WO2019190093A1 - Skin care method using, in combination, application of rf energy to skin and stem cell-derived exosome treatment on skin - Google Patents

Abstract

The present invention provides a skin care method comprising the steps of: (a) applying RF energy to the skin of a mammal; and (b) applying, before or after applying the RF energy, a composition containing stem cell-derived exosomes as active ingredients to the skin of the mammal to which the RF energy is to be applied. The skin care method of the present invention performs, together with RF energy application, stem cell-derived exosome treatment on the skin, thereby reducing skin redness, which is a side effect caused by RF energy application, and exhibiting effects of a reduction in the pain caused by RF energy application, and skin soothing. In addition, when RF energy application and stem cell-derived exosome treatment are performed in combination, according to the skin care method of the present invention, downtime, which is the disappearing time for skin redness and the swelling of the skin that are the side effects caused by RF energy application, can be reduced.

Description

Skin beauty method using RF energy applied to skin and exosome treatment derived from stem cells

The present invention relates to a skin care method using a combination of RF energy applied to the skin and exosome treatment derived from stem cells.

In addition, the present invention relates to a skin beauty method for reducing skin redness, such as side effects due to RF energy application by treating the skin with exosomes derived from stem cells with RF energy application.

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 this regard, it is known to use heat energy for improving the appearance of the skin, beauty of the skin and body shaping. Application of radio frequency (RF) energy is one of the methods of heating skin and subcutaneous tissue. When RF energy is applied to the skin, deep heat is generated inside the skin to improve blood circulation and regenerate skin tissue. In general, RF energy application to the skin is performed using an RF electrode or applicator connected to an RF energy source. By contacting the RF energy transfer surface of the RF electrode to the surface of the skin and applying RF energy, the applied RF energy can transfer heat into the skin and subcutaneous tissue. However, RF energy is mostly reflected from the skin surface and the amount of penetration into the skin is small, so high energy must be applied to the skin to obtain a desired skin beauty effect. The area of the skin that is in contact with the electrode surface then receives more RF energy to form so-called "hot spots". In this case, due to high energy transfer, severe pain occurs in the treatment area, and there is a side effect of reddening or swelling of the skin after the procedure. Severe side effects can cause skin burns due to deep fever. Therefore, a method of cooling the procedure site for preventing skin burns and pain relief when RF energy is applied is used. However, the use of the cooling means has a problem of frostbite concerns and skin cosmetic efficiency at the treatment site.

Cosmetic surgery using RF energy is cheaper than irradiating a laser beam, and may be applied to the fractional technique used in laser beam irradiation. The RF-based fractional technique produces a scanning pattern of micron-sized holes in the skin, similar to a laser, in which electrode tips arranged in a matrix or array are brought into contact with the skin and a high voltage RF pulse is applied to the electrode tips. A small hole for mesotherapy is formed in the lower skin. Since the fractional technique using the RF energy application causes severe pain to the patient, a method of reducing the pain and minimizing side effects by applying RF energy after applying lidocaine cream to the skin has been proposed. However, side effects still occur despite the improvement of the RF treatment equipment and technique. Therefore, there is a need for research in other aspects to minimize the side effects of applying RF energy.

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 differently regulated by 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 material 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 exosomes and incorporating them into medical or cosmetic technologies, and when exosomes derived from stem cells are applied to the skin along with RF energy application, the skin is a side effect of RF energy application. The present invention was completed by confirming the effect of reducing redness and the like.

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.

An object of the present invention is to provide a skin care method using a combination of RF energy applied to the skin and exosomes derived from stem cells.

Another object of the present invention is to provide a skin beauty method for reducing skin redness, which is a side effect of RF energy application by treating exosomes derived from stem cells with RF energy.

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 method of applying the radio frequency (RF) energy to the skin, before the application of the RF energy, during the application of the RF energy or after the application of the RF energy. To reduce the skin redness, such as side effects due to the application of RF energy except for the treatment, comprising applying a composition containing stem cells derived from exosomes as an active ingredient to the skin subjected to the RF energy application Provide beauty methods.

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.). Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and differently regulated by 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 have an effect of reducing skin redness, which is a side effect of RF energy application, and various stem cells that are used in the art or may be used in the future if they do not cause adverse effects on the human body. Of course, you can use the derived exosomes. 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 "RF energy" has a conventional meaning, which is the product of RF power and the time that the RF power is applied or delivered to the skin region to be treated. "RF voltage" and "RF power" are mathematically closely related parameters, and knowing one of them and the load (skin) impedance can determine the other parameter at a given skin impedance at a particular time. On the other hand, by controlling the voltage of the RF generator can control the RF power and RF energy delivered to the skin.

As used herein, the term "skin care" refers to positive effects such as reducing, alleviating and / or ameliorating skin swelling and / or swelling after RF treatment, such as pain, skin redness or swelling after RF treatment, and / or soothing skin. It means to bring effect.

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 the microcurrent is introduced, the manner in which the microcurrent is 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.

In one embodiment of the skin care method of the present invention, (a) applying the RF energy to the skin of the mammal, (b) before or after applying the RF energy, the skin of the mammal to which the RF energy application target Applying a composition comprising a stem cell-derived exosomes as an active ingredient.

In the skin care method of an embodiment of the present invention, the RF energy application may be a fractional RF energy application.

The skin care method of an embodiment of the present invention can shorten downtime, which is a time taken for the skin redness and swelling, which are side effects due to RF energy application, to disappear.

In the skin care method of an embodiment of the present invention, the composition may be a cosmetic composition or a skin external preparation. For example, the cosmetic composition may be a cream or lotion.

On the other hand, in the skin care method of one embodiment of the present invention, when the composition is made of an external preparation for skin and / or cosmetic composition, the component usually used in cosmetics or external preparation for skin within the range that does not impair the effects of the present invention, For example, a moisturizer, antioxidant, oily component, ultraviolet absorber, emulsifier, surfactant, thickener, alcohol, powder component, colorant, aqueous component, water, various skin nutrients, etc. can be suitably blended as needed.

In addition, the composition may be used in combination with a conventional skin improver and / or moisturizer in addition to stem cells derived from exosomes, as long as the action (reduction of side effects due to RF energy application, etc.) is not impaired. For example, the exosomes derived from stem cells may be supported or mixed in at least one of a hydrogel, hyaluronic acid, a hyaluronic acid salt (for example, sodium hyaluronate), or a hyaluronic acid gel. 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 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.

When the external preparation for skin of one embodiment of the present invention is prepared in a cosmetic composition, for example, it may be used for the purpose of reducing skin redness, and the like, and the cosmetic formulation may be prepared in any formulation commonly 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.

Skin cosmetic method of an embodiment of the present invention, (c) performing iontophoresis (iontophoresis) by flowing a microcurrent to the skin of the mammal to which the composition containing the stem cell-derived exosomes as an active ingredient is applied And (d) delivering the stem cell-derived exosomes into the mammalian skin through the microcurrent.

In the skin care method of an embodiment of the present invention, the composition is, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, It can be applied to various forms such as aerosol, mist, foundation, powder and oil paper. For example, the composition may be applied or deposited on at least one side of a mask pack, mask sheet or patch.

In the skin care method of an embodiment of the present invention, the step (b) is (b1) applying the composition directly to the skin of the mammal, (b2) the mask pack, mask sheet is applied or deposited the composition Or by contacting or attaching the patch to the skin of the mammal, or by sequentially proceeding with (b1) and (b2).

In the skin care method of an embodiment of the present invention, at least one surface of the mask pack, the mask sheet or the patch includes a hydrogel, hyaluronic acid, a hyaluronic acid salt (for example, sodium hyaluronate), or a hyaluronic acid gel. At least one of the 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 skin care method of an embodiment of the present invention, step (c) may be performed by contacting or attaching an iontophoresis device to the skin of the mammal.

In the skin care method of an embodiment of the present invention, the iontophoresis device is a flexible battery, lithium ion secondary battery, alkaline battery, dry cell, mercury battery, lithium battery, nickel-cadmium battery, and reverse electrodialysis battery It may include at least one battery selected from the group consisting of.

Skin beauty method of the present invention by treating the skin exosomes derived from stem cells with RF energy applied to the skin to reduce the redness of the side effect of applying RF energy, pain reduction and skin soothing effect by applying RF energy Indicates. In addition, according to the skin care method of the present invention when combined with the RF energy application and stem cell-derived exosomes derived from RF energy application side effects due to RF energy redness and swelling time it takes to reduce the downtime (downtime) and It can have the same positive effect.

In addition, the skin cosmetic method of the present invention when the exosomes derived from stem cells to the skin using the iontophoresis device to reduce the side effects due to the application of RF energy and improve the positive skin cosmetic effect as described above Can be.

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. 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.

FIG. 8A shows the left side of the subject 1 who applies exosomes derived from stem cells and performs iontophoresis according to an embodiment of the present invention after RF energy is applied, and merely a rubber mask after RF energy is applied. The right side of the subject 1 to be applied is shown separately. 8B is a case where the exosomes derived from stem cells according to an embodiment of the present invention and iontophoresis were performed after applying RF energy (left side of the subject 1), and only a rubber mask (control) was applied. It is a figure which shows the redness measurement result of the (right side of the subject 1's face) for each facial zone.

FIG. 9 is a graph comparing the red phase measurement result of the test subject 1 of FIG. 8B.

10 is a result of measuring the redness (redness) on the 10th day after applying the exosomes and physiological saline derived from stem cells according to an embodiment of the present invention to the right and left of the subject 2, respectively, after applying RF energy It is a graph.

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 performed 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: RF energy application to human skin and treatment of exosomes derived from stem cells

Example 5-1: First Facial Clinical Trial

RF energy was applied to the human face using Secret (Eulda Co., Ltd., Gyeonggi-do, Korea). [RF equipment specifications and parameters are as follows: fractional needle 25 pin (1 × 1cm) / depth, 1.0 mm / intensity; RF parameter 60% / RF, 4 MHz; 300 shots, application time 150 ms, application interval 100 ms].

Prior to treatment, the redness of the right and left faces of Subject 1 was measured using a Mark-Vu facial skin analyzer of PS-I Plus, Gyeonggi-do, Korea. After washing the face of Subject 1 (adult female), an anesthetic was applied to the face for 30 minutes, and RF energy was applied to the face according to the manufacturer's instructions. As shown in FIG. 8A, 1 ml of an exosome (exosome prepared in Example 2) derived from stem cells at a concentration of 7.39 × 10 ⁸ particles / mL was placed on the left side of the subject 1 immediately after RF energy was applied. After application, iontophoresis was performed, and a rubber mask was laminated on the right face of the subject 1 and pressed. Iontoporesis was performed by flowing a 0.5 mA microcurrent for 20 minutes to the left face of the stem cell-derived exosomes using an iontophoresis device (IONZYME) (purchased from Environ).

Using the Mark-Vu facial skin analyzer of PS-I Plus, Gyeonggi-do, Korea, the redness of the right and left faces of subject 1 was measured and the red color before the treatment was measured. Compared to Before measurements (FIG. 8B). The percent increase of redness measured at each site was analyzed by the following formula: Red phase increase rate = [(red phase after treatment)-(red phase before treatment)] / (red phase before treatment) ( %). In accordance with the results of the red phase measurement and when applying the RF energy to the skin treated with the exo-derived stem cells of the present invention was evaluated whether to reduce the side effects due to RF energy application to restore to a normal state.

As a result, the F region and the H region of the face where the stem cell-derived exosomes of one embodiment of the present invention were treated and subjected to iontophoresis were simply stacked with a rubber mask and pressed to control regions E and G. It was confirmed that the redness (sideness), which is a side effect due to the application of RF energy, was significantly reduced compared to the region (FIG. 9).

Subject 1 was also asked to answer a questionnaire about pain reduction and skin sedation effects. Subject 1 responded that the stem cell-derived exosomes had less pain due to RF treatment and had a skin soothing effect.

Example 5-2: Second Facial Clinical Trial

Exosome (2 × 10 ⁹ particles / mL concentration) derived from stem cells of one embodiment of the present invention after applying RF energy (Exosome prepared in Example 2) in the same manner as described in Example 5-1 (Suspension) 1 ml was applied to the right side of the subject 2's face, and physiological saline was applied to the left side of the subject 2's face. And a rubber mask (rubber mask) was laminated on the face of the subject 2 for 30 minutes and pressed.

Facial redness of subject 2 was measured using Mark-View Facial Skin Analyzer at 10 days after the above treatment, and compared with the facial redness of subject 2 measured on the day after RF procedure. The percentage change of redness of subject 2 was analyzed by the following formula: Red phase change rate = [(red phase measured on day N)-(red phase on the day after the procedure)] / (red on the day after the procedure) group)(%). As a result, the red face of the face which is a side effect due to the application of RF energy in the right face of the subject 2 treated with the stem cell-derived exosomes of one embodiment of the present invention compared to the left face of the treated subject 2 treated with physiological saline It was confirmed that the redness was significantly reduced (FIG. 10).

Subject 2 was also asked to answer a questionnaire about pain reduction and skin soothing effects. Subject 2 responded that stem cell-derived exosomes had less pain due to RF treatment and had a skin calming effect.

Based on the above experimental results, when applying exosomes derived from stem cells together with applying RF energy to the skin, the redness of the face, which is a side effect of applying RF energy, is reduced, and the pain caused by applying RF energy is reduced. And it can be seen that the skin soothing effect. In addition, co-treatment with stem cell-derived exosomes in conjunction with RF energy application has a positive effect of reducing downtime, which is a time taken for skin redness and swelling to disappear as a side effect of RF energy application. I can see it coming.

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 (13)

(a) applying radio frequency (RF) energy to a mammal's skin; (b) before or after the RF energy application, comprising applying a composition comprising exosomes as an active ingredient to the skin of the mammal subject to the RF energy application, skin due to the application of RF energy other than therapeutic Beauty treatments to reduce side effects. The method of claim 1, A method for skin beauty, which reduces at least one of skin redness or pain due to the application of RF energy. The method of claim 2, Skin beauty method that shortens downtime, which is the time taken for skin redness and swelling to disappear as a side effect of RF energy application. The method of claim 3, A method of skin beauty, further exhibiting a skin soothing effect. The method according to any one of claims 1 to 4, The composition is characterized in that the suspension, skin cosmetic method. The method according to any one of claims 1 to 4, The composition is characterized in that the cosmetic composition or a skin external preparation, skin care method. The method of claim 6, The cosmetic composition is a cream or lotion, skin beauty method. The method according to any one of claims 1 to 4, (c) performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the composition is applied; and (d) delivering the exosomes into the mammalian skin through the microcurrent. Further comprising, skin beauty method. The method of claim 8, The composition is at least selected from the group consisting of patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, aerosols, mists, foundations, powders and oil papers. A skin care method, characterized in that applied to one type. The method of claim 9, The composition is applied to or deposited on at least one side of the mask pack, mask sheet or patch, skin care method. The method of claim 10, (B) step (b1) applying the composition directly to the skin of the mammal, (b2) contacting or attaching a mask pack, mask sheet or patch to which the composition has been applied or deposited Or by performing the steps (b1) and (b2) sequentially. The method of claim 11, The step (c) is performed by contacting or attaching an iontophoresis device to the skin of the mammal. The method of claim 12, 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. , Skin beauty method.

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