WO2019190093A1 - Procédé de soin de la peau utilisant, en combinaison, l'application d'énergie rf sur la peau et le traitement par exosomes dérivés de cellules souches sur la peau - Google Patents

Procédé de soin de la peau utilisant, en combinaison, l'application d'énergie rf sur la peau et le traitement par exosomes dérivés de cellules souches sur la peau Download PDF

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WO2019190093A1
WO2019190093A1 PCT/KR2019/003069 KR2019003069W WO2019190093A1 WO 2019190093 A1 WO2019190093 A1 WO 2019190093A1 KR 2019003069 W KR2019003069 W KR 2019003069W WO 2019190093 A1 WO2019190093 A1 WO 2019190093A1
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skin
energy
exosomes
composition
present
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Korean (ko)
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조병성
이용원
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Exocobio Inc
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Exocobio Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/40—Applying electric fields by inductive or capacitive coupling ; Applying radio-frequency signals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • 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.
  • 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.
  • RF energy is one of the methods of heating skin and subcutaneous tissue.
  • RF energy application to the skin is performed using an RF electrode or applicator connected to an RF energy source.
  • RF energy transfer surface of the RF electrode 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.
  • 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".
  • hot spots 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • RF radio frequency
  • 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.
  • exosome 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.
  • 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.
  • 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.
  • 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.
  • by controlling the voltage of the RF generator can control the RF power and RF energy delivered to the skin.
  • 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.
  • 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.
  • the present invention is not limited thereto, and various methods of iontophoresis may be used.
  • 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.
  • 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.
  • the composition may be a cosmetic composition or a skin external preparation.
  • the cosmetic composition may be a cream or lotion.
  • the component usually used in cosmetics or external preparation for skin within the range that does not impair the effects of the present invention,
  • 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.
  • 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.
  • 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.
  • 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 external preparation for skin of one embodiment of the present invention is prepared in a cosmetic composition
  • a cosmetic composition for example, it may be used for the purpose of reducing skin redness, and the like
  • the cosmetic formulation may be prepared in any formulation commonly prepared in the art.
  • 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.
  • ingredients conventionally used in external preparations for skin and / or cosmetics such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings.
  • a carrier e.g., a carrier.
  • 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.
  • iontophoresis iontophoresis
  • 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.
  • the composition may be applied or deposited on at least one side of a mask pack, mask sheet or patch.
  • 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).
  • 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.
  • step (c) may be performed by contacting or attaching an iontophoresis device to the skin of the mammal.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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 ⁇ g 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.
  • TEM transmitted electron microscopy
  • 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.
  • FIGS. 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.
  • 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. .
  • phosphate-buffered saline purchased from ThermoFisher Scientific
  • exosomes 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 ⁇ m 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 °C or less) and then used for exosome separation. In addition, the filtered culture solution was stored frozen in an cryogenic freezer of -60 °C or less and thawed and then exosomes were separated. Thereafter, exosomes were separated from the culture using a tangential flow filtration device (TFF).
  • TMF tangential flow filtration device
  • Example 1 the exosomes were separated from the culture medium filtered with a 0.22 ⁇ m 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 molecular weight cutoffs
  • 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.
  • 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.
  • Figures 6A to 6E 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.
  • 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.
  • 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).
  • NTA nanoparticle tracking analysis
  • TRPS tunable resistive pulse sensing
  • 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.
  • FIGS. 5A to 5C 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.
  • 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. .
  • 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 purchasedd from System Biosciences
  • anti-CD81 purchasedd from System Biosciences
  • anti-TSG101 purchasedd from Abcam
  • 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.
  • an exosome-human CD63 separation / detection kit purchased from ThermoFisher Scientific
  • PE-mouse anti-human CD63 PE-Mouse anti markers were stained using -human CD63
  • PE-mouse anti-human CD81 purchasedd from BD
  • 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.
  • the processes of the separation method of one embodiment of the present invention are scale-up and suitable for GMP.
  • HS68 cells which are human skin fibroblasts
  • HS68 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.
  • WST-1 reagent purchased from Takara
  • MTT reagent purchased from Sigma
  • CellTiter-Glo reagent purchased from Promega
  • 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
  • RF energy was applied to the human face using Secret (Eulda Co., Ltd., Gyeonggi-do, Korea).
  • 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].
  • 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).
  • IONZYME iontophoresis device
  • Red phase increase rate [(red phase after treatment)-(red phase before treatment)] / (red phase before treatment) ( %).
  • 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.
  • 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.
  • 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)(%).
  • 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.

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Abstract

La présente invention concerne un procédé de soin de la peau comprenant les étapes de : (a) application d'énergie RF à la peau d'un mammifère ; et (b) application, avant ou après l'application de l'énergie RF, d'une composition contenant des exosomes dérivés de cellules souches en tant que substances actives sur la peau du mammifère à laquelle l'énergie RF doit être appliquée. Le procédé de soin de la peau de la présente invention réalise, conjointement avec l'application d'énergie RF, un traitement par des exosomes dérivés de cellules souches sur la peau, de façon à réduire la rougeur de la peau, qui est un effet secondaire causé par l'application d'énergie RF, et à présenter des effets de réduction de la douleur causée par l'application d'énergie RF, et à apaiser la peau. En outre, lorsque l'application d'énergie RF et le traitement par exosomes dérivés de cellules souches sont effectués en combinaison, selon le procédé de soin de la peau de la présente invention, le temps d'arrêt, qui est le temps nécessaire pour la disparition de la rougeur de la peau et du gonflement de la peau qui sont les effets secondaires causés par l'application d'énergie RF, peut être réduit.
PCT/KR2019/003069 2018-03-29 2019-03-15 Procédé de soin de la peau utilisant, en combinaison, l'application d'énergie rf sur la peau et le traitement par exosomes dérivés de cellules souches sur la peau Ceased WO2019190093A1 (fr)

Applications Claiming Priority (4)

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KR20180036305 2018-03-29
KR10-2018-0036305 2018-03-29
KR10-2019-0017770 2019-02-15
KR1020190017770A KR20190114740A (ko) 2018-03-29 2019-02-15 피부에 대한 ｒｆ 에너지 인가와 줄기세포 유래의 엑소좀 처리를 병용한 피부 미용방법

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WO2019190093A1 true WO2019190093A1 (fr) 2019-10-03

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246270A1 (en) * 2008-01-31 2009-10-01 Jones Dennis R Use of Iontophoresis or Electrotherapy and Ultrasound to Deliver Agents for Skin Rejuvenation.
KR20160144946A (ko) * 2016-12-07 2016-12-19 (주)제니트리 피부병변의 치료 또는 미용 목적으로 시술되는 레이저에 의한 피부 과민 반응의 예방 및 개선용 조성물과 이를 이용한 화장료
KR20170044999A (ko) * 2015-10-16 2017-04-26 (주)프로스테믹스 줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법
KR20170082111A (ko) * 2016-01-05 2017-07-13 바이오센서연구소 주식회사 마스크팩에 장착되는 장치, 그를 포함하는 마스크팩 및 키트
KR20170085010A (ko) * 2016-01-12 2017-07-21 주식회사 강스템바이오텍 고함량의 성장인자를 함유한 줄기세포 유래 엑소좀

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246270A1 (en) * 2008-01-31 2009-10-01 Jones Dennis R Use of Iontophoresis or Electrotherapy and Ultrasound to Deliver Agents for Skin Rejuvenation.
KR20170044999A (ko) * 2015-10-16 2017-04-26 (주)프로스테믹스 줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법
KR20170082111A (ko) * 2016-01-05 2017-07-13 바이오센서연구소 주식회사 마스크팩에 장착되는 장치, 그를 포함하는 마스크팩 및 키트
KR20170085010A (ko) * 2016-01-12 2017-07-21 주식회사 강스템바이오텍 고함량의 성장인자를 함유한 줄기세포 유래 엑소좀
KR20160144946A (ko) * 2016-12-07 2016-12-19 (주)제니트리 피부병변의 치료 또는 미용 목적으로 시술되는 레이저에 의한 피부 과민 반응의 예방 및 개선용 조성물과 이를 이용한 화장료

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