Composition comprising mixture of exosomes as active ingredient and use thereof

Abstract

The present invention relates to a composition comprising a mixture of exosomes derived from different cells, as an active ingredient and use thereof. More specifically, the present invention relates to a composition for treating skin diseases and / or improving skin conditions, comprising, as an active ingredient, mixed exosomes or fused exosomes in which natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes are mixed or fused. The composition according to the present invention exhibits an excellent cell proliferation effect and the effect of promoting collagen production, and thus can be effectively used as not only a cosmetic material for improving skin conditions, such as by reducing skin wrinkles, regenerating wounds, and increasing elasticity, but also a pharmaceutical material for preventing or treating skin diseases such as atopic dermatitis.

Description

Composition containing mixed exosomes as an active ingredient and use thereof

[0001] The present invention relates to a composition comprising mixed exosomes derived from different cells as an active ingredient and its use. More specifically, the present invention relates to a composition for treating skin diseases and / or improving skin conditions, comprising mixed exosomes or fused exosomes comprising a mixture of natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as an active ingredient.

[0002] Cell-to-cell interaction is essential for the survival of cells and organelles. Recently, the exchange of information between cells via extracellular vesicles (EVEs) has attracted significant attention. EVEs are double-membrane lipid-encapsulated vesicles secreted by cells. Depending on their size, they can be divided into exosomes (30–150 nm), microvesicles (50–1,000 nm), and large oncosomes (1–10 μm). Exosomes were first identified in the 1980s and were initially considered simple cellular waste products. However, it has since been discovered that exosomes contain various bioactive substances, including proteins, lipids, and genetic material, and that they reflect the properties and conditions of their origin. These exosomes are increasingly being recognized as important not only for their biological function but also for their role in disease pathogenesis and diagnosis. Exosomes originate from various cells, including normal cells, cancer cells, stem cells, and immune cells (Ali Hazrati1et.al.,Biomarker Research, 10(30):1-25, 2022).

[0003] Natural killer cells (NK cells), a representative innate immune cell, function to eliminate host cells infected with tumor cells, bacteria, intracellular parasites, or viruses without prior antigen sensitization. Furthermore, NK cells play a crucial role in both the innate immune response against pathogens or cancer cells infecting the host and the adaptive immune response through cytokine secretion.

[0004] Both resting and activated NK cells continuously produce and secrete exosomes, and exosomes derived from activated NK cells have been reported to activate non-activated NK cells. These exosomes have been found to express common NK cell markers such as CD56, NKG2D, and NCR, as well as killer-associated proteins such as perforin and FAS-L (Lugini L. et al., J. Immunol., 189(6):2833-2842, 2012). Recently, studies using these NK cell-derived exosomes have been attempted to treat various malignancies.

[0005] Meanwhile, stem cells are known to participate in biological functions within the human body, such as promoting angiogenesis, suppressing inflammation, and regulating immunity by regulating the microenvironment of damaged tissue. These biological functions are driven by the secretion of various growth factors, cytokines, proteins constituting the extracellular matrix, and antioxidant proteins from mesenchymal stem cells, which promote the protection and regeneration of damaged tissue.

[0006] Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into various cell types, including osteoblasts, chondrocytes, muscle cells, and adipocytes. Exosomes secreted by MSCs are known to have the effect of reducing inflammation by suppressing the secretion of inflammatory substances and stimulating cells involved in anti-inflammatory processes (Korean Patent Publication No. 10-2021-0122719).

[0007] Accordingly, research is actively underway on the therapeutic effects of various diseases using exosomes secreted by immune cells such as stem cells and NK cells, rather than using the cells themselves.

[0008] Accordingly, the inventors of the present invention have continued research utilizing various cell-derived exosomes to improve skin conditions such as wrinkle improvement, wound regeneration, and increased elasticity, as well as to treat skin diseases such as atopic dermatitis. As a result, the present invention was completed by confirming that treatment with a mixture of NK cell-derived exosomes and MSC-derived exosomes had a synergistic effect in promoting human fibroblast proliferation and collagen production compared to treatment with cell-derived exosomes alone.

[0009] To achieve the above purpose, one aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients.

[0010] Another aspect of the present invention provides a cosmetic composition for improving skin condition, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients.

[0011] Another aspect of the present invention provides a use of the pharmaceutical composition for preventing or treating skin diseases.

[0012] Another aspect of the present invention provides the use of the pharmaceutical composition for use in the manufacture of a medicament for the prevention or treatment of a skin disease.

[0013] Another aspect of the present invention provides a method for preventing or treating a skin disease comprising administering the pharmaceutical composition.

[0014] The composition comprising NK cell-derived exosomes and MSC-derived exosomes according to the present invention as active ingredients exhibits excellent cell proliferation and collagen production promotion effects. Accordingly, the composition of the present invention can be widely utilized as a cosmetic material for improving skin conditions, such as wrinkle reduction, wound regeneration, and increased elasticity, as well as a pharmaceutical material for preventing or treating skin diseases such as atopic dermatitis.

[0015] FIG. 1 is a schematic diagram illustrating an experimental schedule for evaluating the proliferation capacity of human skin fibroblast BJ6 cells following treatment with mixed exosomes derived from human natural killer cells and human mesenchymal stem cells (NK exo + MSC exo) according to one embodiment of the present invention.

[0016] FIG. 2a and FIG. 2b are graphs showing the results of measuring the size of exosomes derived from human natural killer cells (FIG. 2a) and human mesenchymal stem cells (FIG. 2b) using NTA equipment after isolating them according to one embodiment of the present invention.

[0017] Figure 3a is a diagram showing the results of evaluating the proliferation capacity of human skin fibroblast BJ6 cells following treatment with mixed exosomes derived from human natural killer cells and human mesenchymal stem cells (NK exo + MSC exo) according to one embodiment of the present invention. Specifically, these are microscopic photographs showing the degree of proliferation of human skin fibroblast BJ6 cells 24 hours and 48 hours after treatment with mixed exosomes.

[0018] Figure 3b is a diagram showing the results of evaluating the proliferation capacity of human skin fibroblast BJ6 cells following treatment with mixed exosomes derived from human natural killer cells and human mesenchymal stem cells (NK exo + MSC exo) according to one embodiment of the present invention. Specifically, this is a graph showing the degree of fibroblast proliferation quantified through absorbance measurement after treatment with mixed exosomes.

[0019] FIG. 4 is a graph showing the results of measuring the amount of collagen type I secreted from human skin fibroblasts BJ6 following treatment with mixed exosomes derived from human natural killer cells and human mesenchymal stem cells (NK exo + MSC exo) according to one embodiment of the present invention.

[0020] FIG. 5 is a graph showing the results of comparing cell proliferation rates by treating human skin fibroblasts, BJ6, with a mixed exosome (NK exo + MSC exo) derived from human natural killer cells and human mesenchymal stem cells according to one embodiment of the present invention after reacting it with a hyaluronic acid (HA) polymer.

[0021] Figure 6 is a graph showing the results of comparing the level of nitric oxide (NO) production according to the mixing ratio of human natural killer cells and human mesenchymal stem cell-derived exosomes according to one embodiment of the present invention.

[0022] Figures 7a and 7b are diagrams sequentially showing proteins identified through proteomics analysis of exosomes derived from human natural killer cells (Figure 7a) and human mesenchymal stem cells (Figure 7b) according to one embodiment of the present invention, according to the degree of increase. Here, the sequential listing is based on proteins that increased 100-fold compared to normal cells (control group).

[0023] FIG. 8 is a drawing showing the results of FACS analysis confirming the expression level of CD56 and DNAM1, which are NK cell surface markers, for human natural killer cell-derived exosomes according to one embodiment of the present invention.

[0024] FIG. 9a and FIG. 9b are diagrams showing a schematic diagram (FIG. 9a) of fused exosomes obtained using human natural killer cell and human mesenchymal stem cell-derived exosomes according to one embodiment of the present invention based on an extrusion method, and a graph measuring the size thereof (FIG. 9b).

[0025] A composition comprising a mixture of NK cell-derived exosomes and MSC-derived exosomes

[0026] One aspect of the present invention provides a composition comprising a mixture of NK cell-derived exosomes and MSC-derived exosomes.

[0027] Specifically, the present invention provides a composition comprising a mixed form of mixed exosomes capable of simultaneously imparting functions such as anti-inflammatory and anti-itching effects, anti-aging and skin regeneration effects, which are possessed by the natural killer cell-derived exosomes and the mesenchymal stem cell-derived exosomes, respectively.

[0028] The composition containing the above mixed exosomes was confirmed to exhibit a synergistic effect on improving skin compared to single cell-derived exosomes through human fibroblast proliferation and collagen secretion ability, and thus can be usefully utilized as a pharmaceutical material for preventing or treating skin diseases as well as a cosmetic material for improving skin condition.

[0029] Pharmaceutical composition comprising NK cell-derived exosomes and MSC-derived exosomes

[0030] One aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients.

[0031] The term "natural killer cell" or "NK cell" as used herein refers to a lymphocyte that accounts for approximately 15% of peripheral blood lymphocytes and plays a crucial role in the innate immune response. NK cells activate dendritic cells and induce cytotoxic T lymphocytes (CTLs) to specifically react with tumors, thereby eliminating tumor cells. Natural killer cells directly kill malignant tumors such as sarcoma, myeloma, carcinoma, lymphoma, and leukemia. Most NK cells present in the body of a normal person exist in an inactive state and are activated in response to interferon or macrophage-derived cytokines.

[0032] NK cells can be generated from hematopoietic cells, such as hematopoietic stem or progenitor cells, placental or umbilical cord-derived stem cells, induced pluripotent stem cells, or cells differentiated therefrom, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, bone marrow, spleen, liver, etc.

[0033] The term "stem cell" as used herein refers to an undifferentiated cell capable of differentiating into various types of tissue cells. The stem cell may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, tissue-derived stem cells, and combinations thereof. The stem cell may preferably be a mesenchymal stem cell.

[0034] The term "mesenchymal stem cell (MSC)" used herein refers to a cell that maintains stemness and self-renewal and has the ability to differentiate into various mesenchymal tissues, and can be extracted from bone marrow, adipose tissue, umbilical cord blood, synovium, bone tissue, and subpatellar fat. It is known that mesenchymal stem cells play an important role in skin regeneration by secreting various growth factors and cytokines such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) and promoting collagen production from fibroblasts. In addition, exosomes secreted by mesenchymal stem cells are known to have the effect of reducing inflammation by suppressing the secretion of inflammatory substances and stimulating cells involved in anti-inflammation.

[0035] In the present invention, the mesenchymal stem cells may be derived from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerve, skin, amniotic fluid, or amniotic membrane. Preferably, they may be derived from umbilical cord blood.

[0036] The exosomes in the culture medium of the above umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) contain various growth factors such as EGF, VEGF, TGF, HGF, FGF, IGF, and PDGF at higher levels than the exosomes in the culture medium of adipose tissue-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells. Growth factors such as EGF promote the proliferation of fibroblasts, which are skin cells, and promote cell migration and collagen synthesis, and protect cells in an inflammatory environment and suppress inflammatory mechanisms. Therefore, the UCB-MSC-derived exosomes can exhibit excellent skin condition improvement effects such as skin regeneration, improvement of skin elasticity, prevention or improvement of skin wrinkles, prevention or improvement of skin aging, hair growth or restoration of reduced hair follicles, and wound healing effects.

[0037] The term "exosome" as used herein may refer to a nano-sized particle that is naturally secreted by living cells, packaged in a lipid bilayer, and serves as a transporter of information between cells. The size of exosomes is known to be approximately 30 nm to 250 nm in diameter, and although there are some differences depending on the type of cell of origin, it is known that the exosome membrane contains surface proteins (surface markers) such as CD9, CD63, and CD81, and the inside of the exosome contains proteins such as TSG101 and ALIX that can prove that it is of endosomal origin. In addition, it contains proteins including growth factors and cytokines with various functions, as well as nucleic acids such as mRNA and miRNA, and has components and effects that reflect the characteristics of the cell of origin. In particular, stem cell-derived exosomes are known to have the effects of regulating stem cell differentiation, regeneration, growth promotion, and inducing specific immune responses.

[0038] In the present invention, the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes are not limited thereto, but can be obtained from individual cell culture solutions.

[0039] The term "culture medium" as used herein means a composition containing nutrients necessary to maintain cell growth and survival in vitro.

[0040] According to the present invention, exosomes derived from individual cultures of natural killer cells and mesenchymal stem cells can be isolated from the cultures using a commercially available exosome isolation reagent (e.g., ExoQuick-TC™) or the TFF (Tangential Flow Filtration) method. In addition to these methods, exosomes can be obtained from the cultures using ultracentrifugation, ultrafiltration, or SEC (Size Exclusion Chromatography) methods, and the present invention is not limited to the exosome obtaining methods described above.

[0041] More specifically, the NK cell-derived exosomes may be exosomes present within NK cells or secreted into the culture medium of NK cells. Furthermore, the NK cells may be NK cells obtained from blood, but may also be NK cells differentiated from stem cells or induced pluripotent stem cells.

[0042] The above NK cell-derived exosomes may express CD56 and DNAM1 simultaneously at 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more.

[0043] The above NK cell-derived exosomes can be prepared as follows using an exosome extraction method known in the art, but is not limited thereto:

[0044] a) A step of culturing NK cells in a culture medium and then subculturing them in a serum-free and antibiotic-free medium;

[0045] b) a step of recovering the cell culture supernatant;

[0046] c) a step of centrifuging the recovered cell culture supernatant; and

[0047] d) Step of isolating and purifying NK cell-derived exosomes.

[0048] Additionally, the method may further include, but is not limited to, the following steps:

[0049] e) A step of lyophilizing the separated and purified NK cell-derived exosomes.

[0050] Additionally, the method may further include the following steps prior to step a), but is not limited thereto:

[0051] a-1) Step of differentiating NK cells from induced pluripotent stem cells.

[0052] The NK cell-derived exosomes include ANXA6, IGHA2, IGKV2-28, PLCB3, IGLC2, HPR, IGHV1-2, IGHV3-35, IGHA1, C4BPA, IGLV3-10, IGHM, APOA2, APOC1, IGKV2-30, LRG1, PRKACA, CD5L, IGHV5-51, IGKV1-16, IGKV2-29, APOL1, IGHV1-46, IGHV3OR16-12, H3C1, C1QA, IGLV7-46, PON1, IGLV1-47, HRG, SLC26A1, IGHV3-49, IGKV3-11, GNLY, IGHA2, HPX, CA1, IGHV3-53, H2AC1, At least one protein selected from the group consisting of IGHV3-74, C1QB, IGHV3-15, IGLV6-57, IGLV9-49, KLKB1, IGKV-8, HPD, MYH10, C4A, APOC4, CAT, SERPINA6, IGHG3, CETP, IGHV3-66, C4BPB, IGLV2-8, PPP2R1A, PGLYRP2, APOE, ORM1, C8B, C1S, LBP, CLU, H4C1, IGKV2-24, IGKV1-6, IGLV3-9, ARPC1B, PPP1CB, APOA1, GP1BA, ITGAM, PCYOX1, C6, IL2, CFD, C1R, CD63, ITGB2, HLA-B, C3, GPX3, B2M and C1RL proteins is upregulated 4-fold. It may contain proteins that increase abnormally.

[0053] Additionally, the NK cell-derived exosome may comprise a protein in which at least one protein selected from the group consisting of IGHA2, ANXA6, IL2, C4A, CFD, CD5L, C1R, CD63, GNLY, ITGB2, HLA-B, C3, ITGAM, GPX3, B2M, C1RL, PLCB3, and SLC26A1 proteins is increased by four times or more.

[0054] More specifically, the MSC-derived exosomes may be exosomes present within MSCs or secreted into the culture medium of MSCs. In addition, the MSCs may be MSCs obtained from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerve, skin, amniotic fluid, or amniotic membrane, but may also be MSCs differentiated from induced pluripotent stem cells (iPSC-MSCs, iMSCs).

[0055] The above MSC-derived exosomes can be prepared using exosome extraction methods known in the art, including but not limited to:

[0056] a) A step of culturing MSCs in a culture medium and then subculturing them in a serum-free and antibiotic-free medium;

[0057] b) a step of recovering the cell culture supernatant;

[0058] c) a step of centrifuging the recovered cell culture supernatant; and

[0059] d) Step of isolating and purifying MSC-derived exosomes.

[0060] Additionally, the method may further include, but is not limited to, the following steps:

[0061] e) A step of lyophilizing the separated and purified MSC-derived exosomes.

[0062] Additionally, the method may further include the following steps prior to step a), but is not limited thereto:

[0063] a-1) Step of differentiating MSCs from induced pluripotent stem cells.

[0064] The MSC-derived exosomes include IGHG2, IGKV2-30, PPARA, ZNF350, UBR4, VCAN, IDH1, NID2, PGAM4, RGN, MASP2, CP, JCHAIN, RAB11A, PSMA1, IGHV1-2, H3-7, SERPINF1, GLOD4, IGHM, PSMB3, PGD, IGKV3-11, APOA4, SRPX, SERPINH1, APP, C4BPB, MMP2, SDC1, MYL6, ESD, PTX3, HSPA1B, PHGDH, CORO1C, MGAT2, RAB1A, LRP1, MAPRE2, PPP1CB, VASN, PLTP, C4BPB, RPL18, RAN, ITGA4, HMGCS1, LOXL2, IGHV1-46, At least one protein selected from the group consisting of PCYOX1, CFHR5, CD59, FSTL3, C1QB, CORO1A, RPS26, SERPINA3, IGHV4-34, SERPINA4, CAT, CFH, COTL1, IGKV3-7, PON1, COL5A2, H3-3A, B4GALT1, CLTC, PIGR, CCT4, IGHV3OR16-9, RACK1, AGT, CFP, TGFBI, FERMT3, PSMB9, NF2, IGHV3-49, MASP1, COLEC11, HBA1, GSN, APOE, GAPDH, F9, PGK1, GSTP1, PFN1, ADIPOQ, CAP1, RHOA, MFGE8, GGH, CNTN4, PTGR1, TSG101, PRKACA and FBLN2 proteins It may contain proteins that increase by more than four times.

[0065] Additionally, the MSC-derived exosomes may include a protein in which at least one protein selected from the group consisting of HBA1, GSN, APOE, C4BPB, GAPDH, F9, PGK1, GSTP1, PFN1, ADIPOQ, CAP1, RHOA, MFGE8, GGH, CNTN4, CLTC, PTGR1, TSG101, PRKACA, and FBLN2 proteins is increased by four times or more.

[0066] The term "induced pluripotent stem cell (iPSC)" used herein refers to a cell that is induced to have pluripotent differentiation ability through an artificial dedifferentiation process from a differentiated cell, such as a somatic cell, and is also referred to as an induced pluripotent stem cell. The induced pluripotent stem cell can be obtained by reprogramming the cell with a specific dedifferentiation inducing factor, such as Sox2, c-Myc, Klf4, Oct-4, etc., and can differentiate into various organ cells. The induced pluripotent stem cell of the present invention includes an induced pluripotent stem cell derived from all mammals, such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, but may preferably be an induced pluripotent stem cell derived from humans.

[0067] In the present invention, the pharmaceutical composition may contain each of the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients in an amount of about 0.1 wt% to about 90 wt%, specifically about 0.5 wt% to about 75 wt%, and more specifically about 1 wt% to about 50 wt%, based on the total weight of the composition.

[0068] Specifically, the pharmaceutical composition may contain about 1 ㎍ to about 1000 ㎍, about 5 ㎍ to about 500 ㎍, about 10 ㎍ to about 200 ㎍, or about 20 ㎍ to about 150 ㎍ of each of the natural killer cell-derived exosomes and the mesenchymal stem cell-derived exosomes. More specifically, the pharmaceutical composition may contain about 30 ㎍ to 100 ㎍ of each of the natural killer cell-derived exosomes and the mesenchymal stem cell-derived exosomes.

[0069] In the present invention, the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes included as effective ingredients of the pharmaceutical composition may be mixed exosomes in a co-mixed form. Furthermore, the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes may be fused exosomes in a form in which each of the natural killer cell-derived exosomes and the mesenchymal stem cell-derived exosomes is extruded from cells and physically fused.

[0070] In the present invention, the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes may be mixed in a weight ratio of, but not limited to, 1:10 to 10:1, 1:9 to 9:1, 1:4 to 4:1, 3:7 to 7:3, 2:3 to 3:2, or 1:1. Preferably, the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes may be mixed in a weight ratio of 1:1.

[0071] The pharmaceutical composition according to the present invention can be used for the prevention or treatment of skin diseases.

[0072] The above "skin disease" may be selected from the group consisting of, but is not limited to, atopic dermatitis, wounds, skin wrinkles, skin aging, loss of skin elasticity, dry skin, sensitive skin, acne, hair loss, and skin pigmentation and combinations thereof.

[0073] The term "prevention" as used herein can comprehensively mean preventing a disease in advance or reducing the likelihood or frequency of occurrence by administering the pharmaceutical composition in a pharmaceutically effective amount. For example, it can mean reducing the probability of occurrence or the probability of recurrence in a patient who is likely to develop a skin disease or a patient who has previously developed the disease. The "pharmaceutically effective amount" has the same meaning as "therapeutically effective amount," and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, administration route, administration method, number of administrations, treatment period, combination, or concurrently used drugs.

[0074] The term "treatment" as used herein may comprehensively mean improving a disease by administering the pharmaceutical composition in a pharmaceutically effective amount, may provide relief or cure of the symptoms of the disease in a shorter period of time compared to natural healing, and may improve one or most of the symptoms caused by the disease. The pharmaceutically effective amount is the same as described above. The pharmaceutical composition of the present invention may be a composition for treating a skin disease on its own, or may be administered together with other pharmacological ingredients and used as a therapeutic adjuvant for the disease. Accordingly, the term "treatment" includes the meaning of "treatment assistance."

[0075] Meanwhile, the pharmaceutical composition of the present invention is administered in a "therapeutically effective amount." The therapeutically effective amount is the same as described above.

[0076] The term "administration" as used herein means introducing a given substance into an individual by an appropriate method, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto. In addition, the pharmaceutical composition of one embodiment of the present invention may be administered by any device that allows the active substance to travel to the target tissue or cell. Specifically, it may be administered parenterally, and more specifically, it may be administered subcutaneously or transdermally. In addition, the pharmaceutical composition may be directly applied to the skin. When applying the pharmaceutical composition to the skin, it may include directly applying the pharmaceutical composition according to the present invention to the skin or spraying it, depending on its form.

[0077] Here, the subject to which the pharmaceutical composition can be administered may be a mammal, and specifically, a human.

[0078] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition according to the present invention may be administered in one to several divided doses at a dose of 0.001 mg / kg to 100 mg / kg for adults. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0079] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" herein means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity beyond what the subject of application (prescription) can tolerate. The carrier may be present in an amount of about 1% to about 9999% by weight, preferably about 90% to about 9999% by weight, based on the total weight of the pharmaceutical composition of the present invention.

[0080] The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of such carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition, but are not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington's Pharmaceutical Sciences (19th ed., 1995)."

[0081] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalants, and suppositories according to a method known in the art together with a suitable carrier. Preferably, the pharmaceutical composition of the present invention can be prepared as an injection. The injection may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection that is dissolved or suspended, but is not limited thereto. Depending on the type of the injection, the injection may contain at least one of distilled water for injection, vegetable oil (e.g., peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, estradiol benzoate, bismuth subsalicylate, sodium arsenobenzol, or streptomycin sulfate, and may optionally contain a stabilizer or preservative.

[0082] When the pharmaceutical composition of the present invention is manufactured as a topical skin preparation, it can be formulated in the form of an ointment, liquid, cream, spray, patch, etc. At this time, as long as the effects of the present invention are not impaired, ingredients commonly used in cosmetics or topical skin preparations, such as moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powdered ingredients, coloring agents, aqueous ingredients, water, various skin nutrients, etc., can be appropriately blended as needed.

[0083] The pharmaceutical composition of the present invention can also be prepared in the form of an injectable filler. For example, the injectable filler may include one or more carriers selected from the group consisting of alginic acid, carboxymethyl cellulose, chitosan, dextran, collagen, gelatin, pectin, agar, amylose, cyclodextrin, and elastin. When the injectable filler composition includes hyaluronic acid, the hyaluronic acid may have a cross-linked structure.

[0084] Additionally, biodegradable polymer scaffolds may include, for example, hyaluronic acid, polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), polyamino acid, polyanhydride, polyorthoester, and copolymers thereof.

[0085] Additionally, the filler injection according to the present invention may include a local anesthetic, an antihistamine, a vitamin, etc.

[0086] Local anesthetics may include, for example, lidocaine, etidocaine, bupivacaine, tetracaine, mepivacaine, procaine, prilocaine, and ropivacaine, but there are no specific restrictions if they are local anesthetics for injection.

[0087] Antihistamines may include, for example, plokon (piprinhydrinate), chlorpheniramine, diphenylpyraline (piprinhydrinate), diphenhydramine, and cetirizine, but there are no specific restrictions if they are antihistamines for injection.

[0088] The present invention provides a method for preventing or treating skin diseases using the pharmaceutical composition. The present invention also provides a use of the pharmaceutical composition for the manufacture of a medicament for preventing or treating skin diseases. Here, the pharmaceutical composition, skin disease, and prevention and treatment are the same as those described above.

[0089] A method for preventing or treating the above skin disease may include administering a pharmaceutical composition according to the present invention to the skin of a subject in need thereof. Here, the pharmaceutical composition, skin disease, treatment, and prevention are the same as those described above.

[0090] Cosmetic composition comprising NK cell-derived exosomes and MSC-derived exosomes

[0091] Another aspect of the present invention provides a cosmetic composition for improving skin condition, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients.

[0092] The above natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes are as described above.

[0093] The cosmetic composition of the present invention is used for improving skin condition.

[0094] As used herein, the term "improving skin condition" may be one selected from the group consisting of suppressing the occurrence of wrinkles, suppressing skin aging, improving skin elasticity, skin regeneration, wound healing, corneal regeneration, soothing skin irritation, and combinations thereof. Furthermore, it may be characterized by protecting the skin from deterioration or loss of skin cell function, improving the skin condition, or preventing or improving skin diseases.

[0095] The above cosmetic composition can be formulated into a cosmetic formulation commonly manufactured in the art. The cosmetic composition can be formulated into, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. More specifically, the cosmetic composition can be formulated into a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder. In addition, the cosmetic composition can be manufactured into a cosmetic filler.

[0096] Cosmetic fillers can be applied by applying them to the skin surface, and can include, for example, fragrances, xanthan gum, waxes, butters, oils, surfactants, moisturizers, alcohols, etc., and can include any of the components of a cosmetic composition that can be typically included without particular limitation. In addition, when the cosmetic filler composition includes hyaluronic acid, the hyaluronic acid can have a non-crosslinked structure.

[0097] When the formulation of the cosmetic composition according to the present invention is a paste, cream or gel, it may include a carrier component selected from the group consisting of animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and mixtures thereof.

[0098] The formulation of the cosmetic composition according to the present invention may include a carrier component selected from the group consisting of a solvent, a solvating agent, an emulsifying agent, and mixtures thereof, which are solutions or emulsions. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol, sorbitan fatty acid esters, and mixtures thereof.

[0099] When the formulation of the cosmetic composition according to the present invention is a suspension, it may include a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and a carrier component selected from the group consisting of microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth and mixtures thereof.

[0100] The above cosmetic composition may additionally contain various known additives in addition to the carrier depending on the formulation.

[0101] The above additives include emulsifiers, moisturizers, surfactants, chelating agents, antioxidants, bactericides, and stabilizers.

[0102] The emulsifier may include liquid paraffin, cetyl thanoate, stearic acid, etc. The moisturizer may include a polyol selected from the group consisting of glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, sorbitol, and any combination thereof.

[0103] The above chelating agent may include sodium ethylenediaminetetraacetate (EDTA), α-hydroxy fatty acids, lactoferrin, α-hydroxy acids, citric acid, lactic acid, malic acid, bilirubin, biliverdin, and the like.

[0104] The antioxidant may include butylhydroxyanisole, dibutylhydroxytoluene, or propyl gallate. In addition, ingredients that can be incorporated into the cosmetic composition or external skin preparation include fat components, emollients, organic and inorganic pigments, organic powders, ultraviolet absorbers, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, vitamins, and the like.

[0105] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0106] I. Preparation of mixed exosomes and fused exosomes

[0107] Manufacturing Example 1. Obtaining exosomes derived from natural killer cells

[0108] To culture natural killer (NK) cells, feeder cells irradiated with 100 Gy were seeded with NK cells at a 1:2 ratio. The cells were cultured in a medium containing human AB serum, 1% L-glutamine, and IL-15. The cell culture supernatant was then collected, and 1 L of medium was centrifuged at 300 rcf (g). After centrifugation, cell debris was removed.

[0109] To isolate and concentrate exosomes using the TFF method, the NK culture medium was circulated with triple-distilled water for 30 minutes in a TFF device equipped with a filter. All 1× PBS in the device was removed through the permeate line. For concentration, a culture medium corresponding to 1 / 50 to 1 / 10 of the culture medium was added to the tank of the TFF device and circulated through the device to concentrate. For buffer exchange, the solution to be used as the final buffer was diluted at least 10 times the starting volume. To recover exosomes circulating at the minimum flow rate, the valve of the drain line was opened to obtain NK-derived exosomes.

[0110] Manufacturing Example 2. Obtaining exosomes derived from mesenchymal stem cells

[0111] To culture umbilical cord blood-derived mesenchymal stem cells (MSCs), 10% serum and 1% antibiotics were added to DMEM medium and cultured in T75 flasks. The culture medium was then collected, and 1 L of the medium was centrifuged at 300 rcf (g). Cell debris was removed after centrifugation.

[0112] To isolate and concentrate exosomes using the TFF method, the MSC culture medium was circulated with triple-distilled water for 30 minutes in a TFF device equipped with a filter. After circulating, MSC-derived exosomes were obtained using the same method as in Preparation Example 1.

[0113] Manufacturing Example 3. Manufacturing of mixed exosomes derived from natural killer cells and mesenchymal stem cells.

[0114] Natural killer cell-derived exosomes (NK exo) and mesenchymal stem cell-derived exosomes (hMSC exo) obtained by separation from the above Manufacturing Examples 1 and 2 were mixed at 40 μg each. At this time, each was mixed in liquid form in a volume of 20 μL of PBS to prepare cell-derived mixed exosomes.

[0115] Meanwhile, in addition to the 1:1 ratio of the same amount as above, NK exosomes and MSC exosomes were also prepared at mixing ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 and used in the experiment.

[0116] Manufacturing Example 4. Manufacturing of fusion exosomes derived from natural killer cells and mesenchymal stem cells.

[0117] A fused exosome was prepared by fusing the NK-derived exosomes and MSC-derived exosomes obtained by separation from Manufacturing Examples 1 and 2 by the extrusion method (Fig. 9a).

[0118] II. Confirmation of the skin healing effect of mixed exosomes derived from cell culture fluid.

[0119] Example 1. Evaluation of cell proliferation effects following treatment with mixed exosomes derived from NK and MSCs.

[0120] Using the human skin fibroblast BJ6 cell line and Cell Counting Kit-8 (CCK-8), we evaluated the cell proliferation capacity induced by mixed exosomes derived from human natural killer cells (NK cells) and human mesenchymal stem cells (hMSCs). The cell proliferation capacity results of this experiment can be used as an indicator to evaluate the efficacy of improving skin conditions such as anti-aging and wound regeneration, as well as the symptom improvement of skin diseases such as atopic dermatitis.

[0121] Specifically, BJ6 cell line, a human skin fibroblast cell line, was seeded at 1×10 in a 48-well plate. 4The cells were seeded per well and cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture in a medium containing 10% FBS, the existing medium was removed. Afterwards, the test substances in Table 1 below were treated in a medium containing 1% Exosome-Depleted FBS (Thermo Fisher, Cat. No. A27208031) and cultured for an additional 48 hours (Fig. 1).

[0122] Group test substance treatment concentration control group (Vehicle)PBS-NK exoHuman natural killer cell (hNK cell) culture medium-derived exosomes alone40 μgMSC exoHuman mesenchymal stem cell (hMSC) culture medium-derived exosomes alone40 μgNK exo + hMSC exoHoNK cell culture medium-derived exosomes and hMSC culture medium-derived exosomes mixture40 μg + 40 μg

[0123] At this time, the NK-derived exosomes and MSC-derived exosomes isolated in Manufacturing Examples 1 and 2, respectively, which were used as test materials for evaluating cell proliferation capacity, were measured in size using NTA equipment (Nanoparticle Tracking Analysis), and both exosomes were confirmed to fall within the exosome size range of 40 to 200 nm. As shown in Figures 2a and 2b, respectively, the sizes of both NK-derived exosomes and MSC-derived exosomes had peaks between 40 and 200 nm.

[0124] After treatment with the test substance, the proliferation of human skin fibroblasts was observed under a microscope after 48 hours of additional culture (Fig. 3a).

[0125] Additionally, to quantitatively measure cell viability, the CCK-8 solution was diluted 1 / 10 using medium containing 1% Exosome-Depleted FBS, and 200 μL was treated per well. After reacting in an incubator for 2 hours, the absorbance was measured at a wavelength of 450 nm using a spectrophotometer. After absorbance measurement, the cell proliferation rate of each group was calculated as a percentage (%) compared to the control group (Vehicle).

[0126] As shown in Figs. 3a and 3b, the proliferation capacity of skin fibroblasts was significantly increased when treated with a mixture of hNK cell-derived exosomes and hMSC-derived exosomes compared to the hNK-derived exosomes alone and hMSC-derived exosomes alone treatment groups. Specifically, compared to the control group, the cell proliferation rate increased to 176.5% and 182.0% when treated with hNK-derived exosomes alone and hMSC-derived exosomes alone, respectively, whereas the cell proliferation rate was significantly increased to 250.4% when treated with mixed exosomes.

[0127] Through this, we were able to demonstrate a remarkable synergistic effect on cell proliferation by combining NK cell-derived exosomes and hMSC-derived exosomes compared to single-cell-derived exosomes. Ultimately, this suggests that the mixture can be utilized for improving skin conditions, such as anti-aging and wound regeneration, as well as alleviating the symptoms of skin diseases such as atopic dermatitis.

[0128] Example 2. Evaluation of collagen secretion following treatment with mixed exosomes derived from NK and MSC.

[0129] The collagen type I secretion capacity of mixed exosomes derived from human natural killer cells (NK cells) and human mesenchymal stem cells (hMSCs) was evaluated using the human skin fibroblast BJ6 cell line and the Collagen I Alpha 1 ELISA assay kit (R&D systems). The collagen secretion capacity results from fibroblasts in this experiment can be used as an indicator to evaluate the efficacy of improving skin conditions such as anti-aging and wound regeneration, and improving the symptoms of skin diseases such as atopic dermatitis.

[0130] Specifically, BJ6 cell line, a human skin fibroblast cell line, was seeded at 1×10 in a 6-well plate. 5 The cells were seeded per well and cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture in a medium containing 10% FBS, the existing medium was removed. Afterwards, the test substances in Table 1 were treated in a medium containing 1% Exosome-Depleted FBS and cultured for an additional 48 hours.

[0131] To measure the amount of collagen type I secretion, the Collagen I α1 (COL1α1) ELISA measurement sample was diluted 1 / 10 in PBS, and 100 μL was treated per ELISA well. Afterwards, the ELISA assay was performed according to the manufacturer's manual, and finally, the absorbance was measured at a wavelength of 450 nm using a spectrophotometer. At this time, the amount of COL1α1 secretion was quantitatively calculated by deriving a standard curve using the standard stock sample in the kit.

[0132] As a result of the measurement, as shown in Fig. 4, the amount of COL1α1 secretion was significantly increased in the test group treated with a mixture of hNK cell-derived exosomes and hMSC-derived exosomes compared to the hNK-derived exosome-only and hMSC-derived exosome-only treatment groups. Specifically, the basic background COL1α1 secretion amount was confirmed to be at the level of 5.8 ng / mL in the fibroblast-only control group that was not treated with the test substance after 48 hours of culture. In addition, it was confirmed that COL1α1 was secreted at the level of 7.5 ng / mL in the case of treatment with 40 μg of NK cell culture fluid-derived exosomes (NK exo) alone, and at the level of 7.0 ng / mL in the case of treatment with 40 μg of hMSC cell culture fluid-derived exosomes (MSC exo) alone. In contrast, in the group treated with 40 μg of NK cell culture-derived exosomes and 40 μg of a mixture of hMSC cell culture-derived exosomes (NK exo + MSC exo), COL1α1 was secreted at a level of 12 ng / mL.

[0133] Through this, we were able to demonstrate that a mixture of NK cell-derived exosomes and hMSC-derived exosomes can increase collagen secretion levels from dermal fibroblasts. Ultimately, this mixture can be usefully utilized in cosmetics and pharmaceuticals for improving skin conditions, such as anti-aging and wound healing, as well as alleviating the symptoms of skin diseases such as atopic dermatitis.

[0134] Example 3. Evaluation of cell proliferation rate according to treatment with mixed exosomes containing hyaluronic acid polymers derived from NK and MSC.

[0135] In order to observe the effect of mixed exosomes from the above Preparation Example 3 included in HA (Hyaluronic Acid) polymers on cell proliferation, first, NK-derived exosomes and MSC-derived exosomes were mixed with 100 μg of HA polymers. Then, after treatment to human dermal fibroblasts (BJ6), the cell proliferation rate of the fibroblasts was evaluated at 24, 48, and 72 hours. At this time, the cell proliferation rate was calculated as a proliferation rate (%) based on the BJ6 only treatment group (Vehicle) that did not include HA and mixed exosomes, which served as a negative control. Thereafter, the cell proliferation rates were compared between the mixed exosome treatment group that did not include HA and the mixed exosomes that included HA.

[0136] As shown in Fig. 5, it was confirmed that the cell proliferation rate of fibroblasts increased over time at 24, 48, and 72 hours by treatment with mixed NK cell-derived exosomes and hMSC-derived exosomes even in the presence of HA polymers.

[0137] Thus, we demonstrated that mixed exosomes based on HA polymers can be commercialized in various forms. For example, they could be usefully applied to pharmaceuticals and cosmetics, such as injectable treatments for osteoarthritis and HA-containing wrinkle-improving agents.

[0138] Example 4. Evaluation of NO production according to the mixing ratio of NK and MSC-derived exosomes.

[0139] The degree of nitric oxide (NO) production according to treatment with mixed exosomes derived from NK and MSC prepared at various mixing ratios in Manufacturing Example 3 above was evaluated.

[0140] Specifically, Raw 264.7 cells, a macrophage cell line used for NO production evaluation, were obtained from the Korean Cell Line Bank. The Raw 264.7 cells were cultured in a 37°C, 5% CO2 incubator using RPMI-1640 medium (Hyclon) supplemented with 10% fetal bovine serum (FBS; gibco) and penicillin / streptomycin (p / s; gibco).

[0141] Raw 264.7 cells were cultured at 1×10 5 Cells were dispensed into 96-well microplates at a concentration of 10 cells / well, stabilized, and stimulated with LPS (Lipopolysaccharide; Sigma) at a concentration of 5 μg / mL for 1 hour. Then, mesenchymal stem cell exosomes (MSC-exo) and iPSC (induced pluripotent stem cell)-derived natural killer cell exosomes (EiNK-exo) were mixed at various ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, and treated at a total concentration of 100 μg / well for 24 hours (Fig. 6).

[0142] After 24 hours, 50 μL of cell culture supernatant was mixed with an equal volume of Griess reagent (Promega) sulfanilamide solution in a new 96-well microplate and incubated at room temperature for 10 minutes. After the reaction, 50 μL of N-1-napthylethylenediamine dihydrochloride (NED) was co-treated, and the degree of NO production was measured at 540 nm using a microplate reader (ELISA reader). The concentration of NO was calculated by a standard curve for different concentrations of sodium nitrite.

[0143] As shown in Fig. 6, when treated with 5 μg / mL of LPS, it was confirmed that the amount of NO production increased 26-fold compared to the untreated control group. As the ratio of MSC-exo: EiNK-exo decreased from 9:1 to 1:9, that is, as the concentration of EiNK increased, the production of NO decreased, and when the ratio of MSC-exo: EiNK-exo was 1:9, a significant reduction in NO production was confirmed at 84%.

[0144] III. Characterization of NK and MSC-derived exosomes

[0145] Example 5. Proteomic analysis of NK and MSC-derived exosomes using proteomics analysis.

[0146] Proteomic analysis was performed on the NK and MSC-derived exosomes obtained from Manufacturing Examples 1 and 2 above using proteomics analysis. At this time, exosomes from normal cells (293T cells) were used as a control group.

[0147] The results of proteomic analysis of NK and MSC-derived exosomes are presented graphically, with proteins increased by more than fivefold compared to normal cell exosomes, ranked in descending order of increase. Proteins increased by 100-fold compared to normal cell exosomes are presented in descending order.

[0148] In the case of NK-derived exosomes, a total of 167 proteins were identified that were increased by more than 4-fold, including ANXA6, IGHA2, PLCB3, HPR, IGHV1-2, C4BRA, IGHM, AOPA2, AOPC1, LRG1, CD5L, APOL1, H3C1, PON1, HRG, GNLY, HPX, CA1, C8B, C4A, and C1S (Fig. 7a).

[0149] Additionally, in the case of MSC-derived exosomes, proteins that were increased by more than fourfold were identified as HBA1, GSN, APOE, C4BPB, GAPDH, F9, PGK1, GSTP1, PFN1, ADIPOQ, CAP1, RHOA, MFGE8, GGH, CNTN4, CLTC, PTGR1, TSG101, PRKACA, and FBLN2 (Fig. 7b).

[0150] Example 6. Surface marker analysis of NK-derived exosomes

[0151] To identify the surface markers of the NK-derived exosomes obtained from the above Preparation Example 1, FACS analysis was performed using CD56 antibody (BD, Cat. No. 555518) and DNAM1 antibody (Biolegend, Cat. No. 338312). As a result, it was confirmed that CD56, an NK cell marker, and DNAM1, an NK surface-activated receptor, were simultaneously expressed, and the dual expression rate for the surface markers was analyzed to be 95% or higher (Fig. 8).

[0152] Example 7. Size analysis of NK and MSC-derived fusion exosomes

[0153] The sizes of NK-derived exosomes and MSC-derived exosomes obtained from Manufacturing Examples 1 and 2, respectively, were fused through an extrusion method as in Manufacturing Example 4, and then measured using an NTA device.

[0154] As a result, as shown in Fig. 9b, the size of NK- and MSC-derived fused exosomes peaked between 40 and 200 nm. This indicates that the fused exosomes, obtained by fusion of isolated NK-derived exosomes and MSC-derived exosomes through extrusion, fall within the exosome size range of 40 to 200 nm, similar to NK exosomes and MSC exosomes.

Claims

1. A pharmaceutical composition for preventing or treating skin diseases, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as active ingredients.

2. In paragraph 1, A pharmaceutical composition wherein the above natural killer cell-derived exosomes simultaneously express CD56 and DNAM1 at a level of 70% or more.

3. In paragraph 1, The above natural killer cell-derived exosomes were ANXA6, IGHA2, IGKV2-28, PLCB3, IGLC2, HPR, IGHV1-2, IGHV3-35, IGHA1, C4BPA, IGLV3-10, IGHM, APOA2, APOC1, IGKV2-30, LRG1, PRKACA, CD5L, IGHV5-51, IGKV1-16, IGKV2-29, APOL1, IGHV1-46, IGHV3OR16-12, H3C1, C1QA, IGLV7-46, PON1, IGLV1-47, HRG, SLC26A1, IGHV3-49, IGKV3-11, GNLY, IGHA2, HPX, CA1, IGHV3-53, H2AC1, At least one protein selected from the group consisting of IGHV3-74, C1QB, IGHV3-15, IGLV6-57, IGLV9-49, KLKB1, IGKV-8, HPD, MYH10, C4A, APOC4, CAT, SERPINA6, IGHG3, CETP, IGHV3-66, C4BPB, IGLV2-8, PPP2R1A, PGLYRP2, APOE, ORM1, C8B, C1S, LBP, CLU, H4C1, IGKV2-24, IGKV1-6, IGLV3-9, ARPC1B, PPP1CB, APOA1, GP1BA, ITGAM, PCYOX1, C6, IL2, CFD, C1R, CD63, ITGB2, HLA-B, C3, GPX3, B2M and C1RL proteins is increased 4-fold Exosomes containing proteins that increase in number, The above mesenchymal stem cell-derived exosomes expressed IGHG2, IGKV2-30, PPARA, ZNF350, UBR4, VCAN, IDH1, NID2, PGAM4, RGN, MASP2, CP, JCHAIN, RAB11A, PSMA1, IGHV1-2, H3-7, SERPINF1, GLOD4, IGHM, PSMB3, PGD, IGKV3-11, APOA4, SRPX, SERPINH1, APP, C4BPB, MMP2, SDC1, MYL6, ESD, PTX3, HSPA1B, PHGDH, CORO1C, MGAT2, RAB1A, LRP1, MAPRE2, PPP1CB, VASN, PLTP, C4BPB, RPL18, RAN, ITGA4, HMGCS1, LOXL2, IGHV1-46, PCYOX1, CFHR5, CD59, FSTL3, C1QB, CORO1A, RPS26, SERPINA3, IGHV4-34, SERPINA4, CAT, CFH, COTL1, IGKV3-7, PON1, COL5A2, H3-3A, B4GALT1, CLTC, PIGR, CCT4, IGHV3OR16-9, RACK1, AGT, CFP, TGFBI, FERMT3, PSMB9, NF2, IGHV3-49, MASP1, COLEC11, HBA1, GSN, APOE, GAPDH, F9, PGK1, GSTP1, PFN1, ADIPOQ, CAP1, RHOA, MFGE8, GGH, CNTN4, PTGR1, With TSG101, PRKACA and FBLN2 proteins A pharmaceutical composition comprising an exosome comprising a protein in which at least one protein selected from the group consisting of proteins is increased by a factor of four or more.

4. In paragraph 3, The above natural killer cell-derived exosome is an exosome containing a protein in which at least one protein selected from the group consisting of IGHA2, ANXA6, IL2, C4A, CFD, CD5L, C1R, CD63, GNLY, ITGB2, HLA-B, C3, ITGAM, GPX3, B2M, C1RL, PLCB3 and SLC26A1 proteins is increased by 4-fold or more, A pharmaceutical composition, wherein the above mesenchymal stem cell-derived exosome is an exosome comprising a protein in which at least one protein selected from the group consisting of HBA1, GSN, APOE, C4BPB, GAPDH, F9, PGK1, GSTP1, PFN1, ADIPOQ, CAP1, RHOA, MFGE8, GGH, CNTN4, CLTC, PTGR1, TSG101, PRKACA, and FBLN2 proteins increases by 4-fold or more.

5. In paragraph 1, A pharmaceutical composition comprising exosomes co-mixed with natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as an active ingredient.

6. In paragraph 1, A pharmaceutical composition, wherein the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes are obtained from individual cell culture solutions.

7. In paragraph 1, A pharmaceutical composition wherein each of the natural killer cell-derived exosomes and the mesenchymal stem cell-derived exosomes are extruded from a cell and physically fused.

8. In paragraph 1, A pharmaceutical composition wherein the natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes are mixed at a weight ratio of 1:9 to 9:

1.

9. In paragraph 1, A pharmaceutical composition, wherein each of the above natural killer cells and mesenchymal stem cells is differentiated from blood and induced pluripotent stem cells (iPSC).

10. In paragraph 1, A pharmaceutical composition wherein the above mesenchymal stem cells are derived from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerve, skin, amniotic fluid or amniotic membrane.

11. In paragraph 1, A pharmaceutical composition, wherein the skin disease is selected from the group consisting of atopic dermatitis, wounds, skin wrinkles, skin aging, decreased skin elasticity, dry skin, sensitive skin, acne, hair loss, and skin pigmentation, and combinations thereof.

12. A cosmetic composition for improving skin condition, comprising natural killer cell-derived exosomes and mesenchymal stem cell-derived exosomes as effective ingredients.

13. In paragraph 12, A cosmetic composition wherein the improvement in skin condition is selected from the group consisting of inhibition of wrinkle occurrence, inhibition of skin aging, improvement of skin elasticity, skin regeneration, wound healing, corneal regeneration, relief of skin irritation, and combinations thereof.

14. Use of the pharmaceutical composition of paragraph 1 for the prevention or treatment of skin diseases.

15. Use of the pharmaceutical composition of paragraph 1 for manufacturing a drug for preventing or treating skin diseases.

16. A method for preventing or treating a skin disease, comprising a step of administering the pharmaceutical composition of paragraph 1.

Images