KR20190116858A - A composition comprising an exosome and/or extracellular vesicle derived from stem cell as an active ingredient and its application for preventing, alleviating, reducing or removing postinflammatory hyperpigmentation - Google Patents

Abstract

The present invention provides a composition for preventing, alleviating, reducing, or eliminating post-inflammatory hyperpigmentation, comprising, as active ingredients, at least one selected from the group consisting of high-purity exosomes and/or extracellular vesicles derived from stem cells and having a uniform particle size distribution, and fractions containing high-purity exosomes and/or extracellular vesicles isolated from a stem cell culture medium and having a uniform particle size distribution. The composition of the present invention can inhibit the production of TNF-α, IL-6, IL-1β, and nitric oxide (NO) that stimulate melanocytes, and thus can ultimately reduce melanin production, and furthermore, can penetrate deeply into the dermal layer where melanin pigments are deposited, and thus can effectively prevent, alleviate, reduce, or eliminate post-inflammatory hyperpigmentation. In addition, the composition of the present invention, when applied to the skin using iontophoresis, laser beam irradiation, IPL irradiation, and/or RF energy application, allows exosomes and/or extracellular vesicles to penetrate deeply into the dermal layer of the melanin-deposited skin, thereby maximizing the cosmetic effect or therapeutic effect for the skin, and in particular, the composition of the present invention can cause synergistic effects in reducing or eliminating pigments by laser beam irradiation, IPL irradiation, and/or RF energy application.

Description

Prevention, alleviation, reduction or elimination of post-inflammatory hyperpigmentation of compositions comprising stem cell-derived exosomes and / or extracellular vesicles as an active ingredient {A composition comprising an exosome and / or extracellular vesicle derived from stem cell as an active ingredient and its application for preventing, alleviating, reducing or removing postinflammatory hyperpigmentation}

The present invention is a post-inflammatory hyperpigmentation of a composition comprising at least one of exosomes and / or extracellular vesicles, or fractions containing the same, having high purity and uniform particle size separated from a stem cell culture medium as an active ingredient ( It relates to the prevention, alleviation, reduction or elimination of postinflammatory hyperpigmentation.

The present invention also relates to a skin external preparation and a cosmetic composition for preventing, alleviating, reducing or removing post-inflammatory hyperpigmentation comprising the composition.

In addition, the present invention provides a large amount of exosomes and / or extracellular vesicles derived from stem cells of high purity and uniform particle size distribution that can be clinically applied in the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation. The present invention relates to an excellent clinical and commercial technology that can provide a composition containing exosomes and / or extracellular vesicles derived from stem cells having excellent functional activity as an active ingredient in large quantities at low cost.

Postinflammatory hyperpigmentation (PIH) is a synthesis of more melanin in the skin than normal to protect the skin from inflammation from UV rays. In general, post-inflammatory pigmentation is a pathological and physiological reaction resulting from skin inflammatory diseases such as acne, atopic dermatitis, lupus erythematosus, psoriasis and lichen planus, trauma, burns, allergic reactions, physical damage, and drug reactions. to be. In addition, hyperpigmentation after inflammation may also appear as an aftereffect of hypersensitivity to shaving, shaving, chemical exfoliation or other dermatological treatments such as laser, ultrasound, high frequency current, LED light and visible light. Post-inflammatory hyperpigmentation is not only visually unpleasant, but excessive pigmentation can result in pathological psychological reactions that result in loss of confidence in appearance and psychological contraction.

Post-inflammatory hyperpigmentation is a unique skin pigmentation associated with melanin overproduction and deposition. Burns or inflammation of the skin activate melanocytes, which excessively increases melanin synthesis and increases the transfer of melanin to the surrounding keratinocytes, resulting in excessive deposition of melanin on the skin. The trigger of melanocyte activity is known to be stimulated by inflammatory mediators such as reactive oxygen species, cytokines, chemokines, etc. secreted during the inflammatory process. Leukotrienes such as LT-C4, LT-D4, prostaglandins such as PGE2, PGD2, Thromboxane-2, TNF-α, IL-6, IL-1 Oxidized species, such as cytokines and nitric oxides, stimulate melanocyte activity. In melanocytes stimulated and activated by these substances, melanin pigment is oversynthesized and delivered to surrounding keratinocytes, and in the dermis, inflammatory cells are activated. In addition, the melanin pigment is lowered from keratinocytes and placed in the dermis through the base layer destroyed by inflammation, and macrophages of the activated inflammatory cells are fed melanin pigment and become melanocytes (melanophage), which stays in the upper part of the dermis. Dark pigmentation appears on the skin.

Such post-inflammatory hyperpigmentation is difficult to treat because melanin pigment is released into the dermis beyond the epidermal phase and is captured by melanin phagocytic cells. Conventional post-inflammatory hyperpigmentation therapy is to control inflammation with corticosteroids and use photoprotective agents to prevent further pigmentation. Chemical peeling compounds, such as salicylic acid and glycolic acid, may be used to promote skin regeneration and reduce pigmentation. In addition, skin whitening agents such as hydroquinone, azelaic acid, and kojic acid may help to actively remove hyperpigmentation, but there is a problem of highlighting hyperpigmentation sites after inflammation due to nonspecific discoloration of normal skin. have.

The conventional post-inflammatory hyperpigmentation therapy has a problem in that it is fundamentally limited to remove the pigment or causes side effects such as skin cancer and vitiligo or effects such as long-term use. Covering the area with makeup is the only alternative.

On the other hand, a method for regenerating skin tissue or improving or treating skin conditions or diseases using stem cells has been proposed. Although embryonic stem cells or fetal tissue-derived stem cells have excellent differentiation and regenerative treatment ability and low rejection, they are not applicable to clinical practice due to ethical issues and there is a risk of forming tumors. As an alternative, a method of skin regeneration and treatment using adult stem cells has been proposed. However, if adult stem cells of the patient's own adult stem cells are used, there is a risk of developing graft-versus-host disease. There is a complicated and expensive problem because the process of culturing stem cells after harvesting them.

Recently, in view of the problems of stem cells as described above, there is an attempt to regenerate skin tissue or to improve or treat skin conditions or diseases using a culture medium obtained by culturing adult stem cells. However, the adult stem cell culture medium contains various proteins, cytokines, and growth factors secreted by the adult stem cells, while other components such as waste products secreted as the cells grow, antibiotics added to prevent contamination, and animal-derived serum. Because it is included, it is likely to be exposed to various risks when used on the skin. Therefore, in order to use the adult stem cell culture medium, a separate process for removing wastes or various dangerous components is required, and accordingly, it becomes expensive.

Recently, research has been reported that the cell secretome contains various bioactive factors that control the behavior of the cells, and in particular, the exosomes or extracellular cells that have intercellular signaling functions are contained in the cell secretions. 'Extracellular vesicles' are included, and research on their composition and function is being actively conducted.

Cells release various membrane types of endoplasmic reticulum into the extracellular environment, which are commonly referred to as extracellular vesicles (EVs). Extracellular vesicles are called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, may be used separately from exosomes.

Exosomes are tens to hundreds of nanometers of endoplasmic reticulum consisting of a double phospholipid membrane identical to the structure of a cell membrane, and include proteins, mRNAs, miRNAs, DNA, and the like, called exosome cargo. Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and differently regulated by the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. In the case of proliferating stem cell-derived exosomes, characteristics of stem cells related to cell behavior such as cell migration, proliferation and differentiation and tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).

However, despite various studies, including the possibility of treating specific diseases using exosomes and / or extracellular vesicles, the development of application technology related to improving skin penetration ability of exosomes and / or extracellular vesicles The situation is poor, and in particular, grafting with various medical or beauty techniques has not received much attention.

While the inventors have intensively studied new applications of exosomes and / or extracellular vesicles and their incorporation into medical or cosmetic techniques, exosomes and / or extracellular vesicles isolated from stem cell cultures have been used to detect melanocytes. It inhibits the production of stimulating TNF-α, IL-6, IL-1β, and nitric oxide (NO), ultimately reducing melanin production and penetrating into the dermis where the phagocytic cells that collect melanin pigments stay The present invention was completed by confirming that it is effective in preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation.

On the other hand, it is to be understood that the matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and are not cited as an approval that they can be used as the "prior art" of the present invention.

Republic of Korea Patent Publication No. 10-2014-0071273 (2014.06.11) Republic of Korea Patent Publication No. 10-2014-0008364 (2014.01.21) Republic of Korea Patent Publication No. 10-2017-0044767 (2017.04.25)

J Clin Aesthetic Dermatol. 2010 Jul; 3 (7), pp.20 ~ 31 (July 2010)

An object of the present invention is a hyperpigmented post-inflammatory hyperpigmentation of a composition comprising, as an active ingredient, at least one of exosomes and / or extracellular vesicles, or fractions containing the same, having high purity and uniform particle size separated from a stem cell culture medium. To prevent, alleviate, reduce or eliminate deposition.

Another object of the present invention to provide a skin external preparation and cosmetic composition for the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation comprising the composition.

Still another object of the present invention is to obtain a large amount of exosomes and / or extracellular vesicles derived from stem cells of high purity and uniform particle size distribution, exosomes derived from stem cells excellent in the functional activity thus obtained and And / or to provide a composition containing the extracellular vesicles as an active ingredient.

Still another object of the present invention is to provide a method for preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation using the composition.

Another object of the present invention to provide a cosmetic method through the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation using the composition.

Still another object of the present invention is to provide a composition comprising a exosome and / or extracellular vesicles suitable for the above methods as an active ingredient, and a drug delivery system or method therefor.

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby. Further objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the present invention is exosomes and / or extracellular vesicles of uniform high purity particle size distribution derived from stem cells, and exosomes of high purity particle size separated from the stem cell culture medium And / or provides a composition for the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation comprising at least one selected from the group consisting of extracellular vesicle-containing fractions as an active ingredient.

In addition, the present invention can obtain a large amount of exosomes and / or extracellular vesicles derived from stem cells of high purity and uniform particle size distribution that can be clinically applied in the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation. The present invention provides a novel clinical and commercially superior technology that can provide a composition containing exosomes and / or extracellular vesicles derived from stem cells having excellent functional activity as an active ingredient in large quantities at low cost. .

As used herein, the term "extracellular vesicles (EV)" encompasses membrane-derived vesicles (membrane vesicles), ectosomes, shedding vesicles, microparticles, or equivalents thereof. Used in the sense.

As used herein, the term "exosomes" refers to vesicles of a size ranging from tens to hundreds of nanometers (preferably approximately 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of a cell membrane, provided that The particle size of exosomes may vary depending on the stem cell type, isolation method, and measurement method. (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015 ) DOI 10.1007 / s00216-015-8535-3), a protein called exosome cargo, mRNA, miRNA, DNA and the like. Exosome cargo includes a wide range of signaling factors, which are known to be specific for cell types and differently regulated by the environment of the secretory cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Known.

As used herein, the term "inflammatory hyperpigmentation" refers to acne, atopic dermatitis, contact allergic dermatitis, ataxia, psoriasis, lichen planus, lupus erythematosus, schizophrenia, trauma, burns, allergic reactions, physical damage, drugs Hyperpigmentation due to various endogenous or exogenous inflammations such as reactions, mechanical trauma, ionized or non-ionized radiation, lasers, ultrasounds, radiofrequency currents, LED rays, visible light, shaving, chemical exfoliation or other dermatological treatments .

As used herein, the term “Intense Pulsed Light (IPL)” is light that has various wavelength ranges and is periodically emitted in the form of a strong pulse. IPL, for example, is light in the wavelength range of approximately 300-1200 nm (varies depending on the IPL device) and is periodically emitted in the form of a strong pulse. IPL has its physical properties determined by four components: the wavelength range, the pulse width, the spacing between pulses, and the energy used. IPL irradiation equipment uses a lamp flash that emits light at a wavelength of approximately 300-1200 nm and controls the wavelength of the light emitted by the filter. Xenon lamps are commonly used as irradiation lamps of IPL appliances. The amount of irradiation energy of the IPL should be adjusted to the desired level. To adjust the amount of irradiation energy, the lamp flash circuit must be driven and controlled. As a lamp flash driving circuit of an IPL device, a pulse amplitude modulation (PAM) driving circuit system, a pulse width modulation (PWM) driving circuit system, and a mixed method of both are used.

As used herein, the term "RF energy" has a conventional meaning, which is the product of RF power and the time that the RF power is applied or delivered to the skin region to be treated. "RF voltage" and "RF power" are mathematically closely related parameters, and knowing one of them and the load (skin) impedance can determine the other parameter at a given skin impedance at a particular time. On the other hand, by controlling the voltage of the RF generator can control the RF power and RF energy delivered to the skin.

As used herein, the term “iontophoresis” refers to a method of allowing an ionized active ingredient to penetrate the skin with electrical repulsion by changing a skin's electrical environment by applying a potential difference by flowing a microcurrent to the skin to which the active material is applied. Means. The iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into the electrode patch on the skin to introduce a microcurrent into the skin, and a battery is mounted on the electrode patch itself. The manner in which the microcurrent is introduced, the manner in which the microcurrent is introduced into the skin through a patch equipped with reversed electrodialysis means for generating a current through the difference in ion concentration between the high concentration electrolyte solution and the low concentration electrolyte solution. Can be. However, the present invention is not limited thereto, and various methods of iontophoresis may be used.

When the composition of the present invention is applied as a pharmaceutical composition, an external skin preparation or a cosmetic composition, exosomes and / or extracellular vesicles having a high purity particle size distribution contained as an active ingredient are prevented, alleviated or reduced after inflammation. Alternatively, it may have a significant effect on elimination and may solve the safety problem of stem cells or stem cell culture.

In the composition of one embodiment of the invention, the exosomes and / or extracellular vesicles can be obtained by performing the following steps: (a) adding an excipient to the stem cell culture, (b) Filtering the stem cell culture medium to which the excipient is added, (c) separating exosomes and / or extracellular vesicles from the filtered stem cell culture medium using tangential flow filtration (TFF), and (d) An excipient is added to the buffer used for desalting and diafiltration, and TFF (Tangential Flow Filtration) using the excipient-added buffer is used for the isolated exosomes and / or extracellular vesicles. Desalting and diafiltration.

In addition, in the composition of one embodiment of the present invention, the fraction may be obtained by performing the following steps: (a) adding an excipient to the stem cell culture, (b) adding the excipient Filtering the stem cell culture, (c) separating the fraction containing the exosomes and / or extracellular vesicles as an active ingredient from the filtered stem cell culture using TFF (Tangential Flow Filtration), and ( d) Desalting and buffer exchange for the separated fractions by adding an excipient to the buffer used for desalting and diafiltration, and using TFF (Tangential Flow Filtration) using the excipient-added buffer. performing diafiltration).

In the composition of one embodiment of the present invention, when the excipient is added to the buffer solution used for desalting and diafiltration in step (d), exosomes and / or extracellular particles with uniform particle size distribution and high purity Vesicles, or fractions comprising them, can be effectively obtained (see FIG. 6).

On the other hand, "excipient" is usually a substance added for the purpose of giving a suitable firmness or shape to the drug, or to give a certain weight in the case of a small amount of the main agent to be easy to handle, In the present invention, "excipients (also called additives)" known in the art are capable of efficiently separating exosomes and / or extracellular vesicles against impurities such as cell debris, waste, proteins and macroparticles. Give a function. By way of example, and not by way of limitation, excipients may include sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanoose, glucose, Glucose, galactose, fructose, taloose, mannose, tagatose, psycose, erythroose, erythrose, treose, erythritol, arabinose, xylose, lyxose, ribose, li Bloose, Xylulose, Aldoheptoose, Heptulose, Octulose, Gentianose, Umbeliferose, Planteose, Isolknose, Raffinose, Lychnose, Stakiose, Verbas Verbascose, mannitol, maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, starch cracked sugar, starch cracked sugar reducing alcohol, nonionic surfactant (e.g. For example, Tween-20, Triton X-100, Pluonic F-68, etc.), histidine (histidine), glycerol (glycerol) and cholesterol (cholesterol) may be at least one selected from the group consisting of.

In the composition of one embodiment of the present invention, the desalting and buffer exchange may 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 or more preferably 12 times or more relative to the starting volume.

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

In the composition of one embodiment of the present invention, step (c) may further comprise the step of concentrating 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 may be sonicated before TFF.

In the composition of one embodiment of the invention, the exosomes and / or extracellular vesicles or fractions are expressed in TNF-α mRNA expression, IL-6 mRNA expression, IL-1β mRNA expression, iNOS mRNA expression in vitro and Characterized by reducing NO production.

The type of stem cells in the composition of one embodiment of the present invention is not limited, but may preferably be adipose, bone marrow, umbilical cord or cord blood-derived stem cells, more preferably may be adipose-derived stem cells. The type of the adipose derived stem cells is not limited as long as there is no risk of infection by the pathogen and does not cause an immune rejection reaction, but preferably, human adipose derived stem cells.

The composition of one embodiment of the present invention may be prepared into a pharmaceutical composition. When the composition of one embodiment of the present invention is prepared as 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. 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, methyl cellulose, undecided Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. Pharmaceutical compositions of one embodiment of the present invention, powders, pills, tablets, capsules, suspensions, emulsions, syrups, granules, oral preparations such as elixirs, aerosols, external preparations, suppositories, according to a conventional method Or in the form of sterile injectable solutions.

Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance into the patient in any suitable manner, the route of administration of the pharmaceutical composition via any general route as long as the drug can reach the target tissue. Can be. For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration may include transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal 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 compositions of one embodiment of the present invention may be administered by any device in which the active substance may migrate to the target tissue or cell. In addition, an effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to anticipate a disease therapeutic effect.

Solid preparations for oral administration of the pharmaceutical composition of one embodiment of the present invention may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition, the solid dosage form for oral administration may include a lubricant (lubricant) such as silica, stearic acid, magnesium stearate, calcium stearate, talc, polyethylene glycol in addition to the excipient. Liquid formulations for oral administration of the pharmaceutical composition of one embodiment of the present invention may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are simple diluents.

Formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized formulation, or suppository. Formulations for parenteral administration of the pharmaceutical compositions of one embodiment of the invention may also be prepared by injection. Injectables of one embodiment of the present invention may be, but are not limited to, aqueous injections, non-aqueous injections, aqueous suspension injections, non-aqueous suspension injections, or solid injections used by dissolution or suspension. Injectables of one embodiment of the present invention, depending on the type of distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglycerides, diglycerides, propylene glycol, camphor, ester benzoic acid benzoic acid, bismuth succilate It may include at least one of sodium arsenobenzol, or streptomycin, and optionally may include a stabilizer or preservative.

The blending ratio of the pharmaceutical composition of one embodiment of the present invention may be appropriately selected depending on the kind, amount, form, etc. of the additional ingredients as described above. For example, with respect to the total amount of injection, the pharmaceutical composition of the present invention may be included in an amount of about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, suitable dosages of the pharmaceutical compositions of one embodiment of the present invention include the type of disease of the patient, the severity of the disease, the type of formulation, the method of formulation, the age, sex, weight, health condition, diet, excretion rate, time of administration and administration of the patient. It can be adjusted according to the method. For example, when administering the pharmaceutical composition of an embodiment of the present invention to adults, it may be administered in one to several divided doses of 0.001 mg / kg to 100 mg / kg per day.

On the other hand, when the composition of one embodiment of the present invention is prepared as an external preparation for skin, a component usually used in cosmetics or external preparation for skin, for example, a moisturizer, an antioxidant, an oily component, and an ultraviolet ray, within the range not impairing the effects of the present invention. Absorbents, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients and the like can be appropriately blended as necessary.

In addition, the skin external preparation of one embodiment of the present invention is conventional insofar as it does not impair its action (preventing, alleviating, reducing or eliminating hyperpigmentation after inflammation) in addition to exosomes and / or extracellular vesicles derived from stem cells. Antioxidants, sunscreens, sunscreens, sunblocks, skin-whitening agents, anti-inflammatory agents, anti-acne agents, and / or moisturizers used in combination can be used together. For example, the exosomes and / or extracellular vesicles of the present invention, or fractions containing them, may be used in at least one of a hydrogel, hyaluronic acid, hyaluronic acid salt (e.g., sodium hyaluronate), or hyaluronic acid gel. It may be supported or mixed. In the external preparation for skin of an embodiment of the present invention, the type of hydrogel is not limited, but preferably, the hydrogel may be a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. The polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin.

Skin external preparation of one embodiment of the present invention, for example, patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, It can be applied to various forms such as powder and oil paper.

When the external preparation for skin of one embodiment of the present invention is prepared as a cosmetic composition, it is used for the purpose of preventing, alleviating, reducing or eliminating hyperpigmentation after inflammation, and the cosmetic formulation may be any formulation commonly prepared in the art. Can be prepared. For example, patch, mask pack, mask sheet, supple cosmetics, nourishing cosmetics, astringent cosmetics, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , Soaps, shampoos, surfactant-containing cleansing, bathing agents, body lotions, body creams, body oils, body essences, body cleaners, hair dyes, hair tonic and the like, but is not limited thereto.

The external preparation for skin and / or cosmetic composition of one embodiment of the present invention comprises ingredients conventionally used in external preparations for skin and / or cosmetics, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. And a carrier. In addition, in each formulation for the external preparation for skin and / or cosmetic composition, other ingredients may be appropriately selected and blended by those skilled in the art without difficulty according to the kind or purpose of use of the external preparation for skin and / or cosmetic composition.

Another embodiment of the present invention using the cosmetic composition, provides a cosmetic method through the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation.

Cosmetic method of one embodiment of the present invention, (a) applying the cosmetic composition directly to the skin of the mammal, (b) a patch, mask pack or mask sheet to which the cosmetic composition is applied or deposited, the skin of the mammal Contacting or attaching to, or sequentially proceeding with (a) and (b).

The cosmetic method of one embodiment of the present invention may further include 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 an embodiment of the present invention may further include contacting or attaching an iontophoresis device to the skin of the mammal.

In the cosmetic method of one embodiment of the present invention, the iontophoresis device is composed of a flexible battery, a lithium ion secondary battery, an alkaline battery, a battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electric dialysis battery It may include at least one battery selected from the group, or may include a patch, mask pack or mask sheet on which the at least one battery is mounted.

The cosmetic method of an embodiment of the present invention, the laser beam irradiation, IPL (Intense Pulsed Light) irradiation to the skin of the mammal before, with or after the application of the cosmetic composition to the skin of the mammal, The method may further include performing at least one selected from the group consisting of RF (radio frequency) energy application, chemical exfoliation, skin peeling, and freezing treatment. The freezing treatment may be performed as disclosed in Korean Patent Publication No. 10-2017-0044767. However, the cosmetic method of the present invention is not limited thereto, and various methods of reducing the pigment by freezing the local skin region may be used.

In the method for improving the skin condition according to one embodiment of the present invention, at least one of the exosomes and / or extracellular vesicles, or the fraction, may be selected from the group consisting of the laser beam irradiation, the IPL (Intense Pulsed Light) irradiation, and the RF ( radio frequency) can penetrate into the mammal's skin (eg, the dermal and / or subcutaneous layers) through the micropores of the skin of the mammal produced by at least one selected from the group consisting of energy application.

Another embodiment of the invention provides a method of preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation comprising administering to a mammal a therapeutically effective amount of said composition.

The composition of the present invention can inhibit the production of TNF-α, IL-6, IL-1β, and nitric oxide (NO) that stimulate melanocytes and ultimately reduce melanin production, and melanin pigment is deposited It can penetrate deep into the dermis, effectively preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation.

The exosomes and / or extracellular vesicles obtained according to the separation method of the present invention are uniform in particle size distribution, TNF-α and IL- stimulating melanocytes, compared to the extracellular vesicles obtained according to the conventional separation method. 6, IL-1β, and NO (nitric oxide) production inhibitory effect is much better. As described above, the composition of the present invention containing the stem cell-derived exosomes and / or extracellular vesicles with excellent functional activity effectively inhibits the activity of melanocytes and the production of melanin, thereby preventing hyperpigmentation after inflammation, It is far superior to the prior art in terms of mitigation, reduction or elimination effects.

Therefore, the composition of the present invention can be usefully used as a skin external preparation and cosmetic composition for the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation.

In addition, the present invention can obtain exosomes and / or extracellular vesicles derived from stem cells having high purity and uniform particle size, or fractions containing the same in large quantities at low cost. Accordingly, the present invention can provide a composition at low cost in a large amount at low cost with stem cells-derived exosomes and / or extracellular vesicles, or a fraction containing the same as an active ingredient excellent in functional activity. In addition, the compositions of the present invention are capable of scale-up and are also suitable for Good Manufacturing Practice (GMP).

In addition, the compositions of the present invention can be applied to the skin using iontophoresis, laser beam irradiation, IPL irradiation and / or RF energy application to the exosomes and / or extracellular vesicles up to the dermal layer of the melanin-deposited skin. It can penetrate deeply to maximize the skin cosmetic effect or therapeutic effect, and in particular can cause synergistic effects and pigment reduction or removal by laser beam irradiation, IPL irradiation and / or RF energy application.

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

1 is a method for producing exosomes and / or extracellular vesicles, and / or fractions comprising the same from the stem cell culture in accordance with an embodiment of the present invention for separating and purifying exosomes and / or extracellular vesicles A flowchart describing the process.
Figure 2 is a ratio of the total amount of protein contained in the solution for each step of preparing an exosome and / or extracellular endoplasmic reticulum, and / or a fraction comprising the same from stem cell culture according to an embodiment of the present invention (Relative) amount of protein) is measured. The ratio of the total amount of protein in each step is expressed as the relative ratio of the total amount of protein to the stem cell culture. The experimental results show the results obtained in two different batches, respectively.
Figure 3 shows the results of measuring the productivity (purity) and (purity) of the exosomes and / or extracellular vesicles obtained according to an embodiment of the present invention. Productivity of exosomes and / or extracellular vesicles was calculated as "particle count of exosomes and / or extracellular vesicles obtained per mL of stem cell culture (CM)", and the purity of exosomes and / or extracellular vesicles was " Number of particles of exosomes and / or extracellular vesicles per μg of protein 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 exosomes and / or extracellular vesicles obtained in accordance with an embodiment of the present invention. "A" indicates particle size distribution and particle number by tunable resistive pulse sensing (TRPS) analysis. "B" indicates particle size distribution and number of particles by nanoparticle tracking analysis (NTA) analysis. "C" shows the particle image by magnification by means of the transmitted electron microscopy (TEM) analysis. "D" shows western blot results of exosomes and / or extracellular cell bodies obtained according to one embodiment of the invention. "E" shows the results of flow cytometry for CD63 and CD81 in marker analysis for exosomes and / or extracellular cell bodies obtained according to one embodiment of the invention.
FIG. 5 shows NTA analysis results for particle size distribution (uniform distribution) showing that exosomes and / or extracellular vesicles with uniform particle size distribution and high purity are obtained with the addition of excipients. As the amount of excipient added is increased, particle size distribution results with a single peak can be obtained.
Figure 6 shows the NTA analysis results showing the particle size distribution depending on whether or not excipients added during the preparation of the exosomes and / or extracellular vesicles, and / or fractions comprising the same according to an embodiment of the present invention. "A" indicates the addition of the excipients throughout the manufacturing process, B when the cell culture solution is frozen and thawed, and then added the excipients, "C" represents the result of the preparation without the addition of the excipients. "D" shows a result of comparing relative productivity and relative concentration of exosomes and / or extracellular vesicles separated by A to C methods. "E" shows the mean particle size (Mean size) of the exosomes and / or extracellular vesicles separated by the A to C method.
Figure 7 shows the results confirmed that there is no cytotoxicity after the treatment of exosomes and / or extracellular vesicles according to an embodiment of the present invention to HS68 cells, human skin fibroblasts.
FIG. 8 shows that LN-induced expression of TNF-α, IL-6, IL-1β and iNOS mRNA was reduced when raw exo cells were treated with exosomes and / or extracellular vesicles of the present invention with LPS. It is a graph which shows the real-time PCR result confirmed.
Figure 9 shows the results of experiments confirming the effect of reducing the inflammatory cytokine TNF-α formation by exosomes and / or extracellular vesicles according to an embodiment of the present invention. In Figure 9, PBS is phosphate-buffered saline, DEX is dexamethasone, each number represents the exosomes and / or extracellular vesicle throughput (μg / mL).
10 shows the effect of reducing NO formation by exosomes and / or extracellular vesicles isolated according to one embodiment of the present invention, and the effect of reducing NO formation by extracellular vesicles separated by conventional precipitation method (PPT). The experimental results compared are shown. A is the result of NTA analysis of the extracellular vesicles separated by the conventional precipitation method, B is the result of NTA analysis of the exosomes and / or extracellular vesicles separated and purified by the method according to an embodiment of the present invention, C Is a graph comparing the effect of reducing NO formation. The decrease in NO formation was expressed as a relative percentage (%) to the reduction in NO formation by the positive control dexamethasone (Dex).
FIG. 11 shows the results of fluorescence intensity measurements identifying exosomes and / or extracellular vesicles stained with PKH67.
12 is a fluorescence microscope image confirming the degree of delivery of fluorescently stained exosomes and / or extracellular vesicles into porcine skin tissue.
Figure 13 is a confocal fluorescence microscope image confirming the degree of delivery of fluorescently stained exosomes and / or extracellular vesicles into the skin of the mouse.
FIG. 14 is a graph comparing total fluorescence intensities obtained by measuring fluorescence intensities on each image of FIG. 13.
15 is a photograph showing that post-inflammatory hyperpigmentation caused by laser beam irradiation was reduced by treating a composition comprising an exosome and / or extracellular vesicles according to one embodiment of the present invention.
Figure 16 is an ion that flows a microcurrent to the skin (affected) to which the composition is applied after applying a composition comprising exosomes and / or extracellular vesicles according to an embodiment of the present invention on the skin (affected) of the human As a result of performing the topography, the erythema (hyperpigmentation after skin inflammation) of the severely affected dermatitis (lesion) is significantly improved.
17 is a schematic diagram illustrating the relationship between the inflammatory response and the activity and melanogenesis of melanocytes.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, except to exclude other components unless specifically stated otherwise.

Example

Example 1 Culture of Cells

Raw 264.7, a mouse macrophage line, was purchased from Korea Cell Line Bank and cultured. 5% CO in DMEM (purchased from Thermo Scientific) medium containing 10% fetal bovine serum (purchased from Thermo Scientific) and 1% antibiotic-antimycotics (purchased from Thermo Scientific) for cell culture Subcultured at ₂ , 37 ° C.

HS68 cells, which are human dermal fibroblasts, were purchased from ATCC and were treated with 10% fetal bovine serum (obtained from Thermo Scientific) and 1% antibiotic-antimycotics (obtained from Thermo Scientific). Passage was performed in DMEM (purchased from Thermo Scientific) containing medium at 5% CO ₂ , 37 ° C.

Fat-derived stem cells were cultured at 5% CO ₂ , 37 ° C. according to cell culture methods known in the art. Then, washed with phosphate buffer (phosphate-buffered saline (PBS, purchased from Thermo Scientific), and then incubated for 1 to 10 days by replacing with serum-free, phenol-free medium and recovering the supernatant (hereinafter, culture). It was.

An excipient was added to the culture to obtain exosomes and / or extracellular vesicles with uniform particle size distribution and high purity during separation of exosomes and / or extracellular vesicles. Excipients include sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanose, glucose, dextrose, glucose, galactose, fructose, and talo Oss, mannose, tagatose, psycose, erythrose, erythrose, treos, erythritol, arabinose, xylose, rickose, ribose, ribulose, xylulose, aldohepto Oss, Heptulose, Octulose, Gentianose, Umbeliferose, Planteose, Isolknose, Raffinose, Lychnose, Stakiose, Verbascose, Mannitol , Maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, starch cracked sugar, starch cracked sugar reducing alcohol, nonionic surfactant (e.g., Tween-20, Triton X- 100, Pluonic F-68, etc.), Histi 0.01 to 30% by weight of at least one selected from the group consisting of dean (histidine), glycerol (glycerol) and cholesterol (cholesterol) was added. After the addition of excipients, the culture solution was filtered through a 0.22 μm filter to remove impurities such as cell debris, waste and macroparticles. The filtered culture immediately separated the exosomes and / or extracellular vesicles through a separation process. In addition, the filtered culture was stored in a refrigerator (image 10 ℃ or less) and then used for the separation of exosomes and / or extracellular vesicles. In addition, the filtered culture was frozen and stored in a cryogenic freezer of -60 ° C or less and thawed, and then exosomes and / or extracellular vesicles were separated. Thereafter, exosomes and / or extracellular vesicles were separated from the culture using a tangential flow filtration device (TFF).

Example 2: Isolation and Purification of Exosomes and / or Extracellular Vesicles by the TFF Method

In Example 1, TFF (Tangential Flow Filtration) method was used for separation, concentration, desalting and diafiltration of exosomes and / or extracellular vesicles from the culture medium filtered with a 0.22 μm filter. The TFF method was used to sonicate the cultures before separating and concentrating the exosomes and / or extracellular vesicles to release agglomerates of potential exosomes and / or extracellular vesicles. Cartridge filters (also known as hollow fiber filters; GE Healthcare or Spectrum Labs) or cassette filters (Pall or Sartorius or Merck Millipore) were used as filters for the TFF method. TFF filters can be selected by various molecular weight cutoffs (MWCO). Exosomes and / or extracellular vesicles were selectively isolated and concentrated by the selected MWCO, and particles, proteins, lipids, nucleic acids, and small molecule compounds smaller than MWCO were removed.

MWCO 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da TFF filters, or 0.05 μm TFF filters were used to isolate and concentrate the exosomes and / or extracellular vesicles. The culture was concentrated to a volume of 1/100 to 1/25 using the TFF method, while substances smaller than MWCO were removed to separate exosomes and / or extracellular vesicles.

The isolated, concentrated exosomes and / or extracellular vesicle solutions were further subjected to desalting and diafiltration using the TFF method. At this time, desalting and buffer exchange were carried out continuously (discontinuous diafiltration) or at least 4 times, preferably 6 times to 10 times, more preferably, relative to the starting volume. It was performed using a buffer solution having a volume of 12 times or more. An excipient was added to the buffer solution to obtain exosomes and / or extracellular vesicles with uniform particle size distribution and high purity. The results of confirming the effect of obtaining a high yield of exosomes and / or extracellular vesicles with a high purity and uniform particle size distribution according to the excipient treatment is shown in FIG.

Example 3: Characterization of Isolated Exosomes and / or Extracellular Vesicles

The amount of protein in the separated exosomes and / or extracellular vesicles, cultures, and fractions of the TFF separation process was measured using BCA colorimetry (purchased from Thermo Fisher) or FluoroProfile fluorescence (purchased from Sigma). The extent to which the exosomes and / or extracellular vesicles are separated and concentrated and the proteins, lipids, nucleic acids, small molecule compounds, etc. are removed by the TFF method of one embodiment of the present invention is monitored by protein quantification and the results are shown in FIG. 2. It was. As a result, it was found that the protein present in the culture medium was effectively removed by the TFF method of one embodiment of the present invention.

When the exosomes and / or extracellular vesicles were separated by the TFF method of one embodiment of the present invention, the results of comparing the productivity and purity in five independent batches are shown in FIG. 3. Analysis of the results obtained from the five independent batches, it was confirmed that the exosomes and / or extracellular vesicles can be isolated very stably by the TFF method of one embodiment of the present invention.

The isolated exosomes and / or extracellular vesicles were separated from the particle size by nanoparticle tracking analysis (NTA; purchased from Malvern) or tunable resistive pulse sensing (TRPS; purchased from Izon Science). The concentration was measured. The uniformity and size of the isolated exosomes and / or extracellular vesicles were analyzed using a transmitted electron microscopy (TEM). TRPS, NTA, and TEM analysis results of the exosomes and / or extracellular vesicles isolated according to one embodiment of the present invention are shown in FIG. 4.

After separating the exosomes and / or extracellular vesicles by the TFF method, the results of NTA analysis of the size distribution of the exosomes and / or extracellular vesicles depending on the addition of excipients are shown in Figure 5. When the exosomes and / or extracellular vesicles were separated by the TFF method in the absence of excipients, particles having a size of 300 nm or more were found, whereas increasing the amount of excipient added reduced the particles having a size of 300 nm or more. It was confirmed that the size distribution of the exosomes and / or extracellular vesicles was uniform.

The addition of excipients was further investigated in the process of separating exosomes and / or extracellular vesicles by the TFF method. As shown in FIG. 6, when an excipient was added to the entire TFF process, an exosome and / or extracellular vesicles having a uniform size distribution could be obtained (FIG. 6A). On the other hand, in the case of using the culture solution that was stored frozen without the excipient or the TFF without the excipient at all, a non-uniform exosome and / or extracellular vesicles containing a lot of large particles was obtained (Fig. 6B And FIG. 6C).

As a result of comparing the relative productivity and concentration of the isolated exosomes and / or extracellular vesicles, when the excipient was added to the entire TFF, the exosomes and / or extracellular vesicles were obtained with very high productivity. Alternatively, the concentration of extracellular vesicles was also at least five times higher (FIG. 6D). As shown in the NTA analysis, the average size of the isolated exosomes and / or extracellular vesicles was also uniformly confirmed at 200 nm when the excipient was added throughout the TFF process (FIG. 6E).

FIG. 4D shows the presence of CD9, CD63, CD81 and TSG101 markers as a result of Western blot for exosomes and / or extracellular vesicles isolated 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 antibodies to each marker.

Figure 4E confirmed the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer for exosomes and / or extracellular vesicles isolated in accordance with the method of one embodiment of the present invention. To isolate exosomes and / or extracellular vesicles that are positive for CD63, an exosome-human CD63 separation / detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method and PE-mouse anti-human After staining the markers using CD63 (PE-Mouse anti-human CD63) (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD), a flow cytometer (ACEA) was used. Biosciences).

Putting the above results together, the present invention adds a substance known as an excipient during separation and / or purification using tangential flow filtration to produce exosomes and / or extracellular vesicles with high purity and uniform particle size distribution. It was confirmed that the high yield can be separated and purified economically and efficiently. In addition, it can be seen that the processes of the separation method of one embodiment of the present invention are scale-up and suitable for GMP.

Example 4 Measurement of Cytotoxicity Following Treatment with Exosomes and / or Extracellular Vesicles

In order to evaluate the toxicity of the exosomes and / or extracellular vesicles obtained according to the isolation method of one embodiment of the present invention in HS68 cells, which are human skin fibroblasts, cells were treated with exosomes by concentration and the cell proliferation rate was confirmed. HS68 cells were suspended in DMEM containing 10% FBS and then aliquoted to have a confluency of 80-90% and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After 24 hours, the culture solution was removed and treated with exosomes and / or extracellular vesicles prepared in Example 2 by concentration to evaluate the cell viability while culturing for 24 to 72 hours. Cell viability was determined by WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramamar blue reagent ( Measurements were made using alamarBlue reagent (purchased from Thermo Scientific) and a microplate reader (purchased from Molecular Devices).

The control group was based on the number of cells cultured in a normal cell culture medium that was not treated with exosomes and / or extracellular vesicles, and cytotoxicity by the exosomes and / or extracellular vesicles of the invention within the concentration range tested. It was confirmed that this did not appear (Fig. 7).

Example 5 Inflammation Response Measurement Using Macrophage Cell Line

Raw 264.7 cells were suspended in DMEM medium containing 10% FBS and dispensed to have 80-90% confluency in each well of a multiwell plate. Next day, the exosomes and / or extracellular vesicles of the present invention diluted in fresh serum-free medium containing LPS (exosomes and / or extracellular vesicles prepared in Example 2) were treated at an appropriate concentration for 1 to 24 hours. Incubated. After culturing, the culture supernatant was taken, and the inflammatory response was confirmed by measuring NO and inflammatory cytokines present in the culture. Inflammatory responses in the culture were measured using a NO detection kit (purchased from Intron Bio or Promega). ELISA kit (purchased from the R & D system) was performed according to the manufacturer's manual to determine the amount of inflammatory cytokine TNF-α in the group treated with LPS only and the group treated with the exosomes and / or extracellular vesicles of the present invention. Positive control was treated with dexamethasone (purchased from Sigma). In addition, cDNA was prepared from 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 by real-time PCR. The kind and sequence of primers used for real-time PCR are shown in Table 1 below.

Primer Types and Sequences Used in Real-Time PCR gene order Forward primer (5 '→ 3') Reverse primer (5 '→ 3') TNF-α 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-1β 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 extracellular vesicles of the present invention were treated with LPS on Raw 264.7 cells, which are macrophages of mice, TNF-α, an inflammatory cytokine induced by LPS , IL-6 and IL-1β mRNA expression decreased, and the NO expression enzyme iNOS mRNA expression decreased.

Next, as shown in FIG. 9, TNF-, an inflammatory cytokine induced by LPS as a result of treating the exosomes and / or extracellular vesicles of the present invention in the presence of LPS in macrophage RAW264.7 cells of mice. It was confirmed to reduce α production.

These results indicate that exosomes and / or extracellular vesicles of the present invention are useful for the prevention, alleviation, reduction or elimination of post-inflammatory hyperpigmentation, ie TNF-α, IL-6, which stimulate melanocytes, IL-1β, and NO (nitric oxide) to inhibit the production and ultimately has the activity that can reduce melanin production, the exosomes and / or extracellular vesicles of the present invention prevents hyperpigmentation after inflammation, It is strongly suggested that it can be usefully used as an active ingredient of a composition for alleviation, reduction or elimination.

Example 6 Comparison of NO Formation Reduction Effects According to Separation Methods

As an example that does not limit the present invention, the exosomes and / or cells obtained by TFF separation purification of one embodiment of the present invention in order to compare the effect of reducing NO formation of exosomes and / or extracellular vesicles according to the separation method In addition to the extracellular vesicles, extracellular vesicles separated by a conventional precipitation method were prepared. Precipitation was performed according to the protocol of the manufacturer (System Biosciences). The extracellular vesicles separated by the conventional precipitation method (see FIG. 10A) were compared with the exosomes and / or extracellular vesicles separated and purified by the TFF method of one embodiment of the present invention (see FIG. 10B). It was confirmed that the uniformity is low and has various sizes. Also, as shown in FIG. 10C, the exosomes and / or extracellular vesicles separated and purified by the TFF method of one embodiment of the present invention exhibit NO formation to a much higher level than the extracellular vesicles obtained by conventional precipitation methods. It was confirmed to inhibit. These results show that the exosomes and / or extracellular vesicles isolated and purified according to one embodiment of the present invention are superior in terms of uniformity of particle size distribution and inhibition of NO formation compared to the extracellular vesicles isolated according to the conventional method. have.

Thus, the exosomes and / or extracellular vesicles obtained according to the separation method of one embodiment of the present invention may be characterized in terms of their performance or functional activity (e.g., particle size distribution) compared to the extracellular vesicles obtained according to conventional separation methods. Uniformity, inhibition of NO production, stimulation of melanin cell activity and reduction of melanin production, etc.), and the composition of the present invention containing exosomes and / or extracellular vesicles derived from stem cells having excellent functional activity as an active ingredient. Is much superior to the prior art in terms of the effect of preventing, alleviating, reducing or eliminating hyperpigmentation after inflammation.

Example 7 Skin Permeability Testing of Exosomes and / or Extracellular Vesicles of the Invention

PKH67 dye (available from Sigma) was used to prepare fluorescently stained exosomes and / or extracellular vesicles. 1 mM PKH67 was diluted in Diluent C (purchased from Sigma) to prepare a 10 μM PKH67 solution, and then mixed with an appropriate concentration of exosomes and / or extracellular vesicle solutions for 10 minutes with light blocking at room temperature. Reacted. After the reaction was completed, a MW3000 spin column (purchased from Thermo Fisher) was used to remove residual PKH67 dye from the exosomes and / or extracellular vesicles of the invention stained with PKH67 (hereinafter abbreviated as "PKH-exosome"). Used. As a result of confirming using a fluorometer (purchased from Molecular Devices), after removing PKH67 which did not react with the exosomes and / or extracellular vesicles of the present invention, it was confirmed that sufficient intensity of fluorescence was detected in PKH-exosomes (FIG. 11). ).

PKH-exosomes were dispersed in phosphate buffer solution (PBS) at a suitable concentration, for example 1 × 10 ⁵ particles / mL 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 then reacted for an appropriate time, for example, 30 minutes to 1 hour, so that the PKH-exosomes were delivered to the subcutaneous tissue. In addition, PKH-exosomes were applied to the outside of the pig skin and covered with a nonwoven fabric, and then a microcurrent was flowed for a predetermined time, for example, 30 minutes to 1 hour. After the reaction was completed, the pig skin tissue was immersed in 3.7% formaldehyde solution to react overnight, washed three times with phosphate buffer solution for 5 minutes. The washed porcine skin tissue was soaked in 30% sucrose solution and then treated with OCT compound. After washing three times with phosphate buffer solution three times each, a section was made in the longitudinal section using a microtome to place the tissue section on the slide glass. The fabrication of tissue sections can also be performed prior to fixation of the tissue with formaldehyde solution. Fluorescence detected from PKH-exosomes in tissue sections was observed using a fluorescence microscope. The above method confirmed the PKH-exosomes that penetrated the epidermis of porcine skin tissue and delivered to the subcutaneous tissue (FIG. 12). As shown in FIG. 12, the exosomes and / or extracellular vesicles of the present invention can effectively pass through the skin barrier and be delivered deep into the skin tissue and can be effectively absorbed by the skin. Therefore, the exosomes and / or extracellular vesicles contained in the external preparation or cosmetic composition of the present invention as an active ingredient can penetrate deep into the dermal layer in which melanin is deposited, thereby preventing, alleviating, reducing or eliminating hyperpigmentation after inflammation. Etc., it is expected to act effectively.

Next, the skin tissue of the hairless mouse was extracted and placed above 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 solution (PBS) at a suitable concentration, for example 1 × 10 ⁵ particles / mL to 1 × 10 ⁹ particles / mL, and then applied to the outside of the skin tissue of the mouse. In this case, in order to prevent the drying of the PKH-exosomes, the nonwoven fabric was pre-positioned outside the mouse skin tissue, and PKH-exosomes were injected between the nonwoven fabric and the skin tissue. After that, the reaction was carried out for 30 minutes to 1 hour. In addition, after injecting PKH-exosomes between the nonwoven fabric and the skin tissue, a microcurrent was flowed for a predetermined time, for example, 30 minutes to 1 hour. After the reaction, immediately check the PKH-exosomes delivered into the skin tissue using confocal fluorescence microscope (Leica, SP8X), or further react the skin tissue and PKH-exosome solution for 1 to 6 hours. After PKH-exosomes were confirmed by confocal fluorescence microscopy. As a result of confirming the above method, the exosomes and / or extracellular vesicles of the present invention can effectively pass through the skin barrier and be delivered deep into the skin tissue, and can be effectively absorbed by the skin (FIGS. 13 and 14).

Therefore, the exosomes and / or extracellular vesicles contained in the external preparation or cosmetic composition of the present invention as an active ingredient can penetrate deep into the dermal layer in which melanin is deposited, thereby preventing, alleviating, reducing or eliminating hyperpigmentation after inflammation. Etc., it is expected to act effectively.

Example 8 Mitigation of Postpigmentation Hyperpigmentation Caused by Laser Beam Irradiation

A suitable laser beam was irradiated on human skin for the purpose of improving skin condition. The frequency or interval of irradiation of the laser beam can be adjusted according to the skin condition of the patient or the purpose of improving the skin condition.

In each laser beam irradiation, a composition containing the exosomes and / or extracellular vesicles obtained according to the separation method of one embodiment of the present invention may be applied directly to the skin, and an iontophoresis device (fine to skin It can also be applied to the skin using an active transdermal permeable device that allows a current to flow. For example, ampoules containing exosomes and / or extracellular vesicles obtained according to the separation method of one embodiment of the invention before, during, and / or after each laser beam irradiation, including translucent latex compositions And liquid immersion sheet mask (white sheet mask in which the translucent fluid phase is deposited) is used every morning and evening for 3 to 5 days, or the ampoule and liquid immersion sheet mask and transdermal penetration promoting sheet mask (Iontoporesis device) Silver sheet masks, including whether applied is optional) were used every morning and evening for 3 to 5 days.

The photographs immediately after the laser beam irradiation and the application of the composition containing the exosomes and / or extracellular vesicles, for example, three days later, were compared, and the exosomes and / or extracellular vesicles were also compared. The containing composition was compared to the treated and untreated skin areas. When treating the skin with a composition containing exosomes and / or extracellular vesicles with laser beam irradiation, it was confirmed whether the side effects caused by laser beam irradiation were alleviated or restored to normal condition. To this end, the treatment of compositions containing exosomes and / or extracellular vesicles promotes skin tissue regeneration, prevents skin cracking and scab formation, alleviates or reduces hyperpigmentation, restores to normal skin, skin Moisturizing and soothing, reduction of heat damage and recovery of skin damage were evaluated. As a result of evaluating each of the above evaluation items before and after the use of the composition containing the exosomes and / or extracellular vesicles, the effect of alleviating or reducing post-inflammatory hyperpigmentation due to laser beam irradiation after use was compared with before use. It was confirmed that the obtained (see Fig. 15).

Example 9: Treatment of a composition comprising exosomes and / or extracellular vesicles as an active ingredient on human skin

The composition containing the exosomes and / or extracellular vesicles obtained according to the separation method of one embodiment of the present invention (including the semi-transparent latex composition) is contained in the affected areas (hands, necks, arms, etc.) of three severe atopic patients. After applying three times a week for two weeks, the iontophoresis was performed to flow a microcurrent to the affected area where the composition was applied using the iontophoresis device. As a result, the severe itching of the patients was significantly alleviated, and the erythema symptom, which is a hyperpigmentation symptom after skin inflammation, was also significantly improved (FIG. 16). All patients who applied the composition containing the exosomes and / or extracellular vesicles of the present invention alleviated the symptoms of erythema, which is a symptom of hyperpigmentation after severe itching and skin inflammation, so that the symptoms of steroids or antihistamines can be discontinued. Improvements were made.

Therefore, the external preparation for skin or cosmetic composition containing the exosomes and / or extracellular vesicles obtained according to the separation method of one embodiment of the present invention as an active ingredient, hyperpigmentation after inflammation as confirmed through the above clinical trials It can be seen that the effect of the prevention, alleviation, reduction or elimination of.

As mentioned above, although this invention was demonstrated to the said Example, this invention is not limited to this. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

<110> ExoCoBio Inc. <120> A composition comprising an exosome and / or extracellular vesicle          derived from stem cell as an active ingredient and its          application for preventing, alleviating, reducing or removing          postinflammatory hyperpigmentation <130> P18-0046KR <160> 10 <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> <223> IL-6 forward primer <400> 7 gccagagtcc ttcagagaga taca 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 8 attggatggt cttggtcctt agcc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta forward primer <400> 9 gcaacgacaa aatacctgtg gcct 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta reverse primer <400> 10 agttggggaa ctctgcagac tcaa 24

Claims (27)

From a group consisting of exosomes and / or extracellular vesicles with a high purity particle size distribution derived from stem cells, and a fraction containing exosomes and / or extracellular vesicles with a high purity particle size distribution isolated from stem cell culture. A composition for preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation comprising at least one selected as an active ingredient. The method of claim 1,
The exosomes and / or extracellular vesicles are obtained by performing the following steps:
(a) adding an excipient to the stem cell culture medium, (b) filtering the stem cell culture medium to which the excipient is added, and (c) from the filtered stem cell culture medium, TFF (Tangential Flow Filtration) Separating the exosomes and / or extracellular vesicles using the same, and (d) adding an excipient to the buffer used for desalting and diafiltration, and using TFF (Tangential Flow) using the excipient-added buffer. Performing desalting and diafiltration of the isolated exosomes and / or extracellular vesicles. The method of claim 1,
The fraction is obtained by performing the following steps:
(a) adding an excipient to the stem cell culture medium, (b) filtering the stem cell culture medium to which the excipient is added, and (c) from the filtered stem cell culture medium, TFF (Tangential Flow Filtration) Separating the fraction containing the exosomes and / or extracellular vesicles as an active ingredient, and (d) adding an excipient to the buffer used for desalting and diafiltration, and adding the excipient to the buffer. Performing desalting and diafiltration of the separated fractions using TFF (Tangential Flow Filtration) using a solution. The method according to claim 2 or 3,
The desalting and buffer exchange may be performed continuously or intermittently, the composition. The method according to claim 2 or 3,
Desalting and buffer exchange using a buffer solution having a volume of at least four times the starting volume, the composition. The method according to claim 2 or 3,
A composition characterized by using a molecular weight cutoff (MWCO) 100,000 Da, 300,000 Da, 500,000 Da or 750,000 Da, or 0.05 μm TFF filter for tangential flow filtration (TFF). The method according to claim 2 or 3,
The step (c) further comprises the step of concentrating to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration). The method according to claim 2 or 3,
The filtered stem cell culture is characterized in that the ultrasonication (sonication) before TFF, composition. The method according to claim 2 or 3,
The excipients include sucrose, trehalose, lactose, lactulose, maltose, cellobiose, isomaltose, turanose, glucose, dextrose, glucose, galactose, fructose, Taloose, mannose, tagatose, psycose, erythrose, erythrose, treose, erythritol, arabinose, xylose, rickose, ribose, ribulose, xylulose, aldo Heptose, Heptulose, Octulose, Gentianose, Umbeliferose, Planteose, Isolknose, Raffinose, Lychnoose, Stakiose, Verbascose, Mannitol ), Maltitol, lactitol, maltotriose, polyethylene glycol, xylitol, sorbitol, starch cracked sugar, starch cracked sugar reducing alcohol, nonionic surfactant, histidine, glycerol and Cholester At least one member, the composition selected from the group consisting of (cholesterol). The method according to any one of claims 1 to 3,
The exosomes and / or extracellular vesicles or fractions are characterized in that in vitro assays reduce TNF-α mRNA expression, IL-6 mRNA expression, IL-1β mRNA expression, iNOS mRNA expression and NO production, Composition. Exosomes and / or extracellular vesicles derived from stem cells, and
A skin external preparation for preventing, alleviating, reducing or removing post-inflammatory hyperpigmentation comprising at least one selected from the group consisting of exosomes and / or extracellular vesicle-containing fractions isolated from stem cell culture. The method of claim 11,
The exosomes and / or extracellular vesicles or fractions are supported or mixed on at least one of a hydrogel, hyaluronic acid, hyaluronic acid salt, or hyaluronic acid gel. The method of claim 12,
The hydrogel is a skin external preparation, characterized in that the hydrogel obtained by dispersing the gelling polymer in a polyhydric alcohol. The method of claim 13,
The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. The polyhydric alcohol is at least one member selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin. . Exosomes and / or extracellular vesicles derived from stem cells, and
Cosmetic composition for the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation comprising at least one selected from the group consisting of exosomes and / or extracellular vesicle-containing fractions isolated from stem cell culture. The method of claim 15,
The exosomes and / or extracellular vesicles or fractions are supported or mixed in at least one of a hydrogel, hyaluronic acid, hyaluronic acid salt, or hyaluronic acid gel. The method of claim 16,
The hydrogel is a cosmetic composition, characterized in that the hydrogel obtained by dispersing the gelling polymer in a polyhydric alcohol. The method of claim 17,
The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. The polyhydric alcohol is at least one member selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin. . The method according to any one of claims 15 to 18,
At least one selected from the group consisting of patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, aerosols, mists, foundations, powders and oil papers Cosmetic composition, characterized in that applied to the form. The method of claim 19,
Cosmetic composition, characterized in that applied to at least one side of the patch, mask pack or mask sheet. (a) applying the cosmetic composition according to any one of claims 15 to 18 directly to the skin of a mammal; (b) the mammal applying a patch, mask pack or mask sheet to which the cosmetic composition is applied or deposited; A method of cosmetics through the prevention, alleviation, reduction or removal of post-inflammatory hyperpigmentation, comprising contacting or adhering to the skin, or sequentially proceeding with (a) and (b). The method of claim 21,
(c) further comprising performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the cosmetic composition is applied. The method of claim 22,
A method of cosmetics further comprising contacting or attaching an iontophoresis device to the skin of the mammal. The method of claim 23, wherein
The iontophoresis device includes at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery. , The cosmetic method comprising a patch, a mask pack or a mask sheet is equipped with at least one battery. The method of claim 22,
Laser beam irradiation, IPL (Intense Pulsed Light) irradiation, RF (radio frequency) energy application, chemical keratin before, with or after application of the cosmetic composition to the skin of the mammal And performing at least one selected from the group consisting of removal, skin peeling and freezing treatment. The method of claim 25,
At least one of the exosomes and / or extracellular vesicles, or the fraction, is at least one selected from the group consisting of the laser beam irradiation, the IPL (Intense Pulsed Light) irradiation, and the radio frequency (RF) energy application Cosmetic method, characterized in that penetrated into the skin of the mammal through the micropores of the skin of the mammal produced by. A method of preventing, alleviating, reducing or eliminating post-inflammatory hyperpigmentation comprising administering to a mammal a therapeutically effective amount of the composition of any one of claims 1 to 3.

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