KR20190069277A - A composition comprising an exosome and/or extracellular vesicle derived from stem cell as an active ingredient and its application for preventing, suppressing, alleviating or improving sensitive skin - Google Patents

Abstract

Provided is a composition which comprises exosomes and/or extracellular vesicles derived from stem cells and exosomes separated from a stem cell culture medium and/or at least one selected from a group consisting of extracellular vesicle-contained fractions, as an active component and is configured to prevent, suppress, alleviate, and ameliorate the sensitive skin. According to the present invention, the composition can effectively reduce production of inflammatory substances and prevent or suppress oxidative stress, thereby effectively preventing, inhibiting, alleviating, or ameliorating the sensitive skin and preventing, alleviating, ameliorating or treating various sensitive skin diseases. In addition, when the composition of the present invention is directly applied to the skin, skin troubles related to the sensitive skin, skin redness, and erythema can be remarkably alleviated or ameliorated.

Description

[0001] The present invention relates to a composition comprising an exosome derived from stem cells and / or an extracellular endoplasmic reticulum as an active ingredient for the purpose of preventing, inhibiting, alleviating or improving the sensitive skin of an exosome and / or an extracellular vesicle derived from a stem cell as an active ingredient its application for preventing, suppressing, alleviating, or improving sensitive skin}

The present invention relates to a composition for preventing and inhibiting sensitive skin of a composition comprising at least one of exosome derived from stem cells and / or extracellular endoplasmic reticulum, or fraction containing exosome and / or extracellular endoplasmic reticulum isolated from a stem cell culture liquid as an active ingredient , Mitigation or improvement.

The present invention also relates to a composition for preventing, suppressing, alleviating or improving sensitive skin, an external preparation for skin and a cosmetic composition containing the composition. The present invention also relates to a pharmaceutical composition for preventing, alleviating, ameliorating or treating a sensitive skin disease comprising the composition,

Further, the present invention can obtain a large amount of exosome and / or extracellular endoplasmic reticulum derived from stem cells having high purity and uniform particle size distribution which can be clinically applied for prevention, suppression, alleviation or improvement of sensitive skin and the like , And a clinically and commercially superior technique capable of providing a large amount of a composition containing the exosome and / or extracellular endoplasmic reticulum derived from stem cells obtained in this way having excellent functional activity as an effective ingredient at low cost.

Sensitive skin is said to be sensitive to external changes or environmental changes. Generally, sensitive skin is sensitive to substances that are not problematic for most people. Skin which is likely to cause skin troubles, skin barrier function is deteriorated, and it is constitutionally sensitive to allergic substances and irritants. Refers to a skin that is inherently resistant to the skin due to shortage, overwork, physiological change, seasonal change, mental stress, or the like, which is liable to cause temporary skin troubles with stimulants when the physiological function of the skin is weakened. In some cases, sensitive skin is defined as a skin that is more responsive to contact stimuli or allergic responses than the average group.

Factors that cause the skin condition to become sensitive include a decrease in skin barrier function, a decrease in skin irritation threshold, skin dryness, physicochemical stimulation, stress, physical condition, seasonal change, ultraviolet rays, physiology and the like. In addition, there are cases where the skin is sensitized by wrong skin care, or the skin is sensitized by psychological anxiety.

Generally, susceptible skin holders complain of erythema, dry skin, rough skin, stinging and itching. Skin cosmetics for the improvement of sensitive skin are minimizing side effects by preliminarily eliminating substances that may cause irritation. As a more aggressive method, recently developed functional cosmetics and medicinal products are improving the sensitive skin by suppressing the expression of cytokines and inflammatory mediators, and strengthening the skin barrier.

However, non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, neuropeptide antagonists, and COX inhibitors, which are effective for controlling cytokines and inhibiting the expression of inflammatory mediators, have serious side effects and are problematic from the standpoint of stability in cosmetic formulations. In view of these problems, studies on an external preparation for skin and a cosmetic composition for improving sensitive skin using natural materials as raw materials are actively researched. For example, efforts have been made to improve sensitive skin using watermelon endothelial extract, Indian oriental resin extract, lavender extract, black bean extract, and top extract.

However, in the case of cosmetics for the improvement of sensitive skin using these natural substances as raw materials, since the content of active ingredients in natural extracts is small, it is necessary to use a large amount in order to substantially improve the sensitive skin, and most of them are natural materials It is used only for marketing, but it requires more scientific research in terms of preventing, suppressing, alleviating, improving and treating sensitive skin.

Meanwhile, methods for improving or treating skin conditions or diseases using stem cells have been proposed. Embryonic stem cells or embryonic stem cells derived from embryonic stem cells are excellent in their ability to differentiate and regenerate and have a low rejection rate. However, they can not be applied to clinical practice due to ethical problems, and there is a risk of forming tumors. As an alternative, a method for improving or treating skin condition or disease using adult stem cells has been proposed. However, when adult adult stem cells use an adult adult stem cell, there is a risk of causing graft-versus-host disease. In order to treat using adult stem cells, There is a complicated and costly problem in that stem cells are collected and cultured.

In recent years, attempts have been made to improve or treat skin conditions or diseases using a culture solution obtained by culturing adult stem cells in consideration of the problems of stem cells as described above. However, adult stem cell culture contains various proteins, cytokines, and growth factors secreted by adult stem cells. On the other hand, components such as antibiotics and animal-derived serum added to prevent contamination, It is highly likely to be exposed to various risks when used on skin because it is included. Therefore, in order to use the adult stem cell culture solution, a separate process is required to remove waste products and various dangerous components, which is costly.

Recently, studies have shown that secretory cells contain a variety of bioactive factors that control cell behavior. In particular, there are reports that exosome or extracellular (Extracellular vesicle) ', and its components and functions are actively researched.

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.

The exosome is a vesicle of several tens to several hundreds of nanometers in size, consisting of double lipid membranes identical to the cell membrane structure, and contains proteins, mRNAs, miRNAs and DNAs called exosomes 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 a cell-mediated signal transduction mediator that regulates cell motility, 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. In the case of proliferating stem cell-derived exosomes, cell movement such as cell migration, proliferation and differentiation, and stem cell characteristics related to tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).

However, in spite of various studies such as suggesting possibility of treatment of some diseases using exosome and / or extracellular endoplasmic reticulum, more detailed clinical and non-clinical studies are required, and in particular, exosome and / The inventors of the present invention have developed a technique for scientifically identifying a variety of targets to which the exosome and / or the extracellular endoplasmic reticulum can be applied to treat various diseases.

The present inventors have sought to develop a new sensitive skin improving and anti-inflammatory substance capable of solving the problems of anti-inflammation, skin safety, and stability in formulation of cosmetics. Accordingly, the present inventors have conducted intensive studies on the new uses of exosome and / or extracellular endoplasmic reticulum derived from stem cells, and found that exosome and / or extracellular endoplasmic reticulum are effective for prevention, inhibition, And thus the present invention has been completed.

It should be understood, however, that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention, and not as an admission that it can be used as the "prior art "

Korean Registered Patent No. 10-1081178 (2011.11.07) Korean Registered Patent No. 10-1151093 (July 7, 2012) Korean Registered Patent No. 10-1663912 (October 10, 2016) Korean Patent Publication No. 10-2017-0099743 (2017.09.01) Korean Patent Publication No. 10-2016-0116802 (October 10, 2016)

Pin Li et al., Progress in Exosome Isolation Techniques, Theranostics, 2017, 7 (3): 789-804 (2017.01.26) Coumans et al., Methodological Guidelines to Study Extracellular Vesicles, Circulation Research, 2017, 120: 1632-1648 (Jul.

It is an object of the present invention to provide a method for the prevention of sensitive skin of a composition comprising at least one of exosome derived from stem cells and / or extracellular endoplasmic reticulum, or fraction containing exosome and / or extracellular endoplasmic reticulum isolated from a stem cell culture , ≪ / RTI >

It is another object of the present invention to provide a composition for preventing, suppressing, alleviating or improving sensitive skin comprising the composition, an external preparation for skin and a cosmetic composition.

It is another object of the present invention to provide a pharmaceutical composition for preventing, alleviating, improving or treating a sensitive skin disease comprising the composition.

 It is still another object of the present invention to provide a stem cell-derived exosome and / or extracellular endoplasmic reticulum capable of obtaining a large amount of a high purity but uniform particle size distribution from a stem cell, / ≪ / RTI > extracellular < RTI ID = 0.0 > extracellular < / RTI >

Another object of the present invention is to provide a method for preventing, suppressing, alleviating or improving sensitive skin using the composition.

It is still another object of the present invention to provide a cosmetic method using the composition to prevent, inhibit, alleviate or improve sensitive skin.

It is another object of the present invention to provide a method for preventing, alleviating, ameliorating or treating a sensitive skin disease using the composition.

However, the above-described problems of the present invention are illustrative and not intended to limit the scope of the present invention. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention provides a method for producing a stem cell culture, which comprises culturing at least one species selected from the group consisting of exosome derived from stem cells and / or extracellular endoplasmic reticulum, and fraction containing exosome and / As an active ingredient, a composition for preventing, suppressing, alleviating or ameliorating a sensitive skin.

In addition, the present invention can obtain a large amount of exosome and / or extracellular endoplasmic reticulum derived from stem cells having a high purity and a uniform particle size distribution which can be clinically applied for prevention, suppression, alleviation or improvement of sensitive skin, The present invention provides novel clinical and commercial superior technologies that can provide a large amount of a composition containing the exosome and / or extracellular endoplasmic reticulum derived stem cell-derived excellent functional activity thus obtained as an effective ingredient at low cost.

As used herein, the term "Extracellular vesicles (EV)" is intended to encompass membrane vesicles, ectosomes, shedding vesicles, microparticles, It is used as a meaning.

As used herein, the term "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 to the structure of the cell membrane The particle size of the exosome may be variable depending on the type of stem cells to be separated, the method of isolation and the method of measurement) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, ) DOI 10.1007 / s00216-015-8535-3), proteins called exosomal cargo, mRNA, miRNA, and DNA. 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 a cell-mediated signal transduction mediator that regulates cell motility, including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known. The stem cell-derived exosome and / or extracellular endoplasmic reticulum used in the present invention can be isolated from the stem cell culture liquid by various methods known in the art and can be isolated according to the separation method of one specific example described herein (Korean Published Patent Application No. 10-2016-0116802; Pin Li et al., Progress in Exosome Isolation Techniques, Theranostics, 2017, 7 (3): 789-804; Coumans et al., Methodological Guidelines to Study Extracellular Vesicles, Circulation Research, 2017, 120: 1632-1648, etc.).

As used herein, the term "prevent, inhibit, alleviate or ameliorate sensitive skin" is constitutionally sensitive to allergic substances or irritants. Skin troubles are caused by skin, lack of sleep, overwork, Refers to preventing or suppressing symptoms such as erythema, skin dryness, skin roughness, flushing symptoms, stinging, itching, etc. appearing on skin which is likely to occur, or to alleviate or restore to normal skin conditions the symptoms already shown. In addition, "susceptible skin disease" is defined as a symptom of a symptom such as erythema, skin dryness, skin roughness, flushing, burning, itching, and the like, Is used in a broad sense.

The term "iontophoresis" as used herein refers to a method in which an ionized active ingredient is allowed to permeate through the skin by an electrical repulsive force by applying a minute current to the skin to which the effective substance is applied, . The iontophoresis used in one embodiment of the present invention is a method in which a current flows from an external power source to an electrode patch on the skin to introduce minute current into the skin, A method in which minute current is introduced, a method in which minute current is introduced into skin through a patch equipped with a reversed electrodialysis means for generating current through a difference in ion concentration between a high concentration electrolyte solution and a low concentration electrolyte solution, and the like . However, the present invention is not limited thereto, and it is needless to say that various types of iontophoresis can be used.

On the other hand, limited wrinkle improvement and skin regeneration effects using exosomes derived from stem cells have been reported. However, the skin regeneration using the exocytosis in the above-mentioned prior art is not related to the prevention, suppression, alleviation or improvement of the sensitive skin, but to the improvement of the wrinkles or the restoration of the skin elasticity through regeneration of the skin tissue. There have been attempts to regenerate dermis in the skin by using stem cells and stem cell culture to treat wounds and to regenerate skin tissue to improve skin elasticity. However, exosomes and / or cells There has been no known fact that there is an "prevention, inhibition, alleviation or improvement of sensitive skin" effect as defined above when using an outer envelope.

It is possible to clinically apply exosome and / or extracellular endoplasmic reticulum economically separated and purified from a stem cell culture medium to prevent, inhibit, alleviate or improve sensitive skin after culturing stem cells capable of mass culture so far The implemented therapeutic agent has not been developed. For example, fat-derived adipose-derived stem cells can be obtained in large quantities by simple procedures such as liposuction, and about 40 times more stem cells than bone marrow, umbilical cord or cord blood, , It is possible to obtain exosome and / or extracellular endoplasmic reticulum which have high purity and uniform particle size distribution from the adipose stem cell culture medium because there are many cellular debris such as cell debris, waste products, proteins and macro particles in the fat, It is difficult to economically separate in large quantities. Therefore, from the viewpoint of economy, there are technical barriers to massive separation of exosomes having a high purity particle size distribution from a stem cell culture solution.

When the composition of the present invention is applied to a pharmaceutical composition, an external preparation for skin or a cosmetic composition, the exosome and / or the extracellular endoplasmic reticulum derived from stem cells contained as an active ingredient have a significant effect on preventing, suppressing, And it can solve the safety problem of the stem cell itself or stem cell culture fluid. Therefore, the stem cell-derived exosome and / or extracellular endoplasmic reticulum contained in the composition of the present invention is a completely different mechanism from the conventional limited wrinkle improvement and skin regeneration, and thus can prevent, inhibit, alleviate or improve the effect of the sensitive skin And it is evident that this efficacy is entirely unpredictable from the prior art.

The composition for preventing, inhibiting, alleviating or ameliorating the sensitive skin of the present invention may be a composition for preventing or inhibiting, modifying or alleviating the skin of the exosome and / or extracellular endoplasmic reticulum derived from stem cells and a fraction containing exosome and / As an active ingredient.

In the composition of one embodiment of the present invention, the exosome and / or the extracellular endoplasmic reticulum or the fraction may be subjected to an ultracentrifugation, a density gradient centrifugation, an ultrafiltration, a size exclusion (Eg, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome extraction kit a total exosome isolation kit, or a polymer based precipitation, which is used in the art or can be used in the future.

In the composition of one embodiment of the present invention, the exosome and / or extracellular endoplasmic reticulum can be obtained by performing the following steps: (a) adding an excipient to the stem cell culture, (b) (C) separating the exosome and / or extracellular endoplasmic reticulum from the filtered stem cell culture using TFF (Tangential Flow Filtration), and (d) An excipient is added to a buffer solution used for desalting and diafiltration, and TGF (Tangential Flow Filtration) using a buffer solution to which the excipient is added is used to separate the exo and / or extracellular endoplasmic reticulum Performing desalting and diafiltration.

In addition, in the composition of one embodiment of the present invention, the fraction can be obtained by performing the following steps: (a) adding an excipient to the stem cell culture solution, (b) adding the excipient (C) separating the fraction containing the exosome and / or extracellular endoplasmic reticulum as an active ingredient from the filtered stem cell culture liquid by using TFF (Tangential Flow Filtration), and d) adding an excipient to the buffer used for desalting and diafiltration, and desalting and buffer exchange (using TFF (Tangential Flow Filtration) using the buffer containing the excipient) diafiltration.

In the composition according to one embodiment of the present invention, when the excipient is added to the buffer solution used for desalting and diafiltration in the step (d), the exoose having a uniform particle size distribution and high purity and / The vesicle, or fractions containing it, can be effectively obtained (see Fig. 6).

However, the exosome and / or extracellular endoplasmic reticulum and fractions of the present invention are not limited thereto, and those obtained by various separation methods as described above may be used for the composition for preventing, suppressing, alleviating or improving sensitive skin of the present invention Of course. Hereinafter, the separation method of one embodiment described in the present specification can be applied to the exosome and / or the extracellular endoplasmic reticulum or the fraction containing the exosomal and / or extracellular endoplasmic reticulum which can be used for the composition for preventing, suppressing, alleviating or improving the sensitive skin It should be understood that the present invention is not limited thereto.

The term "excipient " means a substance added for the purpose of imparting a suitable hardness or shape to a drug or making it easy to handle with a certain weight when the amount of the main agent is small, In the present invention, the term "excipient (or also referred to as an additive)" as known in the art is used to efficiently distinguish exosome and / or extracellular endoplasmic reticulum from impurities such as cellular debris, waste products, Function. As an example without limiting the invention, excipients include sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanose, dextrose, The present invention relates to a pharmaceutical composition containing at least one of glucose, galactose, fructose, talose, mannose, But are not limited to, bacteriocin, bacteriocin, bacteriocin, bacteriocin, bacteriocin, borax, xylulose, aldoheptose, heptulose, octolose, gentianose, The present invention relates to a method for producing a starch derivative, which comprises administering an effective amount of a surfactant selected from the group consisting of verbascose, manitol, maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, For example, Tween-20, Triton X-100, Pluonic F-68, etc.), histidine, glycerol and cholesterol.

In the composition of one embodiment of the present invention, desalination 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.

In the composition of one embodiment of the present invention, a TFF filter with a molecular weight cutoff (MWCO) of 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da, or a 0.05 탆 TFF filter may be used for TFF.

In the composition of one embodiment of the present invention, 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).

In the composition of one embodiment of the present invention, the filtered stem cell culture solution may be subjected to sonication before TFF.

In a composition according to an embodiment of the present invention, the exosome and / or extracellular endoplasmic reticulum or fraction thereof can be used for the expression of TNF-α mRNA, IL-6 mRNA expression, IL-1β mRNA expression, iNOS mRNA expression, NO production.

In the composition of one embodiment of the present invention, the exosome and / or extracellular endoplasmic reticulum or fraction is isolated from different batches of cell culture, and each batch of exosome and / The ER (or fractions containing each of them) reduce TNF-α mRNA expression, IL-6 mRNA expression, IL-1β mRNA expression, iNOS mRNA expression, and NO production in in vitro assays at the same or similar levels to each other .

In the composition of one embodiment of the present invention, the exosome and / or the extracellular endoplasmic reticulum or the fraction are separated from a cell culture liquid subdivided from a single batch a plurality of times, and each exosomes and / TNF-α mRNA expression, IL-6 mRNA expression, IL-1β mRNA expression, iNOS mRNA expression, and NO production in the in vitro assays at the same or similar levels with each other Can be reduced.

In the composition of one embodiment of the present invention, the exosome and / or the extracellular endoplasmic reticulum or the fraction are separated from the cell culture medium cultured in different production cycles, and the exosome and / or extracellular endoplasmic reticulum Or fractions containing each of them) may reduce TNF- [alpha] mRNA expression, IL-6 mRNA expression, IL-1 [beta] mRNA expression, iNOS mRNA expression, and NO production in an in vitro assay at the same or similar level to each other .

In the composition of one embodiment of the present invention, the type of the stem cells is not limited, but may be preferably mesenchymal stem cells such as fats, bone marrow, umbilical cord or cord blood-derived stem cells, Lt; / RTI > 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 composition of one embodiment of the present invention can be effectively used to prevent, inhibit, alleviate or improve sensitive skin caused by various causes as listed above.

The composition of one embodiment of the present invention may be prepared from a pharmaceutical composition. When the composition of one embodiment of the present invention is made from a pharmaceutical composition, the pharmaceutical composition according to one embodiment of the present invention may be various oral or parenteral formulations.

The pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methylcellulose, But are not limited to, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition according to one embodiment of the present invention may be administered orally or parenterally in the form of powders, pills, tablets, capsules, suspensions, emulsions, syrups, granules, elixirs, aerosols, Or in the form of sterile injectable solutions.

Administration of the pharmaceutical composition according to one embodiment of the present invention means introduction of a predetermined substance into a patient by any appropriate method, and the administration route of the pharmaceutical composition may be administered through any ordinary route so long as the drug can reach the target tissue . For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and examples of the parenteral administration include transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, Administration, topical administration, rectal administration, and the like. However, it is not limited thereto and does not exclude various methods of administration known in the art. In addition, the pharmaceutical composition of one embodiment of the present invention may be administered by any device capable of moving the active substance to a target tissue or cell. An effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect the therapeutic effect of the disease.

The solid preparation for oral administration of the pharmaceutical composition of one embodiment of the present invention may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition, the solid preparation for oral administration may contain a lubricant (lubricant) such as silica, stearic acid, magnesium stearate, calcium stearate, talc, and polyethylene glycol in addition to the excipient. The liquid preparation for oral administration of the pharmaceutical composition of one embodiment of the present invention may contain various excipients such as wetting agent, sweetening agent, fragrance, preservative, etc. in addition to water and liquid paraffin which are simple diluents.

The preparation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a left-over preparation. The preparation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may also be prepared by injection. The injection agent of one embodiment of the present invention may be, but not limited to, an aqueous injection agent, a non-aqueous injection agent, an aqueous suspension injection agent, a non-aqueous suspension injection agent, or a solid injection agent used by dissolving or suspending. Injection agents of one embodiment of the present invention may be administered orally or parenterally depending on the type thereof, for example, distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoic acid estradiol, , Arsenobenzazole sodium, or streptomycin sulfate, and may optionally contain stabilizers or preservatives.

The compounding ratio of the pharmaceutical composition according to one embodiment of the present invention can be appropriately selected depending on the kind, quantity and form of the additional components as described above. For example, for the total amount of the injection, the pharmaceutical composition of the present invention may contain about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, a suitable dose of the pharmaceutical composition of one embodiment of the present invention may be appropriately determined depending on the kind of disease of the patient, the severity of the disease, the type of the formulation, the formulation method, the age, sex, weight, health condition, diet, Can be adjusted according to the method. For example, when the pharmaceutical composition of one embodiment of the present invention is administered to an adult, it may be administered at a dose of 0.001 mg / kg to 100 mg / kg per day for 1 to several times.

On the other hand, when the composition of one embodiment of the present invention is manufactured as an external preparation for skin, it is possible to use a component commonly used in cosmetics or external preparation for skin such as a moisturizer, an antioxidant, Absorbents, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately compounded as needed.

In addition, the external preparation for skin according to one embodiment of the present invention may be used in combination with a conventional agent (for example, an antioxidant, an antiseptic, A sensitive skin improving agent and / or a moisturizing agent may be mixed together and used. For example, the exosome and / or extracellular endoplasmic reticulum of the present invention or a fraction containing the exosome and / or the extracellular endoplasmic reticulum of the present invention can be administered to at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel Supported or mixed. In the external preparation for skin of one embodiment of the present invention, the kind of the hydrogel is not limited, but it may be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. Wherein the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum And 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 the cosmetic composition according to one embodiment of the present invention can be used in the form of, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, , Mist, foundation, powder, oil paper, and the like. For example, the external preparation for skin and / or cosmetic composition may be applied or deposited on at least one surface of the patch, mask pack, or mask sheet.

When the external preparation for skin according to one embodiment of the present invention is made of a cosmetic composition, it is used for the purpose of preventing, suppressing, alleviating or improving the sensitive skin, and the cosmetic preparation can be produced in any formulation conventionally produced in the art . For example, it is possible to use a lotion, a mask pack, a mask sheet, a softener, a nutritional lotion, a convergent lotion, a nutritional cream, a massage cream, an eye cream, a cleansing cream, an essence, an essence, a cleansing lotion, a cleansing foam, But are not limited to, soaps, shampoos, surfactant-containing cleansers, bath salts, body lotions, body creams, body oils, body essences, body cleansers, hair dyeing agents, hair tonics and the like.

The external preparation for skin and / or cosmetic composition according to one embodiment of the present invention includes components commonly used in external preparations for skin and / or cosmetics and includes conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, And may include a carrier. Further, in the respective formulations for the external preparation for skin and / or cosmetic composition, the other ingredients can be mixed and selected without difficulty by the person skilled in the art depending on the kind of the external preparation for skin and / or cosmetic composition or purpose of use.

Another embodiment of the present invention provides a cosmetic method using the above cosmetic composition to prevent, inhibit, alleviate or improve sensitive skin.

The cosmetic method of one embodiment of the present invention comprises the steps of (a) applying the cosmetic composition directly onto the skin of a mammal, (b) applying the patch, mask pack or mask sheet coated or deposited with the cosmetic composition to the skin , Or sequentially advancing the above (a) and (b).

The cosmetic method of one embodiment of the present invention may further comprise performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the cosmetic composition is applied. In addition, the cosmetic method of one embodiment of the present invention may further comprise contacting or attaching the iontophoresis device to the skin of the mammal.

In the cosmetic method according to one embodiment of the present invention, the iontophoresis device is composed of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A mask pack, or a mask sheet on which the at least one battery is mounted.

Another embodiment of the present invention provides a method of preventing, alleviating, ameliorating or treating a sensitive skin disorder, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition.

The composition of the present invention can reduce the production of inflammatory substances and prevent or inhibit oxidative stress, thereby effectively preventing, suppressing, alleviating or ameliorating sensitive skin and preventing, alleviating, ameliorating, or treating various sensitive skin diseases. In addition, when the composition of the present invention is directly applied to human skin, it is possible to remarkably alleviate or improve skin troubles, redness symptoms and erythema associated with sensitive skin. Accordingly, the composition of the present invention can be usefully used as a composition for preventing, suppressing, alleviating or ameliorating sensitive skin, an external preparation for skin and a cosmetic composition.

Further, the present invention can obtain a large amount of exosome and / or extracellular endoplasmic reticulum-derived stem cell-derived or fractions containing the same at a low cost at a high purity and a uniform particle size distribution. Accordingly, the present invention can provide a large amount of a composition containing an exosome and / or an extracellular endoplasmic reticulum derived from a stem cell having excellent functional activity, or a fraction containing the same as an effective ingredient at low cost. In addition, the composition of the present invention is scale-upable and is also suitable for GMP (Good Manufacturing Practice).

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

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing a method of isolating and purifying an exosome and / or an extracellular endoplasmic reticulum in a method for producing an exosome and / or an extracellular endoplasmic reticulum and / or a fraction containing the same according to an embodiment of the present invention Fig.
FIG. 2 is a graph showing the relationship between the total amount of proteins contained in a solution (Relative) and the amount of proteins contained in a solution by a step of preparing exosome and / or extracellular endoplasmic reticulum and / amount of protein). The ratio of the total amount of protein in each step was expressed as a relative ratio of total protein amount to the total stem cell culture 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 the exosome and / or extracellular endoplasmic reticulum obtained according to one embodiment of the present invention. The productivity of the exosome and / or extracellular endoplasmic reticulum was calculated as "the number of particles of exosome and / or extracellular endoplasmic reticulum obtained per mL of stem cell culture (CM)", and the purity of the exosome and / Quot; number of particles of exosome and / or extracellular < RTI ID = 0.0 > vesicle < / RTI > per gram of protein unit contained in the final fraction. The experimental results show the results obtained in five different batches.
Figure 4 shows the results of physical characterization of the exosome and / or extracellular endoplasmic reticulum obtained according to one embodiment of the present invention. "A " represents particle size distribution and particle number by TRPS (tunable resistive pulse sensing) analysis. "B" represents particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis. "C " shows the particle image by transmission electron microscopy (TEM) according to the magnification. "D" represents Western blot results of exosome and / or extracellular cell body obtained according to one embodiment of the present invention. "E" indicates flow cytometric analysis results for CD63 and CD81 in marker analysis for exosome and / or extracellular cell body obtained according to one embodiment of the present invention.
FIG. 5 shows the results of NTA analysis of particle size distribution (uniform distribution) showing that a uniform particle size distribution and high purity of exosome and / or extracellular endoplasmic reticulum upon addition of excipient. As the amount of the excipient added increases, a particle size distribution having a single peak can be obtained.
FIG. 6 shows the results of NTA analysis showing the particle size distribution depending on whether an excipient was added during the production of the exosome and / or the extracellular endoplasmic reticulum and / or fractions containing the same according to an embodiment of the present invention. "A" indicates the case where the excipient is added throughout the production process, and " B " indicates the case where the cell culture solution is stored after freezing, and the excipient is added after thawing. "D" shows the relative productivity and relative concentration of exosomes and / or extracellular endoplasmic reticulum isolated by methods A to C. The "E" shows the average size of the exosome and / or extracellular endoplasmic reticulum isolated by the methods A to C.
FIG. 7 shows the results of confirming that cytotoxicity of HS68 cells, which are human skin fibroblasts, was not exerted after treatment of exosome and / or extracellular endoplasmic reticulum according to one embodiment of the present invention
FIG. 8 shows that the expression level of TNF-a, IL-6, IL-1 beta and iNOS mRNA induced by LPS was decreased when RPS 264.7 cells were treated with LPS and the exosome and / or extracellular endoplasmic reticulum of the present invention And a real-time PCR result.
FIG. 9 shows experimental results of confirming the reduction effect of NO formation, which is one kind of inflammatory reaction, by exoose and / or extracellular endoplasmic reticulum according to one embodiment of the present invention. In Figure 9 DEXA refers to dexamethasone.
Figure 10 shows experimental results confirming the reduction effect of TNF- [alpha] formation, an inflammatory cytokine, by exocose and / or extracellular endoplasmic reticulum in accordance with one embodiment of the present invention. In Fig. 10, PBS represents phosphate-buffered saline, DEX represents dexamethasone, and each numeral represents exosome and / or extracellular vesicle throughput (占 퐂 / mL).
FIG. 11 is a graph showing the results of the isolation and purification of the exo-soma and / or extracellular endoplasmic reticulum according to one embodiment of the present invention in a culture solution produced by two different batches, The results of experiments confirming the reduction effect of NO formation are shown. C is phosphate-buffered saline, DEX is dexamethasone, Harvest 1 and 2 are exosomes isolated and purified respectively from cultures produced in primary and secondary batches 1 and / or Which means that extracellular endoplasmic reticulum was used.
FIG. 12 shows the effect of reducing the formation of NO by the exo and / or extracellular endoplasmic reticulum thus separated and purified by the method according to one embodiment of the present invention by dividing the culture solution produced in a single batch three times And shows the confirmed experimental result. C means phosphate-buffered saline.
Figure 13 shows the effect of reducing the formation of NO by the exo and / or extracellular endoplasmic reticulum thus separated and purified by the method according to one embodiment of the present invention from cultures produced in different production cycles And shows the confirmed experimental result. The degree of decrease in NO formation was expressed as a percentage (%) of NO reduced with respect to the treatment concentration of each exosome and / or extracellular endoplasmic reticulum for NO formed in the control group treated with phosphate buffer and dexamethasone.
FIG. 14 is a graph showing the effect of reducing NO formation by the separated exo and / or extracellular endoplasmic reticulum and the effect of decreasing NO formation by the extracellular endoplasmic reticulum isolated by the conventional precipitation method (PPT) according to one embodiment of the present invention The results of the comparison are shown. A is the result of NTA analysis of the extracellular endoplasmic reticulum isolated by the conventional precipitation method, B is the result of NTA analysis of the exosome and / or extracellular endoplasmic reticulum isolated and purified by the method according to one embodiment of the present invention, and C Is a graph comparing NO formation reduction effects. The degree of reduction of NO formation was expressed as a relative ratio (%) to the degree of NO formation reduction by the positive control dexamethasone (Dex).
Fig. 15 shows the results of fluorescence intensity measurement of exosomes stained with PKH67 and / or extracellular endoplasmic reticulum.
FIG. 16 is an image of a fluorescence microscope confirming the extent of the fluorescence-stained exosome and / or extracellular endoplasmic reticulum transferred into the pig skin tissue. Fluorescence microscope image obtained by diluting the fluoresceinized exosome and / or extracellular endoplasmic reticulum of the present invention in a buffer solution and applying it to the skin surface of the pig after a certain period of time.
FIG. 17 is a confocal fluorescence microscope image showing the extent of fluorescence-stained exosome and / or extracellular endoplasmic reticulum transferred into mouse skin tissue. Fluorescence microscope images obtained by diluting the fluoresceinized exosome and / or extracellular endoplasmic reticulum of the present invention in a buffer solution and applying it to the skin surface of a mouse after a certain period of time.
Fig. 18 is a graph comparing the total fluorescence intensity obtained by measuring the fluorescence intensity on each image from the images of Fig. 17; Fig.
FIG. 19 is a photograph showing a test product containing exosome and / or extracellular endoplasmic reticulum, according to an embodiment of the present invention, on human skin and comparing the skin condition before and after treatment.
FIG. 20 is a graph showing the results obtained by applying a composition comprising exosome and / or extracellular vesicle according to an embodiment of the present invention to human skin (affected part), and then applying ion to the skin (affected part) It is a photograph showing that the erythema of skin (lesion) is remarkably improved as a result of carrying out toporphism.
FIG. 21 is a graph showing the degree of necrosis and apoptosis of HDF cells using Annexin V / PI staining after inducing oxidative stress by hydrogen peroxide in HDF cells of human skin fibroblasts Showing flow cytometry results.
FIG. 22 is a graph showing the death rate of HDF cells obtained by summing up the sections showing early apoptosis and late apoptosis in the flow cytometry analysis of FIG. 21. FIG.
23 shows the results of co-treatment of the exosome and / or extracellular endoplasmic reticulum of the present invention together with LPS and IFN-y for THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages Time PCR results confirming that the expression level of IL-6 mRNA, which is a pro-inflammatory cytokine, is decreased.
24 to 26 show the results of pre-treatment of the exosome and / or extracellular endoplasmic reticulum of the present invention and culturing for a certain period of time on THP-1-derived macrophages obtained by differentiating THP-1 monocytes into macrophages Next, it is a graph showing a real-time PCR result in which the expression levels of IL-6, IL-1 [beta] and TNF- [alpha] mRNAs, which are proinflammatory cytokines, are decreased when LPS and IFN-y are treated.

Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

Example

Example 1: Culture of cells

Mouse macrophage cell line Raw 264.7 was purchased from Korean cell line bank and cultured. For cell culture, DMEM (purchased from Thermo Scientific) medium containing 10% fetal bovine serum (purchased from Thermo Scientific) and 1% antibiotic-antimycotics (purchased from Thermo Scientific) ₂ , and 37 ° C.

HS68 cells, a human dermal fibroblast, were purchased from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (purchased from Thermo Scientific) and 1% antibiotic-antimycotics (purchased from Thermo Scientific) The cells were subcultured in DMEM (purchased from Thermo Scientific) medium containing 5% CO ₂ and 37 ° C. HDF (Human dermal fibroblast) cells and medium were purchased from Cell Biotech (Seoul, Korea) and subcultured at 5% CO ₂ and 37 ° C.

According to the cell culturing method known in the art, adipose-derived stem cells were cultured at 5% CO ₂ and 37 ° C. Then, the cells were washed with a phosphate-buffered saline (PBS, purchased from Thermo Scientific), replaced with serum-free, non-phenol red medium, cultured for 1 to 10 days, and the supernatant Respectively.

In order to obtain a uniform and high purity exosome and / or extracellular endoplasmic reticulum in the process of separating exosome and / or extracellular endoplasmic reticulum, an excipient was added to the culture. Examples of excipients include sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanose, dextrose, glucose, galactose, fructose, But are not limited to, oats, mannose, tagatose, psicose, erythrose, erythroulose, erythritol, erythritol, arabinose, xylose, ricinose, ribose, ribulose, xylulose, But are not limited to, mannitol, mannitol, mannitol, fructose, mannitol, mannitol, fructose, mannitol, (For example, Tween-20, Triton X-ray powder, etc.), maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, 100, Pluonic F-68, etc.), histidine 0.01 to 30% by weight of at least one member selected from the group consisting of histidine, glycerol and cholesterol is added. After the excipient was added, the culture was filtered with a 0.22 탆 filter to remove impurities such as cellular debris, waste products and large particles. The filtered culture immediately separated the exosome and / or extracellular endoplasmic reticulum by a separation process. In addition, the filtered culture was stored in a refrigerator (image 10 ° C or less) and then used for the separation of exosome and / or extracellular endoplasmic reticulum. In addition, the filtered culture solution was stored in an ultra-low temperature freezer at -60 ° C or lower and then thawed, followed by isolation of exosome and / or extracellular elastomer. Thereafter, the exosome and / or extracellular endoplasmic reticulum were separated from the culture medium by using a Tangential Flow Filtration (TFF).

Example 2: Isolation and purification of exosome and / or extracellular endoplasmic reticulum by TFF method

The Tangential Flow Filtration (TFF) method was used in Example 1 to separate, concentrate, desalinate and diafiltrate the exosome and / or extracellular endoplasmic reticulum from the culture filtrated with a 0.22 μm filter. Prior to isolating and concentrating the exosome and / or extracellular endoplasmic reticulum using the TFF method, the culture medium was sonicated to loosen lumps of potential exosome and / or extracellular endoplasmic reticulum. A cartridge filter (also known as GE Healthcare or Spectrum Labs) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used as a filter for the TFF method. The TFF filter can be selected by a variety of molecular weight cutoffs (MWCO). Exosomal and / or extracellular endoplasmic reticulum were selectively isolated and concentrated by selected MWCO, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than MWCO were removed.

MWCO 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da TFF filter, or 0.05 占 퐉 TFF filter was used to separate and concentrate exosome and / or extracellular endoplasmic reticulum. The culture was concentrated to a volume of about 1/100 to 1/25 using the TFF method, while materials smaller than MWCO were removed to separate the exosome and / or extracellular endoplasmic reticulum.

Separated, concentrated exo and / or extracellular ER solutions were further desalted and buffered (diafiltration) using the TFF method. At this time, 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. Excipients were added to the buffer to obtain a uniform particle size distribution and high purity of exosome and / or extracellular endoplasmic reticulum. The results of confirming the effect of obtaining exo and / or extracellular endoplasmic reticulum with high purity and homogeneous particle size distribution in high yield according to the excipient treatment are shown in Fig.

In addition, the exosome and / or extracellular endoplasmic reticulum of the present invention can be isolated from a stem cell culture solution by various methods known in the art besides the above-described separation method (Korean Patent Laid-Open No. 10-2016-0116802 (See, for example, Pin Li et al., Progress in Exosome Isolation Techniques, Theranostics, 2017, 7 (3): 789-804; Coumans et al., Methodological Guidelines to Study Extracellular Vesicles, Circulation Research, 2017, 120: 1632-1648) . For example, the exosomes and / or extracellular endoplasmic reticulum of the present invention can be prepared by ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, , Ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, Or polymer based precipitation. ≪ Desc / Clms Page number 7 >

However, the exosome and / or extracellular endoplasmic reticulum of the present invention are not limited to those separated by the methods as described above, and those obtained by various separation methods which are used in the art or which can be used in the future can be used Of course. It is to be understood that the separation methods as described above are to be understood as an example of a separation method for exosome and / or extracellular endoplasmic reticulum, and the present invention is not limited thereto.

Example 3: Characterization of isolated exosome and / or extracellular endoplasmic reticulum

The amount of protein in the fractions of the isolated exosome and / or extracellular epidermis, culture broth, TFF separation process was measured using the BCA chromogenic method (purchased from Thermo Fisher) or the FluoroProfile fluorescence method (purchased from Sigma). The extent to which the exosome and / or the extracellular endoplasmic reticulum and / or the extracellular endoplasmic reticulum were separated and concentrated and the protein, lipid, nucleic acid, low molecular weight compound, etc. were removed by the TFF method of the present invention was monitored by a protein quantification method, Respectively. 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 the exosome and / or extracellular endoplasmic reticulum were separated by the TFF method of one embodiment of the present invention. As a result of analyzing the results obtained from five independent batches, it was confirmed that the exosome and / or extracellular endoplasmic reticulum can be separated very stably by the TFF method of one embodiment of the present invention.

The isolated exosomes and / or extracellular endoplasmic reticulum can be characterized by particle size and size by nanoparticle tracking analysis (NTA; purchased from Malvern) or tunable resistive pulse sensing (TRPS; purchased from Izon Science) . The uniformity and size of the isolated exosome and / or extracellular endoplasmic reticulum were analyzed using transmission electron microscopy (TEM). The results of the TRPS, NTA and TEM analyzes of the separated exosome and / or extracellular endoplasmic reticulum according to one embodiment of the present invention are shown in FIG.

FIG. 5 shows the results of NTA analysis of the size distribution of exosome and / or extracellular endoplasmic reticulum after addition of excipients after the isolation of exosome and / or extracellular endoplasmic reticulum by the TFF method. Separation of exosomes and / or extracellular endoplasmic reticulum by the TFF method in the absence of excipients revealed particles with a size of 300 nm or more, while increasing the amount of excipient shrinks particles with a size of 300 nm or more It was confirmed that the size distribution of exosome and / or extracellular endoplasmic reticulum became uniform.

The addition of excipients was further investigated in the process of isolating exosome and / or extracellular endoplasmic reticulum by the TFF method. As shown in FIG. 6, when an excipient was added to the entire TFF, exosome and / or extracellular endoplasmic reticulum having a uniform size distribution could be obtained (FIG. 6A). On the other hand, in the case of using the culture solution free of the excipient and freezing, or when the TFF process was carried out without adding any excipient, uneven exosome and / or extracellular endoplasmic reticulum containing large-sized particles were obtained And Fig. 6C).

As a result of comparing the relative productivity and concentration of the separated exosome and / or extracellular endoplasmic reticulum, it was found that exogenous and / or extracellular endoplasmic reticulum could be obtained with very high productivity when the excipient was added to the entire TFF, Or extracellular < / RTI > endoplasmic reticulum was also 5-fold higher (Figure 6D). As shown in the results of the NTA analysis, the average size of the separated exo-soma and / or extracellular endoplasmic reticulum was uniformly observed at 200 nm when the excipient was added to the whole TFF (Fig. 6E).

Figure 4D shows the presence of CD9, CD63, CD81 and TSGlOl markers as a result of Western blotting on isolated exosomes and / or extracellular vesicles according to the method of one embodiment of the present invention. Anti-CD9 (purchased from Abcam), anti-CD63 (purchased from System Biosciences), anti-CD81 (purchased from System Biosciences), and anti-TSG101 (purchased from Abcam) were used as the antibodies for each marker.

FIG. 4E shows the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer on the exosome and / or extracellular endoplasmic reticulum isolated according to the method of one embodiment of the present invention. An exosomal-human CD63 Isolation / Detection Kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method to isolate exosome and / or extracellular endoplasmic reticulum positive for CD63, and PE-mouse anti-human The markers were stained using CD63 (PE-Mouse anti-human CD63) (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD) Biosciences).

The present invention is based on the finding that the present invention provides a method of separating and / or purifying exosomes and / or extracellular endoplasmic reticulum, which has high purity and uniform particle size distribution by adding a substance known as an excipient in separation and / It can be economically and efficiently separated and purified at a high yield. Also, it can be seen that the processes of the separation method according to one embodiment of the present invention are scalable and suitable for GMP.

Example 4: Cytotoxicity measurement by treatment with exosome and / or extracellular endoplasmic reticulum

In order to evaluate the toxicity of exosome and / or extracellular endoplasmic reticulum obtained according to the separation method of one embodiment of the present invention in human skin fibroblast HS68 cells, exosomes were treated at different concentrations to observe the cell proliferation rate. HS68 cells were suspended in DMEM containing 10% FBS, and then the cells were subcultured to have a confluency of 80 to 90% 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 and / or extracellular endoplasmic reticulum prepared in Example 2 for each concentration for 24 to 72 hours. Cell viability was measured using WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramar Blue reagent alamarBlue reagent (purchased from Thermo Scientific) and a microplate reader (purchased from Molecular Devices).

The comparative group was based on the number of cells cultured in the normal cell culture medium without the treatment of exosome and / or extracellular endoplasmic reticulum, and the cytotoxicity by the exosome and / or extracellular endoplasmic reticulum of the present invention within the tested concentration range (Fig. 7).

Example 5 Measurement of Inflammatory Response Using Macrophage Cell Lines

Raw 264.7 cells were suspended in DMEM medium containing 10% FBS and dispensed into each well of a multiwell plate to have a confluency of 80-90%. The next day, the exosome and / or extracellular endoplasmic reticulum of the present invention (exosome prepared in Example 2 and / or extracellular endoplasmic reticulum) diluted in fresh serum-free medium containing LPS was treated for 1 to 24 hours Lt; / RTI > The cultured supernatant was taken and NO and inflammatory cytokines present in the culture were measured to confirm the inflammatory response. The inflammatory response in the culture medium was measured using a NO detection kit (purchased from Intron Bio or Promega). The ELISA kit (purchased from the R & D system) was carried out according to the manufacturer's manual to confirm the amount of TNF-α in the group treated with LPS only and the inflammatory cytokine TNF-α in the group treated with the exosomes and / or extracellular endoplasmic reticulum of the present invention. Dexamethasone (purchased from Sigma) was treated as a positive control. In addition, cDNA was prepared from the total RNA obtained from the Raw 264.7 cells treated as described above, and mRNA changes of iNOS, TNF-α, IL-6 and IL-1β were measured using a real-time PCR method. The GAPDH gene was used as a standard gene for quantifying the above genes. The types and sequences of the primers used in the real-time PCR are shown in Table 1 below.

Primer types and base sequences used in real-time PCR gene order The forward primer (5 '- > 3') The reverse primer (5 '- > 3') TNF-a TCT CAT CAG TTC TAT GGC CCA GAC (SEQ ID NO: 1) GGC ACC ACT AGT TGG TTG TCT TTG (SEQ ID NO: 2) iNOS GCT ACC ACA TTG AAG AAG CTG GTG (SEQ ID NO: 3) CCA TAG GAA AAG ACT GCA CCG AAG (SEQ ID NO: 4) GAPDH GAC ATC AAG AAG GTG GTG AAG CAG (SEQ ID NO: 5) CCC TGT TGC TGT AGC CGT ATT CAT (SEQ ID NO: 6) IL-6 GCC AGA GTC CTT CAG AGA GAT ACA (SEQ ID NO: 7) ATT GGA TGG TCT TGG TCC TTA GCC (SEQ ID NO: 8) IL-1? GCA ACG ACA AAA TAC CTG TGG CCT (SEQ ID NO: 9) AGT TGG GGA ACT CTG CAG ACT CAA (SEQ ID NO: 10)

First, as shown in FIG. 8, when the exosome and / or the extracellular endoplasmic reticulum of the present invention were treated with LPS against Raw 264.7 cells, which are macrophages of mice, LPS-induced inflammatory cytokine TNF-a , The expression level of IL-6 and IL-1? MRNA was decreased, and the expression level of iNOS mRNA of NO production enzyme was decreased. Next, as shown in Fig. 9, treatment of the present exosome and / or extracellular endoplasmic reticulum of RAW264.7 cells, a macrophage of mice, with LPS in the presence of LPS resulted in NO production, an inflammatory reaction induced by LPS Dependent manner. In addition, as shown in Fig. 10, RAW264.7 cells, which are macrophages of mice, were treated with the exosome and / or extracellular endoplasmic reticulum of the present invention in the presence of LPS. As a result, LPS-induced inflammatory cytokine TNF- Production. These results indicate that the exosome and / or the extracellular endoplasmic reticulum of the present invention have an activity of reducing the inflammatory reaction induced by LPS, which is useful for preventing, inhibiting, alleviating or improving the sensitive skin, It is strongly suggested that moxa and / or extracellular endoplasmic reticulum may be usefully utilized as an effective ingredient of a composition for prevention, inhibition, alleviation or improvement of sensitive skin.

As an example that does not limit the present invention, the performance or functional activity of the exosome and / or the extracellular endoplasmic reticulum may be the same regardless of the production batch, production cycle, and subdivision of the culture medium of the stem cell culture liquid Or similar were maintained. As shown in Fig. 11, in each of the stem cell cultures produced from one or two times of stem cell cultures in different batches, the exosome and / or extracellular space of each batch isolated and purified according to the method of one embodiment of the present invention The ERs were found to decrease LPS-induced NO production in RAW264.7 cells in a concentration-dependent manner to substantially the same or similar degree to each other. As shown in FIG. 12, each of the exosomes and / or extracellular endoplasmic reticulum obtained by dividing and purifying the stem cell culture solution produced in one batch according to the method of the embodiment of the present invention three times, In a concentration-dependent manner, the production of NO induced by LPS in RAW 264.7 cells to substantially the same or similar degree. In addition, as shown in FIG. 13, the exocomes and / or extracellular endoplasmic reticulum of each cycle, which are separated and purified periodically according to the method of one embodiment of the present invention, have substantially the same or similar degree to each other as RAW 264.7 Induced LPS-induced NO production in a concentration-dependent manner.

The above results suggest that the composition containing the exosome and / or the extracellular endoplasmic reticulum of the present invention as an active ingredient can reduce the production of inflammatory substances in the skin to effectively alleviate or improve the sensitive skin. The composition of the present invention is expected to be useful for preventing, suppressing, alleviating or improving sensitive skin and for preventing, alleviating, improving or treating sensitive skin diseases.

Example 6: Comparison of reduction effect of NO formation according to separation method

As one example that does not limit the present invention, in order to compare the effect of reducing the formation of NO in the exosome and / or extracellular endoplasmic reticulum according to the separation method, the exosome obtained by the TFF separation purification of one embodiment of the present invention and / Extracellular < / RTI > vesicles isolated by conventional precipitation methods were prepared in addition to the outer vesicle. The precipitation method was performed according to the protocol of the manufacturer (System Biosciences). The extracellular endoplasmic reticulum (see Fig. 14A) isolated by the conventional precipitation method has a particle size distribution (Fig. 14A) as compared with the exosome and / or extracellular endoplasmic reticulum (see Fig. 14B) isolated and purified by the TFF method of one embodiment of the present invention It is confirmed that the uniformity is low and various sizes are available. 14C, the exosome and / or the extracellular endoplasmic reticulum isolated and purified by the TFF method of one embodiment of the present invention has a much higher level of NO formation than the extracellular endoplasmic reticulum obtained by the conventional precipitation method Respectively. These results show that, according to one embodiment of the present invention, the exo and / or extracellular endoplasmic reticulum isolated and purified are superior in terms of uniformity of particle size distribution and inhibition of NO formation compared to extracellular endoplasmic reticulum isolated according to the conventional method have.

Thus, the exosome and / or the extracellular endoplasmic reticulum obtained according to the separation method of one embodiment of the present invention can be used for the purpose of improving the performance or functional activity (for example, The composition of the present invention, which contains the stem cell-derived exosome and / or the extracellular endoplasmic reticulum with excellent functional activity as an active ingredient, is superior to the prevention of the sensitive skin , Suppression, alleviation or amelioration, and prevention, alleviation, improvement, or therapeutic effect of a sensitive skin disease.

Example 7 Effect of Preventing Oxidative Stress by Hydrogen Peroxide

Human skin fibroblast HDF cells were dispensed into each well of a 12-well plate at a density of 6.5 x 10 < ^{4 &} gt ;. The exosomes and / or extracellular endoplasmic reticulum of the present invention (exosome prepared in Example 2 and / or extracellular endoplasmic reticulum) diluted in serum-free medium was treated for each concentration and cultured for 22 hours. Then 1 mM hydrogen peroxide (purchased from Sigma) was further treated and cultured for 2 hours.

In order to confirm that the exosome and / or extracellular endoplasmic reticulum of the present invention can prevent cell death due to oxidative stress induced by hydrogen peroxide, HDF cell viability was measured using a flow cytometer as follows . First, the culture medium was removed and washed with 1 ml of DPBS (Dulbecco's Phosphate Buffered Saline) (purchased from Gibco), 150 μl of trypsin / EDTA (purchased from Gibco) was added and the plate was allowed to stand for 4 minutes in a 5% CO ₂ incubator at 37 ° C . Then, 450 μl of the culture medium was added, and the cells were suspended and centrifuged at 1,500 rpm for 3 minutes. The supernatant was removed, washed twice with 500 μl of DPBS, and then suspended in 25 μl of 1 × Annexin V binding buffer (purchased from BD). 25 μl of 1X Annexin V binding buffer, FITC annexin V (purchased from BD) and PI staining solution (purchased from BD) mixed solution was added to each suspended cell sample and incubated for 15 minutes at room temperature. Respectively. 150 μl of 1 × Annexin V binding buffer per sample was added and the degree of apoptosis was measured with a flow cytometer (NovoCyte).

As a result, it was confirmed that apoptosis induced by hydrogen peroxide-induced oxidative stress was significantly reduced in the group treated with the exosome and / or extracellular endoplasmic reticulum of the present invention (FIGS. 21 and 22). These experimental results strongly suggest that the composition comprising the exosome and / or the extracellular endoplasmic reticulum of the present invention has an anti-oxidative stress effect and can suppress, alleviate or improve the sensitive skin caused by oxidative stress.

Accordingly, it can be seen that the composition of the present invention has an effect of reducing the production of inflammatory substances and preventing or suppressing oxidative stress, and it is possible to prevent, inhibit, alleviate or improve sensitive skin and prevent, Or treatment of the disease.

Example 8: Skin permeability test of the exosome and / or extracellular endoplasmic reticulum of the present invention

PKH67 dyes (purchased from Sigma) were used to prepare fluorescently stained exosomes and / or extracellular endoplasmic reticulum. 1 mM PKH67 was diluted with Diluent C (purchased from Sigma) to prepare a 10 μM PKH67 solution. Then, the solution was mixed with a solution of exosome and / or extracellular ER at an appropriate concentration, Lt; / RTI > After the reaction, an MW3000 spin column (purchased from Thermo Fisher) was used to remove residual PKH67 dye from the exosome and / or extracellular endoplasmic reticulum (hereinafter abbreviated as "PKH-exosomes") of the present invention stained with PKH67 Respectively. After confirming the use of a fluorescence meter (purchased from Molecular Devices), it was confirmed that PKH-exosomes which did not react with the exosome and / or extracellular endoplasmic reticulum of the present invention were removed, and fluorescence of sufficient intensity was detected in the PKH-exosomes ).

The PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, a concentration of 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the skin outer surface of the pig skin tissue. To prevent drying of the applied PKH-exosomes, the nonwoven fabric was covered and allowed to react for an appropriate period of time, for example, 30 minutes to 1 hour to allow the PKH-exosomes to be delivered to the subcutaneous tissues. In addition, the PKH-exosomes were applied to the outer surface of the pig skin and covered with the nonwoven fabric, followed by flowing a microcurrent for a predetermined time, for example, 30 minutes to 1 hour. After the reaction was completed, the pig skin tissue was immersed in a 3.7% formaldehyde solution, reacted overnight, and washed three times for 5 minutes each with phosphate buffered saline. The washed pig skin tissue was immersed in a 30% sucrose solution and treated with OCT compound. After washing three times for 5 minutes with phosphate buffer solution, sections were prepared on a longitudinal section using a microtome, and tissue sections were placed on a slide glass. On the other hand, the preparation of the tissue section may be carried out before the tissue is fixed with formaldehyde solution. Fluorescence detected from PKH-exosomes in tissue sections was observed using fluorescence microscopy. In the above manner, PKH-exosomes transmitted through the epidermis of pig skin tissue and transferred to subcutaneous tissues were confirmed (Fig. 16). As shown in FIG. 16, the exosome and / or extracellular endoplasmic reticulum of the present invention can effectively pass through the skin barrier, deeply transfer into the skin tissue, and can be effectively absorbed to the skin.

Accordingly, the external preparation for skin or cosmetic composition containing the exosome and / or the extracellular endoplasmic reticulum of the present invention as an active ingredient is effective for preventing, suppressing, alleviating or improving sensitive skin and for preventing, alleviating, , It is expected to work effectively.

Next, the skin tissue of the hairless mouse was removed and placed on top of the chamber of the Franz Diffusion Cell. At this time, the inside of the diffusion cell was filled with a phosphate buffer solution. PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the outside of the skin tissue of the mice. At this time, in order to prevent drying of PKH-exosomes, the nonwoven fabric was pre-positioned outside the mouse skin tissue, and PKH-exosomes were injected between the nonwoven fabric and skin tissue. Then, the reaction was carried out for 30 minutes to 1 hour. In addition, the PKH-exosomes were injected between the nonwoven fabric and the skin tissue, and the microcurrent was flowed for a predetermined time, for example, 30 minutes to 1 hour. After completion of the reaction, the PKH-exosomes transferred into the skin tissue were confirmed immediately using a confocal fluorescence microscope (Leica, SP8X) or the skin tissue and the PKH-exosomal solution were further reacted for 1 hour to 6 hours , And PKH-exosomes were identified by confocal fluorescence microscopy. As a result of the above-mentioned methods, the exosome and / or extracellular endoplasmic reticulum of the present invention can effectively pass through the skin barrier and be deeply transferred into the skin tissue and can be effectively absorbed to the skin (FIGS. 17 and 18).

Accordingly, the external preparation for skin or cosmetic composition containing the exosome and / or the extracellular endoplasmic reticulum of the present invention as an active ingredient is effective for preventing, suppressing, alleviating or improving sensitive skin and for preventing, alleviating, , It is expected to work effectively.

Example 9: Treatment of composition comprising exosome and / or extracellular endoplasmic reticulum as an active ingredient for human skin

(Including a semi-transparent liquid composition) containing the exosome and / or extracellular endoplasmic reticulum obtained according to the separation method of one embodiment of the present invention, a liquid immersion sheet mask (a white sheet mask in which the translucent liquid phase is deposited) A "test product" consisting of a promoting sheet mask (silver sheet mask containing iontophoresis device) is placed on the face of a person with a strong redness and skin trouble associated with sensitive skin (hereinafter referred to as "case 1") To evaluate whether skin troubles and flushing symptoms are alleviated or improved. In addition, the test product was applied once to the face of a person with redness (hereinafter referred to as "case 2") to evaluate whether the overall skin tone and redness symptoms improved.

Only one use of the "test product" containing the exosome of the present invention and / or the extracellular endoplasmic reticulum significantly improved flushing and skin troubles in case 1 (see case 1 in FIG. 19) The overall skin tone was improved and flushing was alleviated (see case 2 in FIG. 19). Therefore, the external preparation for skin or cosmetic composition containing the exosome and / or the extracellular endoplasmic reticulum of the present invention as an effective ingredient has the effect of preventing, suppressing, alleviating or alleviating redness symptoms and skin troubles associated with sensitive skin have.

In addition, a composition (including a semi-transparent liquid composition) containing the exosome and / or the extracellular endoplasmic reticulum obtained according to the separation method of one embodiment of the present invention can be administered to three affected atopic patients (hands, neck, arms, etc.) After applying the solution three times a week for 1 to 2 weeks, iontophoresis was performed by using a iontophoresis device to flow a microcurrent into the affected part of the composition. As a result, the severe itching of the patients in relation to the sensitive skin was remarkably alleviated, and the erythema symptoms were also remarkably improved (Fig. 20). Patients applying the composition containing the exosome and / or extracellular endoplasmic reticulum of the present invention alleviated severe itching and erythema and improved symptoms to such an extent that the prescription of the steroid or antihistamine preparation was stopped.

Accordingly, the external preparation for skin or cosmetic composition containing the exosome and / or extracellular endoplasmic reticulum obtained according to the separation method of one embodiment of the present invention as an active ingredient can be used for preventing, Suppressing, alleviating or improving the effect of the present invention.

Example 10: Determination of anti-inflammatory effect using THP-1 monocyte

After the THP-1 monocyte (THP-1 human monocyte) was differentiated into macrophages, the anti-inflammatory effects of the exosome and / or extracellular endoplasmic reticulum of the present invention were confirmed as follows.

THP-1 monocytes were cultured in RPMI supplemented with 10% FBS (Fetal Bovine Serum), 1% penicillin-streptomycin and 100 nM Phorbol 12-myristate 13-acetate (purchased from Sigma) 1640 (purchased from ThermoFisher Scientific) medium at 1640 (purchased from ThermoFisher Scientific), and then seeded in a 12-well plate at a density of 6 × 10 ⁵ cells / mL and cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours to obtain THP-1 Derived macrophage (THP-1 derived macrophage). The differentiated cells were then washed once with PBS and cultured in RPMI 1640 medium without formalin 12-myristate 13-acetate for 24 hours to stabilize the cells.

For the co-treatment group, RPMI 1640 medium without FBS (Fetal Bovine Serum), 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN -gamma (purchased from Peprotech) to activate THP-1 derived macrophages and also to isolate the exosome and / or extracellular endoplasmic reticulum of the present invention (the exosome prepared in Example 2) And / or extracellular endoplasmic reticulum) and cultured for 24 hours. On the other hand, in the case of the pre-treatment experimental group, RPMI 1640 medium containing no FBS (Fetal Bovine Serum) for the THP-1 derived macrophages and dexamethasone (purchased from Sigma) And cultured for 24 hours and treated with 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN-gamma (Peprotech®) and / or extracellular endoplasmic reticulum ) And cultured for 4 hours to activate THP-1-derived macrophages.

CDNA was prepared from the RNA isolated from cultured THP-1 derived macrophages, and cDNA was prepared from the pro-inflammatory cytokines IL-6, IL-1β And TNF-alpha mRNA expression levels were measured. GAPDH was used as a standard gene for quantifying the genes. The types and sequences of the primers used in the PCR are shown in Table 2 below.

Primer types and base sequences used in real-time PCR gene order The forward primer (5 '- > 3') The reverse primer (5 '- > 3') IL-6 AAGCCAGAGCTGTGCAGATGAGTA (SEQ ID NO: 11) TGTCCTGCAGCCACTGGTTC (SEQ ID NO: 12) IL-1? ACCTGTCCTGCGTGTTGAAAGATG (SEQ ID NO: 13) TGGGCAGACTCAAATTCCAGCTTG (SEQ ID NO: 14) TNF-a CTGCCTGCTGCACTTTGGAG (SEQ ID NO: 15) ACATGGGCTACAGGCTTGTCACT (SEQ ID NO: 16) GAPDH CTTTGGTATCGTGGAAGGACTC (SEQ ID NO: 17) GTAGAGGCAGGGATGATGTTCT (SEQ ID NO: 18)

As shown in FIG. 23, the THP-1-derived macrophages obtained after differentiating THP-1 monocytes into macrophages were treated with LPS and IFN-y with the present exosome and / or extracellular endoplasmic reticulum co-treatment), the expression level of IL-6 mRNA, which is a proinflammatory cytokine, was decreased compared to the negative control. Also, as shown in FIGS. 24 to 26, THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages were treated with the exosomes and / or cells of the present invention before LPS and IFN- IL-6, IL-1β and TNF-α mRNA expression levels were decreased in the pre-treatment of the outer endoplasmic reticulum compared to the negative control. The proinflammatory cytokines IL-6, IL-1 [beta] and TNF- [alpha] increase expression in proinflammatory M1 macrophages and decrease expression in anti-inflammatory M2 macrophages. Therefore, the above results suggest that the exosome and / or extracellular endoplasmic reticulum of the present invention can inhibit, alleviate and reduce the inflammatory reaction in the skin and the resulting sensitive skin symptoms, and the experimental results of the above- The result is strongly supported.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> ExoCoBio Inc. <120> A composition comprising an exosome and / or extracellular vesicle          derived from stem cells as an active ingredient and its          application for preventing, suppressing, alleviating or improving          sensitive skin <130> P17-0028KR <150> KR 10-2017-0169648 <151> 2017-12-11 <160> 18 <170> KoPatentin 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 1 tctcatcagt tctatggccc agac 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 2 ggcaccacta gttggttgtc tttg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 3 gctaccacat tgaagaagct ggtg 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 4 ccataggaaa agactgcacc gaag 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 gacatcaaga aggtggtgaa gcag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 ccctgttgct gtagccgtat tcat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-6 forward primer <400> 7 gccagagtcc ttcagagaga taca 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-6 reverse primer <400> 8 attggatggt cttggtcctt agcc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-1beta forward primer <400> 9 gcaacgacaa aatacctgtg gcct 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-1beta reverse primer <400> 10 agttggggaa ctctgcagac tcaa 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-6 (THP-1) forward primer <400> 11 aagccagagc tgtgcagatg agta 24 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6 (THP-1) reverse primer <400> 12 tgtcctgcag ccactggttc 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-1beta (THP-1) forward primer <400> 13 acctgtcctg cgtgttgaaa gatg 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-1beta (THP-1) reverse primer <400> 14 tgggcagact caaattccag cttg 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> TNF-alpha (THP-1) forward primer <400> 15 ctgcctgctg cactttggag 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> TNF-alpha (THP-1) reverse primer <400> 16 acatgggcta caggcttgtc act 23 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) forward primer <400> 17 ctttggtatc gtggaaggac tc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (THP-1) reverse primer <400> 18 gtagaggcag ggatgatgtt ct 22

Claims (27)

Stem cell-derived exosome and / or extracellular endoplasmic reticulum, and
A composition for preventing, inhibiting, alleviating or ameliorating a sensitive skin comprising at least one species selected from the group consisting of exosome isolated from a stem cell culture and a fraction containing an extracellular endoplasmic reticulum. The method according to claim 1,
The exosome and / or the extracellular endoplasmic reticulum or the fraction may be separated by an ultracentrifugation, a density gradient centrifugation, an ultrafiltration, a size exclusion chromatography, an ion exchange Ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer-based Wherein the composition is separated by at least one separation method from polymer based precipitation. The method according to claim 1,
The exosome and / or extracellular endoplasmic reticulum is obtained by performing the following steps:
(a) adding an excipient to the stem cell culture solution, (b) filtering the stem cell culture solution to which the excipient has been added, (c) removing the filtered stem cell culture solution by TFF (Tangential Flow Filtration) (D) adding an excipient to a buffer solution used for desalting and diafiltration, and adding TFF (Tangential Flow) using the buffer solution in which the excipient is added, Filtration, and desalting and diafiltration of the separated exosome and / or extracellular endoplasmic reticulum. The method according to claim 1,
Said fraction being obtained by performing the following steps:
(a) adding an excipient to the stem cell culture solution, (b) filtering the stem cell culture solution to which the excipient has been added, (c) removing the filtered stem cell culture solution by TFF (Tangential Flow Filtration) (D) adding an excipient to a buffer solution used for desalting and buffer exchange (diafiltration), adding a buffer containing the excipient Performing desalting and diafiltration of the separated fractions using TFF (Tangential Flow Filtration) using a solution. The method according to claim 3 or 4,
Wherein the desalting and buffer exchange is carried out continuously or intermittently. The method according to claim 3 or 4,
Characterized in that desalting and buffer exchange are carried out using a buffer solution having a volume at least four times that of the starting volume. The method according to claim 3 or 4,
Characterized in that a TFF filter of molecular weight cutoff (MWCO) 100,000 Da, 300,000 Da, 500,000 Da or 750,000 Da, or 0.05 占 퐉 TFF filter is used for Tangential Flow Filtration. The method according to claim 3 or 4,
Wherein the step (c) further comprises concentrating to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration). The method according to claim 3 or 4,
Wherein the filtered stem cell culture solution is sonicated prior to TFF. The method according to claim 3 or 4,
The excipient may be selected from the group consisting of sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanose, dextrose, glucose, galactose, fructose, Erythritol, arabinose, xylose, ricosos, ribose, ribulose, xylulose, aldose, mannose, mannose, erythrose, erythrose, erythrose, erythritol, But are not limited to, heptose, heptoolose, octolose, gentianus, umbeliferose, planterose, isoleuconose, raffinose, liqueinose, stachyose, verbascose, manitol ), Maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, starch powder, starch-equivalent reduced alcohol, nonionic surfactant, histidine, glycerol and Choleste At least one member, the composition selected from the group consisting of (cholesterol). 5. The method according to any one of claims 1 to 4,
Wherein said exosome and / or extracellular endoplasmic reticulum or said fraction reduces TNF-? MRNA expression, IL-6 mRNA expression, IL-1? MRNA expression, iNOS mRNA expression, and NO production in in vitro assays. Composition. A pharmaceutical composition for preventing, alleviating, ameliorating or treating a sensitive skin disease comprising the composition according to any one of claims 1 to 4. Stem cell-derived exosome and / or extracellular endoplasmic reticulum, and
Wherein the composition comprises at least one species selected from the group consisting of exosome isolated from stem cell culture and fractions containing extracellular endoplasmic reticulum as an effective ingredient for preventing, alleviating, improving or treating a sensitive skin disease. 14. The method of claim 13,
Wherein the exosome and / or extracellular endoplasmic reticulum or fraction is carried or mixed with at least one of hydrogel, hyaluronic acid, hyaluronic acid salt, or hyaluronic acid gel. 15. The method of claim 14,
Wherein the hydrogel is a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. 16. The method of claim 15,
Wherein the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum Wherein said polyhydric alcohol is 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. . Stem cell-derived exosome and / or extracellular endoplasmic reticulum, and
A cosmetic composition for preventing, inhibiting, alleviating or ameliorating a sensitive skin comprising at least one member selected from the group consisting of exosome isolated from a stem cell culture and fractions containing extracellular endoplasmic reticulum. 18. The method of claim 17,
Wherein the exosome and / or the extracellular endoplasmic reticulum or the fraction are carried or mixed in at least one of hydrogel, hyaluronic acid, hyaluronic acid salt, or hyaluronic acid gel. 19. The method of claim 18,
Wherein the hydrogel is a hydrogel obtained by dispersing a gelled polymer in a polyhydric alcohol. 20. The method of claim 19,
Wherein the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum And the polyhydric alcohol is 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. . 21. The method according to any one of claims 17 to 20,
At least one member selected from the group consisting of patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, aerosols, mist, foundations, Wherein the composition is applied to the cosmetic composition. 22. The method of claim 21,
Wherein the cosmetic composition is applied or deposited on at least one surface of the patch, mask pack or mask sheet. (a) applying the cosmetic composition according to any one of claims 17 to 20 directly to the skin of a mammal, (b) applying the patch, mask pack or mask sheet onto which the cosmetic composition is applied or deposited, Comprising the steps of: (a) contacting or adhering to the skin of the skin, or (b) proceeding sequentially with (a) and (b). 24. The method of claim 23,
(c) conducting iontophoresis by flowing a minute current through the skin of the mammal to which the cosmetic composition is applied. 25. The method of claim 24,
Further comprising contacting or attaching an iontophoresis device to the skin of said mammal. 26. The method of claim 25,
The iontophoresis device may include at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, , A patch, a mask pack or a mask sheet on which said at least one battery is mounted. 15. A method of preventing, alleviating, ameliorating, or treating a sensitive skin disorder, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 12.

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