WO2024043716A1 - Composition comprising human cell-derived microvesicles and hyaluronic acid or collagen, and use thereof - Google Patents

Abstract

The present invention relates to a cosmetic or pharmaceutical composition containing, as active ingredients, microvesicles isolated from culture media of natural killer cells, induced pluripotent stem cells, or mesenchymal stem cells, hyaluronic acid, and/or collagen. The composition according to the present invention promotes the proliferation of skin fibroblasts, and therefore, can be effectively used as a cosmetic or pharmaceutical composition for improving, preventing, or treating skin wounds, skin diseases, or skin conditions.

Description

Composition comprising human cell-derived microvesicles and hyaluronic acid or collagen and uses thereof

The present invention relates to microvesicles isolated from cultures of natural killer cells, induced pluripotent stem cells, or mesenchymal stem cells; and a composition for improving skin condition comprising hyaluronic acid and/or collagen.

As modern people's living standards improve and the elderly population increases, interest in and demand for functional cosmetics related to anti-aging, wrinkle improvement, whitening, and UV protection is increasing. However, since most basic cosmetics or functional cosmetics are manufactured with chemical substances, safety issues for the human body have been consistently raised. Accordingly, interest in products containing natural and organic ingredients is increasing, and the market size of related products is also increasing.

On the other hand, a cosmetic composition for improving skin condition containing human embryonic stem cell culture (Korean Patent Publication No. 10-2015-0039343), for improving skin wrinkles or skin aging containing stem cell culture derived from mammals as an active ingredient. A cosmetic composition for inhibition (Korean Patent No. 10-1063299) has been developed. However, there were ethical issues in using human embryonic stem cells, and concerns were raised that their effectiveness was minimal.

In particular, stem cell culture media contains components such as waste products secreted by cells as they grow, antibiotics added to prevent contamination, and animal-derived serum, so there is a high possibility of exposure to various risks when used on the skin. Therefore, in order to use stem cell culture medium, a separate process is required to remove waste products or various dangerous components, which also causes the problem of increased production costs.

Recently, research has been reported that cell secretome contains various bioactive factors that control cell behavior. In particular, cell secretome contains 'exosome' or 'extracellular', which has an intercellular signaling function. Because it contains ‘extracellular vesicles’, research on its components and functions is actively underway. Extracellular vesicles are a general term for membrane-structured vesicles secreted by cells for information exchange between cells, and include exosomes, ectosomes, microvesicles, microparticles, and apoptotic bodies ( apoptotic body), etc.

However, despite various studies being conducted, including the possibility of treating some diseases, there is insufficient research that can be applied to improve or treat skin conditions or diseases. In addition, in the case of exosomes, isolation/purification is complicated and most of them are made up of miRNAs that regulate gene expression rather than mRNAs that can produce proteins. On the other hand, microvesicles mainly contain mRNA that can be directly involved in biological functions, and the isolation method is faster and simpler than that of exosomes. Nevertheless, most secretome research is focused on research using exosomes. Therefore, there is a need to develop a technology that can be applied to the treatment of skin diseases using microvesicles.

Accordingly, while the present inventor was researching to develop a substance to improve skin condition and treat skin diseases using cell secretions (secretome), microvesicles isolated from natural killer cells, induced pluripotent stem cells, or mesenchymal stem cells were discovered. The present invention was completed by confirming that the proliferative ability of skin cells is efficiently promoted.

In order to achieve the above object, one aspect of the present invention provides a cosmetic composition for improving skin condition containing microvesicles isolated from human-derived cells as an active ingredient, and a pharmaceutical composition for preventing or treating skin diseases. .

Another aspect of the present invention provides the use of the pharmaceutical composition for preparing a medicament for preventing or treating skin diseases.

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

Another aspect of the present invention provides a method for preventing or treating skin diseases comprising administering the pharmaceutical composition.

The composition containing microvesicles isolated from natural killer cells, induced pluripotent stem cells, or mesenchymal stem cell culture medium according to the present invention as an active ingredient promoted the proliferation of skin fibroblasts and improved skin wound recovery ability. In addition, when the microvesicles were treated in combination with hyaluronic acid or collagen, the effect of promoting the proliferation of skin fibroblasts was increased compared to treatment with hyaluronic acid or collagen alone. Therefore, the composition according to the present invention can be usefully used as a cosmetic and pharmaceutical composition for improving, preventing or treating skin wounds, skin diseases and skin conditions caused by external damage, and can be used as a new therapeutic agent and cosmetic, and can be used as a new therapeutic agent and cosmetic. It will be useful as an additive in skin ointments and functional cosmetics.

Figure 1 is a diagram showing the process of isolating microvesicles (MVs) from a culture medium of natural killer cells, induced pluripotent stem cells, or mesenchymal stem cells.

Figure 2 is a diagram showing the results of measuring the size of microvesicles (MVs) isolated from the culture medium of natural killer cells (top) or induced pluripotent stem cells (bottom) using dynamic light scattering (DLS).

Figure 3 shows a method of producing a polymer complex mixed with microvesicles (MVs) and hyaluronic acid (HA) or collagen isolated from the culture medium of natural killer cells, induced pluripotent stem cells, or mesenchymal stem cells. This is the drawing shown.

Figure 4 is a diagram illustrating an experimental schedule to confirm the effect of promoting fibroblast proliferation by treatment alone or in combination with microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells. am.

Figures 5A to 5C show microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells in a human fibroblast cell line, treated alone or in combination, and then treated over time (24 hours, 48 hours). Time, 72 hours) This is a diagram showing the results of observing the number of cells under a microscope.

Figure 6 shows microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells in a human fibroblast cell line, treated alone or in combination, by time (24 hours, 48 hours, 72 hours). Time) This is a graph showing the results of measuring cell proliferation rate.

Figure 7 schematizes an experimental schedule to confirm the effect of promoting fibroblast proliferation by treatment alone or in combination with microvesicles (iPSC MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of induced pluripotent stem cells. It is a drawing.

Figures 8a to 8c show the time (24 hours, This is a diagram showing the results of observing the cell number under a microscope (48 hours, 72 hours).

Figure 9 shows the time (24 hours, 48 hours, This is a graph showing the results of measuring the cell proliferation rate (72 hours).

Figure 10 is a schematic diagram of an experimental schedule to confirm the effect of promoting fibroblast proliferation by treatment alone or in combination with microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells. am.

Figures 11a to 11c show microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells in a human fibroblast cell line treated alone or in combination, and then treated by time (24 hours, 48 hours) Time, 72 hours) This is a diagram showing the results of observing the number of cells under a microscope.

Figure 12 shows microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of natural killer cells in a human fibroblast cell line, treated alone or in combination, by time (24 hours, 48 hours, 72 hours). Time) This is a graph showing the results of measuring cell proliferation rate.

Figure 13 shows the results of measuring the cell proliferation rate after treating microvesicles (NK MVs), hyaluronic acid (HA), and collagen isolated from the culture medium of mesenchymal stem cells in a human fibroblast cell line alone or in combination for 48 hours. This is a graph showing .

Figure 14 shows microvesicles (NK MV), hyaluronic acid (HA), and collagen isolated from the culture medium of mesenchymal stem cells in a human fibroblast cell line treated alone or in combination for 24 hours, and then observing the cell number under a microscope. This is a drawing showing the results.

Composition containing microvesicles and polymers

One aspect of the present invention is microvesicles (microvesicles) isolated from human-derived cells; and collagen and/or hyaluronic acid.

Cosmetic composition containing microvesicles

One aspect of the present invention provides a cosmetic composition for improving skin condition containing microvesicles isolated from human-derived cells as an active ingredient. The human-derived cells may be stem cells or immune cells, but are not limited thereto.

The stem cells may be mesenchymal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells, or induced pluripotent stem cells. Preferably, it may be induced pluripotent stem cells or mesenchymal stem cells, but is not limited thereto.

Additionally, the immune cells may be T cells, B cells, natural killer cells, dendritic cells, or macrophages. Preferably, it may be a natural killer cell, but is not limited thereto.

As used herein, the term “induced pluripotent stem cells (iPSCs)” refers to cells created to have pluripotency like embryonic stem cells by inducing dedifferentiation in somatic cells without pluripotency. Induced pluripotent stem cells can also be called pluripotent stem cells. The induced pluripotent stem cells can be generated using somatic cells from a fetus, newborn, child, or adult. The induced pluripotent stem cells may be derived from fibroblasts, keratinocytes, blood cells, or kidney epidermal cells. The induced pluripotent stem cells may be produced by reprogramming somatic cells. Methods for reprogramming somatic cells to produce induced pluripotent stem cells include known methods, such as Takahashi K, Yamanaka S (August 2006), Cell. The method described in vol.126, no.4, pp.663-676 can be used.

Terms used in this specification. “Mesenchymal stem cells (MSCs)” are multipotent stromal cells that can differentiate into various cells, including osteoblasts, cartilage cells, muscle cells, and adipocytes.

As used herein, the term "natural killer cells (NK cells)" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system, and is defined as large granular lymphocyte (LGL) and lymphoid progenitor cells. (common lymphoid progenitor, CLP) constitutes a third cell differentiated from producing B and T lymphocytes. It is a lymphocyte cell that plays a part in the immune response, exists in approximately 10 to 15% of normal blood, and has a high killing ability when reacting with non-self. In the case of cells infected with various viruses, bacterial invasion, or the creation of abnormal cells, natural killer cells can react immediately and non-specifically to remove foreign substances. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and natural killer cells are two types of cells that control the cytotoxic activity of the cells, labeled as “activating receptors” and “inhibitory receptors.” Contains exteroceptors. In addition, microvesicles derived from natural killer cells have the characteristic of removing abnormal cells and simultaneously returning the abnormal cellular environment to normal.

The term “microvesicles (MVs)” used in the present invention is a type of extracellular vesicles with a membrane structure secreted into the extracellular space. The extracellular vesicles contain various biomolecules, such as growth factors, chemokines, cytokines, transcription factors, RNAs (mRNA, miRNA, etc.), and lipids, in the cell from which they are derived. It is a particle-shaped structure encapsulated in a lipid bilayer cell membrane identical to the cell membrane of . Therefore, the extracellular endoplasmic reticulum mediates the transport of proteins, lipids, genetic materials, etc., enables exchange of substances between cells, and acts as a medium for transmitting physiological/pathological signals.

Extracellular vesicles are largely classified into exosomes and microvesicles. Exosomes have a particle diameter of 40-120 nm and are intraluminal vesicles created when the endosomal membrane moves inward during the maturation of multi-vesicular endosomes. Multi-vesicular endosomes are attached to the cell surface and Secreted when combined. CD63, CD9, TSG101, ESCRT, etc. are known as marker proteins of the exosome. Microvesicles are vesicles with a particle diameter of 100 to 1000 nm, whose plasma membrane protrudes outward, separates, and is secreted out of the cell. Markers of the microvesicles include integrin-β, CD40, and selectins.

In the present invention, the human cell-derived microvesicles may be characterized as having a particle size of about 200 nm to about 2,000 nm. Specifically, the microvesicles range from about 200 nm to about 2,000 nm, from about 200 nm to about 1,900 nm, from about 200 nm to about 1,800 nm, from about 200 nm to about 1,700 nm, from about 200 nm to about 1,600 nm, from about 200 nm About 1,500 nm, about 200 nm to about 1,400 nm, about 200 nm to about 1,3000 nm, about 200 nm to about 1,200 nm, about 200 nm to about 1,100 nm, about 200 nm to about 1,000 nm, about 200 nm about 900 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 400 nm, or about 200 nm to about 300 nm. It may have a particle size of nm.

At this time, the microvesicles may not contain exosomes.

In the present invention, the microvesicles are prepared by: i) culturing human-derived cells; ii) obtaining a culture medium; And iii) separating microvesicles from the cell culture medium.

Here, the human-derived cells are the same as described above.

The term “culture” used in the present invention refers to growing cells in appropriately controlled environmental conditions, and the culture process of the present invention can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted by a person skilled in the art depending on the cells selected. The medium refers to a known medium used for culturing cells, and includes all known media for cell culture or modified media thereof.

The human-derived cells are cultured in culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, about 18 hours to about 7 days, or about 1 day to about 3 days. You can.

In one embodiment of the present invention, human-derived induced pluripotent stem cells (iPSCs) are stored in culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, and about 18 hours to about 18 hours. It can be cultured for 7 days or about 1 to about 3 days.

In one embodiment of the present invention, human-derived mesenchymal stem cells (MSCs) are cultured in culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, and about 18 hours to about 18 hours. It can be cultured for 7 days or about 1 to about 3 days.

In one embodiment of the present invention, human-derived natural killer cells are cultured in culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, about 18 hours to about 7 days, or about It can be cultured for 1 to about 3 days.

Microvesicles of the present invention can be extracted from the culture medium by ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, and ion exchange chromatography. It can be separated by separation methods that are used in the industry or may be used in the future, such as chromatography, immunoaffinity capture, microfluidics-based isolation, or polymer-based precipitation. , but is not limited to this.

In one embodiment, the microvesicles of the present invention may be obtained from a pellet collected by centrifuging the human-derived cell culture fluid. Specifically, collecting the culture medium in which the cells were cultured and performing first centrifugation to remove the pellet containing cells and cell debris, and performing second centrifugation on the culture medium from which the pellet was removed to produce microvesicles formed into a pellet. It can be performed as a step to obtain. At this time, the time and temperature at which the second centrifugation is performed may be the same as the time and temperature at which the first centrifugation is performed.

More specifically, the first and second centrifugation may be performed at a temperature of about 0°C to about 10°C, preferably at a temperature of about 3°C to about 5°C. In addition, the time for which the centrifugation is performed may be performed for about 10 to 30 minutes and may be appropriately adjusted depending on the content of the sample. Preferably, it can be performed at about 4°C for 20 minutes.

Additionally, the first centrifugation may be performed at a rate of about 100 g to about 1000 g, about 200 g to about 700 g, or about 300 g to about 450 g. The first centrifugation can be performed at about 300g. Additionally, the secondary centrifugation may be performed at a rate of about 5,000 g to about 40,000 g, about 8,000 g to about 30,000 g, or about 10,000 g to about 20,000 g. Preferably, the first centrifugation can be performed at a speed of about 300 g, and the second centrifugation can be performed at a speed of about 16,000 g.

The cosmetic composition of the present invention may include the microvesicles in an amount of 0.0001 to 10% by weight based on the total weight of the composition, and may specifically include 0.0005 to 10% by weight of the microvesicles, and more specifically, 0.001 to 10% by weight. % of the microvesicles.

Specifically, the composition contains about 1 μg to about 1000 μg of microvesicles, about 5 μg to about 500 μg, about 10 μg to about 200 μg, and about 15 μg. It may contain from about 100 μg or about 20 μg to about 50 μg. More specifically, the composition may include about 40 μg of the microvesicles.

The cosmetic composition of the present invention includes hyaluronic acid or a salt thereof; And/or collagen may be additionally included.

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 has a molecular weight of 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.

In the present invention, the cosmetic composition may include the 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 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 composition may include about 100 μg of hyaluronic acid or a salt thereof.

At this time, the microvesicles and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the microvesicles and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:2.5.

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

Specifically, the 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 composition may include about 100 μg of the collagen.

At this time, the microvesicles and collagen may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the microvesicles and collagen may be mixed at a weight ratio of 1:2.5.

Additionally, the cosmetic composition may include the microvesicles, hyaluronic acid or a salt thereof, and collagen in a weight ratio of 10:1:1 to 1:1:10. Preferably, the cosmetic composition may be a mixture of microvesicles, hyaluronic acid or a salt thereof, and collagen at a weight ratio of 2:1:4 or 2:4:1.

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.

Another aspect of the present invention provides a cosmetic method for improving skin condition using the cosmetic composition according to the present invention. The cosmetic method may include applying the cosmetic composition to the subject's skin. Here, the cosmetic composition is the same as described above.

The subject may be a mammal, and specifically, may be a human. The step of applying to the skin may include directly applying or spraying the cosmetic composition according to the present invention to the skin depending on its form. At this time, the application amount and number of daily uses of the cosmetic composition can be appropriately set depending on the user's age, gender, purpose, severity of symptoms, etc. For example, an appropriate amount of the cosmetic composition is applied 1 to 6 times a day. It can be applied to the skin frequently.

Pharmaceutical composition containing microvesicles

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases containing microvesicles isolated from human-derived cells as an active ingredient. Here, human-derived cells and microvesicles are the same as described above.

The pharmaceutical composition of the present invention may include the microvesicles in an amount of 0.0001 to 10% by weight based on the total weight of the composition, and may specifically include 0.0005 to 10% by weight of the microvesicles, and more specifically, 0.001 to 10%. It may include the microvesicles.

Specifically, the pharmaceutical composition contains about 1 μg to about 1000 μg of microvesicles, about 5 μg to about 500 μg, about 10 μg to about 200 μg, and about 15 μg. It may contain from about 100 μg or about 20 μg to about 50 μg. More specifically, the pharmaceutical composition may include about 40 μg of the microvesicles.

In the present invention, the pharmaceutical composition includes hyaluronic acid or a salt thereof; And/or collagen may be additionally included. Here, hyaluronic acid and collagen are the same as described above.

The “salt of hyaluronic acid” refers to a pharmaceutically acceptable salt of hyaluronic acid within the range having the same efficacy as hyaluronic acid. At this time, “pharmaceutically acceptable salt” refers to a salt that can be used pharmaceutically among salts that are substances in which cations and anions are combined by electrostatic attraction, and has a relatively non-toxic and harmless effect on patients. It refers to any and all organic or inorganic addition salts of the compound that do not reduce the beneficial effects of the compound at a concentration. Such salts include acid addition salts formed by pharmaceutically acceptable free acids or metal salts formed by bases.

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.

At this time, the microvesicles and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the microendoplasmic reticulum and hyaluronic acid or a salt thereof may be mixed at a weight ratio of 1:2.5.

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 microvesicles and collagen may be mixed at a weight ratio of 1:1 to 1:10. Preferably, the microcellular endoplasmic reticulum and collagen may be mixed at a weight ratio of 1:2.5.

Additionally, the pharmaceutical composition may include the microvesicles, hyaluronic acid or a salt thereof, and collagen in a weight ratio of 1:1:10 to 10:1:1. Preferably, the pharmaceutical composition may be a mixture of microvesicles, hyaluronic acid or a salt thereof, and collagen at a weight ratio of 2:1:4 or 2:4:1.

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, chlorpheniramine, diphenylpyraline, piprinhydrinate, diphenhydramine, cetirizine, etc., but there is no particular limitation if it is an injectable antihistamine. .

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.

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. Fabrication of microvesicles and polymer complexes

Preparation Example 1. Microvesicle isolation

Natural killer cells (NK cells) Feeder cells irradiated with 100 Gy were seeded with natural killer cells at a ratio of 1:2. Cells were cultured in medium containing human AB serum, 1% L-glutamine, and IL-15. During subculture, cells were collected using a 240 mm scraper.

In the case of induced pluripotent stem cells (iPSc), the induced pluripotent stem cells were seeded in a flask treated with iMatrix 511 (ATRIXOME) and cultured in medium containing 1% Penicillin-Streptomycin and 10 uM Y-27631. At this time, the medium was changed every day.

Mesenchymal stem cells were purchased from Promo cell and cultured in a T-300 flask culture vessel for one week using Promo cell's stem cell-specific medium and dedicated serum.

The method for isolating microvesicles from cell culture is shown in Figure 1. Specifically, the cell culture supernatants of induced pluripotent stem cells (iPSc), natural killer cells (NK cells), and mesenchymal stem cells were collected after 7, 16, and 23 days of culture, respectively, and incubated at 4°C, 300 g, and 20 g. After centrifugation for a minute, the supernatant was taken to remove cell debris (P1). The obtained supernatant was centrifuged at 4°C, 16,000 g, for 20 minutes, the supernatant was removed, and the pellet was collected (P2). The pellet (P2) was resuspended and the size of the particles was measured using dynamic light scattering (DLS). As a result, microvesicles (NK MVs) and induced pluripotent stem cells (iPSC MVs) derived from the culture medium of natural killer cells were found. The average particle diameters of culture medium-derived microvesicles (iPSC MVs) were found to be 648 nm and 1.415 nm, respectively (Figure 2).

Mesenchymal stem cell-derived exosomes were collected from the mesenchymal stem cell culture supernatant, centrifuged at 300g, and then 4 mL was added to the medium at a 5:1 ratio using an exosome enrichment reagent (Invtrogen, 4478359). , vortexed for 1 minute, and then incubated overnight at 4°C. Afterwards, mesenchymal stem cell-derived exosomes were prepared by centrifugation at 10,000 g for 60 minutes.

Preparation Example 2. Preparation of microvesicle/polymer complex

Natural killer cell-derived microvesicles (NK MVs), induced pluripotent stem cell-derived microvesicles (iPSC MVs), or mesenchymal stem cell-derived microvesicles (MSC MVs) isolated by the method of Preparation Example 1 were mixed with hyaluronic acid or collagen. A microvesicle/polymer complex was fabricated (Figure 3).

Specifically, 400 ㎍ of microvesicles (NK MVs) derived from the natural killer cell culture medium, microvesicles (iPSC MVs) derived from induced pluripotent stem cell culture medium, microvesicles (MSC MVs) derived from mesenchymal stem cell culture medium, or mesenchymal stem cell culture medium-derived 400 ㎍ of exosomes (MSC exo), 1 mg of hyaluronic acid powder (Lifecore, HA1M-5) or 1 mg of collagen powder (Dalimthyssen, BA06091) were added to PBS or D.W. and mixed by vortexing for 2 minutes. . After the mixture was reacted at 4°C for 10 minutes, the foam was removed and the mixture was used as a sample for combined treatment of microvesicles or exosomes and hyaluronic acid or collagen.

II. Confirmation of skin healing effect of cell culture derived microvesicles and polymer complex

Example 1. Confirmation of the effect of natural killer cell culture medium-derived microvesicles and polymer complexes on human fibroblast proliferation

In order to confirm the effect of promoting fibroblast proliferation by treatment with natural killer cell culture medium-derived microvesicles (NK MVs), hyaluronic acid (HA), or collagen, NK MVs and hyaluronic acid were applied to human fibroblast cell lines by the method of Figure 4. After treatment with (HA) and collagen alone or in combination, cell proliferation ability was evaluated.

Specifically, BJ6 cells (Seoul National University, Korea Cell Line Bank), a human fibroblast cell line, were seeded in 48 wells at a concentration of 1 × 10 ⁴ cells/well and cultured for 24 hours in RPMI1640 medium containing 10% FBS. After culturing the inoculated cells for an additional 24 hours, the medium was removed and cultured for an additional 72 hours in RPMI1640 medium containing test substances and 1% FBS.

At this time, the test substances were NK MVs (40 ㎍), hyaluronic acid (HA, 100 ㎍), collagen (100 ㎍), [NK MVs (40 ㎍) + hyaluronic acid (HA, 100 ㎍)] complex, [NK MVs (40 μg) + collagen (100 μg)] complex was used, and the microvesicle/polymer complex was produced in the same manner as Preparation Example 2. Additionally, as a control (vehicle), the polymer was dissolved in PBS and diluted to the same concentration as MVs in RPMI1640 medium containing 1% FBS.

Cell proliferation rate was evaluated by checking the cell number through a microscope (FIGS. 5A to 5C) and measuring absorbance using CCK-8 solution (FIG. 6). The CCK-8 solution was diluted 1/10 in RPMI1640 medium containing 1% FBS, and 200 ㎕ of this was treated per well. After reacting in a 5% CO ₂ cell incubator for 2 hours, absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The cell proliferation rate of each treatment group was calculated and expressed as a percentage (%) compared to the control group (vehicle).

As a result, as shown in FIGS. 5A to 5C and FIG. 6, it was confirmed that cell proliferation ability increased in all treatment groups compared to the control group. When the control group (vehicle) was set at 100% as a relative comparison value, the group treated with hyaluronic acid (HA) or collagen alone showed cell proliferation rates of 131% and 145%, respectively, when treated for 72 hours. In addition, the cell proliferation rate increased by 164%, 192%, and 174% in the NK MVs, [NK MVs + hyaluronic acid] complex, and [NK MVs + collagen] complex treatment groups, respectively.

Example 2. Confirmation of the effect of induced pluripotent stem cell culture medium-derived microvesicles and polymer complexes on human fibroblast proliferation

In order to confirm the skin healing effect by treatment with induced pluripotent stem cell culture medium-derived microvesicles (iPSC MVs), hyaluronic acid (HA), or collagen, NK MVs, hyaluronic acid (HA), and After collagen treatment alone or in combination, cell proliferation ability was evaluated.

At this time, the test substances were iPSC MVs (40 ㎍), hyaluronic acid (HA, 100 ㎍), collagen (100 ㎍), [iPSC MVs (40 ㎍) + hyaluronic acid (HA, 100 ㎍)] complex, [iPSC [MVs (40 μg) + collagen (100 μg)] complex was used. At this time, the microvesicle/polymer complex was produced in the same manner as Preparation Example 2. Additionally, as a control (vehicle), the polymer was dissolved in PBS and diluted to the same concentration as MVs in RPMI1640 medium containing 1% FBS (Figure 7).

As a result, as shown in FIGS. 8A to 8C and FIG. 9, it was confirmed that cell proliferation ability increased in all treatment groups compared to the control group. When the control group (vehicle) was set at 100% as a relative comparison value, the group treated with hyaluronic acid (HA) or collagen alone showed cell proliferation rates of 131% and 145%, respectively, when treated for 72 hours. In addition, the cell proliferation rate was confirmed to be increased by 244%, 192%, and 234% in the iPSC MVs, [iPSC MVs+hyaluronic acid] complex, and [iPSC MVs+collagen] complex treatment groups, respectively. The above results confirmed that the combined use of iPSC MVs or NK MVs and collagen or hyaluronic acid polymers did not interfere with the effect of inducing fibroblast proliferation by iPSC MVs or NK MVs.

Example 3. Confirmation of the effect on human fibroblast proliferation according to the ratio of microvesicles and polymer complexes derived from natural killer cell culture medium

To confirm the skin healing effect of treatment with natural killer cell culture medium-derived microvesicles (NK MVs), hyaluronic acid (HA), or collagen, mix NK MVs, hyaluronic acid (HA), and collagen to the following concentrations. A complex was prepared and the cell proliferation ability of NK MVs, hyaluronic acid (HA), and collagen alone or in combination was evaluated in the same manner as in Example 1.

At this time, the test substances were NK MVs (40 ㎍), hyaluronic acid (HA, 100 ㎍), collagen (100 ㎍), [NK MVs (40 ㎍) + hyaluronic acid (HA, 100 ㎍)] complex, [NK MVs (40 ㎍) + collagen (100 ㎍)] complex, [NK MVs (40 ㎍) + hyaluronic acid (HA, 20 ㎍) + collagen (80 ㎍)] complex, [NK MVs (40 ㎍) +Hyaluronic acid (HA, 80 μg)+Collagen (20 μg)] complex and [NK MVs (40 μg)+Hyaluronic acid (HA, 50 μg)+Collagen (50 μg)] complex were used. Additionally, as a control (vehicle), the polymer was dissolved in PBS and diluted to the same concentration as MVs in RPMI1640 medium containing 1% FBS (FIG. 10).

As a result, as shown in FIGS. 11A to 11C and FIG. 12, it was confirmed that cell proliferation ability increased in all treatment groups compared to the control group. When the control group (vehicle) was set at 100% as a relative comparison value, when treated for 72 hours, the group treated with hyaluronic acid (HA) or collagen alone showed cell proliferation rates of 113% and 130%, respectively. It was confirmed that the cell proliferation rate increased by 154%, 170%, and 171% in the NK MVs, [NK MVs+hyaluronic acid] complex, and [NK MVs+collagen] complex treatment groups, respectively.

In addition, the [NK MVs + hyaluronic acid + collagen] complex treatment group showed differences in proliferation efficiency depending on the ratio of hyaluronic acid and collagen. At this time, it was confirmed that the cell proliferation rate was highest when the mixing ratio of hyaluronic acid and collagen was 50:50 (199%). Through the above results, it was confirmed that the combined use of NK MVs, collagen, and hyaluronic acid polymers can synergistically promote fibroblast proliferation compared to NK MVs alone or the combined use of NK MVs and collagen or hyaluronic acid polymers.

Example 4. Confirmation of the effect on human fibroblast proliferation according to the ratio of microvesicles and polymer complexes derived from mesenchymal stem cell culture medium

In order to confirm the skin healing effect of treatment with mesenchymal stem cell culture medium-derived microvesicles (MSC MVs), hyaluronic acid (HA), or collagen, MSC MVs, hyaluronic acid (HA), and collagen were used at the following concentrations. A composite was prepared by mixing, and the cell proliferation ability of MSC MVs, hyaluronic acid (HA), and collagen alone or in combination was evaluated in the same manner as in Example 1.

At this time, the test substances were MSC MV (40 ㎍), hyaluronic acid (HA, 100 ㎍), collagen (100 ㎍), MSC exo 40 ㎍, [MSC MV (40 ㎍) + hyaluronic acid (HA, 100 ㎍) ] complex, [MSC exo (40 ㎍) + hyaluronic acid (HA, 100 ㎍)] complex, [MSC MV (40 ㎍) + collagen (100 ㎍)] complex, [MSC exo (40 ㎍) + collagen ( collagen, 100 ㎍] complex, [MSC MV (40 ㎍) + collagen (80 ㎍) + hyaluronic acid (HA, 20 ㎍)] complex, [MSC exo (40 ㎍) + collagen (80 ㎍) +hyaluronic acid (HA, 20 ㎍)] complex, [MSC MV (40 ㎍) + collagen (20 ㎍) + hyaluronic acid (HA, 80 ㎍)] complex, [MSC exo (40 ㎍) + collagen (collagen) , 20 ㎍) + hyaluronic acid (HA, 80 ㎍)] complex, [MSC MV (40 ㎍) + collagen (50 ㎍) + hyaluronic acid (HA, 50 ㎍)] complex and [MSC exo (40 ㎍) +Collagen (50 μg)+Hyaluronic acid (HA, 50 μg)] complex was used. Additionally, as a control (vehicle), the polymer was dissolved in PBS and diluted to the same concentration as MVs in DMEM medium containing 1% FBS.

24 hours after sample treatment, 200 ㎕ of CCK-8 solution was applied to each well to measure cell viability, and after completion of the reaction, 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 the MSC cell-derived exosome and microvesicle fusion biocompatible polymer complex, photographs were taken through a microscope for 24 hours to confirm the degree of human fibroblast proliferation in each group (FIG. 14).

As a result, it was confirmed that the cell proliferation rate increased in both the MSC MV, collagen, and hyaluronic acid treatment groups alone and in combination compared to the control group. Specifically, at 24 hours of culture, the MSC exo-treated group and the MSC-MV-treated group increased by 176%, respectively, compared to a 113% increase in the hyaluronic acid-only (100 μg) treatment group and a 130% increase in the collagen-only (100 μg) treatment group. And it was confirmed that the cell proliferation rate increased by 146%. In addition, when collagen and hyaluronic acid were treated in combination, the cell proliferation rate was higher than that of the group treated with MSC exo alone or the group treated with MSC MV alone.

Through the above results, it was confirmed that MSC exo and MSC MV exhibit excellent cell proliferation effects. The above results suggest that MSC exo and MSC MV can ultimately be used to improve skin conditions such as wrinkle improvement and wound regeneration, and to improve symptoms of skin diseases such as atopic dermatitis.

Claims (29)

1. A cosmetic composition for improving skin condition comprising microvesicles isolated from human-derived cells as an active ingredient.
2. According to paragraph 1,

   A cosmetic composition for improving skin condition, wherein the human-derived cells are stem cells or immune cells.
3. According to paragraph 2,

   A cosmetic composition for improving skin condition, wherein the stem cells are mesenchymal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells, or induced pluripotent stem cells.
4. According to paragraph 3,

   A cosmetic composition for improving skin condition, wherein the stem cells are induced pluripotent stem cells or mesenchymal stem cells.
5. According to paragraph 2,

   A cosmetic composition for improving skin condition, wherein the immune cells are T cells, B cells, natural killer cells, dendritic cells, or macrophages.
6. According to clause 5,

   A cosmetic composition for improving skin condition, wherein the immune cells are natural killer cells.
7. According to paragraph 1,

   A cosmetic composition for improving skin condition, wherein the microvesicles have a particle size of 200 to 2,000 nm.
8. In clause 7,

   A cosmetic composition for improving skin condition, characterized in that the microvesicles do not contain exosomes.
9. In clause 7,

   The microvesicles include i) culturing human-derived cells; ii) obtaining a culture medium; And iii) separating microvesicles from the cell culture medium; a cosmetic composition for improving skin condition, which is obtained through.
10. According to paragraph 1,

    The composition includes hyaluronic acid or a salt thereof; And/or a cosmetic composition for improving skin condition, which further comprises collagen.
11. According to clause 10,

    The composition is a cosmetic composition for improving skin condition, wherein microvesicles and hyaluronic acid are mixed at a weight ratio of 1:1 to 1:10.
12. According to clause 10,

    The composition is a cosmetic composition for improving skin condition, wherein microvesicles and collagen are mixed at a weight ratio of 1:1 to 1:10.
13. According to paragraph 1,

    The skin condition improvement is any one selected from the group consisting of suppressing the occurrence of wrinkles, suppressing skin aging, improving skin elasticity, skin regeneration, wound or wound healing, cornea regeneration, alleviating skin irritation, and combinations thereof, and skin A cosmetic composition for improving skin condition, characterized in that it protects the skin from deterioration or loss of cell function or improves the skin condition.
14. A pharmaceutical composition for preventing or treating skin diseases comprising microvesicles isolated from human-derived cells as an active ingredient.
15. According to clause 14,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the human-derived cells are stem cells or immune cells.
16. According to clause 15,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the stem cells are mesenchymal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells, or induced pluripotent stem cells.
17. According to clause 16,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the stem cells are induced pluripotent stem cells or mesenchymal stem cells.
18. According to clause 15,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the immune cells are T cells, B cells, natural killer cells, dendritic cells, or macrophages.
19. According to clause 18,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the immune cells are natural killer cells.
20. According to clause 14,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the microvesicles have a particle size of 200 to 2,000 nm.
21. According to clause 20,

    A pharmaceutical composition for preventing or treating skin diseases, wherein the microvesicles do not contain exosomes.
22. According to clause 20,

    The microvesicles include i) culturing human-derived cells; ii) obtaining a culture medium; and iii) separating microvesicles from the cell culture medium. A pharmaceutical composition for preventing or treating skin diseases.
23. According to clause 15,

    The composition includes hyaluronic acid or a salt thereof; And/or a pharmaceutical composition for preventing or treating skin diseases, which further comprises collagen.
24. According to clause 23,

    The composition is a pharmaceutical composition for preventing or treating skin diseases, wherein microvesicles and hyaluronic acid are mixed at a weight ratio of 1:1 to 1:10.
25. According to clause 23,

    The composition is a pharmaceutical composition for preventing or treating skin diseases, wherein microvesicles and collagen are mixed at a weight ratio of 1:1 to 1:10.
26. According to clause 15,

    The skin disease is characterized in that 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. Pharmaceutical composition for preventing or treating disease.
27. Use of the pharmaceutical composition of claim 14 for manufacturing a medicament for preventing or treating skin diseases.
28. Use of the pharmaceutical composition of claim 14 for preventing or treating skin diseases.
29. A method for preventing or treating skin disease comprising administering the pharmaceutical composition of claim 14.

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