WO2024043713A1 - Composition comprising natural killer cell-derived exosome and biocompatible polymer as active ingredients, and use thereof - Google Patents

Abstract

The present invention relates to: a composition comprising a natural killer cell-derived exosome and a biocompatible polymer as active ingredients; and a use thereof. The composition according to the present invention exhibits excellent cell proliferation and wound healing effects, and thus can be used as both a cosmetic material for improving skin condition, such as by ameliorating skin wrinkles, healing wounds, and increasing elasticity, and a pharmaceutical material for preventing or treating skin diseases such as atopic dermatitis.

Description

Composition containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients and uses thereof

The present invention relates to a composition for treating skin diseases and/or improving skin condition, including a composition containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients.

Exchange of various information between cells (cell-to-cell interaction) is an essential process for the survival of cells and organs made of cells. Recently, information exchange between cells by extracellular vesicles has been attracting attention. Extracellular vesicles are substances with a double lipid membrane structure secreted from cells. Depending on their size, they are classified into exosomes (30-150 nm), microvesicles (50-1,000 nm), and large oncosomes (1-10 μm). It is divided into Among these, exosomes were first identified in the 1980s and were considered simple cellular waste at the time, but since then, exosomes have been recognized as containing various bioactive substances such as proteins, lipids, and genetic materials, and the nature and state of origin from which they were derived are known. It has been confirmed that this is reflected. The importance of these exosomes has been revealed not only in information about biological functions, but also in disease pathogenesis and disease diagnosis. Exosomes are derived from various cells such as normal cells, cancer cells, stem cells, and immune cells (Ali Hazrati1 et.al. , Biomarker Research , 10(30):1-25, 2022).

Natural killer cells (NK cells), a representative innate immune cell, have selective cytotoxicity against target cells and distinguish between normal cells and cancer cells through various immune receptors. The NKG2D receptor, which acts as a master switch to induce the activation of NK cells, is known to mediate the direct cell killing effect of NK cells. Signal transduction through these receptors induces the death of target cells through perforin and granzyme B and exhibits selective cytotoxicity through secretion of cytokines such as IFN-γ and TNF-α.

Dormant 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 were found to express common NK cell markers such as CD56, NKG2D, NCR, and killer-related proteins such as perforin and FAS-L (Lugini L. et.al. , J. Immunol. , 189(6 ):2833-2842, 2012). Recently, research on various malignant tumors has been attempted using these NK cell-derived exosomes.

Meanwhile, Korean Patent Publication No. 10-2019-0003336 discloses the use of exosomes derived from adipose stem cells to treat dermatitis, and Korean Patent No. 10-1663912 discloses the use of exosomes derived from human adipose stem cells for skin whitening and wrinkles. Remedial or regenerative uses are disclosed.

However, research on the use of NK cell-derived exosomes to improve skin condition and treat skin diseases is insufficient.

Accordingly, the present inventor continued research using NK cell-derived exosomes for improving skin conditions such as improving skin wrinkles, wound regeneration, and increasing elasticity, and for treating skin diseases such as atopic dermatitis. As a result, NK cell-derived exosomes; The present invention was completed by confirming that it has excellent cell proliferation and wound healing ability through the development of a complex of biocompatible polymers such as collagen and hyaluronic acid.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases containing NK cell-derived exosomes and biocompatible polymers as active ingredients.

Another aspect of the present invention provides a cosmetic composition for improving skin condition containing NK cell-derived exosomes and biocompatible polymers as active ingredients.

Another aspect of the present invention provides the use of a pharmaceutical composition containing NK cell-derived exosomes and biocompatible polymers as active ingredients for preventing or treating skin diseases.

Another aspect of the present invention provides the use of a pharmaceutical composition containing NK cell-derived exosomes and biocompatible polymers as active ingredients for use in the production of a drug for preventing or treating skin diseases.

Another aspect of the present invention provides a method for preventing or treating skin diseases, comprising administering a pharmaceutical composition containing NK cell-derived exosomes and biocompatible polymers as active ingredients.

The composition containing NK cell-derived exosomes and biocompatible polymers according to the present invention as active ingredients has excellent cell proliferation and wound healing effects. Accordingly, the composition of the present invention can be widely used as a cosmetic material for improving skin conditions such as improving skin wrinkles, wound regeneration, and increasing elasticity, as well as a pharmaceutical material for preventing or treating skin diseases such as atopic dermatitis. there is.

Figure 1 is a diagram showing the results of particle size analysis of NK cell-derived exosomes according to an embodiment of the present invention.

Figure 2a is a diagram showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with exosomes derived from NK cell culture medium. Specifically, this is a photomicrograph of the degree of proliferation of human fibroblasts on the 3rd day after treatment with exosomes derived from NK cell culture medium.

Figure 2b is a diagram showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with exosomes derived from NK cell culture medium. Specifically, this is a graph showing the quantification of the absorbance analysis results measuring the degree of cell proliferation over time (0 h, 24 h, 48 h, and 72 h) after treatment with exosomes derived from NK cell culture medium.

Figure 3 is a diagram showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with NK cell-derived exosomes. Specifically, this is a graph showing the quantification of the absorbance analysis results measuring the degree of cell proliferation over time (24h, 48h, and 72h) after treatment with exosomes derived from NK cell culture medium and exosomes derived from lyophilized NK cells.

Figure 4a is a diagram showing micrographs taken over time of the degree of wound recovery according to treatment with exosomes derived from NK cell culture medium.

Figure 4b is a diagram showing the results of evaluating wound healing efficacy according to exosome treatment derived from NK cell culture medium, and the degree of wound recovery is graphically shown by measuring the gap between cell wound areas.

Figure 5 is a diagram schematically showing the conditions for establishing the mixing ratio of NK cell-derived exosomes and HA complexes for evaluating the cell proliferation ability of NK cell-derived exosomes and biocompatible polymer complexes and the schedule of experiments for evaluating cell proliferation ability.

Figure 6 is a diagram showing the results of evaluating cell proliferation ability according to treatment at various concentrations of NK cell-derived exosomes and HA polymer complex.

Figure 7 is a diagram schematically showing the NK cell-derived exosome and biocompatible polymer complex prepared according to an embodiment of the present invention, showing a schematic diagram of the NK cell-derived exosome complex embedded with collagen polymer.

Figure 8 is a diagram schematically showing the experimental conditions and schedule for evaluating the cell proliferation ability of NK cell-derived exosomes and biocompatible polymer complexes. Here, vehicle, NK cell-derived exosome (40 μg), HA (100 μg), and collagen (100 μg) each contain NK cell-derived exosome and HA complex (HA embedded NK exosome) and NK cell-derived exosome. and collagen complex (Collagen embedded NK exosome) was used as a control.

Figures 9a to 9c are diagrams showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with NK cell-derived exosomes and biocompatible polymer complex. Specifically, human fibroblasts after 24 hours (FIG. 9a), 48 hours (FIG. 9b), or 72 hours (FIG. 9c) of NK cell culture medium-derived exosomes and hyaluronic acid (HA) or collagen polymer complex treatment. This is a photomicrograph showing the degree of proliferation.

Figure 10 is a diagram showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with NK cell-derived exosomes and biocompatible polymer complex. Specifically, this is a graph showing the quantification of the absorbance analysis results of measuring the degree of cell proliferation over time (24h, 48h, and 72h) after treatment of exosomes derived from NK cell culture medium and hyaluronic acid (HA) or collagen polymer complex.

Figure 11 is a diagram schematically showing the experimental conditions and schedule for evaluating the cell proliferation ability of NK cell-derived exosomes and biocompatible dual polymer complex.

Figures 12a to 12c are diagrams showing the results of evaluating the proliferation ability of human fibroblasts according to the treatment time of NK cell-derived exosomes and biocompatible dual polymer complex. Specifically, human cells after 24 hours (FIG. 12a), 48 hours (FIG. 12b), or 72 hours (FIG. 12c) of NK cell culture-derived exosomes and hyaluronic acid (HA) and/or collagen polymer complex treatment. This is a photomicrograph showing the degree of fibroblast proliferation.

Figure 13 is a diagram showing the results of evaluating the proliferation ability of human fibroblasts according to treatment with NK cell-derived exosomes and biocompatible dual polymer complex. Specifically, this graph quantifies the results of absorbance analysis measuring the degree of cell proliferation over time (24h, 48h, and 72h) after treatment with exosomes derived from NK cell culture medium and hyaluronic acid (HA) and/or collagen polymer complex. .

Composition containing NK cell-derived exosomes and polymers

One aspect of the present invention is NK cell-derived exosomes; And a composition comprising a biocompatible polymer is provided.

At this time, the biocompatible polymer may be collagen and/or hyaluronic acid.

Pharmaceutical composition comprising NK cell-derived exosomes and biocompatible polymer

One aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients.

As used herein, the term “natural killer cell” or “NK cell” refers to a lymphoid cell that accounts for approximately 15% of peripheral blood lymphocytes and plays an important role in the innate immune response. do. NK cells activate dendritic cells and induce cytotoxic T lymphocytes (CTL) to respond specifically to tumors to eliminate 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 normal people exist in an inactive state and are activated in response to interferon or macrophage-derived cytokines.

NK cells are hematopoietic cells, such as hematopoietic stems or progenitors, placenta- or umbilical cord-derived stem cells, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, bone marrow, spleen, liver, etc. , induced pluripotent stem cells, or cells differentiated therefrom.

The term “exosome” used herein may refer to nano-sized particles that are naturally secreted by being packaged in a lipid bilayer from living cells, and play a role in transferring information between cells. The size of exosomes is known to be approximately 30 nm to 250 nm in diameter, and although it varies somewhat depending on the type of cell of origin, the exosome membrane contains surface proteins (surface markers) such as CD9, CD63, and CD81, It is known that exosomes contain proteins such as TSG101 and ALIX, which can be proven to be of endosomal origin. In addition, it contains proteins including growth factors and cytokines with various functions, and nucleic acids such as mRNA and miRNA, and has components and efficacy that reflect the characteristics of the cell of origin. In particular, stem cell-derived exosomes are known to have effects such as regulating the differentiation of stem cells, promoting regeneration, growth, and inducing specific immune responses.

In one embodiment of the present invention, the NK cell-derived exosomes may be present within NK cells or may be exosomes secreted in the culture medium of NK cells. Additionally, NK cells may be NK cells obtained from blood, or may be NK cells differentiated from stem cells or induced pluripotent stem cells.

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

a) culturing NK cells in culture medium and then subculturing them in serum-free and antibiotic-free medium;

b) recovering the cell culture supernatant;

c) centrifuging the recovered cell culture supernatant; and

d) Separating and purifying NK cell-derived exosomes.

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

e) Freeze-drying the separated and purified NK cell-derived exosomes.

In addition, the method may further include, but is not limited to, the following steps prior to step a):

a-1) Differentiating NK cells from induced pluripotent stem cells.

As used herein, the term “biocompatible” refers to the property of a material not to cause substantial adverse reactions upon introduction into the host. For example, this means that when a foreign object or substance is introduced into the body, it does not induce harmful reactions such as inflammatory reaction and/or immune reaction. Biocompatibility has a comprehensive meaning such as biodegradability and biostability.

As used herein, the term “biocompatible polymer” refers to a biodegradable polymer that does not induce harmful reactions such as inflammatory and/or immune reactions when introduced into the body, and is a process of decomposition through metabolism in living organisms. It is a polymer substance that changes into a low molecular weight compound. The biocompatible polymer is decomposed through simple hydrolysis or the action of enzymes.

In one embodiment of the present invention, the biocompatible polymer is, but is not limited to, hyaluronic acid, collagen, fucoidan, alginate, heparin, cellulose, gelatin, fibrin, chitosan, dextran, elastin, polyethylene glycol (PEG), It may be selected from the group consisting of polyethyleneimine (PEI), polyvinyl alcohol (PVA), polycaprolactone (PCL), polylactide, polylactic glycolic acid (PLGA), and polygamma glutamic acid.

As used herein, the term "Hyaluronic acid (HA)" is a biopolymer material in which repeating units consisting of N-acetyl-D-glucosamine and D-glucuronic acid are linearly linked, and the molecular weight is 100 kDa. It is a colorless, transparent, high-viscosity linear polysaccharide ranging from 13,000 kDa to 13,000 kDa, and is extracted and extracted from various species and tissues such as vitreous humor of the eye, synovial fluid of joints, and chicken comb using acid solubilization, alkaline solubilization, neutral solubilization, and enzyme solubilization methods. It can be purified and used. Viscosity, elasticity, and moisturizing properties are determined depending on the molecular weight and concentration of hyaluronic acid in the aqueous solution, and as the concentration of hyaluronic acid increases, its properties increase. Hyaluronic acid is known to have different functions depending on its molecular weight.

The hyaluronic acid constituting the composition of the present invention may be 1.0 KDa to 3.0 MDa, 10 KDa to 2.5 MDa, 50 KDa to 2.0 MDa, or 110 KDa to 1.81 MDa.

Additionally, the “salt of hyaluronic acid” may include a cosmetically acceptable salt of hyaluronic acid within the range of having the same efficacy as hyaluronic acid. At this time, the salt of hyaluronic acid may be, for example, sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate.

Additionally, the hyaluronic acid is not limited thereto, but may be a cross-linked hyaluronic acid gel. The crosslinked hyaluronic acid gel is not limited thereto, but includes, but is not limited to, 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis (2,3-epoxypropoxy)butane, 1,4-bis Selected from the group consisting of glycidyloxybutane, 1,2-bis (2,3-epoxypropoxy) ethylene and 1- (2,3-epoxypropyl) -2,3-epoxycyclohexane or combinations thereof It may be a hyaluronic acid gel cross-linked with at least one cross-linking agent.

The term “collagen” used herein is the most commonly found protein in the human body and the most abundant protein in mammals, accounting for approximately 25 to 35% of total proteins. In particular, it is a major component of bones, tendons, and ligaments, and mainly plays a role in maintaining the structure of organs. The collagen can be easily extracted from the skin of mammals such as cows or pigs, and the collagen may include collagen derivatives in addition to pure collagen. Regarding collagen, its origin is not particularly limited, and for example, various collagens derived from mammals, fish, such as bovine bone, bovine skin, pig bone, pig skin, etc. can be used.

Collagen I to collagen IV can be used as the collagen constituting the composition according to the present invention. In one embodiment, the collagen may be collagen I and collagen III. Most preferably, the collagen may be atelocollagen of collagen I. At this time, the atelocollagen refers to collagen from which telopeptide, an antigenic substance that can cause skin hypersensitivity reactions, has been removed.

Additionally, in the present invention, the pharmaceutical composition may include hyaluronic acid or a salt thereof in an amount of 0.1 to 30% by weight based on the total weight of the composition.

Specifically, the pharmaceutical composition contains about 1 μg to about 1000 μg, about 10 μg to about 500 μg, about 30 μg to about 200 μg, or about 50 μg of hyaluronic acid or a salt thereof. It may contain from about 100 μg. More specifically, the pharmaceutical composition may include about 100 μg of hyaluronic acid or a salt thereof.

In the present invention, the pharmaceutical composition may include collagen in an amount of 0.1 to 30% by weight based on the total weight of the composition.

Specifically, the pharmaceutical composition contains about 1 μg to about 1000 μg, about 10 μg to about 500 μg, about 30 μg to about 200 μg, or about 50 μg of collagen. It may contain from about 100 μg. More specifically, the pharmaceutical composition may contain about 100 μg of collagen.

At this time, the exosome and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the exosomes and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:9 to 2:8.

Additionally, the exosomes and collagen may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the exosomes and collagen may be mixed at a weight ratio of 1:9 to 2:8.

Additionally, the pharmaceutical composition may include the exosomes, collagen, and hyaluronic acid or a salt thereof at a weight ratio of 1:1:1 to 1:10:10. Preferably, the pharmaceutical composition may include exosomes, hyaluronic acid or a salt thereof, and collagen in a weight ratio of 1:2.5:2.5.

The pharmaceutical composition according to the present invention can be used for preventing or treating skin diseases.

The “skin disease” may be selected from the group consisting of atopic dermatitis, wounds, skin wrinkles, skin aging, weakened skin elasticity, dry skin, sensitive skin, acne, hair loss, skin pigmentation, and combinations thereof, but is not limited thereto. No.

As used herein, the term “prevention” may comprehensively mean preventing a disease in advance or reducing the possibility or frequency of occurrence of a disease by administering the pharmaceutical composition in a pharmaceutically effective amount. For example, it may be to lower the probability of developing a skin disease or to reduce the probability of recurrence in patients who are likely to develop a skin disease or have previously had it. The “pharmaceutically effective amount” has the same meaning as the “therapeutically effective amount,” including the type of disease, patient’s age, weight, health, gender, patient’s sensitivity to the drug, route of administration, method of administration, number of administrations, treatment period, It can be easily determined by a person skilled in the art according to factors well known in the medical field, such as combination or drugs used simultaneously.

As used herein, the term “treatment” may comprehensively mean improving a disease by administering the pharmaceutical composition in a pharmaceutically effective amount, and may mean alleviating or curing the symptoms of the disease in a shorter time compared to natural cure. It may be something that improves one symptom or most of the symptoms caused by a disease. The pharmaceutically effective amount is the same as described above. The pharmaceutical composition of the present invention may itself be a composition for treating skin diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for the diseases. Accordingly, the term “treatment” includes the meaning of “treatment assistance.”

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.

As used herein, the term "administration" means introducing a predetermined substance into an individual by an appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. . It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, locally, intranasally, intrapulmonaryly, or rectally, but is not limited thereto. Additionally, the pharmaceutical composition of one embodiment of the present invention may be administered by any device that allows the active agent to move to target tissues or cells. Specifically, it may be administered parenterally, and more specifically, may be administered subcutaneously or transdermally. Additionally, the pharmaceutical composition can be applied directly to the skin. When applying the pharmaceutical composition to the skin, the pharmaceutical composition according to the present invention may be applied directly to the skin or sprayed depending on its form.

Here, the subject to whom the pharmaceutical composition can be administered may be a mammal, specifically, a human.

The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can. The dosage of the pharmaceutical composition according to the present invention is 0.001 mg/kg to 100 mg/kg for adults, and can be administered in one to several divided doses. These dosages should not be construed as limiting the scope of the invention in any respect.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Here, “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have any toxicity beyond what the subject of application (prescription) can adapt to. The carrier may be included in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight, based on the total weight of the pharmaceutical composition of the present invention.

The pharmaceutically acceptable carrier may be any carrier that is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. 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).

When the pharmaceutical composition is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. 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 used by dissolving or suspending, but is not limited thereto. Depending on the type of injection, 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 arsenobenzoate, or streptomycin sulfate, and may optionally include a stabilizer or preservative.

When the pharmaceutical composition of the present invention is prepared for external use on the skin, it may be formulated in the form of ointment, liquid, cream, spray, patch, etc. At this time, within the range that does not impair the effect of the present invention, ingredients commonly used in cosmetics or external skin preparations, such as moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants , aqueous ingredients, water, and various skin nutrients can be appropriately mixed as needed.

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

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

Additionally, the filler injection according to the present invention may contain a local anesthetic, antihistamine, vitamin, etc.

Local anesthetics include, for example, Lidocaine, Etidocaine, Bupivacaine, Tetracaine, Mepivacaine, Procaine, and Prilocaine ( Prilocaine, Ropivacaine, etc., but there is no particular limitation as long as it is a local anesthetic for injection.

Antihistamines may include, for example, plokon (Piprinhydrinate), Chlorpheniramine, Diphenylpyraline (Piprinhydrinate), Diphenhydramine, Cetirizine, etc., but are antihistamines for injection. There are no particular restrictions on ramen.

The present invention provides a method for preventing or treating skin diseases using the pharmaceutical composition. Additionally, the present invention provides the use of the pharmaceutical composition for use in the manufacture of a medicament for the prevention or treatment of skin diseases. Another aspect of the present invention provides a use of the pharmaceutical composition for preventing or treating skin diseases. Here, the pharmaceutical composition, skin disease, prevention and treatment are the same as described above.

The method for preventing or treating the skin disease may include administering the 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 described above.

Cosmetic composition containing NK cell-derived exosomes and biocompatible polymers

Another aspect of the present invention provides a cosmetic composition for improving skin condition containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients.

The natural killer cell-derived exosomes and biocompatible polymers are as described above.

The cosmetic composition of the present invention is used to improve skin condition.

As used herein, the term "skin condition improvement" refers to a group consisting of inhibition of wrinkles, inhibition of skin aging, improvement of skin elasticity, skin regeneration, wound healing, cornea regeneration, skin irritation relief, and combinations thereof. It can be one selected from . In addition, it may be characterized by protecting the skin from deterioration or loss of skin cell function, improving skin condition, or preventing or improving skin diseases.

The cosmetic composition may be formulated into a cosmetic formulation commonly prepared in the art. The cosmetic compositions can be formulated as, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations and sprays. It may be converted, but is not limited to this. More specifically, it can be formulated as a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder. Additionally, it can be manufactured as a filler for cosmetics.

Cosmetic fillers can be applied by applying to the skin surface, and may include, for example, fragrance, xanthan gum, wax, butter, oil, surfactant, moisturizer, alcohol, etc. Cosmetics that may typically include Any component of the composition may be included without particular limitation. Additionally, when the cosmetic filler composition includes hyaluronic acid, the hyaluronic acid may have a non-crosslinked structure.

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

The formulation of the cosmetic composition according to the present invention may include a carrier component selected from the group consisting of a solvent that is a solution or emulsion, a solvating agent, an emulsifying agent, and mixtures thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, and mixtures thereof, etc. There is.

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

The cosmetic composition may further include various known additives in addition to the carrier depending on the formulation.

The additives include emulsifiers, moisturizers, surfactants, chelating agents, antioxidants, disinfectants, stabilizers, etc.

The emulsifier may include liquid paraffin, cetyl oltanoate, stearic acid, etc. The moisturizing agent 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.

The chelating agent may include sodium ethylenediaminetetraacetate (EDTA), α-hydroxy fatty acid, lactoferrin, α-hydroxy acid, citric acid, lactic acid, malic acid, bilirubin, biliverdin, etc.

The antioxidant may include butylhydroxyanisole, dibutylhydroxytoluene, or propyl gallate. In addition, ingredients that can be mixed in the cosmetic composition or external skin preparation include fat ingredients, emollients, organic and inorganic pigments, organic powders, ultraviolet absorbers, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, coolants, antiperspirants, and vitamins. etc.

Hereinafter, the present invention will be explained in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to these only.

I. Preparation of exosome and polymer complex

Preparation Example 1. NK cell culture and exosome isolation

To culture NK cells, feeder cells irradiated with 100 Gy were seeded with NK cells at a ratio of 1:2. NK cells were cultured with human AB serum, 1% L-glutamine, and IL-15. Afterwards, the cell culture supernatant was collected and 20 mL of medium was centrifuged at 300 rcf(g). After centrifugation, cell debris was removed. Afterwards, 4 mL of exosome enrichment reagent (Invitrogen, Cat. No. 4478359) was added to the medium at a 5:1 ratio, vortexed for 1 minute, and incubated overnight at 4°C. Afterwards, NK cell-derived exosomes were separated and purified using a centrifuge at 10,000 rcf(g) for 60 minutes.

Preparation Example 2. Preparation of exosome/polymer complex

NK cell-derived exosomes isolated by the method of Preparation Example 1 were mixed with collagen or hyaluronic acid to prepare an exosome/polymer complex (FIG. 9).

Specifically, 400 μg of exosomes derived from the culture medium of the NK cells; and 1 mg of collagen powder (Dalimthyssen, BA06091) or 1 mg of hyaluronic acid powder (Lifecore, HA1M-5) were added to PBS or D.W. and mixed by vortexing for 2 minutes. After reacting the mixture at 4°C for 10 minutes, the bubbles were removed and the mixture was mixed with exosomes; and was used as a sample during combined treatment with collagen or hyaluronic acid.

II. Confirmation of skin healing effect of exosomes and polymer complex derived from cell culture medium

Example 1. Evaluation of cell proliferation effect according to treatment with exosomes derived from NK cell culture medium

Using the human fibroblast BJ6 cell line and Cell Counting Kit-8 (CCK-8), the cell proliferation ability of exosomes derived from natural killer cells (NK cells) was evaluated. The cell proliferation results of this experiment are used as an indicator to evaluate the efficacy of improving skin conditions such as wrinkle improvement and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Specifically, the BJ6 cell line, a human fibroblast, was dispensed into a 96-well plate at 5×10 ³ cells/well and cultured for 24 hours under cell culture conditions of 37°C and 5% CO ₂ . The culture was maintained for 24 hours in a medium containing 10% FBS, and then the existing medium was removed. Afterwards, the test material was treated with medium containing 1% FBS and further cultured for 72 hours. In this experiment, exosomes (50 μg) isolated from the control (Vehicle) and NK cell culture medium were used as test substances.

To measure cell viability, the CCK-8 solution was diluted 1/10 using medium containing 1% FBS, and 100 μL of this 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 measuring the absorbance, the cell proliferation rate of each group was calculated as a percentage (%) compared to the control group.

As a result of the measurement, as shown in Figures 2a and 2b, cell proliferation ability was increased by NK cell-derived exosomes. Specifically, at 72 hours of culture, it was confirmed that the cell proliferation rate increased to 234% in the NK cell culture medium-derived exosome-treated group compared to a 132% increase in the control group (Vehicle) (Figure 2b).

Through this, it was found that the cell proliferation effect can be increased by NK cell-derived exosomes. Accordingly, it was found that it can ultimately be used to improve skin conditions such as improving skin wrinkles and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Example 2. Evaluation of cell proliferation effect according to treatment of lyophilized NK cell-derived exosomes

The cell proliferation ability of NK cell-derived exosomes was evaluated using the human fibroblast BJ6 cell line and Cell Counting Kit-8 (CCK-8) in the same manner as in Example 1. However, in this case, NK cell-derived exosomes used were NK cell culture fluid-derived exosomes and freeze-dried NK cell-derived exosomes, respectively.

As a result of measuring the degree of fibroblast proliferation by treating NK cell culture medium-derived exosomes and lyophilized NK cell-derived exosomes, respectively, as shown in Figure 3, compared to the control group, NK cell culture medium-derived exosomes and lyophilized NK It was confirmed that cell proliferation ability increased to an equivalent level by both cell-derived exosomes.

These results indirectly showed that the characteristics of NK cell-derived exosomes were maintained even in a freeze-dried state. In other words, it shows an excellent cell proliferation effect due to NK cell-derived exosomes without being affected by freeze-drying, so it can be useful for improving skin conditions such as wrinkle improvement and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis. I could see that it was there.

Example 3. Evaluation of wound healing efficacy according to NK cell-derived exosome treatment

The wound healing efficacy of NK cell-derived exosomes was evaluated using the human fibroblast BJ6 cell line and wound healing assay technique. The wound healing assay is a technique that proliferates human fibroblasts on a 35 mm flask and then creates a wound using a tip of 1 mm or less. The wound healing efficacy evaluation results of this experiment are used as an indicator to evaluate the efficacy of improving skin conditions such as wrinkle improvement and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Specifically, the BJ6 cell line, a human fibroblast, was dispensed at 5×10 ⁴ cells/well in a 35 mm flask and cultured for 24 hours under cell culture conditions of 37°C and 5% CO ₂ . After maintaining the culture in medium containing 10% FBS for 24 hours, the existing medium was removed and a 35 mm flask was scraped using a tip to create a wound on the cells. Afterwards, after washing once with PBS, the test material was treated with medium containing 1% FBS and further cultured for 56 hours. In this experiment, exosomes (50 μg) isolated from control (vehicle) and NK cell culture medium were used as test substances.

To measure the wound healing efficacy of cells, images were acquired over time using a microscope as shown in Figure 4a. Afterwards, the degree of wound recovery, i.e., the degree of cell wound healing efficacy, was confirmed by measuring the interval of cell growth on the wounded location (Figure 4b).

Compared to the control group, it was confirmed that the wound healing efficacy was increased by treatment with exosomes derived from NK cell culture medium. Specifically, after 56 hours, it was confirmed that the NK cell culture medium-derived exosome-treated group had a wound healing effect of approximately 1,027 μm compared to the control group.

Through this, it was found that wound healing efficacy can be increased by NK cell-derived exosomes. Accordingly, it was found that it can ultimately be used to improve skin conditions such as improving skin wrinkles and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Example 4. Evaluation of cell proliferation effect according to treatment of NK cell-derived exosomes and hyaluronic acid complex at different concentrations

We attempted to achieve a better cell proliferation effect by utilizing NK cell-derived exosomes and applying biocompatible polymers that are commonly used as cosmetics and regenerative medical materials. Accordingly, in order to establish a combination concentration that shows an excellent cell proliferation effect by treatment with NK cell-derived exosomes and hyaluronic acid (HA) complex, an experiment was designed as shown in Figure 5.

Specifically, HA- and NK cell-derived exosomes were 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, and 2, respectively, based on total weight. NK cell-derived exosomes and HA complexes were prepared by mixing at ratios of :8, 1:9, and 0:10. Afterwards, cell proliferation ability was evaluated according to treatment with NK cell-derived exosomes and HA complex concentrations.

The cell proliferation ability of NK cell-derived exosomes and HA complexes was evaluated using the human fibroblast BJ6 cell line and Cell Counting Kit-8 (CCK-8) in the same manner as in Example 1. The cell proliferation rate of the NK cell-derived exosome and HA complex treatment group at each mixing concentration was calculated as a percentage (%) compared to the control group. At this time, the control group was compared and calculated by fixing it at 100% as the relative comparison value.

As a result of checking the difference in cell proliferation rate according to the treatment of NK cell-derived exosomes and HA complex combination concentration compared to the control group, as shown in Figure 6, the NK cell culture medium-derived exosomes rather than the proliferation effect due to hyaluronic acid (HA) It was found that cell proliferation ability was improved.

Specifically, at 72 hours of culture, in the group treated with NK cell culture medium-derived exosomes alone (HA 0 μg + NK exo 100 μg), there was a 94% increase in the group treated with HA alone (HA 100 μg + NK exo 0 μg). The cell proliferation rate increased to 169%. In addition, the group treated with HA and NK cell-derived exosomes (HA 10 μg + NK exo 90 μg) fused at a mixing ratio of 1:9 showed a cell proliferation effect of 157%, compared to the group treated with NK cell-derived exosomes alone. It was confirmed that it exhibited a similar level of cell proliferation ability (FIG. 6).

Example 5. Evaluation of cell proliferation effect of NK cell-derived exosomes and biocompatible polymer complex

In order to confirm whether the biocompatible polymer applied in Example 4 shows an excellent cell proliferation effect even when using a polymer other than hyaluronic acid, a collagen complex containing NK cell-derived exosomes was prepared using an additional collagen polymer (Figure 7 ). Based on the results obtained in Example 4, NK cell culture medium-derived exosomes and biocompatible polymer complexes were prepared at the optimal mixing ratio and cell proliferation ability was evaluated.

Specifically, to prepare a collagen complex containing NK cell culture medium-derived exosomes, 1 mg of collagen powder and 400 μg of NK cell-derived exosomes were added to 1 mL of distilled water and mixed by vortexing for 2 minutes. . Afterwards, it was maintained at 4°C for 10 minutes, and the bubbles generated by vortexing were removed. Collagen embedded NK cell driven exosome complex prepared according to the above method was used to evaluate cell proliferation ability. At this time, the final combined concentration of the complex was 40 μg of NK cell-derived exosomes and 100 μg of collagen.

Likewise, the NK cell-derived exosome (40 μg) and HA polymer (100 μg) complex tested in Example 4 was prepared to have the same final mixing concentration, and the two NK cell-derived exosome and biocompatible polymer complexes were prepared. The cell proliferation effect was compared and evaluated.

Specifically, in addition to performing the experiment by distributing the BJ6 cell line, which is a human fibroblast, in a 48-well plate at 1 × 10 ⁴ cells/well, the experiment was conducted in the same manner as in Example 1, according to the experimental conditions and schedule as shown in Figure 8. Cell proliferation ability was evaluated over 24 hours, 48 hours, and 72 hours using the BJ6 cell line and Cell Counting Kit-8 (CCK-8). At this time, the control substances used were vehicle, hyaluronic acid alone (HA, 100 μg), NK cell culture medium-derived exosomes (40 μg), and collagen alone (Collagen, 100 μg), and the test substance was hyaluronic acid (HA). , 100 μg) and NK cell culture medium-derived exosomes (40 μg) fusion material and collagen (100 μg) and NK cell culture medium-derived exosomes (40 μg) were used.

To measure the survival rate of cells, the same procedure as in Example 1 was performed except that 200 μL of CCK-8 solution was applied to each well, and after completion of the reaction, the absorbance was measured using a spectrophotometer. After measuring the absorbance, the cell proliferation rate of each experimental group was calculated as a percentage (%) compared to the control group (vehicle). At this time, the control group was compared and calculated by fixing it at 100% as the relative comparison value.

In addition, in order to confirm the difference in cell proliferation rate according to treatment with NK cell culture medium-derived exosomes and biocompatible polymer complex, photographs were taken through a microscope over 24 hours, 48 hours, and 72 hours to confirm the degree of human fibroblast proliferation in each group. (Figures 9a to 9c).

As a result of checking the difference in cell proliferation rate according to the treatment of NK cell-derived exosomes and biocompatible polymer complex compared to the control group, the skin moisturizing and improvement effect was verified, and the biocompatible polymer hyaluronic acid and It was found that cell proliferation ability was improved by the main effect of collagen and NK cell culture medium-derived exosomes (Figure 10).

Specifically, at 72 hours of culture, exosomes derived from NK cell culture medium alone (NK exo 40 μg) increased by 130% in the HA-only (HA 100 μg) treatment group and 131% in the collagen-only (Collagen 100 μg) treatment group. ) In the case of the treatment group, it was confirmed that the cell proliferation rate increased to 157%. In particular, the exosome and HA polymer complex derived from NK cell culture medium showed a cell proliferation rate of 193%, and the exosome and collagen polymer complex derived from NK cell culture medium showed a cell proliferation rate of 189%.

Through this, it was found that excellent cell proliferation effects can be induced by NK cell-derived exosomes, especially NK cell-derived exosomes and biocompatible polymer complexes. Accordingly, it was found that it can ultimately be used to improve skin conditions such as improving skin wrinkles and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Example 6. Evaluation of cell proliferation effect of NK cell-derived exosomes and biocompatible dual polymer complex

In order to confirm whether the biocompatible polymer applied in Example 5 shows a better cell proliferation effect by using additional polymers in addition to hyaluronic acid alone, a dual polymer with additional collagen fusion was used to create a dual polymer containing NK cell-derived exosomes. A composite was prepared (Figure 11). Based on the results derived in Example 4, NK cell culture medium-derived exosomes and biocompatible dual polymer complex were prepared at the optimal mixing ratio and cell proliferation ability was evaluated. At this time, the same method as Example 5 was performed except that hyaluronic acid and collagen were simultaneously contained as biocompatible polymers.

Specifically, the final blended concentration of the composite is as shown in Table 1 below. Samples 1 and 2 were prepared as a control group of the dual polymer composite sample, and samples 3 to 5 were prepared as an experimental group.

Polymer composite sample No.	Hyaluronic acid (HA)	Collagen	NK exosomes (NK exo)
One	100 μg	0	40 μg
2	0	100 μg	40 μg
3	20 μg	80 μg	40 μg
4	80 μg	20 μg	40 μg
5	50 μg	50 μg	40 μg

Specifically, the cell proliferation effects of the five types of NK cell-derived exosomes and biocompatible polymer complexes were compared and evaluated. Cell proliferation ability was evaluated over 24 hours, 48 hours, and 72 hours using the BJ6 cell line and Cell Counting Kit-8 (CCK-8) according to the experimental conditions and schedule as shown in Figure 11 in the same manner as in Example 5. did. At this time, the control substances used were vehicle, hyaluronic acid alone (HA, 100 μg), collagen alone (Collagen, 100 μg), and NK cell culture medium-derived exosomes (40 μg) alone. The test substances are as shown in Table 1 above.

Cell viability was measured in the same manner as in Example 5 using the CCK-8 solution, and absorbance was measured using a spectrophotometer after completion of the reaction. After measuring the absorbance, the cell proliferation rate of each experimental group was calculated as a percentage (%) compared to the control group (vehicle). At this time, the control group was compared and calculated by fixing it at 100% as the relative comparison value.

In addition, in order to confirm the difference in cell proliferation rate according to treatment with NK cell culture medium-derived exosomes and biocompatible polymer complex, photographs were taken through a microscope over 24 hours, 48 hours, and 72 hours to confirm the degree of human fibroblast proliferation in each group. (Figures 12a to 12c).

As a result of checking the difference in cell proliferation rate according to the treatment of NK cell-derived exosomes and biocompatible polymer complex compared to the control group, the skin moisturizing and improvement effect was verified, and the biocompatible polymer hyaluronic acid and It was found that cell proliferation ability was improved by the main effect of collagen and NK cell culture medium-derived exosomes (Figure 13).

Specifically, at 72 hours of culture, NK cell culture medium-derived exosomes alone (NK exo 40 μg) increased by 113% in the HA-only (HA 100 μg) treatment group and 130% in the collagen-only (Collagen 100 μg) treatment group. ) In the case of the treatment group, it was confirmed that the cell proliferation rate increased to 182%. In particular, the exosome and HA polymer complex derived from NK cell culture medium showed a cell proliferation rate of 214%, and the exosome and collagen polymer complex derived from NK cell culture medium showed a cell proliferation rate of 200%.

In addition, three samples from the NK cell culture medium-derived exosome treatment group using a dual polymer complex containing both HA and collagen, namely Sample No. 3 (HA 20 μg + Collagen 80 μg + NK cell-derived exosome 40 μg), sample For sample No.4 (HA 80 μg + Collagen 20 μg + NK cell-derived exosomes 40 μg) and sample No.5 (HA 50 μg + Collagen 50 μg + NK cell-derived exosomes 40 μg), 204% and 194%, respectively. It was confirmed that skin fibroblasts were proliferating at % and 229%.

Through this, it was found that an excellent cell proliferation effect can be induced by NK cell-derived exosomes, especially NK cell-derived exosomes and a biocompatible dual polymer complex. Accordingly, it was found that it can ultimately be used to improve skin conditions such as improving skin wrinkles and wound regeneration, and improving symptoms of skin diseases such as atopic dermatitis.

Claims (11)

1. A pharmaceutical composition for preventing or treating skin diseases containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients.
2. According to paragraph 1,

   The biocompatible polymers include hyaluronic acid, collagen, fucoidan, alginate, heparin, cellulose, gelatin, fibrin, chitosan, dextran, elastin, polyethylene glycol (PEG), polyethyleneimine (PEI), polyvinyl alcohol (PVA), and polycapro. A pharmaceutical composition selected from the group consisting of lactone (PCL), polylactide, polylactic glycolic acid (PLGA), and polygamma glutamic acid.
3. According to paragraph 1,

   The exosome is a pharmaceutical composition obtained from natural killer cell culture medium.
4. According to paragraph 3,

   A pharmaceutical composition wherein the natural killer cells are differentiated from iPSCs.
5. According to paragraph 1,

   The pharmaceutical composition, wherein the skin disease is selected from the group consisting of atopic dermatitis, wounds, skin wrinkles, skin aging, weakened skin elasticity, dry skin, sensitive skin, acne, hair loss, skin pigmentation, and combinations thereof.
6. A cosmetic composition for improving skin condition containing exosomes derived from natural killer cells and biocompatible polymers as active ingredients.
7. According to clause 6,

   The biocompatible polymers include hyaluronic acid, collagen, fucoidan, alginate, heparin, cellulose, gelatin, fibrin, chitosan, dextran, elastin, polyethylene glycol (PEG), polyethyleneimine (PEI), polyvinyl alcohol (PVA), and polycapro. A cosmetic composition selected from the group consisting of lactone (PCL), polylactide, polylactic glycolic acid (PLGA), and polygamma glutamic acid.
8. According to clause 6,

   The skin condition improvement is selected from the group consisting of suppressing the occurrence of wrinkles, suppressing skin aging, improving skin elasticity, skin regeneration, wound or wound healing, corneal regeneration, alleviating skin irritation, and combinations thereof. Composition.
9. Use of the pharmaceutical composition of claim 1 for preventing or treating skin diseases.
10. Use of the pharmaceutical composition of claim 1 to prepare a medicament for the prevention or treatment of skin diseases.
11. A method for preventing or treating skin disease comprising administering the pharmaceutical composition of claim 1.

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