WO2021096089A1 - Novel composition comprising stem cell-derived exosomes and polydeoxyribonucleotide as active ingredients - Google Patents

Abstract

The present invention provides a composition for increasing the effects of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle reduction and/or skin elasticity improvement, the composition comprising, as an active ingredient, a combination of a stem cell-derived exosome and a polydeoxyribonucleotide. The composition of the present invention comprises, as an active ingredient, a combination of a stem cell-derived exosome and a polydeoxyribonucleotide, and thus can exhibit the effects of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle reduction and/or skin elasticity improvement, which are superior to the effects of a composition comprising only a stem cell-derived exosome or a polydeoxyribonucleotide.

Description

New composition containing stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients

The present invention relates to a new composition comprising stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients, and more particularly, by containing a combination of stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients. It relates to a new composition capable of enhancing the effect of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement.

In addition, the present invention relates to a cosmetic composition for improving skin wrinkles, improving skin elasticity or skin regeneration, a cosmetic composition for whitening, and a pharmaceutical composition for skin regeneration, wound healing or promoting wound healing, a quasi-drug or skin external preparation comprising the composition. will be.

When skin aging occurs, skin elasticity decreases and skin wrinkles increase. In particular, when sunlight and ultraviolet rays are directly irradiated on the skin, a lot of free radicals are generated, and the antioxidant defense system of the skin is damaged by these free radicals, increasing wrinkles and relaxing the skin, thereby accelerating skin aging. Therefore, in order to reduce skin wrinkles and maintain elasticity, it is necessary to protect the skin by inhibiting the production of reactive oxygen species and free radicals, inhibiting inflammatory reactions, and promoting skin regeneration from wounds. Substances known to be effective in improving skin wrinkles include adenosine and retinoic acid, but adenosine has little efficacy in clinical practice, and retinoic acid cannot be used in women of childbearing potential and has side effects such as erythema.

Human skin color is determined by the concentration and distribution of melanin inside the skin. Melanin is converted from tyrosine to dopa (DOPA) and dopaquinone by tyrosinase and then synthesized through a non-enzymatic oxidation reaction. When melanin is produced in excess, pigmentation occurs and spots, spots, and freckles appear on the face, neck, and arms, making the appearance unpleasant. Skin whitening agents are being developed to improve spots, freckles, and dark skin tone, but it is difficult to develop skin whitening agents that have excellent whitening effects and no side effects. For example, skin whitening agents that selectively attack melanocytes that produce melanin have excellent whitening effects, but have side effects of skin toxicity.

Recently, studies have been reported that a variety of bioactive factors that regulate cell behavior are included in cell secretomes. In particular, exosomes or extracellular secretions that have intercellular signaling functions in cell secretions Since it contains extracellular vesicles, studies on its components and functions are actively underway.

Cells release vesicles of various membrane types to the extracellular environment, and these vesicles are usually called extracellular vesicles (EVs). Extracellular vesicles are sometimes called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they are used separately from exosomes.

Exosomes are vesicles having a size of tens to hundreds of nanometers consisting of a double phospholipid membrane that has the same structure as a cell membrane, and contains proteins and nucleic acids (mRNA, miRNA, etc.) called exosomes cargo (cargo). Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the derived cell. In the case of proliferating stem cell-derived exosomes, cell behaviors such as cell migration, proliferation and differentiation are regulated, and the characteristics of stem cells related to tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).

In other words, exosomes, which are called cell avatars, contain bioactive factors such as growth factors, similar to cells, and serve as a carrier that carries bioactive factors between cells and cells, that is, the communication between cells and cells. Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .

However, despite various studies such as suggesting the possibility of treating specific diseases using exosomes, the development of technologies that increase the physiological activity of exosomes while maintaining them stably, development of technologies related to improving the usability of exosomes, And the development of technologies such as enhancement of physiological activity by combination with other substances is insufficient.

In particular, studies to use stem cell-derived exosomes with polydeoxyribonucleotide (PDRN) to use for whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement are known. none.

In this regard, Korean Patent Laid-Open Publication No. 10-2019-0114620 discloses a cosmetic composition containing a human fat stem cell culture medium extract and a polydeoxyribonucleotide (PDRN), but the human fat stem cell culture medium is secreted as cells grow. Since it contains components such as waste products, antibiotics added to prevent contamination, and animal-derived serum, it is highly likely to be exposed to various risks when used on skin or wounds.

The inventors of the present invention have been continuing intensive research on new application fields of exosomes and technology that can increase the physiological activity of exosomes through grafting with medical or cosmetic technology. Stem cell-derived exosomes and polydeoxyribonucleotides When combined, it was confirmed that the effect of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement can be effectively enhanced, thereby completing the present invention.

On the other hand, it should be understood that the matters described as background art are only for improving understanding of the background of the present invention, and are not cited as approval that they can be used as "prior art" of the present invention.

An object of the present invention is to enhance the efficacy of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement by containing stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients. It is to provide a new composition.

Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles, improving skin elasticity or regenerating skin, and a cosmetic composition for whitening, including the composition.

Another object of the present invention is to provide a pharmaceutical composition for skin regeneration, wound healing, or wound healing promotion, a quasi-drug or a skin external preparation comprising the composition.

Another object of the present invention is to provide a cosmetic method or a treatment method for whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement using the above composition.

However, the subject of the present invention as described above is exemplary, and the scope of the present invention is not limited thereby. Further, other objects and advantages of the present invention will become more apparent by the detailed description, claims, and drawings of the present invention.

In order to achieve the above object, the present invention is a composition for enhancing the efficacy of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement, stem cell-derived exosomes and polydeoxyribo It provides a composition comprising a combination with a nucleotide as an active ingredient.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane-derived vesicles (membrane vesicles), ectosomes, shedding vesicles, microparticles, or equivalents thereof. It is used in the sense of saying. Depending on the separation environment, conditions, and methods, extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may have a meaning including nanovesicles that do not have the composition of exosomes.

As used herein, the term "exosomes" refers to vesicles having a size of tens to hundreds of nanometers (preferably about 30 to 200 nm) composed of a double phospholipid membrane identical to the structure of the cell membrane (however, the separation target is The particle size of the exosomes may vary depending on the cell type, separation method, and measurement method. (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins and nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known.

Meanwhile, the term "exosome" as used herein has a nano-sized vesicle structure secreted from stem cells and released into the extracellular space, and has a composition similar to that of exosomes (eg, exosomes- It means to include all similar vesicles). The type of stem cells is not limited, but as an example that does not limit the present invention, embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cells-derived mesenchymal stem cells , Or mesenchymal stem cells derived from induced pluripotent stem cells. As an example not limiting the present invention, the adult stem cells are at least one adult selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells. It can be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton jelly, and placenta. Preferably, the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably stem cells derived from fat. The type of the stem cell is not limited as long as there is no risk of infection by a pathogen and does not cause an immune rejection reaction, but may be preferably human-derived stem cells, more preferably human adipose tissue-derived stem cells.

However, if the stem cell-derived exosomes used in the present invention have whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement effects and do not cause adverse effects on the human body, they are used in the art or in the future. It goes without saying that various stem cell-derived exosomes that can be used can be used. Therefore, it should be understood that the stem cell-derived exosomes isolated according to the separation method of the following examples should be understood as an example of the exosomes that can be used in the present invention, and the present invention is not limited thereto.

In the present specification, the term polydeoxyribonucleotide (PDRN) is a material containing a deoxyribonucleotide polymer, and is used to mean a DNA polymer fragment complex or a DNA polymer fragment mixture unless otherwise defined. Polydeoxyribonucleotides are generally prepared by extracting from salmon semen, and in addition, may be prepared by extracting from semen, testis, milt and/or eggs of fish such as salmon, trout, herring, and tuna. For example, polydeoxyribonucleotides can be prepared by extraction from spermatozoa and high heat treatment.

The term "skin elasticity" as used herein refers to a characteristic in which the skin deformed by an external force easily returns to its original shape when the corresponding external force is removed. "Skin wrinkles" refers to fine lines caused by deterioration of the skin, and may be caused by genes, a decrease in collagen and elastin present in the skin dermis, and external environments. Accordingly, the term "skin wrinkle improvement" as used herein refers to inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating wrinkles that have already been generated. In addition, the term "whitening" as used herein includes increasing the brightness of the skin whose brightness has decreased due to an excessive amount of pigments such as melanin, or maintaining the brightness of the skin at a certain level.

A composition comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide of the present invention as an active ingredient may be a cosmetic composition, a pharmaceutical composition, a quasi-drug, or a skin external preparation.

The present invention provides a cosmetic composition for improving skin wrinkles, improving skin elasticity, or regenerating skin, comprising a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient. In addition, the present invention provides a cosmetic composition for whitening comprising a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient.

The cosmetic composition of one embodiment of the present invention may be, for example, a cream or lotion.

In addition, the present invention provides a pharmaceutical composition for promoting skin regeneration, wound healing, or wound healing, comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide as an active ingredient, a quasi-drug or a skin external preparation. For example, the pharmaceutical composition may be in the form of injections, injections, sprays, liquids or patches, and the quasi-drugs or external skin preparations may be liquids, ointments, creams, sprays, patches, gels, or aerosols. I can.

When the composition of one embodiment of the present invention is used as a pharmaceutical composition, it may include a pharmaceutically acceptable carrier, excipient, or diluent. Examples of the carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto. . In addition, the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect skin regeneration, wound healing, and/or wound healing promoting effect.

The blending ratio of the pharmaceutical composition according to an embodiment of the present invention may be appropriately selected according to the type, amount, or form of the additional ingredients as described above. For example, with respect to the total amount of the injection, the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, the appropriate dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted according to the severity of the disease, the type of the formulation, the formulation method, the patient's age, sex, weight, health condition, diet, excretion rate, administration time, and administration method. I can. For example, when administering the pharmaceutical composition of one embodiment of the present invention to an adult, it may be administered in one to several times in a dose of 0.001 mg/kg to 100 mg/kg per day.

On the other hand, when the composition of one embodiment of the present invention is prepared as a quasi-drug, external skin preparation and/or cosmetic composition, it is usually used in a quasi-drug, external skin preparation and/or cosmetic composition within the scope of not impairing the effect of the present invention. Ingredients, for example, moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc. can be appropriately blended as needed.

In addition, in addition to the combination of stem cell-derived exosomes and polydeoxyribonucleotides, the quasi-drug, skin external preparation, and/or cosmetic composition of one embodiment of the present invention has an action (e.g., whitening, skin regeneration, wound healing, Acceleration of wound healing, improvement of skin wrinkles and/or improvement of skin elasticity, etc.) may be used in combination with conventionally used skin improvement agents, whitening agents, wound treatment agents, and/or moisturizers.

The quasi-drug, skin external preparation and/or cosmetic composition of one embodiment of the present invention is, for example, a patch, a mask pack, a mask sheet, a cream, a tonic, an ointment, a suspension, an emulsion, a paste, a lotion, a gel, an oil, a pack, It can be applied to various forms such as spray, aerosol, mist, foundation, powder, and oil paper.

The cosmetic composition of one embodiment of the present invention may be used for the purpose of whitening, improving skin wrinkles, improving skin elasticity, or regenerating skin, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, soft lotion, nutrient lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , Soap, shampoo, surfactant-containing cleansing, bathing agent, body lotion, body cream, body oil, body essence, body cleaner, hair dye, hair tonic, etc., but is not limited thereto.

The quasi-drug, external skin preparation and/or cosmetic composition of one embodiment of the present invention includes components commonly used in quasi-drug, external skin preparations and/or cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments And conventional adjuvants and carriers such as fragrances. In addition, in each formulation for a quasi-drug, external skin preparation and/or cosmetic composition, other ingredients may be appropriately selected and blended by a person skilled in the art without difficulty depending on the type or purpose of use of the quasi-drug, external skin preparation and/or cosmetic composition. have.

Another embodiment of the present invention provides a cosmetic method for controlling the condition of mammalian skin except for treatment by using the cosmetic composition. In the cosmetic method of the present invention, controlling the condition of the skin means improving the condition of the skin and/or controlling the condition of the skin prophylactically, and improving the condition of the skin means visual and/or feeling of the appearance and feel of the skin tissue. Or it means a positive change that can be perceived tactilely. For example, the improvement of the condition of the skin may be improvement of skin elasticity, improvement of skin wrinkles, skin whitening and/or skin regeneration.

The cosmetic method of one embodiment of the present invention includes (a) applying the cosmetic composition directly to the skin of a mammal, (b) applying the patch, mask pack, or mask sheet to which the cosmetic composition is applied or deposited on the skin of the mammal. It includes contacting or attaching to, or sequentially proceeding the above (a) and (b). In step (a), a lotion or cream may be used as a cosmetic composition.

Alternatively, in the cosmetic method of one embodiment of the present invention, (c) after step (b), the patch, mask pack, or mask sheet is removed from the skin of the mammal, and the cosmetic composition is applied to the skin of the mammal. It may further include the step of applying. In step (c), a lotion or cream may be used as a cosmetic composition.

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.

In addition, the present invention comprises the step of administering a therapeutically effective amount of the pharmaceutical composition to a mammal, or applying the quasi-drug or the external preparation for skin to the skin or wound site of a mammal, skin regeneration, wound treatment or It provides a method for accelerating wound healing. The mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.

The composition of the present invention contains a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient, so that whitening, skin regeneration, wound healing, and more excellent than a composition containing only stem cell-derived exosomes or polydeoxyribonucleotides, It may exhibit the effect of promoting wound healing, improving skin wrinkles and/or improving skin elasticity.

Meanwhile, the scope of the present invention is not limited by the above-described effects.

FIG. 1 shows the results of analyzing physical properties of stem cell-derived exosomes obtained according to an embodiment of the present invention. "Fig. 1A" shows the particle size distribution and the number of particles obtained by nanoparticle tracking analysis (NTA). "Fig. 1B" is a photograph of a particle image taken by TEM (transmitted electron microscopy). "Fig. 1C" shows the results of Western blot for the positive markers of the stem cell-derived exosomes obtained according to an embodiment of the present invention. "Fig. 1D" shows the results of Western blot for negative markers of stem cell-derived exosomes obtained according to an embodiment of the present invention. "Fig. 1E" shows the results of flow cytometry for CD9, CD63 and CD81 in the analysis of markers for stem cell-derived exosomes obtained according to an embodiment of the present invention.

Figure 2 shows the result of confirming that there is no cytotoxicity after treatment with the stem cell-derived exosomes according to an embodiment of the present invention on HS68 cells, which are human skin fibroblasts.

3 is a case where the combination of stem cell-derived exosomes and polydeoxyribonucleotides (PDRN+EXO) of the present invention is treated with Scratch-Wound, exosomes alone treatment (EXO) or polydeoxyribo It is a graph showing that the migration of human skin fibroblasts is much increased compared to the case of nucleotide treatment alone (PDRN).

4 is a case where the combination of the stem cell-derived exosomes and polydeoxyribonucleotides of the present invention (PDRN+EXO) on melanin-producing cells is treated with exosomes alone (EXO) or polydeoxyribonucleotides alone (PDRN). ) Is a graph showing that the amount of melanin production is significantly reduced than that of one case.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

Example

Example 1: Culture of cells

HS68 cells, human dermal fibroblasts, are purchased from ATCC and contain 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific). In the containing DMEM (purchased from ThermoFisher Scientific) medium, 5% CO ₂ , it was subcultured at 37°C.

Human dermal fibroblast (HDF) is purchased from ATCC and contains 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific). DMEM (purchased from ThermoFisher Scientific) _{was subcultured in 5% CO 2} , 37°C conditions.

B16F10 cells, mouse melanoma cells, were purchased from ATCC, 10% fetal bovine serum (purchased from ThermoFisher Scientific), 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific), DMEM containing L-glutamine (purchased from ThermoFisher Scientific) and sodium pyruvate (purchased from ThermoFisher Scientific), phenol-free red (purchased from ThermoFisher Scientific) medium in 5% CO ₂ , and subcultured at 37°C. .

Adipose-derived stem cells were cultured under conditions of _{5% CO 2} and 37°C according to a cell culture method known in the art to which the present invention pertains. Then, after washing with phosphate-buffered saline (purchased from ThermoFisher Scientific), it was replaced with a serum-free and phenol red medium, and cultured for 1 to 10 days, and the supernatant (hereinafter, culture solution) was recovered. .

In the process of separating exosomes, 2% by weight of trehalose was added to the culture medium in order to obtain exosomes having a uniform particle size distribution and high purity. After trehalose was added, the culture solution was filtered through a 0.22 μm filter to remove impurities such as cell debris, waste products, and large particles. The filtered culture solution immediately separated exosomes through a separation process. In addition, the filtered culture solution was stored in a refrigerator (image 10° C. or less) and used to separate exosomes. In addition, the filtered culture was frozen and stored in a cryogenic freezer at -60°C or lower, then thawed, and then exosome separation was performed. Thereafter, exosomes were separated from the culture medium using a tangential flow filtration (TFF).

Example 2: Isolation and purification of exosomes by the TFF method

In Example 1, exosomes were separated from the culture medium filtered with a 0.22 μm filter, concentrated, desalted, and buffer exchanged (diafiltration) using a TFF (Tangential Flow Filtration) method. As a filter for the TFF method, a cartridge filter (aka hollow fiber filter; purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used. TFF filters can be selected by various molecular weight cutoffs (MWCO). The exosomes were selectively separated and concentrated by the selected MWCO, and particles, proteins, lipids, nucleic acids, and low-molecular compounds smaller than the MWCO were removed.

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

The separated and concentrated exosome solution was further desalted and buffer exchanged (diafiltration) using the TFF method. At this time, desalting and buffer exchange were performed continuously (continuous diafiltration) or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably with respect to the starting volume. It was carried out using a buffer solution having a volume of at least 12 times. To the buffer solution, 2% by weight of trehalose dissolved in PBS was added to obtain exosomes having a uniform particle size distribution and high purity.

Example 3: Characterization of isolated exosomes

The separated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (　NTA; purchased from Malvern). The uniformity and size of the separated exosomes were analyzed using a transmission electron microscopy (TEM). NTA and TEM analysis results of exosomes isolated according to the separation method of an embodiment of the present invention are shown in FIGS. 1A and 1B.

1C is a result of performing Western blot on the positive markers of exosomes isolated according to the separation method of an embodiment of the present invention, confirming the presence of CD63, CD9, CD81, Alix, and TSG101 markers. Anti-CD63, anti-CD9, anti-CD81, anti-Alix and anti-TSG101 were used as antibodies for each marker, respectively.

1D is a result of performing Western blot on the negative marker of exosomes isolated according to the separation method of an embodiment of the present invention. Anti-GM130 and anti-calnexin were used as antibodies for each marker, respectively. GM130 and Calnexin are negative markers that should not be present in exosomes when characterizing exosomes. As shown in FIG. 1D, GM130 and Calnexin were found to be present in the lysate of adipocytes, but it was confirmed that they were not present in the exosomes isolated according to the isolation method of one embodiment of the present invention. Accordingly, when synthesizing the results of FIGS. 1C and 1D, it can be seen that exosomes isolated according to the separation method of an embodiment of the present invention are exosomes satisfying the characteristic analysis of positive and negative markers.

1E shows the presence of CD9, CD63 and CD81 markers as a result of analyzing exosomes isolated according to the isolation method of one embodiment of the present invention using a flow cytometer. To isolate exosomes that are positive for CD81, an exosome-human CD81 separation/detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method, and PE-mouse anti-human CD9 (PE-Mouse anti -human CD9) (purchase from BD), PE-mouse anti-human CD63 (PE-Mouse anti-human CD63) (purchase from BD) and PE-mouse anti-human CD81 (PE-mouse anti-human CD81) (BD After staining the marker using (purchased from), it was analyzed using a flow cytometer (ACEA Biosciences).

Example 4: Measurement of cytotoxicity according to exosome treatment

In order to evaluate the toxicity of exosomes obtained according to the isolation method of one embodiment of the present invention from HS68 cells, which are human skin fibroblasts, the cells were treated with exosomes at different concentrations, and the proliferation rate of the cells was confirmed. After the HS68 cells were suspended in DMEM containing 10% FBS, they were dispensed to have a confluency of 80 to 90%, and _{cultured in a 37°C, 5% CO 2} incubator for 24 hours. After 24 hours, the culture medium was removed, and the exosomes prepared in Example 2 were treated according to concentration, and the cell viability was evaluated while incubating for 24 to 72 hours. Cell viability was determined by WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramar Blue reagent (purchased from Promega). 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 a general cell culture medium that was not treated with exosomes, and it was confirmed that cytotoxicity by the exosomes of the present invention did not appear within the tested concentration range (FIG. 2).

Example 5: Preparation of a combination of stem cell-derived exosomes and polydeoxyribonucleotides

Stem cell-derived exosomes prepared as in Example 2 were mixed with polydeoxyribonucleotide (product name: "Lablue", manufactured by ExoCobio Co., Ltd.) and reacted at room temperature for 15 minutes (hereinafter, "PDRN-Exo A little mixed solution (PDRN+EXO)").

As an example, not limiting the present invention, the polydeoxyribonucleotide was manufactured by Exocobio Co., Ltd. (Seoul, Korea), but is not limited thereto, and various polydeoxyribonucleotides commercially available. Can be used. The concentration of stem cell-derived exosomes in the composition may be adjusted to, for example, approximately 1 to 10,000 μg/mL in protein concentration.

Example 6: Confirmation of skin regeneration efficacy using skin fibroblasts

In order to evaluate whether the PDRN-exosomal mixture prepared as in Example 5 promotes wound healing in human skin fibroblasts (HDF), a Scratch-Wound Assay was performed. HDF dispersed in DMEM medium containing fetal calf serum was dispensed into culture plates for wound induction (ImageLock Plate; purchased from EssenBio) at a density of 7,500 cells/well, and cultured for 24 hours at 37°C _{and 5% CO 2} After checking the density of% or more, scratch was induced using WoundMaker (purchased from EssenBio). To confirm the skin regeneration efficacy using skin fibroblasts, the experimental groups were classified as follows:

(1) Negative control group (Control): an experimental group treated with a scratch-round only serum-free medium;

(2) Polydeoxyribonucleotide (PDRN): Experimental group in which polydeoxyribonucleotide was diluted in a serum-free medium and treated with scratch-ound (final concentration: polydeoxyribonucleotide 100 μg/mL);

(3) Stem cell-derived exosomes (EXO): Experimental group in which the stem cell-derived exosomes prepared in Example 2 were diluted in serum-free medium and treated with scratch-ound (final treatment concentration: stem cell-derived exosomes 20 μg/mL );

(4) PDRN-exosomal mixture (PDRN + EXO): Experimental group prepared in Example 5 by diluting the PDRN-exosomal mixture prepared in Example 5 in a serum-free medium and treated with scratch-ound (final treatment concentration: 100 μg of polydeoxyribonucleotides /mL + stem cell-derived exosomes 20 μg/mL).

Each of the experimental groups was treated with Scratch-Wound, and then HDF was incubated for 12 hours at _{5% CO 2 and 37°C.}

As a result of measuring the wound healing ability, when the combination of the stem cell-derived exosome of the present invention and polydeoxyribonucleotide (PDRN+EXO) was treated, exosome alone treatment (EXO) or polydeoxyribonucleotide treatment alone ( It was confirmed that the migration of human skin fibroblasts was significantly increased compared to the case of PDRN) (see FIG. 3).

Therefore, the composition comprising the combination of the stem cell-derived exosomes and polydeoxyribonucleotides of the present invention as an active ingredient may be useful for skin regeneration, wound healing, promoting wound healing, improving skin wrinkles and/or improving skin elasticity. It is expected.

Example 7: Melanogenesis inhibitory effect

The whitening effect of the combination of stem cell-derived exosomes and polydeoxyribonucleotides was confirmed through the degree of inhibition of melanin production in mouse melanoma cells. The melanoma cells are cells derived from mouse melanoma, and are cells that secrete a black pigment called melanin. Melanoma cells were dispensed into 48-well plates by 7,000 cells per unit area, and then cultured for 24 hours at _{5% CO 2 and 37°C.}

To confirm the inhibitory effect of melanogenesis using melanoma cells, the experimental groups were classified as follows:

(1) negative control group (Control): a culture medium mixed with α-MSH (melanin synthesis stimulator), but an experimental group treated with melanoma cells;

(2) Arbutin: an experimental group in which 1 mM arbutin, a positive control, was diluted in a culture medium mixed with α-MSH and treated on melanoma cells;

(3) Polydeoxyribonucleotide (PDRN): Experimental group in which polydeoxyribonucleotide was diluted in a culture medium mixed with α-MSH and treated on melanoma cells (final treatment concentration: polydeoxyribonucleotide 600 μg/mL );

(4) Stem cell-derived exosomes (EXO): Experimental group in which the stem cell-derived exosomes prepared in Example 2 were diluted in a culture medium mixed with α-MSH and treated on melanoma cells (final treatment concentration: stem cell-derived exo Some 6 mg/mL);

(5) PDRN-exosomal mixture (PDRN + EXO): Experimental group in which the PDRN-exosomal mixture prepared in Example 5 was diluted in a culture medium mixed with α-MSH and treated on melanoma cells (final treatment concentration: Polyde Oxyribonucleotide 600 μg/mL + stem cell-derived exosomes 6 mg/mL).

Each of the experimental groups was treated with melanoma cells, and then the melanoma cells were cultured for 48 hours under conditions of _{5% CO 2 and 37°C.} Thereafter, the culture solution was recovered and the melanoma cells were washed using a washing solution (PBS; purchased from ThermoFisher).

The recovered culture medium and CCK-8 assay reagent (Dojindo) were mixed, _{incubated for 1 hour and 30 minutes at 37°C and 5% CO 2} , and then the supernatant was transferred to a 96-well plate and absorbance at 405 nm was measured. I did. In addition, after washing the melanoma cells in the 48-well plate with a washing solution, the washed melanoma cells were treated with 1 N NaOH (purchased from MerckMillipore) mixed with 10% DMSO, and the plate was sealed at 80°C. By heating for 30 minutes, melanin in the melanoma cells was extracted. The extracted melanin was calculated by measuring the absorbance at 405 nm.

As a result, the combination of stem cell-derived exosomes and polydeoxyribonucleotides (PDRN+EXO) of the present invention is more effective in melanoma cells than exosomes alone (EXO) or polydeoxyribonucleotides alone (PDRN). It was confirmed that it significantly reduced melanin synthesis (see FIG. 4). Accordingly, a composition comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide of the present invention as an active ingredient has a whitening effect, and can be usefully used as an active ingredient of a cosmetic composition for whitening.

In the above, the present invention has been described with reference to the above embodiments, but the present invention is not limited thereto. Those skilled in the art will be able to make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Claims (15)

1. A cosmetic composition comprising stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients.
2. The method of claim 1,

   Cosmetic composition for whitening, skin wrinkle improvement, skin elasticity improvement or skin regeneration.
3. The method according to claim 1 or 2,

   A cosmetic composition that is a cream or lotion.
4. A pharmaceutical composition for promoting skin regeneration, wound healing or wound healing, comprising stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients.
5. The method of claim 4,

   Pharmaceutical compositions which are in the form of injections, injections, sprays, liquids or patches.
6. An external drug or skin preparation for skin regeneration, wound healing or wound healing promotion, comprising stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients.
7. The method of claim 6,

   Liquid, ointment, cream, spray, patch, gel, or aerosol.
8. A cosmetic method for controlling the condition of the skin of a mammal by using the cosmetic composition according to claim 1 through skin whitening, skin wrinkle improvement, skin elasticity improvement, or skin regeneration except for treatment.
9. The method of claim 8,

   (a) directly applying the cosmetic composition to the skin of a mammal, (b) contacting or attaching the patch, mask pack, or mask sheet on which the cosmetic composition is applied or deposited on the skin of the mammal, or the ( A) and (b) comprising sequentially proceeding, cosmetic method.
10. The method of claim 9,

    In the step (a), a lotion or cream is used as a cosmetic composition, a beauty method.
11. The method of claim 9 or 10,

    (c) removing the patch, mask pack, or mask sheet from the skin of the mammal after step (b), and applying the cosmetic composition to the skin of the mammal.
12. The method of claim 11,

    In the step (c), a lotion or cream is used as a cosmetic composition, a beauty method.
13. The method according to any one of claims 8 to 10,

    The mammal is a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig.
14. Including the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 4 to a mammal, or applying the quasi-drug or skin external preparation of claim 6 to the skin or wound site of the mammal, skin regeneration, wound A method for facilitating treatment or wound healing.
15. The method of claim 14,

    The mammal is a human, dog, cat, rodent, horse, cow, monkey, or pig, a method for promoting skin regeneration, wound treatment or wound treatment.

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