WO2019231133A1 - Composition pour réduire les pores, comprenant des exosomes dérivés de cellules souches en tant que principe actif - Google Patents

Composition pour réduire les pores, comprenant des exosomes dérivés de cellules souches en tant que principe actif Download PDF

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Publication number
WO2019231133A1
WO2019231133A1 PCT/KR2019/005670 KR2019005670W WO2019231133A1 WO 2019231133 A1 WO2019231133 A1 WO 2019231133A1 KR 2019005670 W KR2019005670 W KR 2019005670W WO 2019231133 A1 WO2019231133 A1 WO 2019231133A1
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composition
pores
skin
exosomes
pore
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PCT/KR2019/005670
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English (en)
Korean (ko)
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조병성
이용원
김광일
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주식회사 엑소코바이오
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Priority claimed from KR1020190019116A external-priority patent/KR102646145B1/ko
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Publication of WO2019231133A1 publication Critical patent/WO2019231133A1/fr

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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—Liposomes
    • 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
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • 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
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0428—Specially adapted for iontophoresis, e.g. AC, DC or including drug reservoirs
    • 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
    • A61N1/325—Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body

Definitions

  • 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.
  • 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the present invention is not limited thereto, and various methods of iontophoresis may be used.
  • composition for reducing pores of one embodiment of the present invention comprises an exosome derived from stem cells as an active ingredient.
  • composition for reducing pores 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.
  • the lyophilized agent may further contain ascorbic acid and retinol.
  • 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.
  • the diluent may be water for injection, physiological saline, phosphate buffer, purified water, or deionized water.
  • the diluent may further comprise hyaluronic acid or hyaluronic acid salt (eg, sodium hyaluronate).
  • 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.
  • composition for reducing pores of one embodiment of the present invention may be a cosmetic composition or an external preparation for skin.
  • composition for reducing pores of one embodiment of the present invention 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.
  • 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.
  • 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.
  • 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.
  • it may be administered in one to several divided doses of 0.001 mg / kg to 100 mg / kg per day.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cosmetic formulation may be prepared in any formulation conventionally 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.
  • 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.
  • 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.
  • 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.
  • the pore reduction composition may be applied or deposited on at least one surface of a mask pack, a mask sheet or a patch.
  • 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).
  • 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.
  • step (c) may be performed by contacting or attaching the iontophoresis device to the skin of the mammal.
  • 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. .
  • 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.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • 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 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).
  • 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.
  • 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.
  • concentrations of methionine, mannitol and trehalose in the aqueous solution were each 9 mg / mL.
  • Example 2 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 (°C) 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
  • lyophilized protective agent components 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.
  • lyophilized protective agent components include at least one of methionine, mannitol and trehalose.
  • 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.
  • 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.
  • 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.
  • the exosomes prepared in Example 2 may be used as a composition for pore reduction after suspending in water for injection.
  • Pore size change rate [(pore size measured on N days)-(pore before treatment) Size] / (pore size before treatment) (%).
  • 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.
  • 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].
  • exosomes exosomes prepared in Example 2
  • 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).
  • IONZYME iontophoresis device
  • 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.
  • 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).

Abstract

La présente invention concerne une composition destinée à réduire les pores, comprenant des exosomes dérivés de cellules souches en tant que principe actif, et un procédé d'esthétique destiné à réduire les pores de la peau mettant en œuvre la composition. Le traitement de la peau avec la composition destinée à réduire les pores comprenant des exosomes dérivés de cellules souches en tant que principe actif, selon la présente invention, a un excellent effet esthétique de la peau pour réduire les pores de la peau, en particulier les pores de la peau du visage, améliorant l'aspect du visage.
PCT/KR2019/005670 2018-05-31 2019-05-10 Composition pour réduire les pores, comprenant des exosomes dérivés de cellules souches en tant que principe actif WO2019231133A1 (fr)

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KR1020190019116A KR102646145B1 (ko) 2018-05-31 2019-02-19 줄기세포 유래의 엑소좀을 유효성분으로 포함하는 모공 축소용 조성물

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005306831A (ja) * 2004-04-20 2005-11-04 Sakamoto Yakusoen:Kk 皮膚外用剤
US20150023908A1 (en) * 2011-03-04 2015-01-22 Ahmed H. Al-Qahtani Skin cream
WO2016072821A1 (fr) * 2014-11-07 2016-05-12 한양대학교 에리카산학협력단 Composition pour l'induction de la différenciation d'adipocyte contenant un exosome dérivé de cellules souches, la régénération de tissus adipeux, et la décoloration de la peau ou l'atténuation des rides
WO2017023689A1 (fr) * 2015-07-31 2017-02-09 Zen-Bio, Inc. Compositions d'exosome et leur utilisation pour la réparation de tissus mous
WO2017218942A1 (fr) * 2016-06-16 2017-12-21 Eye Care International, Llc Compositions et méthodes de traitement du syndrome sec et d'autres surfaces épithéliales non kératinisées ayant subi un traumatisme

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005306831A (ja) * 2004-04-20 2005-11-04 Sakamoto Yakusoen:Kk 皮膚外用剤
US20150023908A1 (en) * 2011-03-04 2015-01-22 Ahmed H. Al-Qahtani Skin cream
WO2016072821A1 (fr) * 2014-11-07 2016-05-12 한양대학교 에리카산학협력단 Composition pour l'induction de la différenciation d'adipocyte contenant un exosome dérivé de cellules souches, la régénération de tissus adipeux, et la décoloration de la peau ou l'atténuation des rides
WO2017023689A1 (fr) * 2015-07-31 2017-02-09 Zen-Bio, Inc. Compositions d'exosome et leur utilisation pour la réparation de tissus mous
WO2017218942A1 (fr) * 2016-06-16 2017-12-21 Eye Care International, Llc Compositions et méthodes de traitement du syndrome sec et d'autres surfaces épithéliales non kératinisées ayant subi un traumatisme

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