according to an embodiment of the present invention, the productivity of the exosomes is significantly increased compared to the productivity of the TNF-α and interferon-γ untreated control group. Figure 2A is a graph comparing the number of exosome particles in the culture medium (CM) before performing ultrafiltration with respect to the culture medium (CM), and FIG. 2B is a graph obtained after performing ultrafiltration with respect to the culture medium (CM). It is a graph comparing the number of exosome particles in the solution, and FIG. 2C is a graph showing the comparison results of FIGS. 2A and 2B together.
3 is an untreated exosome obtained by ultracentrifugation after culturing stem cells in a medium not containing TNF-α and interferon-γ, and stem cells in a medium containing TNF-α and interferon-γ; It is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on each of the pre-treated exosomes obtained by ultracentrifugation after culturing.
Figure 4 is a comparative graph showing that when TNF-α and interferon-γ are pretreated according to an embodiment of the present invention, the productivity of the exosomes is significantly increased compared to the productivity of the TNF-α and interferon-γ untreated control group. 4A is a graph comparing the number of exosome particles in the culture medium (CM) before performing ultracentrifugation for the culture medium (CM), and FIG. 4B is a graph obtained after performing ultracentrifugation for the culture medium (CM). It is a graph comparing the number of exosome particles in the solution (EXO), and FIG. 4C is a graph showing the comparison results of FIGS. 4A and 4B together.
5 shows the content (reflecting the content and productivity of exosomes) of the CD63 marker (exosome-specific marker) in the experimental group pretreated with TNF-α and interferon-γ according to an embodiment of the present invention, the untreated control group, interferon It is a comparative graph showing that the content of the CD63 marker significantly increased in the -γ alone treatment group and the TNF-α alone treatment group.
6 is a graph showing the results of inflammatory cytokine analysis using a multiplex panel, when the pre-treated exosomes according to an embodiment of the present invention were treated with respect to RAW 264.7 cells by concentration (based on the number of particles per mL) It indicates that IL-6 induced by LPS is significantly reduced compared to the case of treatment with the treated exosomes (general exosomes).
7 is a graph showing the results of inflammatory cytokine analysis using a multiplex panel, when the pre-treated exosomes according to an embodiment of the present invention were treated with respect to RAW 264.7 cells by concentration (based on the number of particles per mL) It shows that IL-1α induced by LPS is significantly reduced compared to the case of treatment with the treated exosomes (general exosomes).
8 is a process of pre-treatment with TNF-α and interferon-γ in the proliferation phase during the cell culture process, and cells after washing the cells to produce an exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention; It is a schematic diagram showing the process of recovering the culture medium containing the secreted exosomes by culturing in SFM medium or EDM medium.
9 is a graph showing the results of inflammatory cytokine analysis using a multiplex panel, exosomes produced in EDM medium after pretreatment with TNF-α and interferon-γ according to an embodiment of the present invention is produced in SFM medium It shows that IL-6 is significantly reduced compared to the exosomes.
10 is a graph showing the results of inflammatory cytokine analysis using a multiplex panel, exosomes produced in EDM medium after pretreatment with TNF-α and interferon-γ according to an embodiment of the present invention is produced in SFM medium It shows that IL-27 is significantly reduced compared to the exosomes.
11 is a graph showing the results of inflammatory cytokine analysis using a multiplex panel, exosomes produced in EDM medium after pretreatment with TNF-α and interferon-γ according to an embodiment of the present invention is produced in SFM medium It shows that IL-1α is significantly reduced compared to the exosomes.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. The references cited herein are incorporated herein by reference.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Example

Example 1: Cultivation of cells

According to a cell culture method known in the art to which the present invention pertains, 10% FBS (Fetal Bovine Serum; purchased from ThermoFisher Scientific) and 1% penicillin-streptomycin (purchased from ThermoFisher Scientific) are added at _{5% CO 2 , 37°C conditions.} Adipose-derived stem cells were cultured in MEM-α (Minimum Essential Medium alpha; purchased from ThermoFisher Scientific) medium. When not treated with TNF-α and interferon-γ (IFN-γ), stem cells were maintained in culture for 24 hours. When TNF-α and interferon-γ (IFN-γ) were treated, TNF-α and IFN-γ were treated in the culture medium at a concentration of 20 ng/mL, respectively, and the stem cells were cultured for 24 hours (FIG. 8). See "1. Cell Proliferation Stage"). After incubation for 24 hours, both TNF-α and IFN-γ-treated stem cells and TNF-α and IFN-γ-untreated stem cells were washed with phosphate-buffered saline (“2. cells in FIG. 8 )” Washing"), cultured for 24 hours by replacing it with a serum-free medium or EDM medium, and the supernatant (hereinafter, culture solution) was recovered (see "3. Culture and recovery of culture for exosome production" in FIG. 8). On the other hand, EDM medium collects the permeate obtained after tangential flow filtration of human platelet lysates (hPL) to obtain a human platelet lysate from which extracellular vesicles and exosomes are removed, and the obtained platelets It can be prepared by adding the lysate to the basal medium.

In Examples 2 to 5 to be described later, exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy obtained by being produced in a serum-free medium after pretreatment with TNF-α and interferon-γ are described, Examples In 6, it is explained that exosomes produced in EDM medium after pretreatment with TNF-α and interferon-γ have superior anti-inflammatory, wound healing or wound healing promoting efficacy than exosomes produced in SFM medium.

Example 2: Isolation and purification of exosomes

Example 2-1: Separation using ultrafiltration method

Ultrafiltration was used for separation and concentration of exosomes from the culture medium recovered in Example 1. A centrifugal filter (purchased from Millipore) was used as a filter for ultrafiltration. Centrifugal filters can be selected by various molecular weight cutoffs (MWCOs). Exosomes were selectively separated and concentrated by the selected MWCO, and particles smaller than the MWCO, proteins, lipids, nucleic acids, and low molecular weight compounds were removed.

A centrifugal filter of MWCO 30,000 Da (Dalton) was used to separate and concentrate the exosomes. While the culture medium was concentrated to a volume of about 1/200 using ultrafiltration, substances smaller than MWCO were removed to separate exosomes.

On the other hand, as a method of isolating the exosomes from an animal-derived cell culture medium including stem cells, various methods known in the art may be used in addition to the separation method as described above. For example, for the isolation of exosomes, ultracentrifugation, density gradient centrifugation, tangential flow filtration (TFF), size exclusion chromatography, ion ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer A known separation method such as polymer based precipitation may be used. However, the separation method of the exosomes is not limited to the methods described above, and various separation methods that are used in the art or that can be used in the future are of course applicable.

Example 2-2: Isolation of exosomes using ultracentrifugation

In addition, exosomes were separated from the culture solution recovered in Example 1 using ultracentrifugation. The recovered culture solution was centrifuged sequentially at 4° C. to separate exosomes. First, in order to remove cells from the recovered culture medium, centrifugation was performed at 300 × g for 10 minutes, and then the supernatant was taken. Then, in order to remove cell debris from the supernatant, centrifugation was performed at 2,000 × g for 20 minutes, and then the supernatant was taken. In addition, in order to remove microvesicles from the supernatant, centrifugation was performed at 16,500 × g for 10 minutes, and then the supernatant was taken. After centrifugation was performed on the finally obtained supernatant at 120,000 × g for 120 minutes, the supernatant was removed and a pellet was taken, and then the pellet was washed with phosphate-buffered saline, and again 120,000 × g The supernatant was removed by centrifugation for 120 minutes, and the pellet was suspended in a phosphate buffer to separate exosomes.

Example 3: Characterization of isolated exosomes and evaluation of productivity

Example 3-1: Characterization and productivity evaluation of exosomes isolated by ultrafiltration

Exosomes isolated according to Example 2-1 were measured for particle size and concentration by nanoparticle tracking analysis (　NTA; purchased from Malvern). A graph of the NTA results of the exosomes isolated according to Example 2-1 is shown in FIG. 1 . As shown in FIG. 1, the pre-treated exosomes obtained using ultrafiltration after culturing stem cells in a medium containing TNF-α and interferon-γ according to an embodiment of the present invention (“TNF -α, IFN-γ treated") is an untreated exosome obtained using ultrafiltration after culturing stem cells in a medium not containing TNF-α and interferon-γ (“Untreated” in FIG. 1 ) It can be seen that the particle size distribution is uniform compared to that of ).

In addition, as a result of NTA comparative analysis as shown in FIGS. 2A, 2B and 2C, when pretreatment with TNF-α and interferon-γ (in FIGS. 2A, 2B and 2C, “TNF-α, IFN-γ treated Indicated by ") exosome productivity was approximately 2-3 times higher than that of the exosome productivity of the TNF-α and interferon-γ untreated control group (indicated as "Untreated" in FIGS. 2A, 2B and 2C).

That is, when the number of exosome particles in the culture medium (CM) is measured and compared before ultrafiltration is performed on the culture medium (CM), when TNF-α and interferon-γ are pre-treated, the productivity of exosomes is lower than that of TNF-α and The productivity of the interferon-γ untreated control group was significantly increased ( FIGS. 2A and 2C ). In addition, when the number of exosome particles in the solution obtained after ultrafiltration of the culture medium (CM) was measured and compared, when TNF-α and interferon-γ were pretreated, the productivity of exosomes was increased by TNF-α and interferon- The productivity of the γ-untreated control group was significantly increased ( FIGS. 2B and 2C ).

Therefore, the present invention can economically and efficiently produce exosomes with a uniform particle size distribution in high yield by performing a pretreatment process of culturing stem cells in a medium containing a combination of TNF-α and interferon-γ.

Example 3-2: Characterization and productivity evaluation of exosomes isolated by ultracentrifugation

The exosomes isolated according to Example 2-2 were measured for particle size and concentration by nanoparticle tracking analysis (　NTA; purchased from Malvern). A graph of the NTA results of the exosomes isolated according to Example 2-2 is shown in FIG. 3 . As shown in FIG. 3, the pre-treated exosomes obtained using ultracentrifugation after culturing stem cells in a medium containing TNF-α and interferon-γ according to an embodiment of the present invention (“TNF -α, indicated as “IFN-γ treated”) is an untreated exosome obtained by ultracentrifugation after culturing stem cells in a medium not containing TNF-α and interferon-γ (“Untreated” in FIG. 3 ) It can be seen that the particle size distribution is uniform compared to that of ).

In addition, as a result of NTA comparative analysis as shown in FIGS. 4A, 4B and 4C, when pretreatment with TNF-α and interferon-γ (in FIGS. 4A, 4B and 4C, "TNF-α, IFN-γ treated Indicated by ") exosome productivity was approximately 2-3 times higher than that of the exosome productivity of the TNF-α and interferon-γ untreated control group (indicated as "Untreated" in FIGS. 4A, 4B and 4C) was increased by approximately 2-3 times.

That is, when the number of exosome particles in the culture medium (CM) was measured and compared before performing ultracentrifugation with respect to the culture medium (CM), the productivity of exosomes decreased with TNF-α and interferon-γ when TNF-α and interferon-γ were pretreated. The productivity of the interferon-γ untreated control group was significantly increased ( FIGS. 4A and 4C ). In addition, when the number of exosome particles in the solution (EXO) obtained after performing ultracentrifugation on the culture medium (CM) was measured and compared, when TNF-α and interferon-γ were pretreated, the productivity of exosomes was increased by TNF-α and interferon-γ significantly increased compared to the untreated control group ( FIGS. 4B and 4C ).

Therefore, the present invention can economically and efficiently produce exosomes with a uniform particle size distribution in high yield by performing a pretreatment process of culturing stem cells in a medium containing a combination of TNF-α and interferon-γ.

Example 4: Analysis of increased productivity of exosomes by pretreatment of culturing stem cells in a medium containing a combination of TNF-α and interferon-γ

In addition, for accurate exosome productivity comparison analysis, the experimental group was classified as follows according to whether TNF-α and/or interferon-γ were included in stem cell culture for exosome production:

(1) untreated control group (Untreated): an experimental group in which exosomes were obtained according to Example 2-2 after culturing stem cells in a medium not containing TNF-α and interferon-γ;

(2) interferon-γ alone treatment group (IFN-γ treated): an experimental group in which exosomes were obtained according to Example 2-2 after culturing stem cells in a medium containing interferon-γ;

(3) TNF-α alone treatment group (TNF-α treated): an experimental group in which exosomes were obtained according to Example 2-2 after culturing stem cells in a medium containing TNF-α;

(4) TNF-α and interferon-γ pretreatment group (TNF-α, IFN-γ treated): Exosomes according to Example 2-2 after culturing stem cells in a medium containing TNF-α and interferon-γ the experimental group obtained.

After mixing the exosomes of each of the experimental groups (1) to (4) with Exosome-Human CD63 Flow Detection Reagent (Invitrogen™) overnight, PE mouse anti-human CD63 ( Mouse anti-Human CD63) (BD Parmigen™) was reacted and PE mean fluorescence intensity (MFI) was measured using flow cytometry.

On the other hand, since CD63 is a representative positive marker of exosomes, the CD63 content and the exosome content have linearity with each other, and an increase in the CD63 content means a proportional increase in the exosome content. According to this principle, human CD63 protein for each concentration is prepared by serial dilution of human CD63 protein of known concentration, and human CD63 protein for each concentration and exosome-human CD63 flow detection reagent (Exosome-Human CD63 Flow Detection) Reagent) (Invitrogen™) was mixed overnight and then reacted with PE Mouse anti-Human CD63 (BD Parmigen™) and PE mean fluorescence intensity (mean) using flow cytometry. Fluorescence intensity (MFI) was measured. And after generating a standard quantitative analysis graph that satisfies linearity of 0.99 or more by performing linear regression analysis on the human CD63 protein concentration value and the MFI measurement value corresponding thereto, using the generated standard quantitative analysis graph, the experimental group (1) ~ (4) Each CD63 content was determined.

As a result, the exosome content (CD63 content) measured in the experimental group (4) pretreated with TNF-α and interferon-γ of the present invention was the untreated control group of the experimental group (1), and the interferon-γ alone treatment of the experimental group (2). It was confirmed that the exosome content measured in the TNF-α alone treatment group of the group and the experimental group (3) was significantly higher than that of the experimental group (3) (see FIG. 5). Therefore, the present invention can dramatically increase the productivity of exosomes by performing a pretreatment process of culturing stem cells in a medium containing a combination of TNF-α and interferon-γ.

Example 5: Efficacy of reducing cytokine production by pre-treatment exosomes 1

In RAW 264.7 cells, a mouse macrophage cell line, the effect of reducing inflammatory cytokine production of stem cell-derived exosomes pretreated with TNF-α and interferon-γ (IFN-γ) was confirmed as follows. For this purpose, the experimental groups were classified as follows:

(1) negative control group (control): RAW 264.7 cells were not treated with LPS;

(2) LPS: RAW 264.7 cells were treated only with LPS in the experimental group;

(3) positive control group: the experimental group in which RAW 264.7 cells were treated with dexamethasone before LPS treatment (indicated by “DEX” in FIGS. 6 and 7);

(4) Untreated exosomes (general exosomes) low concentration treatment group: Before RAW 264.7 cells were treated with LPS, general exosomes (separated according to the method of Example 2-1) had a low concentration (1.46×10 ^{9 )} particles/mL) in the experimental group;

(5) Untreated exosomes (general exosomes) high concentration treatment group: Before RAW 264.7 cells were treated with LPS, general exosomes (separated according to the method of Example 2-1) had a high concentration (7.34×10 ^{9 )} particles/mL) in the experimental group;

(6) TNF-α and interferon-γ pretreatment (TNF-α, IFN-γ treated) exosome low concentration treatment group: Before LPS treatment in RAW 264.7 cells, pre-treated exosomes (separated according to the method of Example 2-1) ) of the experimental group treated with a low concentration (1.46×10 ^{9 particles/mL);}

(7) TNF-α and interferon-γ pretreatment (TNF-α, IFN-γ treated) exosome high concentration treatment group: RAW 264.7 cells pre-treated exosomes before LPS treatment (isolated according to the method of Example 2-1 ) of the experimental group treated with a high concentration (7.34×10 ^{9 particles/mL).}

RAW 264.7 cells were suspended in DMEM (Dulbecco Modified Eagle Medium; purchased from ThermoFisher Scientific) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, and then placed in a 96-well plate at 2.5×10 ⁴ cells/well. was inoculated at a density of, and after processing RAW 264.7 cells according to each condition of the experimental groups (1) to (7), 37° C., 5% CO ₂ Incubated for 24 hours in an incubator. Then, 100 ng/mL of LPS (purchased from Sigma) was treated and incubated at 37° C., 5% CO _{2 in an} incubator for 24 hours to induce activation of RAW 264.7 cells.

The culture supernatant of RAW 264.7 cells after culturing was collected, and the production amount of IL-6 and IL-1α in the culture supernatant was measured using the LEGENDplex ^TM bead-based immunoassay for mouse inflammation panel (mouse). The anti-inflammatory effect was confirmed by measuring inflammation panel) (purchased from Biolegend) and a NovoCyte Flow Cytometer (purchased from ACEA).

As shown in FIGS. 6 and 7 , as a result of inflammatory cytokine analysis, experimental groups (4) to (7) in which RAW 264.7 cells were pretreated with stem cell-derived exosomes prior to LPS treatment for RAW 264.7 cells were treated by LPS. The induced production of IL-6 and IL-1α was decreased in a concentration-dependent manner of stem cell-derived exosomes. And, in the case of experimental groups (6) and (7) in which RAW 264.7 cells were pretreated with TNF-α and IFN-γ pre-treated exosomes of the present invention before LPS treatment for RAW 264.7 cells, IL-6 induced by LPS and IL-1α production was significantly reduced in a concentration-dependent manner of the TNF-α and IFN-γ pre-treated exosomes ( FIGS. 6 and 7 ).

In particular, in the case of experimental groups (6) and (7) treated with TNF-α and IFN-γ pre-treated exosomes of the present invention, stem cell-derived exosomes (untreated exosomes not pre-treated with TNF-α and IFN-γ) Or general exosomes) significantly reduced the production of IL-6 and IL-1α based on the same concentration compared to the experimental groups (4) and (5) ( FIGS. 6 and 7 ).

These results show that the pretreatment method of the present invention is excellent in terms of enhancing the anti-inflammatory, wound healing or wound healing promoting efficacy of exosomes. That is, the present invention can significantly enhance the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes compared to the conventional exosome production method and exosomes produced therefrom, and anti-inflammatory, wound treatment or wound treatment promoting efficacy This enhanced exosomes can be economically and efficiently produced in high yield, which is commercially and/or clinically useful.

Therefore, the exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention may be usefully utilized as an active ingredient in a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion, quasi-drugs, external application for skin, or functional cosmetic composition. can

Example 6: Evaluation of the efficacy of reducing cytokine production by pre-treatment exosomes 2

After pretreatment with TNF-α and interferon-γ (IFN-γ), exosomes produced using SFM medium in the culture and culture medium recovery process for exosome production (see FIG. 8 ) (hereinafter referred to as “SFM exosomes”) Ham) and the exo-some produced using the EDM medium (hereinafter, referred to as "EDM exosome"), the effect of reducing the production of inflammatory cytokines was confirmed as follows. However, both SFM exosomes and EDM exosomes were isolated according to Example 2-2. For this purpose, the experimental groups were classified as follows:

(1) negative control group (control): RAW 264.7 cells were not treated with LPS;

(2) LPS: RAW 264.7 cells were treated only with LPS in the experimental group;

(3) positive control group: the experimental group in which RAW 264.7 cells were treated with dexamethasone before LPS treatment (indicated by “DEX” in FIGS. 9 to 11);

(4) SFM exosome low concentration treatment group: Before LPS treatment on RAW 264.7 cells, ^{the experimental group in which SFM exosomes were treated with a low concentration (1.09×10 9} particles/mL) (“Low, SFM” in FIGS. 9 to 11) Mark);

(5) SFM exosome high concentration treatment group: Before LPS treatment on RAW 264.7 cells, ^{the experimental group in which SFM exosomes were treated with high concentration (1.09 × 10 10} particles / mL) (“high, SFM” in FIGS. 9 to 11 ) Mark);

(6) EDM exosome low concentration treatment group: Before LPS treatment on RAW 264.7 cells, ^{the experimental group in which EDM exosomes were treated with a low concentration (1.09×10 9} particles/mL) (“Low, EDM” in FIGS. 9 to 11) Mark);

(7) EDM exosome high concentration treatment group: Before LPS treatment on RAW 264.7 cells, ^{the experimental group in which EDM exosomes were treated with high concentration (1.09×10 10} particles/mL) (“High, EDM” in FIGS. 9 to 11 ) Mark).

RAW 264.7 cells were suspended in DMEM (Dulbecco Modified Eagle Medium; purchased from ThermoFisher Scientific) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, and then placed in a 96-well plate at 2.5×10 ⁴ cells/well. was inoculated at a density of, and after processing RAW 264.7 cells according to each condition of the experimental groups (1) to (7), 37° C., 5% CO ₂ Incubated for 24 hours in an incubator. Then, 100 ng/mL of LPS (purchased from Sigma) was treated and incubated at 37° C., 5% CO _{2 in an} incubator for 24 hours to induce activation of RAW 264.7 cells.

The culture supernatant of RAW 264.7 cells after the culture was completed was collected, and the production amount of IL-6, IL-27 and IL-1α in the culture supernatant was measured for LEGENDplex ^TM bead-based immunoassay (bead-based immnnoassay) in mice The anti-inflammatory effect was confirmed by measurement using a mouse inflammation panel (purchased from Biolegend) and a NovoCyte Flow Cytometer (purchased from ACEA).

As shown in FIGS. 9 to 11 , as a result of inflammatory cytokine analysis, the experimental groups (4) to (7) in which SFM exosomes or EDM exosomes were pre-treated to RAW 264.7 cells before LPS treatment for RAW 264.7 cells (4) to (7) were LPS The production amount of IL-6, IL-27 and IL-1α induced by the stem cell-derived exosomes decreased in a concentration-dependent manner. In particular, in the case of the experimental groups (6) and (7) treated with EDM exosomes, compared with the experimental groups (4) and (5) treated with SFM exosomes, IL-6, IL-27 and IL based on the same concentration The production amount of -1α was significantly reduced ( FIGS. 9 to 11 ).

From these results, according to the present invention, after pretreatment with TNF-α and interferon-γ (IFN-γ), EDM exosomes produced using EDM medium in culture and culture medium recovery process for exosome production are anti-inflammatory, wound treatment or It can be seen that it is more excellent in terms of enhancing the effect of promoting wound healing. That is, EDM exosomes have superior anti-inflammatory, wound treatment or wound treatment promoting efficacy than SFM exosomes, and can economically and efficiently produce exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy at high yields. and/or clinically useful.

Therefore, the EDM exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention is useful as an active ingredient in a pharmaceutical composition, quasi-drug, external application for skin, or functional cosmetic composition for promoting anti-inflammatory, wound treatment or wound treatment. can be utilized.

In the above, the present invention has been described with reference to the above examples, but the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Claims (25)

A method for enhancing the efficacy of anti-inflammatory, wound treatment or wound treatment promotion of exosomes, comprising the step of culturing animal-derived cells in a medium containing TNF-α and interferon-γ. According to claim 1,
The TNF-α and interferon-γ are each contained in the medium at a concentration of 1 ng / mL to 100 ng / mL, exosomes anti-inflammatory, wound treatment or wound treatment promoting efficacy strengthening method. 3. The method of claim 2,
The culture is performed for 12 hours to 48 hours, the exosome anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancing method. 4. The method according to any one of claims 1 to 3,
After the culture, washing the animal-derived cells, culturing the washed animal-derived cells in a serum-free medium, collecting the culture medium, and separating the exosomes from the collected culture medium, anti-inflammatory of exosomes, A method for enhancing the efficacy of wound healing or promoting wound healing. 5. The method of claim 4,
The serum-free medium is characterized in that the platelet lysate is added, the exosome anti-inflammatory, wound treatment or wound treatment promoting efficacy strengthening method. A medium for enhancing anti-inflammatory, wound treatment or wound treatment promoting efficacy of animal cell-derived exosomes, comprising TNF-α and interferon-γ. 7. The method of claim 6,
The TNF-α and interferon-γ are each contained in the medium at a concentration of 1 ng/mL to 100 ng/mL, an animal cell-derived exosome anti-inflammatory, wound treatment or wound treatment promoting efficacy medium for enhancing efficacy. Anti-inflammatory, wound treatment or wound treatment promotion, comprising as an active ingredient an exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy obtained by culturing animal-derived cells in a medium containing TNF-α and interferon-γ for composition. 9. The method of claim 8,
The TNF-α and interferon-γ are each contained in a concentration of 1 ng / mL to 100 ng / mL in the medium, anti-inflammatory, wound treatment or a composition for promoting wound treatment. 10. The method of claim 9,
The culturing is characterized in that it is carried out for 12 hours to 48 hours, anti-inflammatory, wound treatment or a composition for promoting wound treatment. 9. The method of claim 8,
After the culture, washing the animal-derived cells, culturing the washed animal-derived cells in a serum-free medium, collecting the culture medium, and separating the exosomes from the collected culture medium, anti-inflammatory, wound A composition for promoting treatment or wound healing. 12. The method of claim 11,
A composition for promoting anti-inflammatory, wound treatment or wound treatment, characterized in that platelet lysate is added to the serum-free medium. 13. The method according to any one of claims 8 to 12,
A composition for anti-inflammatory, wound treatment or wound healing promotion, administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. 13. The method according to any one of claims 8 to 12,
A pharmaceutical composition, a quasi-drug, a composition for external application to the skin or a functional cosmetic composition, anti-inflammatory, wound treatment or wound treatment promoting composition. 15. The method of claim 14,
The pharmaceutical composition is an injection, injection, spray, liquid or patch type, anti-inflammatory, wound treatment or wound treatment promoting composition. 15. The method of claim 14,
The quasi-drug or the external preparation for skin is a liquid, ointment, cream, spray, patch, gel, or aerosol, anti-inflammatory, wound treatment or composition for promoting wound treatment. 15. The method of claim 14,
The functional cosmetic composition is a lotion or cream, anti-inflammatory, wound treatment or a composition for promoting wound treatment. The therapeutically effective amount of the anti-inflammatory, wound treatment or wound treatment promotion composition according to any one of claims 8 to 12 is administered to a mammal, or the anti-inflammatory, wound treatment or wound treatment promotion composition is administered to a mammal. A method of preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or facilitating wound healing, comprising the step of treating at least one of an inflamed site or a wound site in an animal . 19. The method of claim 18,
The anti-inflammatory, wound treatment or composition for promoting wound treatment is administered or treated to at least one site of an inflammatory site or a wound site in a mammal by injection, microneedling, iontophoresis, application, or a combination thereof, A method of preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or facilitating the healing of a wound. 19. The method of claim 18,
The anti-inflammatory, wound treatment or composition for promoting wound treatment is a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, powder And a method for preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or accelerating wound healing, characterized in that it is applied to at least one form selected from the group consisting of oily paper. 21. The method of claim 20,
The anti-inflammatory, wound treatment or composition for promoting wound treatment prevents, suppresses, alleviates, ameliorates or treats an inflammatory disease that is applied or deposited on at least one surface of a mask pack, mask sheet or patch, or treats a wound, or to promote wound healing. 19. The method of claim 18,
Further comprising the step of performing iontophoresis by flowing a microcurrent to at least one of an inflammatory site or a wound site of a mammal treated with the anti-inflammatory, wound treatment or wound treatment promoting composition, A method of preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or facilitating the healing of a wound. 23. The method of claim 22,
The step of performing the iontophoresis is performed by contacting or attaching the iontophoresis device to at least one of an inflammatory site or a wound site of the mammal to prevent, inhibit, alleviate, improve or treat an inflammatory disease. or a method of treating or facilitating wound healing. 24. The method of claim 23,
The iontophoresis device comprises at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry cell, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrostatic dialysis battery. , a method of preventing, inhibiting, alleviating, ameliorating or treating an inflammatory disease, treating a wound, or facilitating the healing of a wound. 19. The method of claim 18,
wherein the mammal is a human, dog, cat, rodent, horse, bovine, monkey, or pig, for preventing, inhibiting, ameliorating, ameliorating or treating an inflammatory disease, treating a wound, or facilitating the treatment of a wound.

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