WO2019231133A1 - Composition for reducing pores, comprising stem cell-derived exosomes as active ingredient - Google Patents

Abstract

The present invention provides a composition for reducing pores, comprising stem cell-derived exosomes as an active ingredient, and a beauty method for reducing skin pores using the composition. Treating the skin with the composition for reducing pores comprising stem cell-derived exosomes as an active ingredient, according to the present invention, has an excellent skin beauty benefit of reducing skin pores, particularly skin pores of the face, improving the appearance of the face.

Description

Pore reduction composition comprising stem cell-derived exosomes as an active ingredient

The present invention relates to a composition for reducing pores, comprising exosomes derived from stem cells as an active ingredient.

The present invention also relates to a cosmetic method for shrinking skin pores by using the composition for shrinking pores.

As interest in the appearance of the face or body increases, beauty demand for improving skin condition and supplementing the lack of appearance is increasing. In particular, cosmetic complaints about the prominent appearance of pores such as the face in the female layer is increasing. Pores are hair growth holes with a diameter of 0.02 ~ 0.05mm, but the size of the pores increases due to physical changes, seasons, age, and stress. Especially when the pores on the face get bigger, it doesn't look good. Therefore, the demand for beauty to reduce pores such as face is increasing in cosmetic field or cosmetic field.

The mechanism by which the pores become bigger and noticeable is not clear, so it is common to improve the appearance by covering the pores with a foundation or by tightening the skin with a skin astringent. However, converging lotion is a temporary tightening of the skin, which is not a fundamental solution to large skin loads and large pores, and the effect is not sufficient. In addition, a method of removing keratin plugs accumulated around the pores is also used, but the effect is temporary, and the skin damage is often caused when the keratin plug is removed, and the pores are often enlarged. As an alternative to this, various pore-reducing agents such as glycolic acid and ascorbic acid derivatives have been developed, but the effects are not satisfactory until now.

Recently, research on the development of pores shrinkage using natural products is active. In the case of the composition for pore reduction using natural materials as a raw material, the amount of the active ingredient in the natural extract is small, so that a large amount of use is required to obtain the pore reduction effect, and most of them are natural materials for marketing. There is a need for more scientific research on the actual efficacy of pore reduction. In addition, functional cosmetics for pore reduction, including the plant extract as an active ingredient may cause a foreign body in the process of evaporating the solution after applying to the skin, there is a problem that the effect duration is short.

On the other hand, recent studies have reported that the cell secretome contains a variety of bioactive factors that control the behavior (behavior) of the cell, especially in the cell secretion 'exosomes (cell) having a signaling function between cells ', And its research on the composition and function is actively underway.

Cells release various membrane types of endoplasmic reticulum into the extracellular environment, which are commonly referred to as extracellular vesicles (EVs). Extracellular vesicles are called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, may be used separately from exosomes.

Exosomes are tens to hundreds of nanometers of endoplasmic reticulum consisting of a double phospholipid membrane identical to the structure of a cell membrane, and include proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known. Exosomes contain specific genetic materials and bioactive factors depending on the nature and state of the cells from which they are derived. Stem cell-derived exosomes, which proliferate, regulate cell behavior such as cell migration, proliferation and differentiation, and reflect stem cell characteristics related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

However, despite various studies, such as suggesting the possibility of the treatment of specific diseases using exosomes, various medical treatments for the development of new formulations that can stably maintain and store exosomes, and the convenience and efficacy of exosomes are enhanced. Grafting with beauty technology has not received much attention.

The present inventors have been intensively researching new applications of stem cell-derived exosomes and grafting with medical or cosmetic technologies, and when pores are treated on the skin with a composition containing stem cell-derived exosomes as an active ingredient, The present invention was completed by confirming the reduction.

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

The present invention is to provide a composition for reducing pores comprising stem cells-derived exosomes as an active ingredient.

Another object of the present invention to provide a functional cosmetic composition for reducing pores and the external preparation for skin containing the composition.

Another object of the present invention to provide a cosmetic method for reducing the skin pores except for the treatment using the composition for reducing pores.

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby. Further objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the present invention provides a composition for reducing pores, including a stem cell-derived exosomes as an active ingredient, and a cosmetic method for reducing skin pores using the same.

As used herein, the term "exosomes" refers to vesicles of a size ranging from tens to hundreds of nanometers (preferably approximately 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of a cell membrane, provided that Particle size of exosomes may vary depending on the cell type, isolation 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 include proteins called exosome cargo (cargo), nucleic acids (mRNA, miRNA, etc.) and the like. Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known.

On the other hand, the term "exosome" as used herein has a nano-size vesicle structure secreted by stem cells and released into the extracellular space, and a vesicle having a composition similar to exosomes (eg, exosomes- Pseudo vesicles). The type of the stem cells is not limited, but as an example, which does not limit the present invention, preferably may be mesenchymal stem cells, for example, fat, bone marrow, umbilical cord or cord blood-derived stem cells, more preferably Fat-derived stem cells. The type of the adipose derived stem cells is not limited as long as there is no risk of infection by the pathogen and does not cause an immune rejection reaction, but preferably, human adipose derived stem cells.

However, the stem cell-derived exosomes used in the present invention are effective in shrinking pores and do not cause adverse effects on the human body, so that various stem cell-derived exosomes can be used in the art or may be used in the future. . Therefore, the exosomes derived from stem cells isolated according to the isolation method of the embodiments described below 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.

As used herein, the term “iontophoresis” refers to a method of allowing an ionized active ingredient to penetrate the skin with electrical repulsion by changing a skin's electrical environment by applying a potential difference by flowing a microcurrent to the skin to which the active material is applied. Means. The iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into the electrode patch on the skin to introduce a microcurrent into the skin, and a battery is mounted on the electrode patch itself. The manner in which microcurrents are introduced, the manner in which microcurrents are introduced into the skin through a patch equipped with reversed electrodialysis means for generating a current through the difference in ion concentration between the high concentration electrolyte solution and the low concentration electrolyte solution. Can be. However, the present invention is not limited thereto, and various methods of iontophoresis may be used.

The composition for reducing pores of one embodiment of the present invention comprises an exosome derived from stem cells as an active ingredient.

The composition for reducing pores according to one embodiment of the present invention may include a lyophilized agent comprising exosomes derived from the 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 composition for reducing pores according to one embodiment of the present invention, the lyophilized agent 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 pore-reducing composition of one embodiment of the present invention may include the lyophilizing agent and a diluent. For example, the diluent may be water for injection, physiological saline, phosphate buffer, purified water, or deionized water. In addition, the diluent may further comprise hyaluronic acid or hyaluronic acid salt (eg, sodium hyaluronate). As an example, the composition may be a suspension.

The pore-reducing composition of one embodiment of the present invention may be administered to the skin by microneedling, iontophoresis or injection.

The composition for reducing pores of one embodiment of the present invention may be a cosmetic composition or an external preparation for skin.

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

The blending ratio of the pharmaceutical composition of one embodiment of the present invention may be appropriately selected depending on the kind, amount, form, etc. of the additional ingredients as described above. For example, with respect to the total amount of injection, the pharmaceutical composition of the present invention may be included in an amount of about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, suitable dosages of the pharmaceutical compositions of one embodiment of the invention are adjusted according to pore size and number, type of formulation, formulation method, age, sex, weight, health condition, diet, excretion rate, time of administration and method of administration of the patient. Can be. 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 divided doses of 0.001 mg / kg to 100 mg / kg per day.

On the other hand, when the pore-reducing composition of one embodiment of the present invention is made of an external preparation for skin and / or a cosmetic composition, a component, for example, a moisturizing agent, which is usually used in cosmetics or external preparation for skin within the range that does not impair the effects of the present invention , Antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients and the like can be appropriately blended as necessary.

In addition, the skin external preparation and / or cosmetic composition of one embodiment of the present invention, in addition to exosomes derived from stem cells, is used together with conventionally used skin improving agents and / or skin astringents, so long as they do not impair its action (pore reduction, etc.). It can be mixed and used. For example, the exosomes derived from stem cells of the present invention may be supported or mixed in at least one of a hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel. In the external preparation for skin and / or cosmetic composition of one embodiment of the present invention, the type of the hydrogel is not limited, but preferably may be a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. 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 cosmetic composition of one embodiment of the present invention may include, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, and aerosols. It can be applied to various forms such as, mist, foundation, powder and oil paper. For example, the external preparation for skin and / or cosmetic composition may be applied or deposited on at least one side of a patch, mask pack or mask sheet.

The cosmetic composition of one embodiment of the present invention is used for the purpose of pore reduction, skin convergence, and the like, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, supple cosmetics, nourishing cosmetics, astringent cosmetics, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , Soaps, shampoos, surfactant-containing cleansing, bathing agents, body lotions, body creams, body oils, body essences, body cleaners, hair dyes, hair tonic and the like, but is not limited thereto.

The external preparation for skin and / or cosmetic composition of one embodiment of the present invention comprises ingredients conventionally used in external preparations for skin and / or cosmetics, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. And a carrier. In addition, in each formulation for the external preparation for skin and / or cosmetic composition, other ingredients may be appropriately selected and blended by those skilled in the art without difficulty according to the kind or purpose of use of the external preparation for skin and / or cosmetic composition.

In addition, the present invention provides a cosmetic method for shrinking the pores except for the treatment using the composition for reducing pores.

The cosmetic method of shrinking the pores of one embodiment of the present invention comprises the steps of: (a) preparing a composition for pore reduction comprising an exosome derived from stem cells as an active ingredient, and (b) a mammal for the pore reduction composition Treating the skin.

In the cosmetic method of shrinking pores of one embodiment of the present invention, the composition for shrinking pores may be administered to the skin by microneedling, iontophoresis or injection.

The cosmetic method for shrinking the pores of one embodiment of the present invention, (c) iontophore by flowing a microcurrent to the skin of the mammal treated with the pore-shrinkable composition comprising the stem cell-derived exosomes as an active ingredient And performing (d) iontophoresis, and (d) delivering the stem cell-derived exosomes into mammalian skin through the microcurrent.

In the cosmetic method for shrinking pores of one embodiment of the present invention, the composition for shrinking pores may be, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, It can be applied to various types of oils, packs, sprays, aerosols, mists, foundations, powders and oil papers. For example, the pore reduction composition may be applied or deposited on at least one surface of a mask pack, a mask sheet or a patch.

In the cosmetic method for shrinking the pores of one embodiment of the present invention, the step (b) is (b1) applying the composition for pore reduction directly to the skin of the mammal, (b2) the composition for pore reduction Contacting or attaching the applied or deposited mask packs, mask sheets or patches to the skin of the mammal, or by sequentially proceeding with (b1) and (b2).

In the cosmetic method for shrinking the pores of one embodiment of the present invention, at least one surface of the mask pack, mask sheet or patch is hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or At least one of the hyaluronic acid gels may be applied. The type of the hydrogel is not limited, but may be preferably a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer and polyhydric alcohol may be exemplified in the foregoing description.

In the cosmetic method of shrinking the pores of one embodiment of the present invention, step (c) may be performed by contacting or attaching the iontophoresis device to the skin of the mammal.

In the cosmetic method for shrinking the pores of one embodiment of the present invention, the iontophoresis device is a flexible battery, a lithium ion secondary battery, an alkaline battery, a battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electric And at least one cell selected from the group consisting of dialysis cells.

Treating the skin with a pore-reducing composition comprising the stem cell-derived exosomes of the present invention as an active ingredient exhibits an excellent effect in skin beauty to reduce the appearance of skin pores, in particular the skin pores of the face, thereby improving the appearance of the face. .

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

1 is a flowchart illustrating a process for separating and purifying exosomes in a method for producing exosomes from stem cell culture according to one embodiment of the present invention.

Figure 2 shows the results of measuring the relative amount of protein (Relative amount of protein) contained in the solution for each step (step) to prepare an exosome from the stem cell culture in accordance with an embodiment of the present invention. The ratio of the total amount of protein in each step is expressed as the relative ratio of the total amount of protein to the stem cell culture. The experimental results show the results obtained in two different batches, respectively.

Figure 3 shows the results of measuring the productivity (purity) and (productivity) of the exo-some obtained in accordance with an embodiment of the present invention. The productivity of the exosomes was calculated as "the number of particles of exosomes per mL of stem cell culture (CM)", and the purity of the exosomes was calculated as "the number of particles of exosomes per μg of protein contained in the final fraction". It was. The experimental results show the results obtained in five different batches.

4A to 4E show the results of physical characterization of the exosomes obtained according to one embodiment of the present invention. 4A shows particle size distribution and particle number by tunable resistive pulse sensing (TRPS) analysis. 4B shows particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis. FIG. 4C shows the particle image by magnification by means of the transmitted electron microscopy (TEM) analysis. 4D shows Western blot results of exosomes obtained according to one embodiment of the invention. 4E shows the results of flow cytometry for CD63 and CD81 in marker analysis for exosomes obtained according to one embodiment of the invention.

5A-5C show NTA analysis results for particle size distribution showing that exosomes with uniform particle size distribution and high purity are obtained with trehalose addition. As the amount of trehalose added increases, particle size distribution results with a single peak can be obtained.

6A to 6C show NTA analysis results showing particle size distribution depending on whether trehalose is added in the preparation of exosomes according to one embodiment of the present invention. FIG. 6A shows the addition of trehalose throughout the manufacturing process, FIG. 6B shows freezing of the cell culture and thawing after thawing, FIG. 6C shows trehalo. The result obtained without adding oss is shown. FIG. 6D shows the results of comparing relative productivity and relative concentration of exosomes isolated by the methods of FIGS. 6A to 6C. 6E shows the mean particle size of the exosomes isolated by the methods of FIGS. 6A-6C.

Figure 7 shows the results confirming that there is no cytotoxicity after treating the stem cell-derived exosomes according to one embodiment of the present invention to HS68 cells, which are human skin fibroblasts.

Figure 8 is a photograph showing the good properties of the lyophilized exosomes according to the method of one embodiment of the present invention.

9a to 9g are different from the combination of the lyophilized protective agent components and after performing the lyophilization, it is a photograph of the properties of lyophilized exosomes for each lyophilized protective agent combination.

10 is a graph showing the results of measuring the pore size of the face at 37 days after treating the facial pore reduction composition comprising the stem cell-derived exosomes according to one embodiment of the present invention as an active ingredient on the face of test subject 1.

11 is a graph showing the results of measuring the pore size of the face at 35 days after treatment of the composition for pore reduction comprising the stem cell-derived exosomes according to an embodiment of the present invention on the face of test subject 2.

12 is a graph showing the results of measuring the pore size of the face at 20 days after treating the facial pore reduction composition including the stem cell-derived exosomes according to one embodiment of the present invention as an active ingredient on the face of the subject 3.

Figure 13 after the Pico laser beam was applied to the stem cell-derived exosomes and vitamin C according to an embodiment of the present invention on the right and left sides of the subject 4, respectively, 18 days after further performing iontophoresis It is a graph of the result of measuring pore size.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, except to exclude other components unless specifically stated otherwise.

Example

Example 1 Culture of Cells

HS68 cells, which are human dermal fibroblasts, were purchased from ATCC and were prepared by 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific). Passage was carried out in DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO ₂ at 37 ° C.

Fat-derived stem cells were cultured at 5% CO ₂ , 37 ° C. according to cell culture methods known in the art. Then, washed with phosphate-buffered saline (purchased from ThermoFisher Scientific), replaced with serum-free, phenol-free medium, cultured for 1 to 10 days, and the supernatant (hereinafter, culture) was recovered. .

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

Example 2: Isolation and Purification of Exosomes by TFF Method

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

MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da TFF filter was used to isolate and concentrate the exosomes. The culture solution was concentrated to a volume of 1/100 to 1/25 by using the TFF method, while exosomes were separated by removing substances smaller than MWCO.

The separated and concentrated exosome solution was further subjected to desalting and diafiltration using the TFF method. At this time, desalting and buffer exchange were carried out continuously (discontinuous diafiltration) or at least 4 times, preferably 6 times to 10 times, more preferably, relative to the starting volume. It was performed using a buffer solution having a volume of 12 times or more. To the buffer solution was added 2% by weight of trehalose dissolved in PBS to obtain exosomes with uniform particle size distribution and high purity. The results of confirming the effect of obtaining a high purity and uniform particle size distribution of exosomes according to the trehalose treatment in a high yield are shown in Figures 6A to 6E.

Example 3: Characterization of Isolated Exosomes

The amount of protein in the isolated exosomes, cultures, and fractions of TFF separation was measured using BCA coloration (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence (purchased from Sigma). Exosome is isolated and concentrated by the TFF method of one embodiment of the present invention, and the degree of protein, lipid, nucleic acid, low molecular weight compounds, etc. is monitored by protein quantitation and the results are shown in FIG. As a result, it was found that the protein present in the culture medium was effectively removed by the TFF method of one embodiment of the present invention.

When the exosomes were separated by the TFF method of one embodiment of the present invention, the results of comparing the productivity and purity in five independent batches are shown in FIG. 3. Analysis of the results obtained from the five independent batches, it was confirmed that the exosomes can be isolated very stably by the TFF method of one embodiment of the present invention.

The isolated 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 the isolated exosomes were analyzed using a transmitted electron microscopy (TEM). TRPS, NTA, TEM analysis results of the exosomes isolated in accordance with one embodiment of the present invention are shown in Figures 4A to 4C.

After the exosomes were separated by the TFF method, the results of NTA analysis of the size distribution of the exosomes depending on whether trehalose was added are shown in FIGS. 5A to 5C. Trehalose concentrations were increased to 0%, 1% and 2% by weight (from top to bottom in FIGS. 5A-5C) and were repeated three times. When trehalose is not present, particles having a size of 300 nm or more are identified, while increasing the amount of trehalose added decreases the particles having a size of 300 nm or more and makes the size distribution of exosomes uniform. .

The effect of the addition of trehalose in the process of separating exosomes by the TFF method was further investigated. 6A to 6C, when 2% by weight of trehalose dissolved in PBS was added to the entire process of manufacturing the exosomes, an exosome having a uniform size distribution was obtained (FIG. 6A). On the other hand, when the TFF process was performed by adding trehalose only in the desalting and buffer exchange process, the TFF process was used without the addition of trehalose. In case of progress, a non-uniform exosome containing a lot of large particles was obtained (FIGS. 6B and 6C).

As a result of comparing the relative productivity and concentration of the isolated exosomes, when trehalose was added to the entire process of manufacturing the exosomes, the exosomes were obtained with very high productivity, and the concentration of the obtained exosomes was more than five times higher. (FIG. 6D). As shown in the NTA analysis results, the average size of the separated exosomes was also uniformly confirmed at 200 nm when trehalose was added to the entire process of manufacturing the exosomes (FIG. 6E).

Figure 4D shows the results of Western blot for exosomes isolated according to the method of one embodiment of the present invention, confirming the presence of CD9, CD63, CD81 and TSG101 markers. 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 antibodies to each marker.

Figure 4E confirmed the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer for the exosomes isolated in accordance with the method of one embodiment of the present invention. To isolate exosomes positive for CD63, an exosome-human CD63 separation / detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method, and PE-mouse anti-human CD63 (PE-Mouse anti markers were stained using -human CD63) (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD) and analyzed using a flow cytometer (ACEA Biosciences). It was.

Summarizing the above results, the present invention confirms that exosomes with high purity and uniform particle size distribution can be efficiently and efficiently separated and purified in high yield by adding trehalose in the manufacturing process using tangential flow filtration. Could. In addition, it can be seen that the processes of the separation method of one embodiment of the present invention are scale-up and suitable for GMP.

Example 4 Measurement of Cytotoxicity According to Exosome Treatment

In order to evaluate the toxicity of exosomes obtained according to the isolation method of an embodiment of the present invention in HS68 cells, which are human skin fibroblasts, cells were treated with exosomes at different concentrations, and cell proliferation rates were confirmed. HS68 cells were suspended in DMEM containing 10% FBS and then aliquoted to have a confluency of 80-90% and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After 24 hours, the culture solution was removed and the exosomes prepared in Example 2 were treated for each concentration, and cultured for 24 to 72 hours to evaluate cell viability. Cell viability was determined by WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramamar blue reagent ( Measurements were made using alamarBlue reagent (purchased from ThermoFisher Scientific) and a microplate reader (purchased from Molecular Devices).

The comparison group was based on the number of cells cultured in the normal cell culture medium not treated with exosomes, it was confirmed that no cytotoxicity by the exosomes of the present invention within the concentration range tested (Fig. 7).

Example 5: Lyophilization of Exosomes

Example 5-1: Lyophilization Conditions

Lyophilization protectors including methionine, mannitol and trehalose were 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 [produced by Biof.D.C (Hwasun-gun, Jeollanam-gun, Korea)] containing ascorbic acid and retinol at 0.5 mg / mL, respectively. In this embodiment, the lyophilized protective agent is added to the solution containing ascorbic acid and retinol, but an aqueous solution may be prepared by adding the lyophilized protective agent to water for injection, purified water, physiological saline, or deionized water. The concentrations of methionine, mannitol and trehalose in the aqueous solution were each 9 mg / mL.

After mixing the exosomes (5 × 10 ⁸ particles) prepared in Example 2 to the aqueous solution containing a lyophilized protective agent and freeze using the lyophilization equipment (Manufacturer: VIRTIS, ITME No .: 34424) under the conditions of Table 1 below Drying was performed. Lyophilization was carried out in the order of conditions 1, 2, 3, 4, 5, 6, 7 and 8 of Table 1.

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

After lyophilization of the exosomes by treatment with a lyophilized protective agent including methionine, mannitol and trehalose, it was confirmed that the color was milky and exhibited a good property of maintaining a porous sponge shape (FIG. 8). ). That is, in the lyophilization method of the exosome of the present invention, a lyophilized product exhibiting excellent properties could be obtained by lengthening the drying time under reduced pressure and constituting the lyophilization protective agent with a combination of methionine, mannitol and trehalose.

Example 5-2 Comparison of Properties of Exosome Products According to Lyophilized Protective Agent Components

On the other hand, the properties of exosomes when the exosomes were lyophilized using various lyophilized protective agents including at least one of methionine, mannitol and trehalose (hereinafter referred to as lyophilized protective agent components) were compared. According to the method described in Example 5-1, seven kinds of aqueous solutions in which a lyophilized protective agent component alone, a combination of two components, or three component combinations were added. The concentration of each lyophilized protective agent component in the aqueous solution was adjusted to 9 mg / mL. According to the lyophilization conditions and methods of Example 5-1, lyophilization (5 × 10 ⁸ particles) prepared in Example 2 was mixed with an aqueous solution containing each combination of lyophilized protective agent components and then lyophilized. .

The appearance of lyophilized exosome products was photographed and evaluated (FIGS. 9A-9G). According to the good and bad cake shape of lyophilized exosome product, the best was 5 points and the worst was 1 point. The results of evaluating the properties of the lyophilized exosome products by combination of the lyophilized protective agent are shown in Table 2 below.

Comparison of Characteristics of Lyophilized Exosome Products by Components of Lyophilized Protective Agent Lyophilized Protectant Composition Mannitol Trehalos Methionine Methionine + Trehalose Methionine + Mannitol Trehalose + mannitol Methionine + mannitol + trehalose (this invention) Score One One 4 3 4 2 5 drawing Figure 9a Figure 9b Figure 9c Figure 9d Figure 9e Figure 9f Figure 9g

As shown in FIGS. 9A to 9G and Table 2, the exo-drying of the exosomes using the lyophilized protective agent composed of the combination of methionine, mannitol and trehalose of the present invention showed the best product appearance. The exosomes lyophilized by the combination of one or two of the three components were found to be poorer than the product appearance of the present invention.

Example 6: First Facial Clinical Trial of Pore Reducing Composition

A freeze-dried product containing the stem cell-derived exosomes (lyophilized exosomes prepared in Example 5-1) at a concentration of 5 × 10 ⁸ particles / vial was mixed with purified water to prepare a 2 mL aqueous solution. Using the stem cell-derived exosome-containing pore reduction composition prepared as described above, the microneedle therapy system (MTS) equipped with a very thin needle was used twice every two weeks at the face part of the subjects 1 to 3, which were stressed due to the pores. MTS equipment used was MY-M of Bombtech Electronics Co., Ltd., located in Gangseo-gu, Seoul, Korea.

On the other hand, in the present embodiment was used as a composition for reducing pores after mixing the lyophilized exosomes prepared in Example 5-1 with an aqueous solution, alternatively the exosomes prepared in Example 2 (for example, 1 × 10 ⁸ particles Exosome of / mL or more) may be used as a composition for pore reduction after suspending in water for injection.

The pore size of each subject's face was measured using a Mark-Vu facial skin analyzer of PS-I Plus, Gyeonggi-do, Korea. The percentage change of pore (% change of pore) measured in each part of the face (forehead, nose, left and right cheeks) was analyzed by the following formula: Pore size change rate = [(pore size measured on N days)-(pore before treatment) Size] / (pore size before treatment) (%).

For Subject 1, it was confirmed that the pore size was significantly reduced in the forehead and left cheek at 37 days after the treatment compared to the pore size before treatment with the composition for reducing pores of the present invention (FIG. 10). In addition, in the case of Subject 2, it was confirmed that the pore size was significantly reduced in both the forehead, the nose, and the left and right cheeks on the 35th day after the treatment compared to the pore size before the pore reduction composition treatment of the present invention (FIG. 11). In the case of the subject 3, it was confirmed that the pore size was significantly reduced in the left and right cheeks on the 20th day after the treatment compared to the pore size before the pore reduction composition treatment of the present invention (FIG. 12).

In summary, the skin pores reduction composition comprising stem cell-derived exosomes as an active ingredient significantly reduces the size of the pores of the face, thereby improving the appearance of the face by reducing the pores. It can be seen that it can exhibit a cosmetic effect.

Example 7: Secondary Clinical Trial of Pore Reducing Composition

Pico lasers were irradiated on the face of a person using Pico Plus, Pico Plus, Lutronic, Inc., Gyeonggi-do, Korea. [Laser parameters are as follows: wavelength 1064 nm; Wavelength energy 0.7 J / cm ² ; Laser spot size 7 mm; Laser irradiation time 450 ps (picosecond); Frequency 10 Hz; Number of surveys 3000 shots].

After washing the face of the subject 4, an anesthetic was applied to the face for 30 minutes and irradiated with Pico laser. Immediately after the laser beam irradiation, 1 ml of exosomes (exosomes prepared in Example 2) derived from stem cells were applied to the right face of Subject 4 with a concentration of 7.39 × 10 ⁸ particles / mL of the stock solution (suspension), and left of Subject 4 A proper amount of vitamin C solution (1% vitamin phosphate solution) was applied to the face. Then, iontophoresis was performed on the right and left faces of the subject 4, respectively. Iontoporesis was performed in a manner of flowing a 0.5 mA microcurrent for 20 minutes to the face coated with exosomes derived from stem cells using an iontophoresis device (IONZYME) (purchased from Environ).

The pore size was measured and compared in the same manner as in Example 6 using Mark-View Facial Skin Analyzer at 18 days after the above treatment. As a result, the right face of the stem cell-derived exosomes of one embodiment of the present invention was confirmed that the pores shrinkage effect is remarkable compared to the left face treated with vitamin C solution (Fig. 13).

According to the results of the experiment, the treatment of pores reduction composition comprising stem cell-derived exosomes as an active ingredient to the skin and iontophoresis significantly reduced the size of the pores of the face to maximize the pore reduction effect more It can be seen that.

As mentioned above, although this invention was demonstrated to the said Example, this invention is not limited to this. Those skilled in the art can 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 (17)

Comprising a stem cell-derived exosomes as an active ingredient, a composition for shrinking pores. The method of claim 1, Exosome derived from the stem cells, and a lyophilized formulation comprising methionine, mannitol and trehalose as an active ingredient, a composition for shrinking pores. The method of claim 2, The lyophilizing agent further contains ascorbic acid and retinol, pores shrinkage composition. The method of claim 2, Further comprising a diluent, pore shrinkage composition. The method of claim 4, wherein The diluent is water for injection, physiological saline, phosphate buffer solution, purified water, or deionized water, characterized in that for reducing pores. The method of claim 5, The diluent further comprises hyaluronic acid or hyaluronic acid salt, pore shrinkage composition. The method according to any one of claims 1 to 6, A composition for shrinking pores, which is a suspension. The method according to any one of claims 1 to 6, A composition for shrinking pores, which is administered to the skin by microneedling, iontophoresis or injection. The method according to any one of claims 1 to 6, A composition for reducing pores, which is a cosmetic composition or an external preparation for skin. (a) preparing a pore-reducing composition comprising stem cells-derived exosomes as an active ingredient, and (b) treating the mammalian skin with the pore-reducing composition, except for therapeutic use. Beauty method to shrink pores. The method of claim 10, The pore-reducing composition is administered to the skin by microneedling, iontophoresis or injection, cosmetic method for shrinking pores except for treatment. The method of claim 10, (c) performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the composition for reducing pores comprising the stem cell-derived exosomes as an active ingredient, and (d) Beauty method for reducing the pores, except for therapeutic use, further comprising the step of delivering the stem cell-derived exosomes inside the mammalian skin through microcurrent. The method of claim 12, The pore-reducing composition is a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, powder and oil paper group A cosmetic method for shrinking pores except for treatment, characterized in that applied to at least one form selected from. The method of claim 13, The pore-reducing composition is applied to or deposited on at least one surface (mask) of the mask pack, mask sheet or patch, a cosmetic method for shrinking pores except for treatment. The method of claim 14, The step (b) is (b1) applying the pore reduction composition directly to the skin of the mammal, (b2) the mask pack, mask sheet or patch to which the pore reduction composition is applied or deposited, the mammal A cosmetic method for shrinking pores except for treatment, which is performed by contacting or adhering to the skin of the skin or by sequentially performing the steps (b1) and (b2). The method of claim 15, Wherein (c) step is performed by contacting or attaching the iontophoresis device to the skin of the mammal, the cosmetic method for shrinking pores except for therapeutic use. The method of claim 16, 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 battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery. , Beauty method to shrink pores except for treatment.

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