Composition for treating inflammatory skin diseases, comprising stem cell-derived extracellular vesicles having enhanced efficacy

Abstract

The present invention relates to a use of extracellular vesicles derived from three-dimensional spheroid-type cell aggregates prepared by a method of the present invention for preventing, ameliorating or treating inflammatory skin diseases. It was demonstrated that the extracellular vesicles derived from three-dimensional spheroid-type cell aggregates according to the present invention suppress the expression of inflammatory cytokines and restore skin barrier-related proteins in animal models of atopic dermatitis. This indicates that the extracellular vesicles of the present invention simultaneously improve immune homeostasis and the skin barrier, and thus may be widely used in the fields of prevention, amelioration, and treatment of inflammatory dermatitis.

Description

Composition for treating inflammatory skin diseases containing stem cell-derived extracellular vesicles with enhanced efficacy

[0001] The present invention relates to the use of extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by the method of the present invention for the prevention, improvement, or treatment of inflammatory skin diseases.

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by the combined action of genetic and environmental factors, and is known to affect approximately 3–10% of adults and 20% of children worldwide. Although AD is not a life-threatening disease, it significantly reduces the quality of life by causing persistent itching, eczematous lesions, and skin inflammation.

[0003] Currently, the treatment of AD relies primarily on corticosteroids or immunosuppressants, and dupilumab, a monoclonal antibody targeting interleukin (IL)-4 and IL-13, is used for patients with moderate to severe disease. However, local side effects such as skin atrophy, infection, and changes in acne patterns have been reported with long-term use, and new cases of adverse effects, such as psoriasis—a Th1-mediated disease—are also being reported. Furthermore, because dupilumab acts primarily on Th2 cytokines, its immunomodulatory range is limited.

[0004] Against this backdrop, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are garnering attention as an alternative to cell-based therapies. MSC-EVs have the advantage of maintaining the therapeutic properties of stem cells while avoiding the potential risks associated with cell therapy, such as immunogenicity and tumorigenesis. Indeed, MSC-EVs have demonstrated immunomodulatory effects in various animal models, proving their potential for treating inflammatory diseases, including atopic dermatitis.

[0005] However, EVs produced by conventional two-dimensional (2D) culture methods have low production yields and functional non-uniformity between batches, which limits their clinical application. To overcome this, various EV production technologies have been proposed, and among them, a micropatterned well-based three-dimensional (3D) spherical aggregate culture system has been reported to increase EV production and enhance the expression of therapeutic substances such as miRNAs and cytokines.

[0006] According to previous studies, MSCs cultured in 3D maintained their original phenotype better and secreted more therapeutic EVs compared to the conventional 2D method. In addition, 3D bioprocessing has enabled the scalability of EV production, reduced variability between donors and batches, and improved therapeutic effects in various animal models of atopic dermatitis.

[0007] Accordingly, for atopic dermatitis involving complex autoimmune responses, there is a need to stably produce EVs with higher quality and therapeutic efficacy and to develop novel treatment strategies that can overcome the limitations of existing therapies. Furthermore, it is necessary to elucidate the mechanisms of action and conduct comparative analyses of EV-based therapies that have mechanisms of action differentiated from existing drugs.

[0008] Accordingly, in order to solve the problems of the prior art as described above, the inventors of the present invention completed the present invention by confirming the therapeutic effect of WJ-MSC-derived extracellular vesicles produced using a three-dimensional static culture system on atopic dermatitis.

[0009] Therefore, the objective of the present invention is to provide a composition for the prevention, improvement, or treatment of inflammatory skin diseases comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates prepared by the method of the present invention.

[0010] Another objective of the present invention is to provide a method for treating inflammatory skin diseases comprising the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by the method of the present invention to an individual in need thereof.

[0011] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating cells and vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0012] In addition, the present invention provides a cosmetic composition for the prevention or improvement of inflammatory skin diseases comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0013] In addition, the present invention provides a cosmetic composition for strengthening the skin barrier comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0014] In addition, the present invention provides a quasi-drug composition for the prevention or improvement of inflammatory skin diseases, comprising a 3D spheroid-shaped cell aggregate-derived extracellular vesicle prepared by (a) a step of preparing a 3D spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the 3D spheroid-shaped cell aggregate.

[0015] The present invention also provides a method for treating an inflammatory skin disease, comprising the step of administering an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate to an individual requiring it, wherein the extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate is prepared through: (a) a step of preparing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating the extracellular vesicle from the three-dimensional spheroid-shaped cell aggregate.

[0016] The three-dimensional spheroid-shaped cell aggregate-derived extracellular vesicles according to the present invention inhibited the expression of inflammatory cytokines and restored skin barrier-related proteins in an animal model of atopic dermatitis. This implies that the extracellular vesicles of the present invention simultaneously improve immune homeostasis and the skin barrier, and thus can be utilized in various ways in the fields of prevention, improvement, and treatment of inflammatory dermatitis.

[0017] Figure 1 is a diagram showing the experimental schedule from the time of atopic dermatitis to the time of sacrifice in an animal model of atopic dermatitis using DFE or DNCB.

[0018] Figures 2a and 2b show the results of confirming the typical circular structure and average particle size by observing the morphology of a 3D-static-spheroid EV according to the present invention using an electron microscope.

[0019] FIG. 2c is a figure showing the results of analyzing the expression of surface markers in a 3D-static-spheroid EV according to the present invention.

[0020] FIG. 2d is a figure showing the results of confirming the purity of a 3D-static-spheroid EV according to the present invention.

[0021] Figure 3 is a figure showing the results of evaluating skin lesions according to 3D-static-spheroid EV treatment according to the present invention in a DFE-induced atopic dermatitis model.

[0022] Figure 4 is a figure showing the results of evaluating skin lesions according to 3D-static-spheroid EV treatment according to the present invention in a DNCB-induced atopic dermatitis model.

[0023] Figure 5 is a figure showing the results of analyzing the tissue of a DFE-induced atopic dermatitis model treated with a 3D-static-spheroid EV according to the present invention through H&E staining.

[0024] Figure 6 is a figure showing the results of analyzing the tissue of a DNCB-induced atopic dermatitis model treated with a 3D-static-spheroid EV according to the present invention through H&E staining.

[0025] Figure 7 is a figure showing the results of analyzing the number of mast cells in a DFE-induced atopic dermatitis model treated with 3D-static-spheroid EVs according to the present invention.

[0026] Figure 8 is a figure showing the results of analyzing the number of mast cells in a DNCB-induced atopic dermatitis model treated with 3D-static-spheroid EVs according to the present invention.

[0027] Figure 9 is a figure showing the results of analyzing serum IgE levels in a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0028] Figure 10 is a figure showing the results of analyzing serum IgE levels in a Uchu-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0029] Figure 11a shows the results of fluorescent imaging of lymph nodes and spleen of a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0030] Figure 11b shows the results of measuring the weight of the spleen and lymph nodes of a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0031] Figure 12 shows the results of analyzing the increased CD4+ / CD8+ ratio in a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0032] Figure 13 is a figure showing the results of analyzing cytokine expression in the skin and spleen of a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0033] Figure 14 is a figure showing the results of analyzing cytokine expression in the skin and spleen of a DNCB-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0034] Figure 15 is a figure showing the results of an analysis of TSLP expression in a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0035] Figure 16 shows the results of analyzing the mRNA expression of skin barrier recovery proteins and the expression of the JAK / STAT3 pathway in a DFE-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0036] Figure 17 shows the results of analyzing the protein and mRNA expression of filaggrin and ocludin in a DNCB-induced atopic dermatitis model administered with a 3D-static-spheroid EV according to the present invention.

[0037] Figure 18a is a figure showing the results of analyzing the mRNA expression of IRAK1, TNF-α, and IFN-γ upon 3D-static-spheroid EV treatment according to the present invention.

[0038] Figures 18b and 18c show the results of analyzing the mRNA and protein expression of IRAK1, TNF-α, and IFN-γ in HaCaT cells transfected with a miR-146a mimic or inhibitor.

[0039] Figure 18d shows the results of confirming whether IRAK1 binds to the 3'-UTR in HaCaT cells transfected with a miR-146a mimic or inhibitor.

[0040] The present invention will be described in detail below.

[0041] According to an aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating cells and vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0042] In the present invention, the cell may be used without limitation as long as it is a cell capable of separating extracellular vesicles, and may be a cell isolated from a biological organism in nature. Additionally, the cell may be of any type of animal or plant origin, including human and non-human mammals, and may be various types of immune cells, tumor cells, or stem cells; preferably, the stem cell may be a mesenchymal stem cell, a pluripotent stem cell, an induced pluripotent stem cell, or an embryonic stem cell.

[0043] In the present invention, the three-dimensional culture refers to culturing in a state in which a three-dimensional arrangement is formed within a test tube. Unlike two-dimensional culture, three-dimensional culture allows for growth in all directions in vitro and creates an environment that is more similar to an in vivo cell environment.

[0044] In the present invention, the three-dimensional culture of step (a) can be performed by any three-dimensional cell culture technology known in the art to which the present invention belongs, for example, by cell culture using a microwell array culture, porous microsphere culture, hanging drop culture, low attachment plate culture, membrane-based cell-detachment culture, thermal lifting culture, centrifugation culture, semisolid medium culture, etc. Preferably, the three-dimensional culture may be dynamic culture or static culture, and more preferably, static culture. In the present invention, when the three-dimensional culture of step (a) is performed as static culture, the devices required for shaking culture are not required, making the culture easier, and enabling mass culture in a GMP (Good Manufacturing Practices) facility.

[0045] In the present invention, the three-dimensional culture of step (a) may be cultured for 1 to 10 days, preferably for 2 to 4 days. In particular, in the present invention, when the culture of step (a) is cultured for 2 to 4 days, the viability of cells present in the three-dimensional spheroid-shaped cell aggregate is maintained at a high level, and the culture time is relatively shorter compared to the conventional process for manufacturing three-dimensional spheroid-shaped cell aggregates, so three-dimensional spheroid-shaped cell aggregates and extracellular vesicles derived therefrom can be rapidly manufactured.

[0046] In the present invention, the three-dimensional culture of step (a) may be cultured by dispensing mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well, preferably at a density of 100 to 600 cells / well, and more preferably at a density of 100 to 500 cells / well, or 200 to 500 cells / well.

[0047] In the present invention, the step of separating the extracellular vesicles in step (b) may be by physical separation or chemical separation. The physical separation may be the extrusion of a sample containing cells or cell aggregates, and the chemical separation may be the treatment with a chemical substance capable of separating extracellular vesicles from cells or cell aggregates. For example, the separation step of the present invention may be performed using a method selected from the group consisting of ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical decomposition, and treatment of physical stimulation that applies force to the cell externally, and may be a physicochemical method of separation through binding of ions with specific biomarkers by ion or affinity chromatography, or a method of separating by destroying cells and binding only extracellular vesicles using polymers and reagents, such as Hansabiomed Life Sciences’ Immunofinity EV Capture or Creative Biolabs’ Exosome Purification Reagent, or Microgentas’ ExoCAS-2 reagent or method, and preferably may be separated by a Tangential Flow Filtration (TFF) method, but is not limited thereto.

[0048] In the present invention, the extracellular vesicle may be one that highly expresses various substances exhibiting efficacy in preventing, improving, or treating inflammatory skin diseases compared to known extracellular vesicles, for example, preferably the extracellular vesicle of the present invention may be one that highly expresses one or more selected from the group consisting of miR-146a, miR-27a, miR-132, miR-184, miR-210, and miR-301b compared to extracellular vesicles derived from spheroid-type cell aggregates cultured in three-dimensional dynamic culture of mesenchymal stem cells; or one or more selected from the group consisting of miR-27a, miR-146a, and miR-146b compared to extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, or may be one that highly expresses VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor).

[0049] In addition, the above extracellular vesicles may be incorporated into the cell and internalized upon treatment with the cell, and in the case of internalization, clinically significant substances highly expressed in the extracellular vesicles may be effectively delivered to the cell, resulting in high expression in the cell.

[0050] In the present invention, extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates exhibiting preventive, improving, or therapeutic effects for inflammatory skin diseases can be used interchangeably with “3D-static-spheroid-EVs.” Additionally, in comparison, extracellular vesicles derived from three-dimensionally dynamically cultured spheroid-shaped cell aggregates can be used interchangeably with “3D-dynamic-PEG-spheroid-EVs.”

[0051] In the present invention, “extracellular vesicles derived from spheroid-shaped cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells” may include, without limitation, extracellular vesicles isolated from spheroid-shaped cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells, and preferably, may be extracellular vesicles disclosed in a registered patent (Application No. 10-2016-0053026, Method for producing extracellular vesicles derived from stem cells).

[0052] In the present invention, the three-dimensional spheroid-shaped cell aggregate may have an average diameter of 74.43 ± 7.756 μm, preferably having a size range of 55 to 95.0 μm, and the average diameter may be 74.43 μm and the coefficient of variation (CV) may be 9.59% as a result of measuring the size distribution of 155 of the three-dimensional spheroid-shaped cell aggregates. The kurtosis of the size distribution of the three-dimensional spheroid-shaped cell aggregate of the present invention may be higher compared to the “spheroid-shaped cell aggregate obtained by three-dimensional dynamic culture of mesenchymal stem cells.” Accordingly, the size of the three-dimensional spheroid-shaped cell aggregate may be smaller and have a relatively uniform size distribution compared to the “spheroid-shaped cell aggregate obtained by three-dimensional dynamic culture of mesenchymal stem cells.”

[0053] In the present invention, the microwell is made of TMSPMA (3-(Trimetoxysily) propylmethacrylate), HEA (Hydroxyethyl acrylate), GMA (Glycidyl methacrylate), EGDMA (diethyleneglycol dimethacrylate), THFA (Tetrahydrofurfuryl acrylate), HMAA (Hydroxymethul acrylamide), and PEA (Phenyl acrylate). epoxyacrylate), HOFHA (6-Hydroxy-2, 2,3,3,4,4,5,5-octafluoro), EOPT (Polyethoxylated(4)pentaerythritoltetraacrylate), HPA (Hydroxypropyl acrylate), BMA (Buthylmethacrlate), PETIA (Pentaerythritol triacrylate), HDDA (Hexan) diol diacrylate), EGPEA (Ethyleneglycol phenyletheracrylate), It may be coated with any one selected from the group consisting of BM (Benzylmethacrylate), HPPA (Hydroxyphenoxypropyl acrylate), BHPEA (2-(4-Benzoyl-3-hydroxyphenoxy)ethylacrylate), HEMA (Hydroxyethyl methacrylate), HPMA (N-(2-Hydroxypropyl) methacrylamide) and MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), preferably coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), but is not limited thereto.

[0054] The microwell of the present invention may have a diameter of 200 to 800 μm, preferably 300 to 800 μm, and more preferably 400 to 800 μm.

[0055] In addition, the microwell may be a flat microwell with no depth, or, in the case of a microwell that forms depth, may have a structure of 100 to 1000 μm, preferably 100 to 900 μm, and more preferably 200 to 900 μm.

[0056] Mesenchymal stem cells cultured by the above structure may maintain a high viability even after a period of culture. Preferably, a microarray containing 1,000 to 100,000 of the above microwells can be fabricated to increase the production yield of cell aggregates.

[0057] When extracellular vesicles are manufactured using the “method for manufacturing extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates” of the present invention, the extracellular vesicles can be mass-produced rapidly and efficiently, along with the manufacturing advantages associated with static culture. In particular, the method for manufacturing extracellular vesicles of the present invention is characterized by being suitable for GMP application.

[0058] The inflammatory skin disease of the present invention may be one or more selected from the group consisting of atopic dermatitis, pruritus, acne, psoriasis, allergic dermatitis, contact dermatitis, exfoliative dermatitis, seborrheic dermatitis, seborrheic scalp dermatitis, lichen planus, rosacea, erythema, lupus, panniculitis, urticaria, folliculitis, and rash, and preferably may be atopic dermatitis.

[0059] It is desirable that the extracellular vesicles of the present invention restore or strengthen skin barrier function.

[0060] It is desirable that the extracellular vesicles of the present invention alleviate thickening of the dermis or epidermis.

[0061] It is preferable that the extracellular vesicles of the present invention inhibit Th1 / Th2 / Th17 / Th22 inflammatory cytokines; or the JAK / STAT3 signaling pathway.

[0062] The pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, a suitable carrier, excipient, and diluent conventionally used in the preparation of the pharmaceutical composition. The pharmaceutical composition of the present invention may further comprise other pharmaceutical active ingredients or active mixtures.

[0063] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as patches, coatings, powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the product is prepared using diluents or excipients such as commonly used fillers, fillers, binders, humectants, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, for example, humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.Witepsol, Macrogol, Tween 61, cacao oil, laurin oil, glycerozelatin, etc. can be used as bases for suppositories.

[0064] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0065] The pharmaceutical composition of the present invention may be administered to mammals, such as rats, mice, livestock, and humans, by various routes. All modes of administration are expected, for example, orally, transdermally, rectally, or intravenously, intramuscularly, subcutaneously, or by injection.

[0066] The definitions of terms for the excipients, binders, disintegrants, lubricants, synergists, flavorings, etc. of the present invention include those described in literature known in the art that have the same or similar functions.

[0067]

[0068] According to another aspect of the present invention, the present invention provides a cosmetic composition comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates. The cosmetic composition may be a cosmetic composition for the prevention or improvement of inflammatory skin diseases; or a cosmetic composition for strengthening the skin barrier.

[0069] In this specification, "cosmetic composition" refers to a composition formed for the purpose of manufacturing cosmetics, and can be broadly interpreted to include compositions for external use. The cosmetic composition according to the present invention may be manufactured in any formulation conventionally produced in the art. For example, the cosmetic composition may have a formulation such as a lotion, such as a softening lotion or a nourishing lotion; a spray-type lotion; a fluid, such as a facial lotion or a body lotion; a cream, such as a nourishing cream, a moisturizing cream, or an eye cream; a stick; an essence; a cosmetic ointment; a spray; a gel; a pack; a sunscreen; a makeup base; a foundation, such as a liquid or spray type; a powder; a makeup remover, such as a cleansing lotion or cleansing oil; a cleansing agent, such as a cleansing foam, a soap, or a body wash, but is not limited thereto.

[0070] The cosmetic composition of the present invention can be used according to a conventional method of use, and the frequency of use may be varied depending on the user's skin condition or preference.

[0071]

[0072] According to another aspect of the present invention, the present invention provides a quasi-drug composition for the prevention or improvement of inflammatory skin diseases, comprising: (a) a step of producing a 3D spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the 3D spheroid-shaped cell aggregate.

[0073] In this specification, "quasi-drug" refers to an article that exhibits an effect of treating, alleviating, managing, or preventing a disease, but has a milder effect on the human body than a pharmaceutical. It excludes articles used for pharmaceutical purposes under the Pharmaceutical Affairs Act, but includes articles classified according to criteria separately established by the Ministry of Health and Welfare. Specifically, it may be, but is not limited to, external skin preparations or personal hygiene products.

[0074] When the composition of the present invention is added to a quasi-drug composition for the purpose of preventing or improving inflammatory skin diseases, the composition may be added as is or used together with other quasi-drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use. The above external skin preparation is not particularly limited thereto, but may be prepared and used in the form of, for example, an ointment, lotion, spray, patch, cream, powder, suspension, gel, or gel.

[0075]

[0076] According to another aspect of the present invention, the present invention provides a method for treating an inflammatory skin disease, comprising the step of administering an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate to an individual in need thereof, wherein the extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate is prepared through the following steps: (a) preparing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) separating the extracellular vesicle from the three-dimensional spheroid-shaped cell aggregate.

[0077] In a specific embodiment of the present invention, the individual may be an individual expected to develop an inflammatory skin disease; an individual that has developed the disease; or an individual that has been determined to be cured, but is not limited thereto.

[0078]

[0079] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0080] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0081]

[0082] [Experimental Example]

[0083] Experimental Example 1. 3D Spheroid Culture of Human Umbilical Cord-Derived Mesenchymal Stem Cells and EV Production

[0084] 1-1. Preparation of Mesenchymal Stem Cells

[0085] Human umbilical cord-derived mesenchymal stem cells (hereinafter WJ-MSC, Samsung Medical Center, Seoul, Korea) at the passage 5 stage were obtained and cultured in a 37°C, 5% CO2 incubator. The growth medium used was α-modified eagle's medium (α-MEM, GIBCO, NY, USA) containing 10% fetal bovine serum (FBS) (GIBCO, NY, USA) and 50 μg / mL gentamicin (GIBCO, NY, USA). WJ-MSCs at the passage 6 stage were used to construct 3D spheroid-shaped cell aggregates.

[0086]

[0087] 1-2. Preparation of 3D Spheroidal Cell Aggregate Culture Medium

[0088] The WJ-MSC prepared in Example 1-1 above was washed with PBS, and trypsin (TrypLE TMThe cells were treated with (Express, GIBCO, NY, USA) and reacted in a CO2 incubator for 5 minutes. Afterward, fresh serum-free medium was added to neutralize trypsin and recover the cells, and a cell pellet was obtained using a centrifuge. Next, fresh serum-free medium was added to prepare a cell suspension, and the cells were counted. After counting the cells, 60 ml of the cell suspension was uniformly dispensed into a microarray containing microwells coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer) with diameters and depths of 500 μm × 200 μm, at a density of 400 cells / well. The cells were kept static to induce spontaneous spheroid-shaped cell aggregate formation, and cultured in a CO2 incubator at 37°C for a total of 4 days to prepare a 3D spheroid-shaped cell aggregate culture medium (hereinafter referred to as 3D-static-spheroid culture medium).

[0089]

[0090] 1-3. Isolation of extracellular vesicles derived from 3D spheroidal cell aggregates

[0091] The 3D-static-spheroid culture medium prepared in Examples 1-2 was recovered, centrifuged at 2,500g for 10 minutes to remove cellular foreign matter, and filtered through a 0.22 μm syringe filter. Subsequently, the 3D-static-spheroid culture medium was passed through a 300 kDa hollow fiber membrane (Pall, NY, USA) using a Tangential Flow Filtration (TFF) system to remove proteins and isolate extracellular vesicles, and then purified once more with physiological saline to obtain high-purity extracellular vesicles derived from the 3D-static-spheroid of the present invention (hereinafter, 3D-static-spheroid EV).

[0092]

[0093] Experimental Example 2. Analysis of EV Characteristics

[0094] EV characterization followed the guidelines of the International Society for Extracellular Vesicles (MISEV 2018 and 2023) and the standards of the Ministry of Food and Drug Safety of Korea. The structure and morphology of EVs were imaged using transmission electron microscopy (TEM). Specifically, 3D static spheroid EVs were fixed for 2 hours in 1% OsO4 dissolved in 0.1 M phosphate buffer (PB). An EM grid was adsorbed onto the extracellular vesicle droplets with the formvar side facing downward for 1 minute. Subsequently, the samples were blotted with filter paper and reacted with 2% uranyl acetate for 15 seconds. After removing excess uranyl acetate, the EM grid was observed using TEM (JEM-1011, JEOL, Japan). Additionally, the size distribution, surface protein markers, purity, functional characteristics, and stability of the EVs were analyzed.

[0095]

[0096] Experimental Example 3. Induction of an Atopic Dermatitis Animal Model

[0097] As shown in Figure 1, an atopic dermatitis model was induced using DFE or DNCB.

[0098]

[0099] 3-1. DFE (Dermatophagoides farinae extract)-induced atopic dermatitis model

[0100] After depilating the dorsal hair of BALB / c mice, atopic-like lesions were induced by treating them once a week with 1% 1-chloro-2,4-dinitrobenzene (dissolved in acetone:olive oil = 3:1). After 2 days, Dermatophagoides farinae extract (DFE, dissolved in PBS) was applied in the same manner (Citeq BV, Netherlands). The above process was repeated for 2 weeks.

[0101]

[0102] 3-2. DNCB (2,4-dinitrochlorobenzene)-induced atopic dermatitis model

[0103] After depilating C57BL / 6 mice, 1% 2,4-dinitrochlorobenzene (DNCB, Sigma-Aldrich Chemical Co. MO, USA; dissolved in acetone:olive oil = 3:1) was applied to the skin once daily during the first week. After a 4-day rest period, a 0.5% DNCB solution was applied to the dorsal skin for 21 days. Prior to sample treatment, a 4% sodium dodecyl sulfate solution was used to enhance skin barrier disruption.

[0104]

[0105] 3-3. Classification and Treatment of Experimental Groups

[0106] Mice were randomly divided into the following four groups (n=6 each):

[0107] - Control / Healthy (Control Group)

[0108] - Placebo (PBS processing)

[0109] - EV(EV 6×10 8 (Subcutaneous injection in dogs)

[0110]

[0111] The DFE-induced atopic dermatitis model was sacrificed on day 35, and the DNCB-induced atopic dermatitis model was sacrificed on day 25. Blood was collected from mice via orbital blood sampling, and skin from the dorsal area was collected for histological and molecular analysis.

[0112]

[0113] Experimental Example 4. Evaluation of Clinical Skin Lesions

[0114] Starting from the first week after inducing atopic dermatitis, the lesions were evaluated three times a week using the clinical visual scoring method. Four symptoms—erythema, dryness / scarring, edema, and erosion—were each evaluated on a scale of 0 (none) to 3 (severe), and the totals were reported.

[0115]

[0116] Experimental Example 5. Histopathological analysis

[0117] 5-1. Basic Tissue Staining

[0118] After sacrificing an atopic dermatitis model, the skin of the ears and back was fixed in 4% paraformaldehyde and embedded in paraffin. The samples were sliced ​​to a thickness of 6 μm, and epidermal thickness and inflammatory cell infiltration were observed using an H&E staining kit (ab245880, Abcam), and mast cell infiltration was analyzed using Toluidine Blue staining (NovaUltra™, IW-3013, IHC World).

[0119]

[0120] 5-2. Immunofluorescence Staining

[0121] Dorsal skin tissue from an atopic dermatitis model was fixed with 4% paraformaldehyde and blocked with 10% goat serum. The tissues were incubated overnight at 4°C with rabbit anti-TSLP (1:400, Abcam) and goat anti-filagrin (1:500, Abcam) antibodies. After washing with PBS, the tissues were reacted with DyLight-labeled anti-goat IgG (1:200, 594 nm, Abcam) and DyLight-labeled anti-rabbit IgG (1:200, 488 nm, Vector Laboratories). The tissues were imaged using a fluorescence microscope, and positively stained cells were quantified using ImageJ software.

[0122]

[0123] Experimental Example 6. Molecular biological analysis

[0124] 6-1. ELISA Analysis

[0125] Proteins were extracted from 0.1 g (n=6) of back tissue from an atopic dermatitis model by homogenizing with RIPA buffer and a protease inhibitor cocktail, and the supernatant was collected after centrifugation (12,000 xg, 15 min, 4°C). After protein quantification using the Bradford method, IL-4, IL-13, IL-22, IL-31, IgE, TNF-α, and IFN-γ were analyzed using an ELISA kit (Invitrogen / BD Biosciences).

[0126]

[0127] 6-2. RT-qPCR Analysis

[0128] RNA extraction and cDNA synthesis were performed according to standardized procedures. Total RNA (500 ng) was reverse transcribed into cDNA using oligo dT primers, and real-time qPCR was performed using Universal SYBR Green Master Mix (Applied Biosystems). Amplification conditions were as follows: 95°C for 15 minutes, (95°C 30 sec, 60°C 30 sec, 72°C 30 sec) x 40 cycles. Gene expression was normalized to GAPDH and 2 -ΔΔCt It was calculated in this way.

[0129]

[0130] 6-3. Western blot analysis

[0131] Proteins extracted from skin tissue of an atopic dermatitis model were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with skim milk for 1 hour. The mixture was reacted with primary antibodies (p-p65, p65, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, p-ERK, ERK, p-p38, p38, β-actin) at a ratio of 1:1000 or 1:5000 at 4°C. After reacting with secondary antibodies (HRP-conjugated) for 1 hour, detection was performed using ECL reagent (GE Healthcare) and an Amersham Imager 600.

[0132]

[0133] Experimental Example 7. Cell experiment (HaCaT cells)

[0134] 7-1. Cell Transformation

[0135] HaCaT cells were grown to a confluent state of 30–60% and transformed into miR-146a mimics, miR-146a inhibitors, or negative controls using Lipofectamine 3000 reagent (Invitrogen). After 6 hours of transformation, the medium was replaced with fresh DMEM containing 10% FBS.

[0136]

[0137] 7-2. Analysis of Luciferase Reporter

[0138] A firefly luciferase reporter plasmid containing the 3′-UTR of the IRAK1 gene was used. HaCaT cells were co-transformed with 50 nM miR-146a mimic or scrambled miRNA. Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) 48 hours after transformation.

[0139]

[0140] 7-3. FACS Analysis

[0141] After preparing a single-cell suspension from the spleen, red blood cells were removed, and the cells were stained with a Mouse T lymphocyte set (BD Biosciences) containing CD3, CD4, and CD8 antibodies. The data were analyzed using a BD FACS Lyric cytometer and processed with Flow Suite software.

[0142]

[0143] Experimental Example 8. EV Labeling and In Vivo Fluorescence Imaging Analysis

[0144] EVs were fluorescently labeled using the ExoGlow-Protein EV Labeling Kit (System Biosciences). The labeled EVs were treated with ExoQuick-TC, and excess dye was removed by centrifugation. After subcutaneous injection of the EVs, lymph nodes and spleens were extracted from sacrificed mice, and fluorescence was measured using the IVIS Imaging System.

[0145]

[0146] Experimental Example 9. Statistical Analysis

[0147] All statistical analyses were performed using GraphPad Prism 8 and ANOVA, with p < 0.05 considered statistically significant. Data were expressed as mean ± standard error (SEM).

[0148]

[0149] [Example]

[0150] Example 1. Comparative analysis of particle size, surface markers, and miRNA expression of 3D-EVs

[0151] The morphology of the 3D-static-spheroid EV prepared in Experimental Example 1 above was observed using an electron microscope, and the results are shown in Figures 2a and 2b.

[0152] As shown in Fig. 2a, the 3D-static-spheroid EV exhibited a typical circular shape, and as shown in Fig. 2b, it was confirmed that the average diameter was 124±4.7 nm.

[0153]

[0154] In addition, the surface protein markers and purity of the 3D-static-spheroid EV prepared in Experimental Example 1 were analyzed, and the results are shown in Figures 2c and 2d, respectively.

[0155] As shown in Figures 2c and d, the CD63+ subgroup was found to be more abundant in the 3D-static-spheroid EV than in the CD9+ or CD81+ subgroups. Additionally, since no organelle marker proteins were detected, it was confirmed that the purity of the 3D-static-spheroid EV is high.

[0156]

[0157] In a prior study, it was confirmed that the 3D-static-spheroid EV prepared in Experimental Example 1 above, compared to the 2D-EV, the 3D-static-spheroid EV highly expresses miR-27a, miR-146b, and miR-146a, which are miRNAs effective in angiogenesis / neurogenesis and immune modulation, and highly expresses integrin 1 / 2 and VEGF / R2 (Vascular endothelial growth factor / R2), which are proteins effective in angiogenesis / neurogenesis.

[0158] In addition, compared to the 3D-dynamic-PEG spheroid EV prepared in Experimental Example 1, it was confirmed that the 3D-static-spheroid EV highly expresses miR-146a, a miRNA effective in angiogenesis / neurogenesis and immunomodulation, miR-27a, miR-132, miR-184, and miR-210, miRNAs effective in angiogenesis / neurogenesis, and miR-301b, which has anti-tumor efficacy, and highly expresses integrin 1 / 2 and VEGF / R2, proteins effective in angiogenesis / neurogenesis.

[0159]

[0160] Example 2. Effect of 3D-Static-Spheroid EV Administration on Improvement of Atopic Clinical Indicators

[0161] In this embodiment, the effect of the 3D-static-spheroid EV prepared in Experimental Example 1 on improving symptoms of atopic dermatitis was confirmed. For this experiment, the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model of Experimental Example 3 were used. Atopic dermatitis symptoms were evaluated using the method of Experimental Example 4. The results of evaluating skin lesions in the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model are shown in Figures 3 and 4, respectively.

[0162] As shown in Fig. 3, EV treatment group (EV 6×10 8 It was confirmed that the atopic score and ear thickness were significantly reduced compared to the placebo treatment group (subcutaneous injection in dogs).

[0163] As shown in Fig. 4, EV treatment group (EV 6×10 8 It was confirmed that the atopic score was significantly reduced compared to the placebo treatment group (subcutaneous injection of dogs).

[0164] In other words, it was confirmed that the 3D-static-spheroid EV prepared in Experimental Example 1 significantly reduced atopic dermatitis scores in various atopic dermatitis models, which means that the 3D-static-spheroid EV prepared in Experimental Example 1 has an excellent effect in improving symptoms of atopic dermatitis.

[0165]

[0166] Example 3. Inhibitory effect on skin thickness and mast cell infiltration following 3D-static-spheroid EV administration

[0167] In this embodiment, the effect of the 3D-static-spheroid EV prepared in Experimental Example 1 on improving symptoms of atopic dermatitis was confirmed through histological analysis. For this experiment, back and ear tissues obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model of Experimental Example 3 were used. Histological analysis of the skin tissues was performed using H&E staining. The results of H&E staining of the skin tissues obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model are shown in Figures 5 and 6, respectively.

[0168] As shown in Fig. 5, EV treatment group (EV 6×10 8 It was confirmed that skin thickness and ear thickness were significantly reduced compared to the placebo treatment group (subcutaneous injection of dogs).

[0169] As shown in Fig. 6, EV treatment group (EV 6×10 8 It was confirmed that the skin thickness of dogs (subcutaneous injection) was significantly reduced compared to the placebo treatment group.

[0170] In other words, it was confirmed that the 3D-static-spheroid EV prepared in Experimental Example 1 significantly reduced skin tissue and ear thickness in various atopic dermatitis models, which means that the 3D-static-spheroid EV prepared in Experimental Example 1 has excellent effects in improving atopic dermatitis symptoms even from a histological perspective.

[0171]

[0172] Mast cell activity was further analyzed in back and ear tissues obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model of Experimental Example 3 above. The results of Toluidine Blue staining of skin tissues obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model are shown in Figures 7 and 8, respectively.

[0173] As shown in Figures 7 and 8, the EV treatment groups (EV 6×10⁻⁶) of the two atopic dermatitis models 8 It was confirmed that the number of mast cells in the back and ear tissues of dogs (subcutaneously injected) was significantly reduced compared to the placebo treatment group.

[0174] In other words, it was confirmed that the 3D-static-spheroid EV prepared in Experimental Example 1 significantly reduced the number of mast cells in various atopic dermatitis models, which means that the 3D-static-spheroid EV prepared in Experimental Example 1 has excellent anti-inflammatory effects even from a histological perspective.

[0175]

[0176] Example 3. Inhibitory effect on skin thickness and mast cell infiltration following 3D-static-spheroid EV administration

[0177] In this example, the effect of the 3D-static-spheroid EV prepared in Experimental Example 1 on serum IgE levels was confirmed. For this experiment, blood obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model of Experimental Example 3 was used. Serum IgE levels were analyzed using the ELISA method, and the results are shown in Figures 9 and 10, respectively.

[0178] As shown in Figures 9 and 10, it was confirmed that serum IgE levels were significantly reduced in both atopic dermatitis models upon EV treatment.

[0179]

[0180] Example 4. Analysis of changes in serum IgE levels following 3D-static-spheroid EV administration

[0181] In this example, the effect of the 3D-static-spheroid EV prepared in Experimental Example 1 on spleen and lymph node analysis and the CD4+ / CD8+ ratio was confirmed.

[0182] First, labeled EV imaging and tissue weights were measured within the spleen and lymph nodes of the DFE-induced atopic dermatitis model. The spleen and lymph nodes obtained from the DFE-induced atopic dermatitis model of Experimental Example 3 were used. The weights of the spleen and lymph nodes were measured, and labeled EVs were imaged using the IVIS Imaging System. The imaging results of the spleen and lymph nodes obtained from the DFE-induced atopic dermatitis model are shown in Fig. 11a, and the weight measurement results are shown in Fig. 11b.

[0183] As shown in Fig. 11a, fluorescence of the labeled EV was observed in the lymph nodes and spleen of an atopic dermatitis model. The fluorescence intensity of the labeled EV increased over time and was found to be strongest at 24 hours after administration of the labeled EV.

[0184] As shown in Figure 11b, the weight of the spleen and lymph nodes in the atopic dermatitis model increased compared to the control group, but it was confirmed that they decreased significantly with EV treatment.

[0185]

[0186] The effect of the 3D-static-spheroid EV prepared in Experimental Example 1 on the CD4+ / CD8+ ratio was confirmed. For this experiment, a spleen obtained from the DFE-induced atopic dermatitis model of Experimental Example 3 was used. The erythrocyte-removed spleen single-cell suspension was stained with a mouse T lymphocyte set, and the CD4+ / CD8+ ratio was analyzed. The results of the analysis of the CD4+ / CD8+ ratio are shown in Figure 12.

[0187] As shown in Figure 12, the DFE-induced atopic dermatitis model had a very high CD4+ / CD8+ ratio, but it was confirmed that it decreased to the level of the control group following EV treatment.

[0188]

[0189] Example 5. Lymph node and spleen changes and T-cell modulation effects induced by 3D-static-spheroid EVs

[0190] In this embodiment, the cytokine regulatory effect of the 3D-static-spheroid EV prepared in Experimental Example 1 was confirmed through histological analysis. For this experiment, dorsal skin tissue and spleen obtained from the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model of Experimental Example 3 were used, and protein and mRNA expression were analyzed using ELISA and RT-qPCR analysis methods. The results of analyzing the expression of cytokines (IL-4, IL-13, IL-31, TNF-α, IFN-γ, IL-1β, IL-6, IL-17, IL-22) in the dorsal skin tissue or spleen of the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model are shown in Figures 13 and 14, respectively.

[0191] As shown in Figure 13, in the EV treatment group, levels of Th2-related cytokines (IL-4, IL-13, IL-31, and IL-5) were reduced in all tissues, and levels of Th1-related cytokines (TNF-α, IFN-γ) were also reduced in all tissues.

[0192] As shown in Figure 14, the EV treatment group showed reduced levels of Th2-related cytokines (IL-4, IL-13, IL-31) and inflammatory cytokines (IL-6, IL-1β). Additionally, the EV treatment group showed reduced levels of Th1-related cytokines (TNF-α, IFN-γ), IL-17, and IL-22.

[0193]

[0194] In addition, the TSLP regulatory effect of the 3D-static-spheroid EV prepared in Experimental Example 1 was confirmed. For this experiment, the DFE-induced atopic dermatitis model of Experimental Example 3 was used, and protein expression was analyzed by immunofluorescence staining and Western blot analysis. The results of the analysis of TSLP expression in the DFE-induced atopic dermatitis model are shown in Figure 15.

[0195] As shown in Figure 15, the DFE-induced atopic dermatitis model confirmed that the expression of TSLP was significantly increased. The EV-treated group confirmed that the expression of TSLP decreased to the level of the normal group.

[0196]

[0197] Example 6. Analysis of the Inhibitory Effects of 3D-Static-Spheroid EVs on Inflammatory and Th-Related Cytokines

[0198] In this embodiment, the skin barrier restoration and related signaling pathways of the 3D-static-spheroid EV prepared in Experimental Example 1 were identified. For this experiment, the DFE-induced atopic dermatitis model and the DNCB-induced atopic dermatitis model from Experimental Example 3 were used. Skin barrier restoration proteins (filagrin, ocludin) and related signaling pathways were identified through immunofluorescence staining, RT qPCR, and Western blot analysis. The results of analyzing the mRNA expression of skin barrier restoration proteins and the expression of the JAK / STAT3 pathway in the DFE-induced atopic dermatitis model are shown in Fig. 16. Additionally, the results of analyzing the protein and mRNA expression of filaggrin and ocludin in the DNCB-induced atopic dermatitis model are shown in Fig. 17.

[0199] As shown in Figure 16, the tissue expression levels of filaggrin, a key protein for maintaining the skin barrier, and ocludin, a component of tight junctions, were measured, and it was confirmed that the levels of these proteins in the EV-treated group decreased to the level of the normal group. In addition, in mice with atopic dermatitis induced, the expression of p-JAK1, JAK2, and STAT3 in the EV-treated group was significantly reduced compared to the placebo group, which means that the JAK / STAT3 pathway is inhibited.

[0200] As shown in Figure 17, the expression of filaggrin and ocludin was analyzed to evaluate skin barrier recovery, and it was confirmed that the expression of the respective proteins was significantly restored in the EV-treated group compared to the placebo group.

[0201] The above results suggest that the 3D-static-spheroid EV prepared in Experimental Example 1 induces the restoration of skin barrier function, which can be achieved not only through the normalization of filaggrin and ocludin expression but also through the inhibition of the JAK / STAT3 pathway.

[0202]

[0203] Example 7. Mechanism of inflammation regulation by 3D-static-spheroid EVs through miR-146a-mediated IRAK1 / NF-κB pathway inhibition

[0204] In this example, the inhibition of inflammation through the miR-146a / NF-κB pathway by the 3D-static-spheroid EV prepared in Experimental Example 1 was confirmed. Specifically, HaCaT cells were transfected with a miR-146a mimic, a miR-146a inhibitor, or a negative control, and mRNA and protein expression were analyzed, along with luciferase analysis. The results of analyzing the mRNA and protein expression of IRAK1, TNF-α, and IFN-γ in HaCaT cells transfected with a miR-146a mimic or inhibitor are shown in Figure 18.

[0205] As shown in Figure 18a, it was confirmed that the EV-treated group downregulated the expression of IRAK1, an inflammation-related target, and simultaneously reduced the levels of Th1 cytokines TNF-α and IFN-γ.

[0206] As shown in Figures 18b and 18c, when HaCaT cells were transfected with a miR-146a mimic, the expression of IRAK1, TNF-α, and IFN-γ decreased, whereas when a miR-146a inhibitor was administered, the expression of these factors increased.

[0207] As shown in Fig. 18d, luciferase reporter analysis confirmed that miR-146a directly binds to the 3'-UTR of IRAK1 and inhibits its expression.

[0208] The above results confirmed that miR-146a, which is abundant in the 3D-static-spheroid EV prepared in Experimental Example 1, is involved in regulating inflammation, which implies that miR-146a is a key regulatory factor. Furthermore, it suggests that the EV of Experimental Example 1 regulates the inflammatory response of atopic dermatitis through the miR-146a / IRAK1 / NF-κB pathway.

[0209]

[0210] In summary, the inventors confirmed that 3D-static-spheroid extracellular vesicles suppress the expression of Th1 / Th2 / Th17 / Th22 inflammatory cytokines in an animal model of atopic dermatitis, and induce the restoration of skin barrier proteins and the inhibition of the JAK / STAT3 signaling pathway. This implies that extracellular vesicles can simultaneously induce the restoration of immune homeostasis and the improvement of the skin barrier, and thus the extracellular vesicles of the present invention can be utilized in various ways in the field of treating inflammatory skin diseases, including atopic dermatitis.

[0211]

[0212] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0213]

[0214] The present invention is a technology developed through the Seoul Metropolitan Government Seoul Economic Promotion Agency 2023 Biomedical Technology Commercialization Support Project, BT230029, Development of a treatment for chronic skin ulcers using freeze-dried extracellular vesicles.

Claims

1. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating cells and vesicles from the above three-dimensional spheroid-shaped cell aggregate; comprising an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through this step, Pharmaceutical composition for the prevention or treatment of inflammatory skin diseases.

2. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wherein the stem cells are one or more selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells.

3. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wherein the three-dimensional culture of step (a) is a static culture in accordance with claim 1.

4. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wherein the three-dimensional culture of step (a) is cultured by dispensing mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well.

5. A pharmaceutical composition for the prevention or treatment of an inflammatory skin disease according to claim 1, wherein the inflammatory skin disease is one or more selected from the group consisting of atopic dermatitis, pruritus, acne, psoriasis, allergic dermatitis, contact dermatitis, exfoliative dermatitis, seborrheic dermatitis, seborrheic scalp dermatitis, lichen planus, rosacea, erythema, lupus, panniculitis, urticaria, folliculitis, and rash.

6. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wherein, in claim 1, the extracellular vesicles restore or strengthen skin barrier function.

7. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wherein, in claim 1, the extracellular vesicles alleviate thickening of the dermis or epidermis.

8. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases according to claim 1, wherein the extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates inhibit Th1 / Th2 / Th17 / Th22 inflammatory cytokines; or the JAK / STAT3 signaling pathway.

9. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; a cosmetic composition for the prevention or improvement of inflammatory skin diseases comprising extracellular vesicles derived from a three-dimensional spheroid-shaped cell aggregate prepared through the above step.

10. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; a cosmetic composition for strengthening the skin barrier comprising extracellular vesicles derived from a three-dimensional spheroid-shaped cell aggregate prepared through the above step.

11. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; a quasi-drug composition for the prevention or improvement of inflammatory skin diseases comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates.

12. Includes the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates to an individual requiring them, and The above-mentioned extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate comprises: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells three-dimensionally in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the above three-dimensional spheroid-shaped cell aggregate; a method for treating an inflammatory skin disease.

Images