KR20190069291A - Cosmetic composition for moisturizing skin comprising an exosome derived from stem cell as an active ingredient - Google Patents

Abstract

According to the present invention, a composition for moisturizing the skin increases the expression or production of AQP3 to thus activate the movement of moisture through the skin, thereby obtaining a moisturizing effect by moisturizing the skin and restoring skin function. Therefore, according to the present invention, the composition for moisturizing the skin exhibits the effects of moisturizing the skin and preventing, suppressing, alleviating or ameliorating skin irritation caused by dryness of the skin and also exhibits excellent effects in skin care, such as improving skin condition which has deteriorated due to skin dryness or restoring the skin condition to a normal state.

Description

[0001] The present invention relates to a cosmetic composition for skin moisturizing comprising an exosome derived from stem cells as an active ingredient,

The present invention relates to a novel skin moisturizing composition comprising stem cells derived exosomes as an effective ingredient.

The present invention also relates to a functional cosmetic composition for moisturizing the skin and an external preparation for skin comprising the composition.

The skin consists of three layers of epidermis, dermis and subcutaneous fat tissue. The epidermis is divided into the stratum corneum, the transparent layer, the granular layer, and the basal layer from the outside to the inside. The stratum corneum, which is the outermost layer of the epidermis, is the most important structure of skin barrier function, consisting of flattened corneocytes and SC intercellular lipid.

The stratum corneum of the epidermis contains 10% to 20% of water and exists at the outermost part of the human body to inhibit the evaporation of water from the body and to prevent excessive moisture from penetrating from the outside. However, when the water content of the stratum corneum is less than 10%, the skin becomes rough and the function of the skin barrier is lost.

Skin barrier protects against external stimuli such as dry environment, ultraviolet rays, pollutants, etc., and protects against excessive divergence of body water through the skin. This protective function is only maintained when the stratum corneum is normally formed .

Dry skin is the most common dermatologic problem in the elderly. Moisturizing and barrier functions are deteriorated in aged skin due to decrease of moisturizing protein and decrease of lipid synthesis ability during skin aging. However, one of the main features of skin aging caused by ultraviolet rays and the like is known as a decrease in function of aquaporin.

Aquaporin proteins are widely expressed in bacteria, plants, and animals, and are selective pores that allow water, glycerol, and small solutes to travel through the cell membrane. To date, 13 kinds of aquaporin proteins have been identified in mammals. Aquaporin is a group of aquaglyceroporin (AQP3, AQP7, AQP5) that transports not only water but also small solutes such as glycerol (AQP0, AQP1, AQP2, AQP4 and AQP5) , AQP10), and the unorthodox AQP group (AQP6, AQP8, AQP11, AQP12), which is under study for its specific function.

Of the above aquaporin proteins, Aquaporin 3 (AQP3) is the most relevant to skin. AQP3 is expressed in mouse and human keratinocytes, only from the basal layer to the granule layer, and disappears in the stratum corneum. Skin without AQP3 has a problem in that the skin elasticity is lowered and recovery of skin barrier function is lowered. AQP3 is present in skin epithelial cells and plays an important role in opening and closing the water passage in the cell membrane. When AQP3 is increased, the movement of moisture becomes active and the moisture is moved to the deep part of the skin, thereby lubricating the skin.

Recently, moisture of the stratum corneum has decreased due to an artificial temperature control of heating and cooling, skin stress due to various stresses and environmental pollution, frequent washing and natural skin aging, and the skin dryness has been increased, . However, currently used skin moisturizing cosmetic has a disadvantage that the moisturizing effect is temporary and can not maintain the skin moisturizing effect continuously.

Considering these problems, researches on the development of skin moisturizers using natural products are actively conducted. In the case of a composition for skin moisturizing using such a natural product as a raw material, since a content of an active ingredient in a natural extract is small, it is necessary to use a large amount in order to obtain a skin moisturizing effect. There is a need for more scientific research on the practical effects of skin moisturization. In particular, the development of a moisturizing agent that moisturizes the skin by moisturizing the skin by increasing the expression or production of AQP3 to obtain a moisturizing effect by restoring the skin function is insufficient.

Recently, there have been reported studies on the secretion of various bioactive factors that regulate cell behavior, and in particular, exosome (exosome), which has intercellular signal transduction, , And the research on the composition and function thereof is actively under way.

Cells release various membrane-type vesicles in the extracellular environment, and these release vesicles are commonly referred to as extracellular vesicles (EVs). The extracellular endoplasmic reticulum is sometimes referred to as cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes and, in some cases, differentiated from exosomes.

Exosome is an endoplasmic reticulum of several tens to several hundreds of nanometers in size, composed of double lipid membranes identical to the cell membrane structure, and contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosomal cargo inside. Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells. Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known. Exosomes contain specific genetic material and bioactivity factors depending on the nature and condition of the derived cells. The proliferating stem cell-derived exosomes regulate cell behavior such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

However, in spite of various studies such as the possibility of treatment of specific diseases using exosome, there have been many studies to develop a new formulation capable of stably maintaining and storing exosome, various medical treatments Or beauty technology has not received much attention.

The present inventors have conducted intensive studies on new functional cosmetics which have no adverse effects on the human body and have excellent skin moisturizing effect, and have found that exosome derived from stem cells increases the expression and activation of AQP3, It is confirmed that it is effective for moisturizing the skin by transferring the moisture deep into the skin, thus completing the present invention.

It should be understood, however, that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention, and not as an admission that it can be used as the "prior art "

Korean Registered Patent No. 10-1724530 (Apr. Korean Patent Publication No. 10-2018-0025332 (Aug.

It is an object of the present invention to provide a novel skin moisturizing composition comprising stem cells derived exosome as an active ingredient.

Another object of the present invention is to provide a functional cosmetic composition for skin moisturizing comprising the composition and an external preparation for skin.

It is still another object of the present invention to provide a cosmetic method for controlling the condition of mammalian skin by moisturizing the skin except for the treatment using the skin moisturizing composition.

However, the above-described problems of the present invention are illustrative and not intended to limit the scope of the present invention. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The inventors of the present invention have conducted intensive studies on the development of a new composition for moisturizing the skin. As a result, it has been confirmed that the exosome derived from stem cells increases the expression and production of AQP3, which plays an important role in water movement in the skin, Derived cosmetic composition containing an exosome as an active ingredient has been developed.

As used herein, the term "exosomes " refers to an endoplasmic reticulum of several tens to several hundred nanometers (preferably about 30 to 200 nm) in size, consisting of double lipid membranes identical in structure to the cell membrane The particle size of the exosome can be varied according to the cell type, the separation method and the measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, DOI 10.1007 / s00216-015-8535-3). Exosomes contain proteins called exosomal cargo (cargo), nucleic acids (mRNA, miRNA, etc.). Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells. Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.

As used herein, the term "exosome" refers to a vesicle having a nano-sized vesicle structure secreted from the stem cells and released into the extracellular space and having a composition similar to that of exosome (for example, exosome- Pseudo-bezacl). The type of the stem cell is not limited, but may be, for example, a mesenchymal stem cell, for example, a fat, a bone marrow, an umbilical cord or a cord blood derived stem cell, Derived stem cells. The type of the adipose-derived stem cell is not limited as long as it does not cause a risk of infection by a pathogen and does not cause an immune rejection reaction, but it may be preferably a human adipose-derived stem cell.

However, it is needless to say that the exosomes derived from stem cells used in the present invention can be used for skin moisturizing and exogenous stem cell-derived exosomes which are used in the art or can be used in the future as long as they do not adversely affect the human body . Therefore, it should be understood that the exosomes derived from the stem cells isolated according to the separation method of the following Examples should be understood as an example of exosome that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "skin moisturizing" means that the homeostasis of the living body is maintained by appropriately controlling moisture loss (moisture evaporation) of the skin and the like. "Skin trouble" means that the skin turns red due to external stimuli or physical changes, and itching appears, and in the case of severe cases, small protuberance or rash occurs.

The term "iontophoresis" as used herein refers to a method in which an ionized active ingredient is allowed to permeate through the skin by an electrical repulsive force by applying a minute current to the skin to which the effective substance is applied, . The iontophoresis used in one embodiment of the present invention is a method in which a current flows from an external power source to an electrode patch on the skin to introduce minute current into the skin, A method in which minute current is introduced, a method in which minute current is introduced into skin through a patch equipped with a reversed electrodialysis means for generating current through a difference in ion concentration between a high concentration electrolyte solution and a low concentration electrolyte solution, and the like . However, the present invention is not limited thereto, and it is needless to say that various types of iontophoresis can be used.

The skin moisturizing composition of one embodiment of the present invention contains exosome derived from stem cells as an effective ingredient.

In the skin moisturizing composition of one embodiment of the present invention, the stem cell-derived exosome may increase the expression or production of AQP3 (Aquaporin 3).

The skin moisturizing composition according to one embodiment of the present invention may include a lyophilized preparation containing the above exosome derived from stem cells and methionine, mannitol and trehalose as an active ingredient. The weight ratio of methionine, mannitol, and trehalose is 1: 1: 1.

In the skin moisturizing composition of one embodiment of the present invention, the lyophilized preparation may further contain ascorbic acid and retinol. For example, the weight ratio of methionine, mannitol, trehalose, ascorbic acid, and retinol is 9: 9: 9: 0.5: 0.5.

The skin moisturizing composition of one embodiment of the present invention may include the lyophilized preparation and a diluent. For example, the diluent may be water for injection, physiological saline, phosphate buffered solution, purified water, or deionized water. In addition, the diluent may further comprise hyaluronic acid or hyaluronate (for example, sodium hyaluronate). As an example, the composition may be a suspension.

The skin moisturizing composition of one embodiment of the present invention may be a cosmetic composition or a skin external preparation. For example, the cosmetic composition may be a cream or lotion.

In the skin moisturizing composition of one embodiment of the present invention, the stem cell-derived exosome is characterized in that it increases the skin hydration.

On the other hand, when the composition for skin moisturizing according to one embodiment of the present invention is made of an external preparation for skin and / or a cosmetic composition, components which are usually used in cosmetics or external skin preparations, such as moisturizers , An antioxidant, an oily component, an ultraviolet absorber, an emulsifier, a surfactant, a thickener, an alcohol, a powder component, a colorant, an aqueous component, water, various skin nutrients and the like.

In addition, the external preparation for skin and / or cosmetic composition according to one embodiment of the present invention may contain, in addition to exosomes derived from stem cells, a conventional skin remedy and / or cosmetic composition insofar as its action (skin moisturization, skin condition improvement, A moisturizing agent may be mixed and used. For example, the stem cell-derived exosome of the present invention may be carried on or mixed with at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel. In the external preparation for skin and / or cosmetic composition according to one embodiment of the present invention, the type of the hydrogel is not limited, but it is preferably a hydrogel obtained by dispersing the gel polymer in a polyhydric alcohol. Wherein the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum And the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerin.

The external preparation for skin and / or the cosmetic composition according to one embodiment of the present invention can be used in the form of, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, , Mist, foundation, powder, oil paper, and the like. For example, the external preparation for skin and / or cosmetic composition may be applied or deposited on at least one surface of the patch, mask pack, or mask sheet.

The cosmetic composition of one embodiment of the present invention is used for the purpose of moisturizing skin, preventing skin dryness, preventing skin trouble due to skin dryness, or improving skin condition, and cosmetic formulations can be prepared in any formulations conventionally produced in the art . For example, it is possible to use a lotion, a mask pack, a mask sheet, a softener, a nutritional lotion, a convergent lotion, a nutritional cream, a massage cream, an eye cream, a cleansing cream, an essence, an essence, a cleansing lotion, a cleansing foam, But are not limited to, soaps, shampoos, surfactant-containing cleansers, bath salts, body lotions, body creams, body oils, body essences, body cleansers, hair dyeing agents, hair tonics and the like.

The external preparation for skin and / or cosmetic composition according to one embodiment of the present invention includes components commonly used in external preparations for skin and / or cosmetics and includes conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, And may include a carrier. Further, in the respective formulations for the external preparation for skin and / or cosmetic composition, the other ingredients can be mixed and selected without difficulty by the person skilled in the art depending on the kind of the external preparation for skin and / or cosmetic composition or purpose of use.

The present invention also relates to a skin mask composition comprising a first mask pack, a mask sheet or a patch which is applied or immersed with a skin moisturizing composition containing an exosome derived from a stem cell as an active ingredient, A skin moisturizing exosome kit comprising an iontophoresis device for flowing an electric current. The stem cell-derived exosomes increase the expression or production of AQP3 (Aquaporin 3).

For example, the skin moisturizing exosome kit of one embodiment of the present invention is characterized in that the first mask pack, the mask sheet or the patch is contacted or adhered onto the skin of the mammal, and the first mask pack, the mask sheet or the patch By placing the iontophoresis device, the first mask pack, the mask sheet or the patch can be contacted or attached to be used in such a manner that a minute current is supplied to the skin of the mammal to which the skin moisturizing composition is applied.

The composition for moisturizing the skin has been conventionally used as far as it does not damage its action (skin moisturization, prevention of dry skin, prevention of skin trouble due to skin dryness, improvement of skin condition, skin beauty, etc.) A skin-improving agent and / or a moisturizing agent may be mixed together and used. For example, the composition for moisturizing the skin may be carried or mixed with at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

The iontophoresis device for skin moisturizing according to one embodiment of the present invention is characterized in that the iontophoresis device comprises a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel- A dialysis cell, or a second mask pack, a mask sheet, or a patch with the at least one battery mounted thereon.

In the skin moisturizing exosome kit of one embodiment of the present invention, the second mask pack, the mask sheet or the patch may be laminated on the first mask pack, the mask sheet or the patch.

In one embodiment of the present invention, at least one surface of the first mask pack, the mask sheet or the patch is coated with a hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate) Or hyaluronic acid gel may be applied. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

According to the skin moisturizing exosome kit of the present invention, the microcurrent flows from the iontophoresis device so that the stem cell-derived exosomes can effectively penetrate the epidermis and penetrate deeply into the skin. Accordingly, it can more effectively contribute to moisturizing the skin.

In addition, the present invention provides a cosmetic method for controlling the condition of a mammalian skin by moisturizing the skin using a composition for moisturizing the skin, except for therapeutic use. In the cosmetic method of the present invention, conditioning of the skin means improvement of the condition of the skin and / or prevention of the condition of the skin, and improvement of the condition of the skin means visual and / Or a tactile perceptible positive change. For example, improving the condition of the skin may be moisturizing the skin, preventing dryness of the skin, preventing skin trouble due to drying of the skin, or moist and shiny skin condition.

The cosmetic method of one embodiment of the present invention comprises the steps of: (a) applying the skin moisturizing composition directly to the skin of a mammal; (b) applying a patch, mask pack or mask sheet on which the skin moisturizing composition is applied or immersed Contacting or adhering to the skin of a mammal, or sequentially advancing (a) and (b) above. In the step (a), lotion or cream may be used as the skin moisturizing composition.

Alternatively, the cosmetic method of one embodiment of the present invention may comprise: (c) removing the patch, mask pack or mask sheet from the skin of the mammal after the step (b), and applying the skin moisturizing composition to the mammal And applying the composition to the skin. In the step (c), lotion or cream may be used as the skin moisturizing composition.

The cosmetic method of one embodiment of the present invention may further comprise performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the skin moisturizing composition is applied. In addition, the cosmetic method of one embodiment of the present invention may further comprise contacting or attaching the iontophoresis device to the skin of the mammal.

In addition, the cosmetic method according to one embodiment of the present invention is a cosmetic method comprising: a first mask pack, a mask sheet or a patch which is coated or immersed with a skin moisturizing composition containing an exosome derived from a stem cell as an active ingredient; May be carried out using a skin moisturizing exosome kit comprising an iontophoresis device that delivers a microcurrent to the skin of an applied mammal.

For example, the cosmetic method of one embodiment of the present invention is a cosmetic method comprising the steps of: (a) applying a first mask pack, a mask sheet, or a patch to which a skin moisturizing composition containing an exosome derived from stem cells as an active ingredient is applied or immersed, (B) placing the iontophoresis device on the first mask pack, mask sheet or patch, and (c) contacting the first mask pack, mask sheet or patch with the skin, Or exfoliating a skin of a mammal to which the composition for skin moisturizing is applied; and (d) applying the stem cell-derived exosome to the skin of the mammal . The stem cell-derived exosomes increase the expression or production of AQP3 (Aquaporin 3).

In at least one surface of the first mask pack, the mask sheet or the patch, at least one surface of the first mask pack, the mask sheet or the patch may be coated with a hydrogel, hyaluronic acid, hyaluronate (e.g., sodium hyaluronate) At least one of the gels may be applied. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

In the cosmetic method according to one embodiment of the present invention, the iontophoresis device includes a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, Or a second mask pack, a mask sheet or a patch having the at least one battery mounted thereon.

In the cosmetic method according to one embodiment of the present invention, the step (b) may be performed by laminating the second mask pack, the mask sheet or the patch on the first mask pack, the mask sheet or the patch.

In the cosmetic method according to one embodiment of the present invention, the microcurrent flows from the iontophoresis device so that the stem cell-derived exosome can effectively penetrate the epidermis and deeply penetrate into the skin. Accordingly, it can more effectively contribute to moisturizing the skin.

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

The stem cell-derived exosomes contained in the skin moisturizing composition of the present invention increase the expression of AQP3, which plays an important role in water migration in the skin. Accordingly, the skin moisturizing composition of the present invention increases the expression or production of AQP3, thereby activating the movement of moisture in the skin, thereby moisturizing the skin and obtaining a moisturizing effect by restoring the skin function. Namely, the skin moisturizing composition containing the stem cell-derived exosome of the present invention as an active ingredient has a functional activity useful as a moisturizing functional cosmetic, that is, an activity to induce the expression of AQP3, And can be usefully utilized as an effective ingredient of a functional cosmetic composition for moisturizing.

Accordingly, the composition for moisturizing the skin of the present invention exhibits an effect of preventing, suppressing, alleviating or improving the skin moisturizing effect and skin irritation caused by dryness of the skin and improving the skin condition which is deteriorated due to drying of the skin, And exhibits excellent effects in skin care.

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

FIG. 1 shows the results of physical property analysis of exosomes obtained according to one embodiment of the present invention. "Figure 1A " shows the particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis. "Figure 1B " shows particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis. "Fig. 1C " shows particle images by transmission electron microscopy (TEM) according to magnification. "Figure 1D " shows the Western blot results of exosomes obtained according to one embodiment of the present invention. ≪ RTI ID = 0.0 > 1E < / RTI > shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.
FIG. 2 shows the result of confirming the absence of cytotoxicity after treatment of exosome according to one embodiment of the present invention with human skin fibroblast HS68 cells.
FIG. 3 is a graph showing a result of significantly increasing the moisture content in the stratum corneum of the skin on both right and left sides when the cosmetic composition for skin moisturizing according to one embodiment of the present invention is applied to the left and right faces of a person.
FIG. 4 is a graph showing the results of a significant increase in the rate of change of the moisture content in the stratum corneum of the skin on both right and left sides when the cosmetic composition for skin moisturizing according to one embodiment of the present invention is applied to the right and left faces of a person.
Fig. 5 shows fluorescence intensity measurement results of exosomes stained with PKH67.
FIG. 6 is an image of a fluorescence microscope confirming the extent of fluorescence-stained exosomes transferred into pig skin tissue.
FIG. 7 is a confocal fluorescence microscope image showing the extent of fluorescence-stained exosomes transferred into the mouse skin tissue.
Fig. 8 is a graph showing a comparison of the total fluorescence intensity obtained by measuring the fluorescence intensity on each image in Fig. 7; Fig.
9 is a perspective view showing a configuration of an exosome kit 100 for skin moisturizing according to one embodiment of the present invention.
Fig. 10 is a photograph showing the skin condition before and after treatment of human skin with the exosome kit for skin moisturizing of the present invention.
Fig. 11 shows the result that skin moisturization is remarkably improved when the skin moisturizing exosomal kit of the present invention is applied to human skin.
12 and 13 are graphs showing that the expression level of AQP3 (Aquaporin 3) increases as a result of treating stem cells-derived exosomes with human epidermal keratinocytes (HEK).
14 is a photograph showing the favorable structure of the lyophilized exosome according to the method of one embodiment of the present invention.
FIGS. 15A to 15G are photographs showing the appearance of exosome according to the combination of freeze-dried protective agent components after the lyophilization and the lyophilization of the combination of the freeze-drying protective agent components were performed differently.

Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

Example

Example 1: Culture of cells

HS68 cells, a human dermal fibroblast, were purchased from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific) The cells were subcultured in DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO ₂ at 37 ° C.

According to the cell culturing method known in the art, adipose-derived stem cells were cultured at 5% CO ₂ and 37 ° C. Then, the cells were washed with a phosphate-buffered saline (purchased from ThermoFisher Scientific), replaced with serum-free, non-phenol red medium, cultured for 1 to 10 days, and the supernatant .

Trehalose was added to the culture medium in an amount of 2% by weight in order to obtain an exosome having a uniform particle size distribution and high purity in the process of separating exosome. After the addition of trehalose, the culture was filtered with a 0.22 μm filter to remove impurities such as cellular debris, waste products and large particles. The filtered cultures were immediately separated to isolate exosomes. In addition, the filtered culture was stored in a refrigerator (image below 10 ° C) and used for exosome isolation. In addition, the filtered culture was frozen in an ultra-low temperature freezer at -60 ° C or lower, and thawed, followed by exosome isolation. Then, the exosomes were separated from the culture medium by using Tangential Flow Filtration (TFF).

Example 2: Isolation and purification of exosome by TFF method

In Example 1, a TFF (Tangential Flow Filtration) method was used for separating, concentrating, desalting and diafiltration exosomes from a culture filtrated with a 0.22 μm filter. For the TFF method, a cartridge filter (also called a hollow fiber filter (purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used. The TFF filter can be selected by a variety of molecular weight cutoffs (MWCO). Exosomes were selectively isolated and concentrated by selected MWCOs, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than MWCO were removed.

To separate and concentrate the exosomes, TFF filters of MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da were used. The culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, and substances smaller than MWCO were removed to separate the exosomes.

Separated and concentrated exosomal solutions were further desalted and buffered (diafiltration) using the TFF method. At this time, the desalination and buffer exchange are performed by continuous diafiltration or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably, Was performed using a buffer solution having a volume of 12 times or more. To the buffer solution, 2% by weight of trehalose dissolved in PBS was added to obtain an exosome having a uniform particle size distribution and high purity.

Example 3: Characterization of isolated exosomes

Separated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (NTA; purchased from Malvern) or tunable resistive pulse sensing (TRPS; purchased from Izon Science). The uniformity and size of isolated exosomes were analyzed by transmission electron microscopy (TEM). The results of TRPS, NTA and TEM analysis of the exosomes isolated according to one embodiment of the present invention are shown in Figs. 1A to 1C.

FIG. 1D shows the presence of CD9, CD63, CD81 and TSG101 markers as a result of performing Western blotting on isolated exosomes according to the method of one embodiment of the present invention. Anti-CD9 (purchased from Abcam), anti-CD63 (purchased from System Biosciences), anti-CD81 (purchased from System Biosciences), and anti-TSG101 (purchased from Abcam) were used as the antibodies for each marker.

FIG. 1E shows the presence of CD63 and CD81 markers as a result of analysis using flow cytometry on exosomes isolated according to the method of one embodiment of the present invention. Human CD63 isolation / detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method to isolate the positive exosomes for CD63 and PE-mouse anti-human CD63 (PE-Mouse anti markers were stained using the PE-mouse CD63 (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD), and analyzed using a flow cytometer (ACEA Biosciences) Respectively.

Meanwhile, the exosomes derived from stem cells used in the present invention are not limited to the exosomes of the above-mentioned embodiments, and it is possible to use various stem cell-derived exosomes which are used in the art or can be used in the future Of course. It is to be understood that the exosomes derived from the stem cells isolated according to the above embodiments are to be understood as an example of exosomes derived from stem cells which can be used in the present invention, and the present invention is not limited thereto.

Example 4: Measurement of cytotoxicity by treatment with exosome

In order to evaluate the toxicity of exosomes obtained according to the isolation method of one embodiment of the present invention in human skin fibroblast HS68 cells, exosomes were treated by concentration to the cells and the proliferation rate of the cells was confirmed. HS68 cells were suspended in DMEM containing 10% FBS, and the cells were mixed with 80 ~ 90% confluency and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, the culture solution was removed, and the cell survival rate was evaluated by culturing the exosome prepared in Example 2 for each concentration and culturing for 24 to 72 hours. Cell viability was measured using WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramar Blue reagent alamarBlue reagent (purchased from ThermoFisher Scientific) and a microplate reader (purchased from Molecular Devices).

The comparative group was based on the number of cells cultured in the normal cell culture medium not treated with exosome, and it was confirmed that the exosome-induced cytotoxicity did not appear within the tested concentration range (FIG. 2).

Example 5: Freeze-drying of exosomes

Example 5-1: Freeze-drying conditions

A lyophilized protective agent containing methionine, mannitol and trehalose was prepared for lyophilization of exosomes. An aqueous solution to which the lyophilized protective agent was added was prepared in 1 mL of an aqueous solution containing ascorbic acid and retinol each at 0.5 mg / mL (manufactured by Biofuji Co., Ltd. (manufactured by Hapon County, Jeonnam, Korea)]. In this embodiment, the lyophilized protective agent is added to a solution containing ascorbic acid and retinol. Alternatively, an aqueous solution may be prepared by adding a lyophilized protective agent to injection water, purified water, physiological saline, or deionized water. The concentrations of methionine, mannitol and trehalose in the aqueous solution were adjusted to be 9 mg / mL, respectively.

(5 × 10 ⁸ particles) prepared in Example 2 were mixed with an aqueous solution containing a lyophilized protective agent, and then freeze-dried using the freeze-drying equipment (VIRTIS, ITME No .: 34424) Drying was carried out. The lyophilization was carried out in the order of conditions 1, 2, 3, 4, 5, 6, 7 and 8 in Table 1 below.

Freeze-drying conditions Total time (min) 4320 Condition Time (min) Temperature (℃) Pressure (mmHg) One 700 -50 760 2 60 -50 760 3 999 -50 0 4 999 -50 0 5 999 -50 0 6 370 -50 0 7 120 -20 0 8 73 10 0

The lyophilized protective agent containing methionine, mannitol and trehalose was treated to freeze-dry the lyophilized lyophilized product. As a result, it was confirmed that the color was milky white and exhibited a good shape maintaining a porous sponge shape ). That is, in the method of lyophilization of exosome of the present invention, the lyophilized product exhibiting excellent properties can be obtained by lengthening the drying time under reduced pressure and by using a combination of methionine, mannitol and trehalose as the lyophilized protective agent.

Example 5-2: Comparison of properties of exosome product according to freeze-dried protective agent composition

On the other hand, the properties of exosomes in the case of lyophilization of exosomes were compared using various lyophilized protective agents containing at least one of methionine, mannitol and trehalose (hereinafter referred to as a lyophilized protective agent component). According to the method described in Example 5-1, 7 types of aqueous solutions were prepared by adding the lyophilized protective agent component alone, a combination of two components, or a combination of three components. The concentration of each freeze-dried protective agent component in the aqueous solution was adjusted to be 9 mg / mL. According to the freeze-drying conditions and methods of Example 5-1, the exoose (5 × 10 ⁸ particles) prepared in Example 2 was mixed with an aqueous solution containing each combination of freeze-dried protective agent components, followed by lyophilization .

The external shape properties of the lyophilized exosome products were photographed and evaluated (Figs. 15A to 15G). According to the good and bad shape of the cake of the freeze dried exosome product, the best was 5 points, and the worst was 1 point, and the outward shape property was evaluated relative. The results of evaluating the properties of the exosome product according to the combination of the freeze-dried protector components are shown in Table 2 below.

Comparison of Properties of Exosome Products by Freeze-Drying Protector Ingredients Composition of freeze-dried protector composition Mannitol Trehalose Methionine Methionine +
Trehalose Methionine +
Mannitol Trehalose +
Mannitol Methionine + mannitol + trehalose (present invention) Rating One One 4 3 4 2 5 drawing 15A 15B 15C 15D 15E 15F 15g

As shown in FIGS. 15A to 15G and Table 2, when the freeze-dried preservative consisting of a combination of methionine, mannitol and trehalose according to the present invention was used to freeze-dry the exosome, It was found that the exosomes obtained by lyophilization of the combination of the three components lacking one or two components were inferior to those of the present invention.

Example 6: Cosmetic composition formulation of one embodiment of the present invention

(Preparation of lotion)

The composition of the cosmetic composition (lotion) was prepared by diluting the stock solution of 1704 μg / mL prepared in Example 2 with the components listed in Table 3, and suspending the same. The final cosmetic composition was prepared to contain exosomes at a concentration of 2 x 10 < ⁴ > particles / mL. The content of each component is shown in Table 3 below.

Ingredients and content of lotion ingredient Content (% by weight) The exosome prepared in Example 2 One glycerin 7.375 Caprylic / capric triglyceride 6 Cetyl ethyl hexanoate 5 Propanediol 5 Phenyl trimethicone 3.5 Stearic acid 3 1,2-hexanediol 2 Panthenol 2 Cetearyl olivate 1.8 Sorbitan olivate 1.2 Diisostearyl malate One Fructan One Ammonium acryloyldimethyltaurate / VP copolymer 0.3 Arachidyl alcohol 0.25 Behenyl alcohol 0.15 Arachidyl glucoside 0.1 Hydrogenated lecithin 0.1 Shea Butter 0.09 Xanthan gum 0.05 Lavender Oil 0.02 Bergamot oil 0.02 Ceramide NP (Ceramide NP) 0.02 Orange peel oil 0.02 Phytosphingosine 0.015 Palmitoyl tetrapeptide-7 0.01 Palmitoyl tripeptide-1 0.01 Purified water Balance

(Preparation of cream)

The exosome stock solution of 1704 μg / mL prepared in Example 2 was diluted and mixed with the ingredients listed in Table 4 to suspend the cosmetic composition (cream). The final cosmetic composition was prepared to contain exosomes at a concentration of 2 x 10 < ⁴ > particles / mL. The content of each component is shown in Table 4 below.

Composition and content of cream ingredient Content (% by weight) The exosome prepared in Example 2 One Caprylic / capric triglyceride 12 glycerin 9.75 Butylene glycol 7 Cetearyl alcohol 3.5 Hydrogenated vegetable oil 3.5 1,2-hexanediol 2 Stearic acid 2 Phenyl trimethicone 2 Jojoba seed oil 2 Beads wax 1.7 Glyceryl stearate 1.5 Stearyl alcohol 1.45 Glyceryl stearate S One Cetyl alcohol One Panthenol One Shea Butter 0.88 Sorbitan sesquioleate 0.5 Polyglyceryl-2 stearate 0.45 Ammonium acryloyldimethyltaurate / Vpicopolymer 0.3 Hydrogenated lecithin 0.1 Bergamot oil 0.04 Ceramide Nephew 0.04 Phytosphingosine 0.03 Ivigo de fruit extract 0.0225 Hanchuncho extract 0.0225 Lavender Oil 0.02 Orange peel oil 0.02 Copper tripeptide-1 0.01 Palmitoyl tetrapeptide-7 0.01 Palmitoyl tripeptide-1 0.01 Calamine 0.001 Purified water Balance

(Preparation of mask pack)

The stock solution of the exosome prepared at the concentration of 1704 μg / mL prepared in Example 2 was diluted and mixed with the ingredients listed in Table 5 below to suspend. The obtained cosmetic composition was applied or immersed to prepare a mask pack. The exosomes were applied or immersed in a mask pack at a concentration of 4 x 10 < ³ > particles / mL. The content of each component is shown in Table 5 below.

Components and content of mask pack ingredient Content (% by weight) The exosome prepared in Example 2 0.1 glycerin 3.070045 Dipropylene glycol 3.03 Methyl propanediol 2 Caprylic / capric triglyceride 1.500025 Olive oil 0.8 1,2-hexanediol 0.7235 Polysorbate 60 0.7 Sorbitan stearate 0.6475 Arginine 0.13 Carbomer 0.13 Hydroxyethylcellulose 0.13 Allantoin 0.1 Trehalose 0.1 Green tea extract 0.07 Butylene glycol 0.07 Ginger extract 0.07 Spanish licorice root extract 0.07 Coptis root extract 0.07 Sucrose cocoate 0.0525 Ethylhexyl glycerin 0.05 Dipotassium glycyrrhizate 0.03 Disodium iodide 0.02 Tocopheryl acetate 0.02 Panthenol 0.02 Ceramide Nephew 0.000001 Sodium hyaluronate 0.01 Xylityl glucoside 0.004 Anhydro xylitol 0.0028 Xylitol 0.0012 Glucose 0.0004 Hydrogenated lecithin 0.000005 Spices 0.012 cholesterol 0.000001 Purified water Balance

Example 7: Test of moisturizing effect on human skin

Twenty-one women in their twenties to thirties, who had no acute or chronic physical disease and had normal skin dryness, were selected as test subjects. The milky lotion and light pink cream of Example 6 were administered twice a day (morning and evening) And applied to the right and left faces. The skin moisture content of the facial area was measured with a moisture meter (Corneometer CM825) (Courage-Khazaka eletronic GmbH, Germany) before the use of the lotion and cream, two weeks after use, and four weeks after use. Skin moisturisation tests were performed on the lotions and creams. All 21 subjects completed the tests without dropouts.

Measurements (A.U.) of the corneometer at the left and right test sites were 51.50 and 51.14 after 2 weeks of use of the lotion and cream, respectively, and 55.50 and 54.66 after 4 weeks of use, respectively (FIG. 3). In addition, changes in the water content of the stratum corneum at each test site were identified as 57.81% and 59.07% after 2 weeks of using the lotion and cream, respectively, and 70.09% and 70.01% after 4 weeks of use, respectively (FIG. Both the left and right test sites exhibited a skin (horny layer) moisture content that increased from two weeks after use of the lotion and cream to a statistically significant level (P < 0.05) before use, It is thought that it can help.

On the other hand, there was no report of a specific skin adverse reaction during the period of using the lotion and cream, and no abnormal findings were observed on the physical examination by a dermatologist.

Therefore, it can be seen that the cosmetic composition of the present invention exhibits excellent effects in moisturizing the skin.

Example 8 Skin permeability test of exosome derived from stem cells contained in cosmetic composition for skin moisturizing

PKH67 dye (purchased from Sigma) was used to prepare fluorescently stained exosomes. 1 mM PKH67 was diluted in Diluent C (purchased from Sigma) to prepare a 10 μM PKH67 solution, mixed with an appropriate concentration of exosome solution, and allowed to react at room temperature for 10 minutes in the absence of light. After the reaction, an MW3000 spin column (purchased from ThermoFisher Scientific) was used to remove the remaining PKH67 dye from the exosomes stained with PKH67 (hereinafter abbreviated as "PKH-exosomes"). After confirming the use of a fluorescence analyzer (purchased from Molecular Devices), PKH67 not reactive with exosomes was removed, and fluorescence of sufficient intensity was detected in PKH-exosomes (FIG. 5).

PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the outer surface of the pig skin. To prevent drying of the applied PKH-exosomes, the nonwoven fabric was covered and allowed to react for an appropriate period of time, for example, 30 minutes to 1 hour to allow the PKH-exosomes to be delivered to the subcutaneous tissues. In addition, the PKH-exosomes were applied to the outer surface of the pig skin and covered with the nonwoven fabric, followed by flowing a microcurrent for a predetermined time, for example, 30 minutes to 1 hour. After the reaction was completed, the pig skin tissue was immersed in a 3.7% formaldehyde solution, reacted overnight, and washed three times for 5 minutes each with phosphate buffered saline. The washed pig skin tissue was immersed in a 30% sucrose solution and treated with OCT compound. After washing three times for 5 minutes with phosphate buffer solution, sections were prepared on a longitudinal section using a microtome, and tissue sections were placed on a slide glass. On the other hand, the preparation of the tissue section may be carried out before the tissue is fixed with formaldehyde solution. Fluorescence detected from PKH-exosomes in tissue sections was observed using fluorescence microscopy. In this way, PKH-exosomes transmitted to the subcutaneous tissue through the epidermis of the pig skin tissue were identified. As a result, it was confirmed that the exosomes were more effectively passed through the epidermis, deeply transferred into the skin tissue, and more effectively absorbed to the skin when active current was delivered (active transdermal delivery) (see FIG. 6).

Next, the skin tissue of the hairless mouse was removed and placed on top of the chamber of the Franz Diffusion Cell. At this time, the inside of the diffusion cell was filled with a phosphate buffer solution. PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the outside of the skin tissue of the mice. At this time, in order to prevent drying of PKH-exosomes, the nonwoven fabric was pre-positioned outside the mouse skin tissue, and PKH-exosomes were injected between the nonwoven fabric and skin tissue. Then, the reaction was carried out for 30 minutes to 1 hour. In addition, the PKH-exosomes were injected between the nonwoven fabric and the skin tissue, and the microcurrent was flowed for a predetermined time, for example, 30 minutes to 1 hour. After completion of the reaction, the PKH-exosomes transferred into the skin tissue were confirmed immediately using a confocal fluorescence microscope (Leica, SP8X) or the skin tissue and the PKH-exosomal solution were further reacted for 1 hour to 6 hours , And PKH-exosomes were identified by confocal fluorescence microscopy. As a result, it was confirmed that the exosomes were more effectively passed through the epidermis and deeply transferred into the skin tissues and more effectively absorbed to the skin when active transdermal delivery was performed (see FIGS. 7 and 8) ).

The results of the above experiment are summarized as follows. When the exosome derived from stem cells is applied to the skin by applying a microcurrent to the skin by iontophoresis, the exosome derived from the stem cell effectively passes through the epidermis, And can be effectively absorbed against the skin. Accordingly, it can more effectively contribute to moisturizing the skin. Therefore, when exosomes derived from stem cells are applied to the skin using the skin moisturizing exosome kit including the iontophoresis device (device for flowing a microcurrent to the skin), the skin penetration ability is further improved, It is possible to maximize the moisturizing effect or the skin cosmetic effect.

Example 9: Treatment of exosome kit for skin moisturizing against human skin

9, the skin moisturizing exosomatic kit 100 according to one embodiment of the present invention comprises a liquid immersion sheet mask 10 (a composition containing a stem cell-derived exosome, Sheet mask) and an percutaneous permeation promoting sheet mask (silver sheet mask) 20 which is an iontophoresis device. A stem cell-derived exosome (12) is applied or deposited on the surface or inside of the liquid immersion sheet mask (10). A battery 22 for generating a microcurrent is mounted on the percutaneous permeation enhancing sheet mask 20 and a conductive pattern 24 electrically connected to the cell 22 is attached to the percutaneous permeation enhancing sheet mask 20, So that the micro current generated from the battery 22 can be uniformly transmitted to the entirety of the percutaneous permeation enhancing sheet mask 20. [ The conductive pattern 24 is formed by printing or plating a conductive metal such as gold, silver, or copper.

The "skin moisturizing exosome kit 100" having the above-described structure is applied to a face of a person (hereinafter referred to as "case 1") having a flushing symptom and skin troubles associated with itching due to skin dryness and skin dryness To assess whether skin troubles and flushing symptoms are alleviated or improved. In addition, the skin moisturizing exosome kit 100 of the present invention was applied once to the face of a person with flushing symptom (hereinafter referred to as "case 2") to evaluate whether the overall skin tone and flushing symptom improved. A liquid immersion sheet mask 10 containing 5 × 10 ⁵ stem cell-derived exosomes 12 was adhered uniformly to the entire face of cases 1 and 2 in accordance with the eyes and mouth parts, The activated percutaneous permeation enhancing sheet mask 20 was laminated on the liquid immersion sheet mask 10 for about 25 to 30 minutes.

As a result, the skin moisturizing exosomal kit 10 comprising the liquid-immersion sheet mask 10 containing the stem cell-derived exosomes 12 at a concentration of 5 x 10 ⁵ particles / mask and the percutaneous permeation- (Case 1), the overall skin tone was improved and the flushing was alleviated even in "case 2" (see FIG. 10) See case 2 in FIG. 10). Thus, the skin moistening exotherm of the present invention comprising the liquid immersion sheet mask 10 on which the composition containing the stem cell-derived exosome as an active ingredient is immersed and the percutaneous permeation promoting sheet mask (iontophoresis device) The kit 100 may be said to have the effect of preventing, suppressing, alleviating or alleviating the skin moisturizing effect and the redness symptoms and skin troubles associated with skin dryness.

Example 10: Test of moisturizing effect on human skin

The acute and chronic physical disease-free healthy women 20 (man 35 ~ 59 years) for the stem cells a bit derived from the exo (Example 2 little ready-exo-in) containing a skin disease 1 × 10 ⁷ of the particles / mL (Translucent oil-in-water) and the skin moisturizing exosome kit 100 of the present invention were used once every three days in the evening to test the skin moisturizing effect. The amount of skin moisture was measured by a moisture meter (Corneometer CM825) (Courage-Khazaka eletronic GmbH, Germany) before use of the ampoule and exosome kit 100, 2 weeks after use, 4 weeks after use, Respectively.

After the facial cleansing, the contents of the ampoule were evenly applied to the face, the liquid immersion sheet mask 10 was brought into close contact with the eyes and the mouth, and the entire surface of the face was evenly adhered. Then, an activated percutaneous permeation- The mask 20 was laminated on the liquid immersion sheet mask 10 and used for about 25 to 30 minutes.

As a result of evaluating the skin moisture content before and after use of the skin moisturizing exosome kit 100 of the present invention, a significant improvement effect was obtained after 2 weeks of use and after 4 weeks of use as compared with before use ). In addition, the rate of change in skin moisture content was 10% on average for the other clinical treatments, and 21.383% for the skin moisturizing exosome kit 100 of the present invention for 4 weeks was very good compared to the average 11).

After the test, a Global Assessment of Efficacy was conducted. The subjects to be tested were 5 stages of excellent skin moisturization (4), good (3), normal (2), poor (1) and very poor (0) after using the skin moisturizing exosome kit (100) To answer the questionnaire. In the questionnaire survey, the mean value and standard deviation of the questionnaire value and the percentage of the subjects to the answer were obtained for the skin moisturizing item. As a result of a survey on the skin moisturizing exosome kit (100) of the present invention, 100% After completion of the test, the subjects were asked to respond to the questionnaire about feeling of use of the skin moisturizing exosome kit (100) of the present invention. (4), good (3), normal (2), poor (1), and very poor (0) for the skin moisturizing, smoothness, spreading, absorbency, Respectively. The mean value and the standard deviation of the questionnaire value and the percentage of the subjects to the answer were obtained for each evaluation item. As a result of the questionnaire evaluation for the skin moisturizing exosome kit (100) of the present invention, 100% of the test subjects were evaluated as above all the evaluation items.

On the other hand, no specific skin adverse reaction was reported during the period of using the skin moisturizing exosome kit 100 of the present invention, and no abnormality was found on physical examination by a dermatologist.

Accordingly, it can be seen that the skin moisturizing exosome kit 100 of the present invention exhibits superior skin moisturizing effect as compared with the conventional clinical treatments, as confirmed by the above-mentioned clinical test.

Example 11: Confirmation of inducing effect of AQP3 expression

The effect of the stem cell-derived exosomes of the present invention on AQP3 (aquaporin 3) expression was confirmed in human epidermal keratinocytes (HEK). To this end, human epidermal keratinocyte (purchased from HEK, Gibco) was dispensed into a multi-well plate and cultured. Then, an adipocyte stem cell culture obtained from Cell Biotech (Seoul, Korea) (The first batch exosome) derived from the stem cells or the exosome (the second batch exosome) derived from the stem cells prepared according to the first and second embodiments were treated according to the method of Example 1 and Example 2, respectively. CDNA was prepared from the RNA isolated from the cells and the amount of mRNA change of AQP3 (Aquaporin 3), which is an important protein in skin moisturization, was measured using a real-time PCR method. The types and sequences of the primers used in the real-time PCR are shown in Table 6 below.

Primer types and base sequences used in real-time PCR gene order The forward primer (5 '- &gt; 3') The reverse primer (5 '- &gt; 3') AQP3 CCG TGA CCT TTG CCA TGT G (SEQ ID NO: 1) TAG CAA AGA TGC CGG CTG TG (SEQ ID NO: 2)

As a result of measuring the amount of mRNA change of AQP3, the exosome derived from stem cells of the present invention increased the expression amount of AQP3 mRNA in the cells. 12, the expression of AQP3 in human epidermal keratinocytes was increased by 7.7% when treated with 30 μg / mL of exosomes derived from the stem cells of the first batch compared to the negative control, The expression of AQP3 in human epidermal keratinocytes was increased by 69.4% when treated with 300 μg / mL of exosomes derived from stem cells compared to the negative control. As shown in Fig. 13, the expression of AQP3 in human epidermal keratinocytes was increased by 6.2% when treated with 3 痢 / mL of exosome derived from stem cells of the second batch, compared with the negative control, The expression of AQP3 in human epidermal keratinocytes was 29.7% higher than that of negative control at 6 μg / mL of cell-derived exosome treatment.

From the above experimental results, it can be confirmed that the exosome derived from stem cells increases the expression of AQP3, which plays an important role in the water movement in the skin. That is, the skin moisturizing composition of the present invention increases the expression or production of AQP3, thereby activating the skin movement of moisture, thereby moisturizing the skin and obtaining a moisturizing effect by restoring the skin function.

Therefore, the skin moisturizing composition containing the stem cell-derived exosome as an active ingredient has a functional activity useful as a moisturizing functional cosmetic, that is, an activity of inducing the expression of AQP3, and the stem cell- And can be usefully utilized as an effective ingredient of a cosmetic composition.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

100: Exosome kit
10: Liquid immersion sheet Mask 12: Stem cell-derived exosome
20: transdermal permeation enhancing sheet mask 22:
24: conductive pattern

<110> ExoCoBio Inc. <120> Cosmetic composition for moisturizing skin comprising an exosome          derived from stem cell as an active ingredient <130> P18-0069KR <150> KR 10-2017-0168838 <151> 2017-12-10 <150> KR 10-2018-0025135 <151> 2018-03-02 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Aquaporin 3 (AQP3) forward primer <400> 1 ccgtgacctt tgccatgtg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aquaporin 3 (AQP3) reverse primer <400> 2 tagcaaagat gccggctgtg 20

Claims (28)

A composition for moisturizing skin comprising an exosome derived from a stem cell as an active ingredient. The method according to claim 1,
Wherein said stem cell-derived exosome increases the expression or production of AQP3 (Aquaporin 3). 3. The method of claim 2,
A composition for skin moisturization which increases skin hydration. The method according to claim 1,
A composition for skin moisturization, comprising a lyophilized preparation containing the above exosome derived from stem cells and methionine, mannitol and trehalose as an active ingredient. 5. The method of claim 4,
Wherein the freeze-dried preparation further contains ascorbic acid and retinol. 5. The method of claim 4,
Wherein the composition comprises the lyophilized preparation and a diluent. The method according to claim 6,
Wherein the diluent is water for injection, physiological saline, phosphate buffer solution, purified water, or deionized water. 8. The method of claim 7,
Wherein the diluent further comprises hyaluronic acid or hyaluronic acid salt. 9. The method according to any one of claims 1 to 8,
At least one member selected from the group consisting of patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, aerosols, mist, foundations, Wherein the composition is applied to the skin. 10. The method of claim 9,
Wherein the composition is applied or deposited on at least one surface of the patch, mask pack or mask sheet. 9. The method according to any one of claims 1 to 8,
A composition for moisturizing skin, which is a cosmetic composition or a skin external preparation. 12. The method of claim 11,
Wherein the cosmetic composition is a cream or a lotion. A first mask pack, a mask sheet or a patch which is applied or deposited with a skin moisturizing composition containing an exosome derived from a stem cell as an active ingredient,
And an iontophoresis device for applying a microcurrent to the skin of the mammal to which the first mask pack, mask sheet or patch is applied. 14. The method of claim 13,
Wherein said stem cell-derived exosomes increase the expression or production of AQP3 (Aquaporin 3). 15. The method of claim 14,
Wherein the first mask pack, the mask sheet or the patch is contacted or adhered onto the skin of the mammal, and the iontophoresis device is placed on the first mask pack, the mask sheet or the patch, Or patches are contacted or adhered to flow micro currents to the skin of a mammal to which the skin moisturizing composition is applied. 16. The method according to any one of claims 13 to 15,
The iontophoresis device includes at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A second mask pack, a mask sheet or a patch, on which at least one kind of battery is mounted; and a skin moisturizing exosomatic kit. 17. The method of claim 16,
Wherein the second mask pack, the mask sheet or the patch is laminated on the first mask pack, the mask sheet or the patch.  (a) applying the skin moisturizing composition according to any one of claims 1 to 8 directly to the skin of a mammal, (b) applying the skin moisturizing composition to the skin, A method of cosmetic treatment wherein the condition of the mammalian skin is regulated through skin moisturization except for treatment, comprising contacting or adhering the sheet to the skin of the mammal, or sequentially advancing (a) and (b) above. 19. The method of claim 18,
In the step (a), lotion or cream is used as a skin moisturizing composition. 19. The method of claim 18,
(c) removing the patch, mask pack or mask sheet from the skin of the mammal after step (b), and applying the skin moisturizing composition to the skin of the mammal. 21. The method of claim 20,
In the step (c), lotion or cream is used as a skin moisturizing composition. 19. The method of claim 18,
Further comprising performing iontophoresis by flowing a minute current through the skin of the mammal to which the skin moisturizing composition is applied. 23. The method of claim 22,
Further comprising contacting or attaching an iontophoresis device to the skin of said mammal. 19. The method of claim 18,
Wherein said mammal is a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig. (a) contacting or adhering a first mask pack, a mask sheet or a patch, onto which a skin moisturizing composition containing or coated with exosome derived from stem cells is applied or immersed, onto the skin of a mammal; and
(b) positioning the iontophoresis device on the first mask pack, mask sheet or patch,
(c) performing iontophoresis in which the first mask pack, the mask sheet or the patch is contacted or adhered to flow a microcurrent into the skin of the mammal to which the composition for moisturizing the skin is applied;
(d) delivering the stem cell-derived exosomes to the interior of the mammalian skin through the microcurrent; and controlling the state of the mammalian skin by moisturizing the skin except for the treatment. 26. The method of claim 25,
Wherein said stem cell-derived exosomes increase the expression or production of AQP3 (Aquaporin 3). 27. The method of claim 25 or 26,
The iontophoresis device includes at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A second mask pack, a mask sheet or a patch, on which said at least one battery is mounted. 28. The method of claim 27,
Wherein the step (b) is performed by laminating the second mask pack, the mask sheet or the patch on the first mask pack, the mask sheet or the patch.

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