WO2018155979A1 - Stem cell-derived nanovesicle, and hair growth promoting and restoring composition containing same - Google Patents

Abstract

The present invention relates to a stem cell-derived microvesicle and a hair growth promoting composition containing the same and, more specifically, to a cosmetic composition and a pharmaceutical composition, which are for treating hair loss or promoting hair growth and contain, as an active ingredient, a stem cell-derived nanovesicle prepared by a method comprising the steps of: (a) suspending, in an isotonic solution, a stem cell culture solution and a hair growth promoting ingredient; (b) degrading cells in the solution of step (a); and (c) obtaining a nanovesicle by ultracentrifuging the solution of step (b). The method of the present invention has an effect of treating or alleviating hair loss symptoms through hair loss preventing and hair growth promoting effects, thereby being useful in the preparation of cosmetics and therapeutic agents which exhibit hair growth promoting effects.

Description

 【Specification】

 [Name of invention]

 Stem cell-derived nano-vesicles and a composition for promoting hair growth and hair growth comprising the same

 This application claims the priority of Korean Patent Application No. 10-2017-0024992, filed February 24, 2017, the entirety of which is a reference of the present application. The present invention relates to a stem cell-derived nano-vesicles and a composition for promoting hair growth and hair growth comprising the same, and more specifically, a) passing the stem cells through a filter; b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles; c) separating and purifying the uniform size of the nano-vesicles from the nano-vesicles obtained in step b); And d) a method comprising the step of encapsulating the hair growth promoting component in the nano vesicles separated and purified in step c), for hair loss treatment or hair growth and hair growth promotion comprising the stem cell-derived nano vesicles as an active ingredient. It relates to cosmetic compositions and pharmaceutical compositions.

Background Art

 Mesenchymal enjoyment cells are cells that have the ability to differentiate into various mesenchymal tissues while maintaining sel f-renewal and stem cell maintenance. It has been found to synthesize materials for regeneration.

In addition, mesenchymal stem cells show immune regulation by inhibiting part of the immune system, inhibiting the activity and proliferation of T and B lymphocytes, preventing the activity of NK cel l, avoiding CTL-mediated lysi s, and dendritic cells. It is capable of coordinating the substances secreted by (dendr it ic cel l) or macrophage (al lo ^¬ transplantat ion) or xenotransplantation (xeno-1 r ansp 1 ant at i on) ° ll It is known to be usable.

Since mesenchymal stem cells were first identified in the bone marrow by Fr iedenstein et al. In 1966, it has been confirmed that they can differentiate into chondrocytes, bone cells, and muscle cells. There is a lot of research on this. In addition, mesenchymal stem cells are currently found in addition to bone marrow, adipose tissue, cord blood cord blood, synovial membrane, synovial f luid, periosteum, dermis, trabecular bone, muscle, etc., each with a similar phenotype However, there are various differences in the proliferation and differentiation potential.

 Recently, it has been known that mesenchymal stem cells are effective for hair growth by extracting fat-derived stem cells from alopecia patients and injecting them directly into the scalp. Hair loss, on the other hand, occurs through natural processes or as a side effect of the use of certain therapeutic drugs designed to alleviate symptoms such as cancer. Androgenetic alopecia (AGA), which is characterized by scalp alopecia, especially in older men and women, is common. Or alopecia areata (AA), which is usually repairable to microinflammatory and called local alopecia, and alopecia associated with chemotherapy or radiation therapy (ie, secondary alopecia).

 Drug remedies are generally used to address these hair loss. Various drugs, such as vitamins and hormones, have been tried, and hair growth agents commercially available under the trade name "Minoxidil" described in US Pat. Nos. 3, 461,461 and 4,596, 812 are mainly used.

 The effects of a commercially available product as a hair growth or hair loss preventing agent on the hair include hair growth stage induction effect, hair growth stage extension effect, 5 α-reductase inhibitory effect, blood circulation promoting effect, bactericidal effect, anti dandruff effect, moisturizing effect, antioxidant Although the effects, such as conventional hair loss prevention, hair growth promoting effect is not well divided. In addition, when these products are used on the scalp, the components for promoting hair growth are not effectively delivered to the body, and hair loss prevention or hair growth promoting effects are not sufficiently exhibited.

[Detailed Description of the Invention]

 [Technical problem]

Accordingly, the present inventors have completed the present invention by finding a method for more effectively delivering drugs into the body, separating nanovesicles from stem cells, and effectively delivering therapeutic or functional substances using the same. Therefore, the object of the present invention a) passing the stem cells through the filter;

 b) centrifuging the cell solution through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) prepared by the method comprising the step of encapsulating the components for promoting hair growth in nano-vesicles separated and purified in step c), hair loss prevention or hair growth and cosmetics for promoting hair growth and hair growth comprising a stem cell-derived nano-vesicles as an active ingredient It is to provide a composition. It is also an object of the present invention to provide a cosmetic composition for the prevention of hair loss or hair growth and hair growth is composed of the vesicle-derived nano-vesicles prepared by the above method. It is also an object of the present invention to provide a cosmetic composition for hair loss prevention or hair growth and hair growth promotion consisting essentially of the stem cell-derived nano-vesicles prepared by the above method. Another object of the present invention

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

d) hair loss treatment or hair growth and hair growth promoting pharmaceutics comprising the stem cell-derived nano vesicles as an active ingredient prepared by the method comprising the step of encapsulating the components for promoting hair growth in the nano-vesicles separated and purified in step c). To provide a composition. It is also another object of the present invention is to provide a hair treatment or hair growth and hair ^growth, the pharmaceutical compositions for promoting the body consisting of stem cells derived from nano-vesicles prepared by the above method.

Another object of the present invention is to provide a pharmaceutical composition for treating hair loss or promoting hair growth and hair growth consisting essentially of the stem cell-derived nano-vesicles prepared by the above method. Another object of the present invention to provide a use of stem cell-derived nano-vesicles prepared by the following method for the treatment of hair loss or hair growth and hair growth promoting agent will be:

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c). Another object of the present invention is to provide a method for treating hair loss or hair growth and hair growth, characterized in that the effective amount of the stem cell-derived nano-vesicles prepared by the following method is administered to a subject in need thereof.

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the uniform size of the nano-vesicles from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c).

Technical Solution

 In order to achieve the above object, the present invention

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentri fuge) to obtain nano-vesicles;

 c) separating and purifying the uniform size of the nano-vesicles from the nano-vesicles obtained in step b); And

 d) hair loss prevention or hair growth and hair growth promoting cosmetics prepared by the method comprising the step of encapsulating the components for promoting hair growth in the nano-vesicles separated and purified in step c) as an active ingredient. The composition is provided.

In another aspect, the present invention provides a cosmetic composition for preventing hair loss or hair growth and hair growth is composed of the stem cell-derived nano-vesicles prepared by the above method. In another aspect, the present invention provides a cosmetic composition for hair loss prevention or hair growth and hair growth promotion consisting essentially of the stem cell-derived nano-vesicles prepared by the above method. In order to achieve the other object, the present invention

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) hair loss treatment or hair growth and hair growth promoting pharmaceutics comprising the stem cell-derived nano vesicles as an active ingredient prepared by a method comprising the step of encapsulating the components for promoting hair growth in the nano-vesicles separated and purified in step c). To provide a composition.

 In another aspect, the present invention provides a pharmaceutical composition for treating hair loss or hair growth and hair growth comprising the stem cell-derived nano-vesicles prepared by the above method.

 In another aspect, the present invention provides a pharmaceutical composition for hair loss treatment or hair growth and hair growth promotion consisting essentially of the cell-derived nano-vesicles prepared by the above method. In order to achieve another object, the present invention provides a use of stem cell-derived nano-vesicles prepared by the following method for the preparation of a hair growth treatment or hair growth and hair growth promoting agent:

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c). In order to achieve another object, the present invention provides a method for treating hair loss or hair growth and hair growth, characterized in that for administering an effective amount of the stem cell-derived nano-vesicles prepared by the following method to a subject in need thereof:

a) passing the stem cells through the filter; b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c). Hereinafter, the present invention will be described in detail. The present invention

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) prepared by the method comprising the step of encapsulating the components for promoting hair growth in nano-vesicles separated and purified in step c), hair loss prevention or hair growth and cosmetics for promoting hair growth and hair growth comprising a stem cell-derived nano-vesicles as an active ingredient To provide a composition.

 In another aspect, the present invention provides a cosmetic composition for preventing hair loss or hair growth and hair growth is composed of the stem cell-derived nano-vesicles prepared by the above method.

In another aspect, the present invention provides a cosmetic composition for hair loss prevention or hair growth and hair growth promotion consisting essentially of the stem cell-derived nano-vesicles prepared by the above method. Each step is explained in full detail. Step (a) is characterized in that to pass the stem cells to the filter. In one embodiment of the present invention, the stem cells cultured in serum-free medium are passed through the stem cells three times or more through a filter having a size of ΙΟμη. 'Stem cells' of the present invention include any undifferentiated or partially undifferentiated cell having the ability to proliferate (self-renewal) and differentiate (plastic). These stem cells are characterized by specific cell lineage (1 ineage), an end-point cell during development. Replaces cells. Stem cells also include stem cells with unlimited self-renewal and pluripotency plasticity and progenitor cells with multipotent or unipotency plasticity. The stem cells in the present invention is characterized in that the mesenchymal stem cells, preferably may be mesenchymal stem cells derived from brain, liver, lung, umbilical cord blood, fetal blood, kidney, adipose tissue, placenta, amniotic membrane or bone marrow have. In the present invention, 'Mesenchymal Stem Cells (MSCs)' is often referred to as mesenchymal stromal cells and refers to pluripotent undifferentiated cells. Mesenchymal stem cells have non-homogeneous populations of stem cells with the ability to self-renewal and mesoderm lineage and different embryonic lineages such as endoderm and ectoderm. Mesenchymal stem cells can differentiate into various types of cells including osteoblasts, chondrocytes, adipocytes, and the like. In addition, mesenchymal stem cells (Embryonic stem cells (ESCs), induced Pluri otent Stem Cells (iPSCs) and other tissue-specific stem cells, compared with other tissue-specific stem cells, tissue regeneration, blood production assistance ( 1 111 01) 01 ^ It is an ideal candidate for the clinical application of regenerative medicine because of its wide availability, low ethical issues and immunogenicity, including support and immunomodulation. Mesenchymal stem cells can be isolated from brain, liver, lung, fetal blood, umbilical cord blood, kidney, adipose tissue and placenta. Preferably it may be isolated from human bone marrow. Step b) is characterized in that to obtain a nano-vesicle by centrifugation (ultracentrifuge) the cell solution passed through the filter. In one embodiment of the present invention, the stem cells are suspended in PBS, passed through the ΙΟμηι size filter and 5μπι polycarbonate filter, and then subjected to ultracentrifugation, to obtain nano-vesicles (Example 2-1 and See FIG. 1). Then, the size of the nano-vesicles obtained using a dynamic light scattering particle size analyzer was observed, and the characteristics of the nano-vesicles were observed using a ΤΕΜ microscope. The nano-vesicles had a size of 25 to 100 nm, and the spherical and phospholipid bilayers (Example 2-2, see FIGS. 2A and 2B) The step (c) is of a uniform size in the nano-vesicle obtained in the step (b) It is characterized by separating and purifying the nano-vesicles. The 'nanovesi cle' of the present invention refers to a vesicle structure having a nano unit size, and is distinguished from nano liposomes consisting of lipid bilayers in terms of being surrounded by a lipid monolayer, It has a structure in which the inside and the outside of the nanovesicle is separated by a lipid bilayer composed of cell membrane components, and has cell membrane lipids, cell membrane proteins, nucleic acids, and cellular components. It effectively contains biomaterials that are difficult to synthesize artificially and can be delivered to specific tissues through membrane proteins, and mediates the transport of mRNA, miRNA and proteins between cells, and plays an important role in signal transduction and interaction between cells. Nanovesicles also represent heterogeneous populat ions, depending on the cell from which they are derived, the number of cells, the size of the cells, and the composition of the antigen. More preferably, it may be a nano-vesicle having a size of 50 to 200nm. In one embodiment of the present invention, in order to separate and purify the nano-vesicles, the nano-vesicles were separated by using a density gradient method, which is a method of ultracentrifugation after adding 50% euprip and 10% Opti-Prap and removing nuclear material. The nano vesicles having a certain quality were separated and purified (see Example 2-3). Step d) is characterized in that to encapsulate the hair growth promoting component in the nano-vesicles separated and purified in step c). In the present invention, 'hair loss' refers to a phenomenon in which hair falls off the scalp or a condition in which the hair is genital or thin, and 'promoting hair growth' means not only promoting the production of new hair, but also allowing the existing hair to grow healthy. Another term used in the art has the same meaning as wool or hair growth promotion. In the present invention, 'hair growth promoting ingredient' is an insulin-like growth factor (IGF-1), fibroblast growth factor (β-f ibroblast growth factor, β-FGF), thymosin P 4 (thymosin β 4), biotin and vitamin C. The 'insulin-like growth factor (IGF-1)' is a hormone having a structure similar to insulin, and is a hair follicle growth-promoting factor produced by DHT (Dihydrotestosterone). It plays an important role in pediatric growth and continues to function in adults to maintain body maintenance. The 'fibroblast growth factor (β-F ibroblast growth factor, β-FGF)' is a growth factor that stimulates fibroblasts to induce strong proliferation, pituitary gland, brain, kidney, adrenal gland, placenta, bone matrix, cartilage, It is widely distributed in endothelial cells and fibroblasts. It stimulates fibroblasts located in the dermis, promotes repair of damaged tissues and collagen synthesis, making the scalp healthy and preventing hair loss. The thymosin p 4 is a 4.9 kDa in vivo protein, and regulates cell migration to promote cell migration, thereby causing vascular endothelial cell migration and wound healing. It is a protein involved in angiogenesis, wound healing, hair growth, and cancer metastasis by promoting the movement of cells, hair source cells and / or cancer cells. The biotin (Biot in) is a kind of vitamin B complex required for the growth of animals and plants and is known as a hair nutrient for helping hair loss treatment and prevention, and has a coenzyme donation to make keratin protein, which makes hair healthy. . The 'vitamin C' has the effect of preventing hair loss by inhibiting the hair follicle destruction substance, and has the effect of thickening thin hair by promoting collagen synthesis. In the present invention, the method for encapsulating the hair growth promoting component in the nano-vesicles may use a well-known encapsulation technique. Preferably, an osmotic pressure method, an ultrasonic grinding method, an electrophoretic method may be used, and most preferably an ultrasonic grinding method. It is possible to encapsulate the hair growth promoting component using. Encapsulation in the present invention means that the desired drug is encapsulated into the nano-vesicles while substantially maintaining physical properties such as structure and size of the nano-vesicles. In one embodiment of the present invention the structure of the nano-vesicles separated and purified in step c) Or to maintain physical properties such as size, in order to more efficiently enclose the component for promoting hair growth, to separate the purified nano-vesicles in the component solution for promoting hair growth, and to incubate for 2 hours in a 37 ° C incubator ， Ultrasonic The method of grinding (soni cat ion) (40kHz, 40 ^° C, 1 hour), the electrophoresis method (200V) was carried out, respectively, and then the filling rate of each solution was measured and compared. As a result, when the component for promoting hair growth was encapsulated by the ultrasonic grinding method, it was confirmed that the physical properties of the nano-vesicles were most effectively maintained, and that the largest amount of hair growth material could be encapsulated (see Example 3). In the present invention, the cosmetics are hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair agent, eyelash hair restorer, eyelash nutrient, pet It is characterized in that the animal shampoo or pet rinse. The formulation of the cosmetic composition of the present invention is not particularly limited and may be appropriately selected as desired. For example, the cosmetic composition of the present invention may be prepared in a formulation such as skin external ointment, flexible cosmetics, nourishing cosmetics, nutrition cream, massage cream, essence, pack, emulsion dog, oil gel and the like. At this time, the skin external ointment may contain petrolatum, polyoxyethylene oleyl ether phosphate, etc., in addition to the medicinal extract of the present invention, the flexible cosmetics, in addition to the medicinal extract of the present invention, polyhydric alcohols (propylene glycol, glycerin, etc.) ) And surfactants (polyethylene oleyl ether, polyoxyethylene, hardened castor oil, etc.) and the like. In addition, the nutrient cream and nutrition cream may include oils (squalene, petrolatum, octyldodecanol, etc.) and wax components (stearyl alcohol, beeswax, etc.) in addition to the medicinal extract of the present invention, the essence is in addition to the composition of the present invention Polyhydric alcohols, such as glycerin and propylene glycol, may be included. In addition to the composition of the present invention, the massage cream may include oils such as liquid paraffin, vaseline, isononylisononanoate, and the pack or general emulsified cosmetics may be used in addition to the medicinal extracts of the present invention. It may be prepared in the form of a wash-of f pack containing pigments such as zinc and titanium dioxide. In addition, the cosmetic composition of the present invention, in addition to the components described above in each formulation, water, oil, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, or fragrances, etc. It can mix and use suitably as needed. In addition, the present invention

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) hair loss treatment or hair growth and hair growth promoting pharmaceutics comprising the stem cell-derived nano vesicles as an active ingredient prepared by the method comprising the step of encapsulating the components for promoting hair growth in the nano-vesicles separated and purified in step c). To provide a composition.

 In another aspect, the present invention provides a pharmaceutical composition for treating hair loss or hair growth and hair growth comprising the stem cell-derived nano-vesicles prepared by the above method.

The present invention also provides a pharmaceutical composition for hair loss treatment or hair growth and hair growth is composed essentially of the stem cell-derived nano-vesicles prepared by the above method. The pharmaceutical composition according to the present invention may contain a stem cell-derived nano-vesicles alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may further contain a sibling or a diluent. As used herein, 'pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may include various drug delivery materials used for oral administration to the peptide formulation. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent and the like in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referred to as described in the following documents ^ (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company East on, PA, 1995). The compositions of the present invention can be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. Can be. The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

 In the case of preparations for oral administration, the compositions of the present invention are formulated using powders, granules, tablets, pills, sugar tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. Can be. For example, oral formulations can obtain tablets or tablets of sugars by combining the active ingredients with solid brothers and then grinding them, adding suitable auxiliaries and processing them into granular mixtures. Examples of suitable excipients include sugars and corn starch, wheat starch, rice starch and potato starch, including lactose, textrose, sucrose, solbi, manny, xili, erythritol and malty. Layering agents such as saloses, gelatin, polyvinylpyrrolidone, and the like, including starch, salose, methyl salose, sodium carboxymethylcellose and hydroxypropylmethyl-cellulose. In some cases, crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a fragrance emulsifier, and a preservative.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, East on, PA, 1995, a prescription generally known in all pharmaceutical chemistries. The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a long term administered multiple dose protocol in a single ion dose treatment protocol. have. Pharmaceutical of the Invention The composition may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of the pharmaceutical composition of the present invention may be from about 0.01 «g to 10,000 mg, most preferably 0.1 / g to 500 mg per kg of patient body weight per day. However, the pharmaceutical composition and dose are determined by taking into consideration the various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the formulation method, route of administration and frequency of treatment. In view of this, one of ordinary skill in the art will be able to determine the appropriate effective dosage of the compositions of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown. In one embodiment of the present invention, the jaw of the back of the mouse is removed, and then divided into three groups, each group of 5 ml of PBS, minoxidil 3% or stem cell-derived endoplasmic reticulum solution, respectively, once a day for 2 weeks. As a result of observing the hair growth effect, it was confirmed that the hair growth effect was the best in the group coated with the stem cell-derived nano-vesicle solution prepared in the present invention (see Example 4 and FIG. 3). The present invention provides the use of the stem cell-derived nano-vesicles prepared by the following method for the preparation of a hair growth or hair growth and hair growth promoting agent:

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c). The present invention provides a method for treating hair loss or hair growth and hair growth, characterized by administering an effective amount of the stem cell-derived nano-vesicles prepared by the following method to a subject in need thereof.

 a) passing the stem cells through the filter;

b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles; c) separating and purifying the uniform size of the nano-vesicles from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c). The term 'effective amount' of the present invention, when administered to a subject, refers to an amount of improving or treating, preventing, detecting, diagnosing, or inhibiting a disease caused by hair loss, and a condition showing an effect of inhibiting or reducing disease caused by hair loss. Preferably it may be a mammal, especially an animal including a human, and may be a cell, tissue, organ or the like derived from the animal. The subject may be a patient in need of the effect.

 The term 'treatment' of the present invention refers generically to ameliorating a disease caused by hair loss or a symptom of a disease caused by hair loss, which may include treating, substantially preventing, or ameliorating such a disease. It may include, but is not limited to, alleviating, healing or preventing one or most of the symptoms resulting from the disease caused by hair loss. The term 'compri sing' of the present invention is used in the same way as 'containing' or 'with features', and in the composition or method, additional component elements or method steps not mentioned are used. Do not exclude The term 'consi sting of' means to exclude additional elements, steps or components, etc., unless otherwise specified. The term 'essent ial ly consi st ing of' includes, in the scope of a composition or method, a component element or step described, as well as a component element or step that does not substantially affect its basic properties, and the like. Means that.

Advantageous Effects

Accordingly, the present invention provides a composition for promoting hair growth and hair growth comprising nano-vesicles derived from stem cells. The method of the present invention prevents hair loss. The hair growth and hair growth promoting effect has the effect of treating or ameliorating the hair loss symptoms, it can be usefully used in the manufacture of cosmetics and therapeutics exhibiting hair growth and hair growth promoting effect. [Brief Description of Drawings]

 La and lb shows a schematic diagram of a method for producing mesenchymal cell-derived nano-vesicles.

 Figure 2a is a graph showing the size distribution of the nano-vesicles obtained by passing the mesenchymal stem cells through the filter, followed by ultracentrifugation.

 Figure 2b shows the observation of the morphology of mesenchymal stem cell-derived nano-vesicles using a TEM microscope.

 Figure 3 shows that the stem cell-derived nano-vesicles containing the hair growth component to the mouse to confirm the hair growth efficacy.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in detail.

 However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following examples.

<Example 1>

 Stem cell serum free culture method

 In order to establish the optimized culture medium and culture conditions in the culture medium used for the serum-free culture of stem cells, the experiment was carried out as follows.

First, in order to equalize the initial cell conditions, frozen cells were cultured using DMEM / FBS culture medium until reaching 80% confluency under 5% C0 ₂ conditions, and then StemPro, MesenCul t, Xmedi a, 10 ml of Nutr iStem and MSCGM-CD were added and seeded in a T75 flask (f lask) that had been coated before use with a coating reagent for each medium, referring to the protoc of each medium.

When passage 5 was reached, some cells were taken and used for flow cytometry and differentiation tests (bone, cartilage, fat). CD markers were identified through f low cytometry, and the expression rates of three positive markers (CD44, CD73 and CD90) and two negative markers (CD34 and CD45) were measured. Then, bone differentiation was confirmed by Al izar in RED staining, cartilage differentiation was confirmed by Alcian blue staining, and fat differentiation was confirmed by Oi l red 0 staining. When passage 10 was reached, some cells were taken in the same manner as above to perform flow cytometry and differentiation tests (bone, cartilage, fat). Then, by measuring the PDT (populat ion doubl- ing time) based on the number of cells obtained in each passage, the medium was identified to maintain the lowest PDT value constantly, and the cell viability was checked for each passage to maintain 80% or more. It was confirmed.

Reviewing the results of each PDT, cell viability, flow cytometry and differentiation test, 10 ⁶ cel ls / ml cells were cultured in a flask using Xmedia (Xcel l therapeut ics), and 10L bioreactor Culture conditions were set.

<Example 2>

 Manufacturing method of nano endoplasmic reticulum using stem cells <2-1> Preparation of stem-derived nano endoplasmic reticulum

Secured frozen cells were dispensed into T75 flasks, 10 ml of Xmedia (Xcel l therapeut ics) was added, and then cultured until reaching 80% confluency under 5% C0 ₂ conditions. Then, cells were detached using Trypsin / EDTA, and then 3XK) ⁵ cells were cultured in a new T75 flask containing Xmedia.

In order to separate the endoplasmic reticulum from the cell, a filter having a hole of ΙΟ μπι size was put in a filter support of a specially designed centrifuge bottle (bott le), and then assembled with a main body and washed with alcohol and PBS. Then, 5xl0 ⁷ eel ls / ml of cultured fungal cells was suspended in 1 ml PBS (phosphate buf fered sal ine) solution. After the suspension was injected into the body, ultracentrifugation was performed for 1 minute and 45 seconds at 13000 g, and repeated three times under the same conditions. Secondly, put a polycarbonate filter having a 5 μπι size hole in the filter support, wash with alcohol and PBS as above, and then put the cells back into the body, repeating three times for 1 minute and 45 seconds at 13000g. Separation was performed. This is shown in FIGS. La and lb.

<2-2> Size and Characterization of Nanovesicles

 In order to measure the size of the nano-vesicles prepared in Example 2-1 and to analyze the characteristics were carried out as follows.

The protein contained in the nano-vesicles obtained in Example 2-1 was distilled in lml PBS at a concentration of 5 yg / ml, placed in a cuvette, and then subjected to a dynamic light scattering particle size analyzer. (Marvern Zetasi zer Nano).

 As a result, as shown in Figure 2a, the size of the nano-vesicles was confirmed to be 25 to 100nm, it was confirmed that the average size is about 50nm. In addition, in order to analyze the characteristics of the nano-vesicles obtained in Example 2-1, the nano-vesicle solution suspended in PBS was added to a glow-discharged carbon-coated copper grid (3) Adsorbed for a minute. The grid was then washed with distilled water and then stained with 2% uranlacetate for 1 minute. TEM microscope, JEMlOlUeol, Japan) was used to observe the nano endoplasmic reticulum using a transmission electron microscope.

 As a result, as shown in Figure 2b, the stem cell-derived nano-vesicles could be confirmed that the phospholipid consisting of a middle layer, it was confirmed that the spherical form. Through this, the stem cell-derived nano-vesicles of the present invention was spherical, consisting of a phospholipid bilayer, it could be confirmed that having an average size of 50nm.

<2-3> Nano-vesicle separation and purification method

 Since nanomaterials other than the vesicles were mixed in the nanovesicles separated in Example 2-1, the nanovesicles were separated and purified by the following method.

 First, the cell suspension is passed through a 5 μηι polycarbonate filter washed with alcohol and PBS as in the above example. Then, add 50 μl Optiprap, 400 μl of 10% Optiprep and 1 ml of the suspension through the filter into 2.2 ml ultracentr i fuge tube, respectively, for 1 hour at 100, 000 g. Ultracentrifugation was performed during the process. After centrifugation, nanovesicles were obtained in the layer between 50% Optiprep and 10% Doptoprap.

 Through this, nano-vesicles having a certain quality were separated and purified by removing more than 90% of the nuclear material.

<Example 3>

 How to embed hair growth efficacy substances into nano vesicles

Growth factors and vitamins that have been previously proven to have hair growth In order to screen the formulations encapsulated in nanovesicles (vitamin) and inorganic components, experiments were carried out as follows.

First, IGF-1, β-FGF, Thymosin β4, Biotin, and Vitamin C were selected as the hair growth components to be administered to the nanovesicles. 50-200 nm nano-vesicles ltng / ml were dispensed in PBS, 1) incubation for 2 hours in a 37 ^° C. incubator, 2) sonication for 1 hour in an ultrasonic bath (40 kHz, 40 ^° C) (sonication), 3) electrophoresis (200V) three processes were performed.

 Through the above three processes, as a result of evaluating the encapsulation rate by the method of encapsulating the hair regrowth effect material in the nano-vesicles, IGF-1, β-FGF, biotin, vitamin C as the hair regrowth effect material by the sonication method When encapsulated in the nanovesicles, it was confirmed that 3 ~ 6pg of hair growth material per lOyg nanovesicles (protein) can be encapsulated.

<Example 4>

 Hair Regrowth Effect of Nanovesicles Encapsulated with Hair Growth Efficacy

 In order to confirm the hair growth effect of the stem cell-derived nano-vesicles prepared in Example 3 was carried out as follows.

C57BL / 6 mice were purchased, and the experiments were carried out at 7 weeks of age, with adaptation for 1 week at 25 ^° C and 40% humidity. Mice were placed in a desiccator containing ether and anesthetized for 1 minute, and the back of the mouse was removed using a hair remover and classified into three groups. One day later, PBS, 3% minoxidil, and stem cell-derived endoplasmic reticulum solutions were applied to mice without back wounds. Each sample was applied 5ml once a day for 2 weeks, after 3 days, 7 days, 14 days after the change of the hair growth of the mouse was observed. PBS was used as a control group, and minoxidil, which was previously used for hair loss treatment, was used to compare effects.

 As a result, as shown in Figure 3, when the minoxidil and stem cell-derived nano-endoplasmic reticulum solution was applied than when applying the PBS, the hair growth effect was better, and stem cell-derived nano-vesicles than when minoxidil was applied The hair growth effect was remarkably good when the solution was applied.

 Through this, it was confirmed that the stem cell-derived nano-vesicles containing the hair growth promoting component of the present invention has a remarkable effect on hair growth promotion and hair loss treatment.

Industrial Applicability As described above, the method of the present invention has the effect of treating or improving hair loss symptoms through hair loss prevention, hair growth and hair growth promoting effects, and through this, to prepare cosmetics and therapeutic agents exhibiting hair growth and hair growth promoting effects. It can be useful.

Claims

[Range of request]

 [Claim 1]

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) prepared by the method comprising the step of encapsulating the components for promoting hair growth in nano-vesicles separated and purified in step c), hair loss prevention or hair growth and cosmetics for promoting hair growth and hair growth comprising a stem cell-derived nano-vesicles as an active ingredient Composition.

[Claim 2]

 The cosmetic composition according to claim 1, wherein the stem cells are mesenchymal stem cells.

[Claim 3]

 The method of claim 1, wherein the stem cells are brain, liver, lung, umbilical cord blood, fetal blood, kidney, adipose tissue, placenta, amniotic membrane and bone marrow mesenchymal stem cells derived from one organ selected from the group consisting of Cosmetic composition.

[Claim 4]

 According to claim 1, wherein the step a) cosmetic composition, characterized in that passed three or more times through the filter of the size ΙΟμπι.

[Claim 5]

 According to claim 1, wherein the size of the nano-vesicles of step b) is a cosmetic composition according to gong.

[Claim 6]

The method according to claim 1, wherein the component for promoting hair growth in step c) includes insulin-like growth factor (IGF-1), fibroblast growth factor (β-f ibroblast growth factor, β-FGF), Consisting of thymosin β 4, biotin and vitamin C Cosmetic composition, characterized in that selected from the group.

[Claim 7]

 The cosmetic composition of claim 1, wherein the cosmetic composition comprises hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow regrowth, eyelash regrowth, eyelash A cosmetic composition which is a nutrient, a pet shampoo or a pet rinse.

[Claim 8]

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) hair loss treatment or hair growth and hair growth promoting pharmaceutics comprising the stem cell-derived nano vesicles as an active ingredient prepared by a method comprising the step of encapsulating the components for promoting hair growth in the nano-vesicles separated and purified in step c). Composition.

[Claim 9]

 Use of stem cell-derived nanovesicles prepared by the following method for the preparation of a hair growth or hair growth and hair growth promoting agent:

 a) passing the stem cells through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c).

[Claim 10]

Hair loss treatment or hair growth and hair growth promoting method characterized by administering to the subject in need thereof an effective amount of the stem cell-derived nano-vesicles prepared by the following method method:

 a) passing the pleasant cell through the filter;

 b) centrifuging the cell solution passed through the filter in step a) (ul tracentr i fuge) to obtain nano-vesicles;

 c) separating and purifying the nano-vesicles of uniform size from the nano-vesicles obtained in step b); And

 d) encapsulating the component for promoting hair growth in the nano-vesicles separated and purified in step c).