WO2019088656A1 - Composition de charge contenant des exosomes stabilisés - Google Patents

Composition de charge contenant des exosomes stabilisés Download PDF

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WO2019088656A1
WO2019088656A1 PCT/KR2018/013011 KR2018013011W WO2019088656A1 WO 2019088656 A1 WO2019088656 A1 WO 2019088656A1 KR 2018013011 W KR2018013011 W KR 2018013011W WO 2019088656 A1 WO2019088656 A1 WO 2019088656A1
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exosome
hyaluronic acid
derived
exosomes
filler composition
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PCT/KR2018/013011
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Korean (ko)
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이용원
김광일
조병성
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주식회사 엑소코바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
    • A61L2300/802—Additives, excipients, e.g. cyclodextrins, fatty acids, surfactants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/24—Materials or treatment for tissue regeneration for joint reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the present invention relates to a stabilized exosome filler composition, and more particularly, to a filler composition that contains exosome in a hyaluronic acid-based filler composition to increase the stability of the exosome.
  • the present invention also relates to a stabilized exosome filler composition capable of increasing the stability of exosome and adding a cosmetic effect by hyaluronic acid-based filler.
  • exosomes which have intercellular signal transduction
  • researches on its components and functions are actively under way.
  • Extracellular vesicles Cells release various membrane-type vesicles in the extracellular environment, and these release vesicles are commonly referred to as extracellular vesicles (EVs).
  • the extracellular endoplasmic reticulum is sometimes referred to as cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes and, in some cases, differentiated from exosomes.
  • Exosome is an endoplasmic reticulum of several tens to several hundreds of nanometers in size, composed of double lipid membranes identical to the cell membrane structure, and contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosomal cargo inside.
  • Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells.
  • Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.
  • Exosomes contain specific genetic material and bioactivity factors depending on the nature and condition of the derived cells.
  • the proliferating stem cell-derived exosomes regulate cell behavior such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
  • the exosome called the avatar of cells contains bioactivity factors such as growth factors similar to cells, and when the exosome is stored at room temperature, there arises a problem of stability in which the activity of growth factors and the like is lowered.
  • the exosome is stored in a simple refrigerator, the physical properties and stability of the exosome are changed in total, and the activity of the bioactive factors contained in the exosome may be lowered depending on the storage time and method.
  • Hyaluronic acid or hyaluronan is a biopolymer material in which a repeating unit composed of N-acetyl-D-glucosamine and D-glucuronic acid is linearly connected, and exists at a high concentration in connective tissues and skin.
  • hyaluronic acid is found in chicken eggs, body fluids, ocular vitreous humor, joint synovial fluid, animal cushioning tissue, and placenta.
  • the hyaluronic acid or its salt has excellent biocompatibility, biodegradability and stability and is utilized as a wrinkle improving filler, an arthritis improving or treating agent, a wound healing agent, an eye surgery aid, an adhesion inhibitor, a drug delivery system and the like.
  • Fillers using hyaluronic acid or its salts are formulations that are injected into a patient to reduce the appearance of, for example, facial lines and wrinkles.
  • the hyaluronic acid-based filler has the effect of treating the soft tissue condition by replacing the endogenous matrix polymer lost in the tissue by injecting into the soft tissues such as facial wrinkles and depressions or by improving the function of the existing matrix polymer.
  • hyaluronic acid-based fillers can be applied locally to small body regions, such as the lips, nose, forehead, balls, chest, and genitals, based on volume enhancement effects, .
  • hyaluronic acid-based fillers can be injected into the joints of patients with degenerative arthritis to improve or improve symptoms.
  • the present inventors have intensively studied the new application field of exosome and medical or cosmetic technology, and found that when exosome is contained in a hyaluronic acid-based filler composition, the stability of exosome increases and hyaluronic acid- It is confirmed that the natural cosmetic effect can be added, and the present invention has been completed.
  • Another object of the present invention is to provide a stabilized exosome filler composition capable of increasing the stability of exosome and adding a cosmetic effect by hyaluronic acid-based filler.
  • the present invention provides a method for producing a hyaluronic acid, a hyaluronic acid salt or a hyaluronic acid gel, which comprises mixing at least one of hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate)
  • hyaluronic acid salt for example, sodium hyaluronate
  • a stabilized exosome filler composition comprising exosomes which are mixed and held in at least one species.
  • exosomes refers to an endoplasmic reticulum of several tens to several hundred nanometers (preferably about 30 to 200 nm) in size, consisting of double lipid membranes identical in structure to the cell membrane The particle size of the exosome can be varied according to the cell type, the separation method and the measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, DOI 10.1007 / s00216-015-8535-3). Exosomes contain proteins called exosomal cargo (cargo), nucleic acids (mRNA, miRNA, etc.).
  • Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells.
  • Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.
  • biological solution means a liquid solution of biogenic origin, in which the exosome is dispersed, suspended, precipitated, suspended or mixed, and includes, for example, a cell culture solution, a cell culture supernatant, Cell culture supernatant, whole blood, serum, cord blood, plasma, multiple fluid, brain and cerebrospinal fluid, placenta extract, and bone marrow aspirate.
  • a cell culture solution a cell culture supernatant, Cell culture supernatant, whole blood, serum, cord blood, plasma, multiple fluid, brain and cerebrospinal fluid, placenta extract, and bone marrow aspirate.
  • a “ biological solution” may be cultured or incubated under conditions that release and / or secrete exosomes, and may be frozen and thawed.
  • exosome is intended to mean an exosome that is secreted from cells of various animals, plants, bacteria, fungi, algae and the like, preferably stem cells, (E. G., Exosome-like vesicles) having a nano-sized bezacl structure and a composition similar to exosome.
  • exosome used in the present invention does not cause an adverse effect on the human body without increasing or at least reducing the cosmetic effect by the hyaluronic acid-based filler
  • various exosomes which are used in the art or can be used in the future can be used Of course. Therefore, it should be understood that the exosome isolated according to the separation method of the following embodiments should be understood as an example of exosome that can be used in the present invention, and the present invention is not limited thereto.
  • the stabilized exosome filler composition according to one embodiment of the present invention is prepared by mixing at least one of hyaluronic acid, hyaluronic acid salt, and hyaluronic acid gel with at least one of hyaluronic acid, hyaluronic acid salt, and hyaluronic acid gel And the like.
  • the stabilized exosome filler composition of one embodiment of the present invention may further comprise a non-crosslinked hyaluronic acid gel and / or a crosslinked hyaluronic acid gel.
  • the crosslinked hyaluronic acid gel may be prepared by mixing 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis (2,3-epoxypropoxy) butane, 1,4-bisglycidyloxybutane, At least one crosslinking agent selected from the group consisting of 1,2-bis (2,3-epoxypropoxy) ethylene and 1- (2,3-epoxypropyl) -2,3-epoxycyclohexane, May be a zero cross-linked hyaluronic acid gel.
  • BDDE 1,4-butanediol diglycidyl ether
  • 1,4-bis (2,3-epoxypropoxy) butane 1,4-bisglycidyloxybutane
  • the exosome may be an exosome derived from a stem cell, an exosome derived from a plant, or an exosome derived from a yeast.
  • the stem cell-derived exosome may be exosomes derived from fat, bone marrow, umbilical cord or cord blood stem cells.
  • the plant-derived exosome may be an exosome derived from rose stem cells or an exosome derived from edelweis.
  • the exosome derived from the rosette stem cell may be one which has been separated and purified from a culture of a rosaceous embryo or a leaf-derived callus, or a culture of a rosaceous cambium or procambium-derived callus.
  • &Quot; Rosa spp. &Quot; is a generic term for dicotyledonous plants, rosewoods, rosacea, and roses, including both wild species and improved horticultural crops.
  • the exosome derived from edelvase may be one which has been separated and purified from a culture solution of plant cells obtained from leaves or stem tissues of Edelweiss, or from a culture of callus induced by scarring of cotyledon germinated from Edelweiss seeds. &Quot; Edelweiss " means a plant of the genus Leontopodium sp. , And may be Leontopodium japonicum as an example without limiting the present invention.
  • the exosome derived from yeast may be exosome derived from wine yeast or exosome derived from galactomiesces.
  • the exosome derived from the wine yeast may be one separated and purified from a wine yeast culture solution, a wine yeast fermentation product or wine.
  • Wine yeast includes both exogenous wine yeast (wild wine yeast) and cultured wine yeast (dry wine yeast).
  • wild wine yeasts include Candida, Pichia, Hanseniaspora, Zygosaccharomyces, Metschnikowiaceae ), Kloeckera (Kloeckera) and the like, and the dry wine yeast used to produce a certain quality of wine is Saccharomyces cerevisiae.
  • the exosome derived from the galactomycese may be one separated and purified from the galactomycese culture broth or the galactomycetes fermentation product.
  • "Galactomycetes” is a fungi belonging to the Dipodascaceae family and refers to galactomycetes yeasts used in the brewing or fermentation process.
  • the exosome can be obtained by performing the following steps: (a) adding trehalose to the biological solution, (b) adding the trehalose (C) separating the exosomes from the filtered biological solution using TFF (Tangential Flow Filtration), and (d) removing the buffer used for desalting and buffer exchange (diafiltration). , Adding trehalose to the solution, and performing desalting and buffer exchange (diafiltration) on the separated exosome using TFF (Tangential Flow Filtration) using the buffer solution to which the trehalose is added .
  • TFF Tangential Flow Filtration
  • exosomes having a uniform particle size distribution and high purity can be effectively obtained (FIGS. 6A to 6C) 6E).
  • trehalose in the pre-filtration step (step (b) before exocase separation by TFF) and desalting by TFF and buffer exchange step (step (d)) after separation of exosome, a high purity particle size distribution Uniform exosome can be obtained with high yield.
  • Trehalose provides the ability to efficiently isolate exosomes for impurities such as cellular debris, waste products, proteins and macromolecules.
  • the desalting and buffer exchange can be performed continuously or intermittently. Desalting and buffer exchange can be carried out using a buffer solution having a volume of at least 4 times, preferably 6 times to 10 times, more preferably 12 times the starting volume.
  • a molecular weight cutoff (MWCO) 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da TFF filter or 0.05 ⁇ ⁇ TFF filter can be used.
  • the step (c) may further include a step of concentrating the solution to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration).
  • the biological solution may be a stem cell culture solution.
  • the type of the stem cell is not limited, but may be a mesenchymal stem cell, for example, a fat, a bone marrow, an umbilical cord or a cord blood-derived stem cell, more preferably a fat-derived stem cell.
  • the type of the adipose-derived stem cell is not limited as long as it does not cause a risk of infection by a pathogen and does not cause an immune rejection reaction, but it may be preferably a human adipose-derived stem cell.
  • the exosome used in the present invention is not limited to the exosome obtained according to the separation method described above, and it is needless to say that various exosomes which are used in the art or can be used in the future can be used. It is to be understood that the exosome isolated according to the above separation method should be understood as an example of exosome that can be used in the composition of the present invention, and the present invention is not limited thereto.
  • the exosome is sterilized by using a sterilizing filter before mixing with at least one of hyaluronic acid, hyaluronic acid salt, and hyaluronic acid gel .
  • the stabilized exosome filler composition of one embodiment of the present invention is preferably applied in an injectable administration mode. However, it is not limited thereto and does not exclude various methods of administration known in the art.
  • the stabilized exosome filler composition of one embodiment of the present invention can be prepared with a pharmaceutical composition for wound healing, scar treatment, improvement or treatment of arthritis and the like.
  • Another embodiment of the present invention provides a cosmetic method for controlling the condition of mammalian skin except for the treatment using the stabilized exosome filler composition.
  • conditioning of the skin means improvement of the condition of the skin and / or prevention of the condition of the skin
  • improvement of the condition of the skin means visual and / Or a tactile perceptible positive change.
  • improvement of skin condition may be improvement of skin wrinkles, skin regeneration, skin moisturization, skin elasticity improvement, volume increase (for example, volume-up, skin distension, etc.).
  • Another embodiment of the present invention provides a method for wound healing, scar treatment, arthritis improvement or treatment using the stabilized exosome filler composition.
  • the stabilized exosome filler composition of the present invention has the advantage of enhancing the stability of exosome and adding the natural cosmetic effect of hyaluronic acid-based filler.
  • the stabilized exosome filler composition of the present invention can provide a long-lasting cosmetic or therapeutic effect derived from exosome as the stability of exosome increases, Can be suitably applied to the implementation of hyaluronic acid-based fillers that are convenient and less painful to the patient.
  • the present invention is capable of mixing the exosomes separated and purified by the inherent separation method into a hyaluronic acid-based filler at a low concentration, and it is easier to mix homogeneously than when a large number of exosome particles are required.
  • the present invention can prevent the problem of increasing the total volume of the hyaluronic acid-based filler composition that may occur when the exosome is mixed for cosmetic or therapeutic effects.
  • FIG. 1 is a flow chart illustrating a process for separating and purifying exosomes in a method for producing exosomes from a biological solution according to one embodiment of the present invention.
  • FIG. 2 is a graph showing a result of measuring a relative amount of protein contained in a solution for each step of preparing a biological solution, for example, a stem cell culture solution, according to an embodiment of the present invention .
  • the ratio of the total amount of protein in each step was expressed by the relative ratio of total protein amount to the total biological solution.
  • the experimental results show the results obtained in two different batches, respectively.
  • FIG. 3 shows the results of measuring the productivity and purity of exosomes obtained according to one embodiment of the present invention.
  • the productivity of exosomes was calculated as "the number of particles of exosome obtained per mL of biological solution, eg, a stem cell culture (CM),” and the purity of exosome was calculated as "the number of exosomes per microgram of protein contained in the final fraction Quot; number of particles "
  • the experimental results show the results obtained in five different batches.
  • 4A to 4E show results of physical property analysis of exosomes obtained according to one embodiment of the present invention.
  • 4A shows particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis.
  • Figure 4B shows particle size distribution and number of particles by NTA (nanoparticle tracking analysis) analysis.
  • 4C shows particle images by transmission electron microscopy (TEM) according to magnification.
  • 4D shows the Western blot results of exosomes obtained according to one embodiment of the present invention.
  • &Quot; Figure 4E shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.
  • Figures 5A-5C show the results of NTA analysis on particle size distribution showing that a uniform and high-purity exosome is obtained with trehalose addition. As the amount of trehalose added increases, a particle size distribution having a single peak can be obtained.
  • 6A to 6C show results of NTA analysis showing particle size distribution according to whether trehalose was added in the process of producing exosome according to one embodiment of the present invention.
  • 6A shows a case where trehalose is added throughout the production process
  • Fig. 6B shows a case where trehalose is added after the cell culture solution is stored in a frozen state
  • the results are shown without adding os.
  • 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 size of exosomes isolated by the methods of FIGS. 6A to 6C.
  • FIG. 7 shows the result of confirming the absence of cytotoxicity after treatment of exosome according to one embodiment of the present invention with HS68 cells of human skin fibroblasts.
  • FIG. 8 is a photograph of an exosome according to an embodiment of the present invention mixed with a hyaluronic acid filler and then photographed using NTA equipment.
  • Fig. 9 is a Western blot result showing that the stabilized exosome filler composition of the present invention is stably maintained without degradation of the surface protein of exosome, CD63, by trypsin treatment.
  • HS68 cells a human dermal fibroblast, were purchased from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific) The cells were subcultured in DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO 2 at 37 ° C.
  • adipose-derived stem cells were cultured at 5% CO 2 and 37 ° C. Then, the cells were washed with a phosphate-buffered saline (purchased from ThermoFisher Scientific), replaced with serum-free, non-phenol red medium, cultured for 1 to 10 days, and the supernatant .
  • a phosphate-buffered saline purchased from ThermoFisher Scientific
  • Trehalose was added to the culture medium in an amount of 2% by weight in order to obtain an exosome having a uniform particle size distribution and high purity in the process of separating exosome.
  • the culture was filtered with a 0.22 ⁇ m filter to remove impurities such as cellular debris, waste products and large particles.
  • the filtered cultures were immediately separated to isolate exosomes.
  • the filtered culture was stored in a refrigerator (image below 10 ° C) and used for exosome isolation.
  • the filtered culture was frozen in an ultra-low temperature freezer at -60 ° C or lower, and thawed, followed by exosome isolation. Then, the exosomes were separated from the culture medium by using Tangential Flow Filtration (TFF).
  • TMF Tangential Flow Filtration
  • Example 1 a TFF (Tangential Flow Filtration) method was used for separating, concentrating, desalting and diafiltration exosomes from a culture filtrated with a 0.22 ⁇ m filter.
  • a cartridge filter also called a hollow fiber filter (purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used.
  • the TFF filter can be selected by a variety of molecular weight cutoffs (MWCO). Exosomes were selectively isolated and concentrated by selected MWCOs, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than MWCO were removed.
  • MWCO molecular weight cutoffs
  • TFF filters of MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da were used.
  • the culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, and substances smaller than MWCO were removed to separate the exosomes.
  • Separated and concentrated exosomal solutions were further desalted and buffered (diafiltration) using the TFF method.
  • the desalination and buffer exchange are performed by continuous diafiltration or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably, Was performed using a buffer solution having a volume of 12 times or more.
  • 2% by weight of trehalose dissolved in PBS was added to obtain an exosome having a uniform particle size distribution and high purity.
  • 6A to 6E show the effect of obtaining an exosome having a high purity and a uniform particle size distribution with trehalose treatment at a high yield.
  • the amount of protein in the fractions of the isolated exosome, culture medium, and TFF separation process was measured using BCA colorimetry (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence (purchased from Sigma).
  • BCA colorimetry purchased from ThermoFisher Scientific
  • FluoroProfile fluorescence purchased from Sigma.
  • the extent to which the exosome was isolated and concentrated by the TFF method of the present invention and the removal of proteins, lipids, nucleic acids, and low-molecular compounds was monitored by a protein determination method, and the results are shown in FIG. As a result, it was found that the protein present in the culture solution was very effectively removed by the TFF method of one embodiment of the present invention.
  • FIG. 3 shows the results of comparing the productivity and purity in five independent batches when isolating exosomes by the TFF method of one embodiment of the present invention. As a result of analyzing the results obtained from the five independent batches, it was confirmed that the exosome can be separated very stably by the TFF method of one embodiment of the present invention.
  • 5A to 5C show results of NTA analysis of the size distribution of exosome according to whether or not trehalose was added after the exosome was separated by the TFF method.
  • concentration of trehalose was increased to 0% by weight, 1% by weight and 2% by weight (from top to bottom of FIGS. 5A to 5C) and repeated three times.
  • particles having a size of 300 nm or more were identified, while particles having a size of 300 nm or more were reduced by increasing the amount of trehalose, and the size distribution of the exosome was uniformized .
  • FIG. 4D shows the presence of CD9, CD63, CD81 and TSG101 markers as a result of performing Western blotting on isolated exosomes according to the method of one embodiment of the present invention.
  • Anti-CD9 purchased from Abcam
  • anti-CD63 purchasedd from System Biosciences
  • anti-CD81 purchasedd from System Biosciences
  • anti-TSG101 purchasedd from Abcam
  • FIG. 4E shows the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer on exosomes isolated according to the method of one embodiment of the present invention.
  • Human CD63 isolation / detection kit purchased from ThermoFisher Scientific
  • PE-mouse anti-human CD63 PE-Mouse anti markers were stained using the PE-mouse CD63 (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD), and analyzed using a flow cytometer (ACEA Biosciences) Respectively.
  • the exosome used in the hyaluronic acid-based filler composition of the present invention is not limited to the exosome of the above-mentioned embodiments, and various exosomes which are used in the art or can be used in the future can be used, to be. It is to be understood that the exosomes separated according to the above embodiments are to be understood as an example of exosomes that can be used in the hyaluronic acid-based filler composition of the present invention, and the present invention is not limited thereto.
  • exosomes were treated by concentration to the cells and the proliferation rate of the cells was confirmed.
  • HS68 cells were suspended in DMEM containing 10% FBS, and the cells were mixed with 80 ⁇ 90% confluency and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. After 24 hours, the culture solution was removed, and the cell survival rate was evaluated by culturing the exosome prepared in Example 2 for each concentration and culturing for 24 to 72 hours.
  • WST-1 reagent purchased from Takara
  • MTT reagent purchased from Sigma
  • CellTiter-Glo reagent purchased from Promega
  • Aramar Blue reagent alamarBlue reagent purchased from ThermoFisher Scientific
  • 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 exosome, and it was confirmed that no cytosoxicity by exosome was observed within the tested concentration range (FIG. 7).
  • the exosome prepared in Example 2 was mixed with at least one of hyaluronic acid, hyaluronic acid salt, and hyaluronic acid gel to prepare a stabilized exosome filler composition.
  • hyaluronic acid salt sodium hyaluronate may be used, but not limited thereto, and various hyaluronic acid salts used in the art can be used.
  • the hyaluronic acid gel may be a non-crosslinked hyaluronic acid or a crosslinked hyaluronic acid gel.
  • the crosslinked gel for example, restylene can be used.
  • a hyaluronic acid-based filler composition in which at least one of hyaluronic acid, hyaluronic acid salt, and hyaluronic acid gel was mixed was sterilized using a heat sterilization or sterilization filter.
  • a filler composition of stabilized exosomes was prepared by mixing exosomes in a hyaluronic acid-based filler composition in which at least one of sterilized hyaluronic acid, hyaluronic acid salt, or hyaluronic acid gel was mixed. At this time, the exosomes can be further sterilized using a sterilization filter before mixing with the hyaluronic acid-based filler composition.
  • the concentration of exosomes in the stabilized exosome filler composition can be adjusted to, for example, from about 1 x 10 5 particles / mL to 1 x 10 11 particles / mL (or about 0.001 to 1000 ⁇ g / mL for protein concentrations) .
  • the exosomes prepared in Example 2 were mixed with a hyaluronic acid filler at a concentration of 1 ⁇ 10 8 particles / mL, and then immediately mixed with the hyaluronic acid-based filler composition using NTA equipment (FIG. 8).
  • NTA equipment NTA equipment
  • the hyaluronic acid-based filler mixed with exosomes was stored at -20 ° C for 30 days, and then photographed using NTA equipment to confirm that the exosome was well maintained in the hyaluronic acid-based filler composition 8).
  • the last photograph on the right side of FIG. 8 confirms that there is almost no particle in the filler having no exosome mixed therein as a negative control photograph without exosome.
  • the stabilized exosome filler composition of the present invention can be suitably applied to the realization of hyaluronic acid-based filler which is easy to inject and less painful to the patient, with long-lasting cosmetic or therapeutic effect .
  • Example 6 Experiment to confirm the increase of stability of exosome by hyaluronic acid
  • the CD63 protein is a membrane protein specifically present on the surface of exosomes. It is an important marker protein used when confirming whether or not the endoplasmic reticulum secreted or released from cells is an exosome. If the CD63 protein is not detected in the endoplasmic reticulum isolated from the stem cell culture, it can be judged that the exosome is not present in the culture solution or that the exosome has lost its inherent characteristics due to the stability problem. Therefore, the stability of exosomes can be evaluated by detecting the CD63 marker protein under specific conditions. In order to confirm the stability enhancement effect of exosome by hyaluronic acid in the stabilized exosome filler composition of the present invention, the following experiment was conducted.
  • the protease, trypsin, And CD63 an exosomal surface marker protein
  • the stabilized exosome filler composition of the present invention was prepared by mixing 30 mg of hyaluronic acid at a concentration of 24 mg / mL in exosomes obtained by concentrating 8 times the exosome prepared in Example 2.
  • trypsin purchased from Sigma
  • 5 ⁇ L of trypsin was treated with the exo single sample and the sample in which the exosomes were mixed with hyaluronic acid, followed by reaction at 37 ° C. for 30 minutes.
  • Protease inhibitor cocktail purchased from Sigma was then treated to inhibit the activity of trypsin.
  • Hyaluronidase obtained from Sigma, which degrades hyaluronic acid, was treated to a final concentration of 1.25 mg / mL and then incubated at 37 < 0 > C for 30 minutes Lt; / RTI > Negative controls without trypsin treatment were also electrophoresed on the exosomal samples.
  • the same amount of protein was developed by electrophoresis in 8% SDS-polyacrylamide gel, and then electroblotted on PVDF membrane. The membrane was blocked with TBS-T buffer (5% skim milk) in TBS-T buffer (0.1% Tween-20 in TBS) to block non-specific binding between anti-CD63 antibody and protein at room temperature for 1 hour.
  • the anti-CD63 antibody was firstly treated at 4 ° C overnight, washed three times with TBS-T buffer for 10 minutes, and incubated with anti-rabbit immunoglobulin G (Jackson Immuno Research Laboratories Inc.) And then washed three times with TBS-T buffer for 10 minutes.
  • CD63-specific protein expression was confirmed on the final PVDF membrane using a chemiluminescent reagent (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific).
  • the stabilized exosome filler composition (middle lane in FIG. 9) of the present invention stably maintains CD63, the surface protein of exosomes, without being decomposed by trypsin treatment.
  • the results of the tryptic treatment of the stabilized exosomal filler composition of the present invention with the CD63 protein Western blot results are almost the same as the sample without trypsin treatment in the exosomes (left lane of FIG. 9).
  • the stabilized exosome filler composition of the present invention has an effect of increasing the stability of exosome. Therefore, the stabilized exosome filler composition of the present invention can maintain the cosmetic or therapeutic effect derived from exosome for a long time as the stability of exosome increases, and the cosmetic or therapeutic effect can be maintained for a long time Can be suitably applied to the implementation of hyaluronic acid-based fillers that are convenient and less painful to the patient.

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Abstract

La présente invention concerne une composition de charge contenant des exosomes stabilisés, la composition de charge comprenant : de l'acide hyaluronique et/ou un sel d'acide hyaluronique et/ou un gel d'acide hyaluronique ; et des exosomes conservés par mélange avec l'acide hyaluronique et/ou le sel d'acide hyaluronique et/ou le gel d'acide hyaluronique. La composition de charge contenant des exosomes stabilisés selon la présente invention permet de conserver des effets cosmétiques ou thérapeutiques dérivés des exosomes pendant une longue durée grâce à l'augmentation de la stabilité exosomale. La composition de charge peut être appliquée de manière appropriée à l'utilisation d'une charge à base d'acide hyaluronique, qui est pratique à injecter et moins douloureuse pour un patient étant donné que les effets cosmétiques ou thérapeutiques sont conservés pendant longtemps.
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EP3827810A4 (fr) * 2018-09-06 2022-05-25 Exocobio Inc. Composition cosmétique contenant un exosome dérivé de galactomyces comme ingrédient actif
EP3922313A4 (fr) * 2019-02-01 2022-12-21 Da Vinci Universale Co., Ltd. Nouveau composant capable de réguler une fonction biologique
CN112870339A (zh) * 2019-12-05 2021-06-01 珠海天使干细胞生物科技有限公司 一种细胞外泌体在眼贴膜中的应用
EP4129317A4 (fr) * 2020-06-04 2024-04-10 Exocobio Inc Composition pour l'anti-inflammation, la cicatrisation de plaies ou la stimulation de la cicatrisation des plaies, comprenant des exosomes dérivés de cellules souches de rose en tant que principe actif
CN113499477A (zh) * 2021-05-12 2021-10-15 浙江大学医学院附属邵逸夫医院 搭载间充质干细胞外泌体的细胞外基质凝胶及其制备方法
WO2022259526A1 (fr) * 2021-06-11 2022-12-15 株式会社フルステム Agent favorisant la production d'exosome et procédé favorisant la production d'exosome
CN113713176B (zh) * 2021-09-02 2022-09-13 首都医科大学附属北京口腔医院 一种水凝胶及其制备方法与应用
CN113713176A (zh) * 2021-09-02 2021-11-30 首都医科大学附属北京口腔医院 一种水凝胶及其制备方法与应用
CN114099772A (zh) * 2021-11-29 2022-03-01 陕西科美致尚生物科技有限公司 一种用于面部塑形填充的材料及其制备方法
CN114939098A (zh) * 2022-05-19 2022-08-26 明德南加(成都)生物技术有限公司 一种负载外泌体的水凝胶及其制备方法和应用
CN114939098B (zh) * 2022-05-19 2023-09-26 明德南加(成都)生物技术有限公司 一种负载外泌体的水凝胶及其制备方法和应用
WO2024085419A1 (fr) * 2022-10-21 2024-04-25 주식회사 엑티브온 Procédé d'extraction d'exosome dérivé de plante, exosome ainsi isolé et son utilisation cosmétique
CN115581810A (zh) * 2022-10-26 2023-01-10 济宁医学院附属医院 一种富含外泌体的水凝胶及其制备方法和应用
CN115581810B (zh) * 2022-10-26 2023-12-22 济宁医学院附属医院 一种富含外泌体的水凝胶及其制备方法和应用
CN117205129B (zh) * 2023-11-08 2024-02-23 北京尧景基因技术有限公司 高山火绒草类囊泡的提取方法及其在制备皮肤细胞胶原再生、修复产品中的应用
CN117205129A (zh) * 2023-11-08 2023-12-12 北京尧景基因技术有限公司 高山火绒草类囊泡的提取方法及其在制备皮肤细胞胶原再生、修复产品中的应用

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