WO2019050240A1 - New use of exosome kit comprising stem cell-derived exosome - Google Patents

Abstract

The present invention provides an exosome kit comprising an iontophoresis device with a mask pack, a mask sheet or a patch to which a composition comprising a stem cell-derived exosome as an active ingredient is applied or immersed in for preventing, suppressing, alleviating, ameliorating, or treating dermatitis. The exosome kit of the present invention exhibits excellent effects in skin care such as ameliorating dermatitis symptoms and skin condition that has deteriorated thereby and recovering the same to a normal condition, and exhibits effects of alleviating and ameliorating a diseased skin condition due to dermatitis or recovering the diseased skin condition to a normal condition. Particularly, the exosome kit of the present invention allows the exosomes applied to or immersed in the mask pack, the mask sheet or the patch to effectively pass through the epidermis and to permeate deeply into the skin by an iontophoresis action, and thus exhibits an effect of alleviating or ameliorating the dermatitis symptoms and the deteriorated skin condition related thereto.

Description

New Uses of Exosome Kit Containing Exosomes from Stem Cells

The present invention relates to a new use of an exosome kit comprising exosomes derived from stem cells.

Specifically, the present invention provides a method for preventing, suppressing, alleviating, ameliorating, or treating dermatitis, which comprises applying a mask pack, a mask sheet or a patch containing a stem cell-derived exosome as an active ingredient, Lt; RTI ID = 0.0 > iontophoresis < / RTI > device.

The skin of the human body is an organ that physically and chemically protects the body from the outside and performs the biochemical functions necessary for the metabolism of the whole body. In general, inflammatory skin diseases that appear on human skin are called dermatitis. Relatively common dermatitis includes atopic dermatitis, contact dermatitis, and seborrhoic dermatitis.

Contact dermatitis is a dermatitis caused by contact with foreign substances. Contact dermatitis is characterized by irritant contact dermatitis and allergic contact dermatitis, depending on the mechanism by which one substance can cause both of these reactions simultaneously. Most of the substances that cause contact dermatitis are organic compounds. When the dermatitis-inducing substance comes into contact with the skin sensitized to these substances, it is known that the memory cell senses it and secretes various chemicals to cause inflammation.

Atopic dermatitis is a chronic inflammatory skin disease characterized by itchy and eczematous lesions. According to the results of previous studies, it is known that various factors are involved in the onset of atopic dermatitis. When atopic dermatitis is induced, inflammatory cells such as eosinophil, neutrophil and mast cell are observed in the lesion and immunoglobulin IgE produced from mast cells increases. In addition, IL-4, IL-5, and IL-13, which are inflammatory cytokines, are amplified and amplified by this process. Recently, TSLP (Thymic stromal lymphopoietin) has been associated with the severity of dermatitis and it is known to cause itching symptoms in atopic dermatitis. In addition, seborrheic dermatitis is a chronic inflammatory skin disease that occurs due to increased activity of sebaceous gland, which occurs in the scalp and face, especially around eyebrows, nose, lips, ears, underarms, chest, groin and the like.

There are efforts to develop dermatitis treatments through inflammation and immunosuppression. Drug remedies developed to date include steroids, antihistamines, and immunosuppressants such as cyclosporin A. However, these drugs have serious problems such as skin atrophy, vasodilation, pigment desalination, hypersensitivity of injection site, tolerance and neutropenia.

Recently, there have been reported studies on the secretion of various bioactive factors that regulate cell behavior, and in particular, exosome (exosome), which has intercellular signal transduction, , And the research on the composition and function thereof is actively under way.

Cells release various membrane-type vesicles in the extracellular environment, and these release vesicles are commonly referred to as extracellular vesicles (EVs). The extracellular endoplasmic reticulum is sometimes referred to as cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes and, in some cases, differentiated from exosomes.

Exosome is an endoplasmic reticulum of several tens to several hundreds of nanometers in size, composed of double lipid membranes identical to the cell membrane structure, and contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosomal cargo inside. Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells. Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known. Exosomes contain specific genetic material and bioactivity factors depending on the nature and condition of the derived cells. The proliferating stem cell-derived exosomes regulate cell behavior such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

However, in spite of various studies such as the possibility of treatment of specific diseases using exosome, there have been many studies to develop a new formulation capable of stably maintaining and storing exosome, various medical treatments Or beauty technology has not received much attention.

The present inventors have conducted intensive studies on the development of new formulations capable of stably maintaining and storing exosomes and the combination of medical or cosmetic techniques, and have found that exosomes which can be applied to prevent, inhibit, alleviate, The kit was developed to complete the present invention.

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

It is an object of the present invention to provide an exosome kit comprising exosome derived from stem cells and an application thereof. Specifically, the object of the present invention is to provide a mask pack, a mask sheet or a patch which is coated or immersed with a composition containing an exosome derived from a stem cell as an active ingredient for the prevention, inhibition, alleviation, improvement or treatment of dermatitis, And an iontophoresis device for use in the treatment of cancer.

Another object of the present invention is to provide a cosmetic method for controlling the condition of mammalian skin through prevention, suppression, alleviation or improvement of dermatitis except for therapeutic use using the exosomic kit.

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

In order to achieve the above object, the present invention provides a method for preventing, suppressing, alleviating, ameliorating, or treating dermatitis, which comprises applying a mask pack containing a composition containing an exosome derived from stem cells as an active ingredient, Or patch, and an iontophoresis device.

As used herein, the term " exosomes " refers to an endoplasmic reticulum of several tens to several hundred nanometers (preferably about 30 to 200 nm) in size, consisting of double lipid membranes identical in structure to the cell membrane The particle size of the exosome can be varied according to the cell type, the separation method and the measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, DOI 10.1007 / s00216-015-8535-3). Exosomes contain proteins called exosomal cargo (cargo), nucleic acids (mRNA, miRNA, etc.). Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells. Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.

As used herein, the term " exosome " refers to a vesicle that has a nano-sized vesicle structure secreted from various stem cells and released into the extracellular space and has a composition similar to exosome (e.g., - pseudo-bezacl). The type of the stem cell is not limited, but may be, for example, a mesenchymal stem cell, for example, a fat, a bone marrow, an umbilical cord or a cord blood derived stem cell, Derived stem cells. The type of the adipose-derived stem cell is not limited as long as it does not cause a risk of infection by a pathogen and does not cause an immune rejection reaction, but it may be preferably a human adipose-derived stem cell.

However, the stem cell-derived exosome used in the present invention is effective for preventing, suppressing, alleviating, ameliorating or treating dermatitis and not causing adverse effects on the human body, so long as the stem cell-derived exosome can be used in a variety of stem cell- Of course, exosomes can be used. Therefore, it should be understood that the exosome isolated according to the separation method of the following embodiments should be understood as an example of exosome that can be used in the present invention, and the present invention is not limited thereto.

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

The exosome kit of one embodiment of the present invention comprises a first mask pack, a mask sheet or a patch which is applied or immersed with a composition containing an exosome derived from a stem cell as an active ingredient, And an iontophoresis device for flowing an electric current.

For example, an exosome kit according to one embodiment of the present invention is characterized in that the first mask pack, the mask sheet or the patch is brought into contact with or attached to the skin of the mammal, and the ion mask By positioning the phoresis device, the first mask pack, mask sheet or patch can be contacted or attached to be used in such a way that micro-currents flow to the skin of the mammal to which the composition is applied.

In addition to the exosome, the composition may be used in combination with a conventionally used dermatitis remedy and / or moisturizer to the extent that its action (alleviation or improvement of dermatitis symptoms) is not impaired. For example, the composition may be supported on or mixed with at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. Wherein the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum And the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerin.

The iontophoresis device according to one embodiment of the present invention may be applied to a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, Or a second mask pack, a mask sheet, or a patch having the at least one battery mounted thereon.

In the exosome kit according to one embodiment of the present invention, the second mask pack, the mask sheet or the patch may be laminated on the first mask pack, the mask sheet or the patch.

In at least one surface of the first mask pack, the mask sheet or the patch, at least one surface of the first mask pack, the mask sheet or the patch may be coated with a hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate) At least one of the gels may be applied. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

According to the exosome kit of one embodiment of the present invention, the micro current flows from the iontophoresis device so that the exosomes can effectively penetrate the epidermis and penetrate deeply into the skin.

The exosome kit of one embodiment of the present invention can be effectively used for preventing, suppressing, ameliorating, alleviating or treating various dermatitis accompanied by itch, and is preferably used for the treatment of contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, Allergic contact dermatitis, contact urticaria, atopic dermatitis, seborrheic dermatitis, autoimmune dermatitis, autoimmune progesterone dermatitis, dermatitis, acne or eczema.

Another embodiment of the present invention provides a cosmetic method for controlling the condition of a mammalian skin by preventing, suppressing, alleviating or improving dermatitis except for therapeutic use using the exosomic kit. In the cosmetic method of the present invention, conditioning of the skin means improvement of the condition of the skin and / or prevention of the condition of the skin, and improvement of the condition of the skin means visual and / Or a tactile perceptible positive change. For example, improvement of the condition of the skin may be mitigation or improvement of dermatitis related symptoms.

The cosmetic method of one embodiment of the present invention can be carried out using the exosome kit as described above.

For example, the cosmetic method of one embodiment of the present invention comprises the steps of (a) contacting a first mask pack, a mask sheet or a patch with a composition containing an exosome derived from a stem cell as an active ingredient, (B) positioning the iontophoresis device on the first mask pack, mask sheet or patch, and (c) contacting the first mask pack, mask sheet or patch with the iontophoresis device, Performing iontophoresis to flow a microcurrent into the skin of the mammal to which the composition is applied; and (d) delivering the exosomes through the microcurrent to the inside of the mammalian skin.

In at least one surface of the first mask pack, the mask sheet or the patch, at least one surface of the first mask pack, the mask sheet or the patch may be coated with a hydrogel, hyaluronic acid, hyaluronate (e.g., sodium hyaluronate) At least one of the gels may be applied. The type of the hydrogel is not limited, but it may preferably be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

In the cosmetic method according to one embodiment of the present invention, the iontophoresis device comprises a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, Or a second mask pack, a mask sheet or a patch having the at least one battery mounted thereon. The step (b) may be performed by laminating the second mask pack, the mask sheet or the patch on the first mask pack, the mask sheet or the patch.

The exosome kits of the present invention exhibit excellent effects on dermatitis symptoms and skin cosmetics such as improving or restoring the dermatological conditions deteriorated due to the dermatitis symptoms, and it is possible to alleviate or improve the condition of the skin condition due to dermatitis, . Particularly, the exosome kit of the present invention can prevent the exosomes applied or deposited on the mask pack, the mask sheet or the patch by the iontophoresis action to effectively penetrate the epidermis and penetrate deeply into the skin, Thereby exhibiting an effect of improving or alleviating the skin condition.

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

FIG. 1 shows the results of physical property analysis of exosomes obtained according to one embodiment of the present invention. &Quot; Figure 1A " shows the particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis. &Quot; Figure 1B " shows particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis. &Quot; Fig. 1C " shows particle images by transmission electron microscopy (TEM) according to magnification. &Quot; Figure 1D " shows the Western blot results of exosomes obtained according to one embodiment of the present invention. ≪ RTI ID = 0.0 > 1E < / RTI > shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.

FIG. 2 shows the result of confirming the absence of cytotoxicity after treatment of exosomes obtained according to one embodiment of the present invention in human skin fibroblast HS68 cells.

FIG. 3 shows that the amount of mRNA expression of TNF-α, IL-6, IL-1β and iNOS induced by LPS was decreased when the exosome used in the exosome kit of the present invention was treated with LPS for RAW 264.7 cells And a real-time PCR result.

FIG. 4 shows experimental results showing that NO formation, which is a type of inflammatory reaction, is reduced by the exosome used in the exosome kit of the present invention. In FIG. 4, PBS is phosphate-buffered saline, DEX is dexamethasone, EXO is exosome, CM is conditioned medium, and CM-EXO is adipocyte- (Exosome-depeleted conditioned media).

FIG. 5 shows experimental results confirming that TNF-.alpha. Formation, which is an inflammatory cytokine, is reduced by exosomes used in the exosome kit of the present invention. In FIG. 5, PBS represents phosphate-buffered saline, DEX represents dexamethasone, and each numeral represents exosome throughput (μg / mL).

FIG. 6 shows the results of co-treatment of exosomes used in the exosomal kit of the present invention together with LPS and IFN-y for THP-1-derived macrophages obtained after differentiating THP-1 monocytes into macrophages Time PCR results confirming that the mRNA expression level of the pro-inflammatory cytokine IL-6 was decreased.

FIGS. 7 to 9 show the results of pre-treatment of the exosomes used in the exosomal kit of the present invention and culturing for a predetermined period of time on THP-1-derived macrophages obtained after differentiating THP-1 monocytes into macrophages Next, it is a graph showing real-time PCR results in which the amount of mRNA expression of IL-6, IL-1 [beta] and TNF- [alpha], which are proinflammatory cytokines, is decreased when LPS and IFN-y are treated.

FIG. 10 shows fluorescence intensity measurement results of exosomes stained with PKH67.

Fig. 11 is a fluorescence microscope image showing the degree of transfer of fluorescent stained exosomes into pig skin tissue. Fig.

Fig. 12 is a confocal fluorescence microscope image showing the extent of fluorescence-stained exosomes transferred into the mouse skin tissue.

13 is a graph showing a comparison of the total fluorescence intensity obtained by measuring the fluorescence intensity on each image in Fig.

14 is a photograph showing the skin condition before and after the treatment of human skin with the exosomal kit of the present invention.

FIG. 15 is a graph showing the results of application of iontophoresis for applying a microcurrent to a skin (affected part) coated with the composition after applying the composition containing exosome used in the exosome kit of the present invention to human skin As a result, it is a photograph showing that the erythema of the skin (the affected part) which has a severe dermatitis is remarkably improved.

16 is a perspective view showing a configuration of the exosome kit 100 according to one embodiment of the present invention.

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

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

Example

Example 1: Culture of cells

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

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

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

Trehalose was added to the culture medium in an amount of 2% by weight in order to obtain an exosome having a uniform particle size distribution and high purity in the process of separating exosome. After the addition of trehalose, the culture was filtered with a 0.22 μm filter to remove impurities such as cellular debris, waste products and large particles. The filtered cultures were immediately separated to isolate exosomes. In addition, the filtered culture was stored in a refrigerator (image below 10 ° C) and used for exosome isolation. In addition, the filtered culture was frozen in an ultra-low temperature freezer at -60 ° C or lower, and thawed, followed by exosome isolation. Then, the exosomes were separated from the culture medium by using Tangential Flow Filtration (TFF).

Example 2: Isolation and purification of exosome by TFF method

In Example 1, a TFF (Tangential Flow Filtration) method was used for separating, concentrating, desalting and diafiltration exosomes from a culture filtrated with a 0.22 μm filter. For the TFF method, a cartridge filter (also called a hollow fiber filter (purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used. The TFF filter can be selected by a variety of molecular weight cutoffs (MWCO). Exosomes were selectively isolated and concentrated by selected MWCOs, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than MWCO were removed.

To separate and concentrate the exosomes, TFF filters of MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da were used. The culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, and substances smaller than MWCO were removed to separate the exosomes.

Separated and concentrated exosomal solutions were further desalted and buffered (diafiltration) using the TFF method. At this time, the desalination and buffer exchange are performed by continuous diafiltration or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably, Was performed using a buffer solution having a volume of 12 times or more. To the buffer solution, 2% by weight of trehalose dissolved in PBS was added to obtain an exosome having a uniform particle size distribution and high purity.

Example 3: Characterization of isolated exosomes

Separated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (NTA; purchased from Malvern) or tunable resistive pulse sensing (TRPS; purchased from Izon Science). The uniformity and size of isolated exosomes were analyzed by transmission electron microscopy (TEM). The results of TRPS, NTA and TEM analysis of the exosomes isolated according to one embodiment of the present invention are shown in Figs. 1A to 1C.

FIG. 1D shows the presence of CD9, CD63, CD81 and TSG101 markers as a result of performing Western blotting on isolated exosomes according to the method of one embodiment of the present invention. Anti-CD9 (purchased from Abcam), anti-CD63 (purchased from System Biosciences), anti-CD81 (purchased from System Biosciences), and anti-TSG101 (purchased from Abcam) were used as the antibodies for each marker.

FIG. 1E shows the presence of CD63 and CD81 markers as a result of analysis using flow cytometry on exosomes isolated according to the method of one embodiment of the present invention. Human CD63 isolation / detection kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's method to isolate the positive exosomes for CD63 and PE-mouse anti-human CD63 (PE-Mouse anti markers were stained using the PE-mouse CD63 (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD), and analyzed using a flow cytometer (ACEA Biosciences) Respectively.

Meanwhile, the exosomes derived from stem cells used in the present invention are not limited to the exosomes of the above-mentioned embodiments, and it is possible to use various stem cell-derived exosomes which are used in the art or can be used in the future Of course. It is to be understood that the exosomes isolated according to the above embodiments are to be understood as an example of exosomes derived from stem cells which can be used in the present invention, and the present invention is not limited thereto.

Example 4: Measurement of cytotoxicity by treatment with exosome

In order to evaluate the toxicity of exosomes obtained according to the isolation method of one embodiment of the present invention in human skin fibroblast HS68 cells, exosomes were treated by concentration to the cells and the proliferation rate of the cells was confirmed. HS68 cells were suspended in DMEM containing 10% FBS, and the cells were mixed with 80 ~ 90% confluency and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, the culture solution was removed, and the cell survival rate was evaluated by culturing the exosome prepared in Example 2 for each concentration and culturing for 24 to 72 hours. Cell viability was measured using WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega), or Aramar Blue reagent alamarBlue reagent (purchased from ThermoFisher Scientific) and a microplate reader (purchased from Molecular Devices).

The comparison group was based on the number of cells cultured in the normal cell culture medium not treated with exosome, and it was confirmed that the cytosolic effect of exosome derived from stem cells did not appear within the tested concentration range (FIG. 2).

Example 5 Measurement of Inflammatory Response Using Macrophage Cell Lines

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

Primer types and base sequences used in real-time PCR

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

First, as shown in FIG. 3, when RAW 264.7 cells, which are macrophages of mice, were treated with exosomes used in the exosomic kit of the present invention together with LPS, TNF-a, an inflammatory cytokine induced by LPS , IL-6 and IL-1β decreased and mRNA expression of iNOS, a NO-producing enzyme, decreased.

Next, as shown in FIG. 4, RAW 264.7 cells, which are macrophages of mice, were treated with exosomes used in the exosomal kit of the present invention in the presence of LPS, and as a result, NO production, which is an inflammatory reaction induced by LPS, Dependent manner. As shown in FIG. 5, RAW 264.7 cells, macrophages of mice, were treated with exosomes used in the exosomal kit of the present invention in the presence of LPS. As a result, LPS-induced inflammatory cytokine TNF-a production . These results indicate that the exosome used in the exosomal kit of the present invention has an activity of reducing the inflammatory reaction induced by LPS, which is useful for preventing, inhibiting, improving, alleviating or treating dermatitis, It strongly suggests that exosome kits may be useful for the prevention, inhibition, amelioration, palliation or treatment of dermatitis.

Example 6: Determination of anti-inflammatory effect using THP-1 monocyte

After the THP-1 monocyte (THP-1 human monocyte) was differentiated into macrophages, the anti-inflammatory effect of exosomes used in the exosome kit of the present invention was confirmed as follows.

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

For the co-treatment group, RPMI 1640 medium without FBS (Fetal Bovine Serum), 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN -γ (purchased from Peprotech) was activated to activate THP-1 derived macrophages and the exosome used in the positive control group dexamethasone (purchased from Sigma) or the exosome kit of the present invention (exosome prepared in Example 2) ) And cultured for 24 hours. On the other hand, in the case of the pre-treatment experimental group, RPMI 1640 medium containing no FBS (Fetal Bovine Serum) for the THP-1 derived macrophages and dexamethasone (purchased from Sigma) The exosomes used in the kit (the exosomes prepared in Example 2) were cultured and cultured for 24 hours and then treated with 100 ng / mL LPS (purchased from Sigma) and 20 ng / mL IFN-y (purchased from Peprotech) And incubated for 4 hours to activate THP-1-derived macrophages.

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

Primer types and base sequences used in real-time PCR

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

As shown in FIG. 6, for the THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages, the exosomes used in the exosomal kit of the present invention were treated with LPS and IFN- co-treatment), mRNA expression of IL-6, a proinflammatory cytokine, was decreased compared to negative control. In addition, as shown in FIGS. 7 to 9, THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages were used in the exosome kit of the present invention before LPS and IFN- IL-6, IL-1β and TNF-α mRNA expression levels were decreased in the pre-treatment of exosomes compared to negative control. The proinflammatory cytokines IL-6, IL-1 [beta] and TNF- [alpha] increase expression in proinflammatory M1 macrophages and decrease expression in anti-inflammatory M2 macrophages.

Therefore, the above results suggest that the exosome used in the exosome kit of the present invention can inhibit, alleviate and reduce the inflammatory reaction and the dermatitis caused thereby, and the exosome kit of the present invention can prevent, , Which may be useful for improving, alleviating, or treating a disease.

Example 7: Skin permeability test of exosome used in the exosome kit of the present invention

PKH67 dye (purchased from Sigma) was used to prepare fluorescently stained exosomes. 1 mM PKH67 was diluted in Diluent C (purchased from Sigma) to prepare a 10 μM PKH67 solution, mixed with an appropriate concentration of exosome solution, and allowed to react at room temperature for 10 minutes in the absence of light. After the reaction, an MW3000 spin column (purchased from ThermoFisher Scientific) was used to remove the remaining PKH67 dye from the exosomes stained with PKH67 (hereinafter abbreviated as "PKH-exosomes"). After confirming the use of a fluorescence meter (purchased from Molecular Devices), PKH67 not reacted with exosomes was removed, and fluorescence of sufficient intensity was detected in PKH-exosomes (FIG. 10).

PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the outer surface of the pig skin. To prevent drying of the applied PKH-exosomes, the nonwoven fabric was covered and allowed to react for an appropriate period of time, for example, 30 minutes to 1 hour to allow the PKH-exosomes to be delivered to the subcutaneous tissues. In addition, the PKH-exosomes were applied to the outer surface of the pig skin and covered with the nonwoven fabric, followed by flowing a microcurrent for a predetermined time, for example, 30 minutes to 1 hour. After the reaction was completed, the pig skin tissue was immersed in a 3.7% formaldehyde solution, reacted overnight, and washed three times for 5 minutes each with phosphate buffered saline. The washed pig skin tissue was immersed in a 30% sucrose solution and treated with OCT compound. After washing three times for 5 minutes with phosphate buffer solution, sections were prepared on a longitudinal section using a microtome, and tissue sections were placed on a slide glass. On the other hand, the preparation of the tissue section may be carried out before the tissue is fixed with formaldehyde solution. Fluorescence detected from PKH-exosomes in tissue sections was observed using fluorescence microscopy. In this way, PKH-exosomes transmitted to the subcutaneous tissue through the epidermis of the pig skin tissue were identified. As a result, it was confirmed that the exosomes were more effectively passed through the epidermis and deeply transferred into the skin tissue and more effectively absorbed to the skin when active current was delivered (active transdermal delivery) (see FIG. 11). As shown in Fig. 11, the exosomes used in the exosome kit of the present invention can be effectively passed through the epidermis and deeply transferred into the skin tissue, and can be effectively absorbed to the skin. Therefore, the exosomic kit of the present invention is expected to be usefully used for preventing, suppressing, improving, alleviating or treating dermatitis.

Next, the skin tissue of the hairless mouse was removed and placed on top of the chamber of the Franz Diffusion Cell. At this time, the inside of the diffusion cell was filled with a phosphate buffer solution. PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 × 10 ⁵ to 1 × 10 ⁹ particles / mL, and then applied to the outside of the skin tissue of the mice. At this time, in order to prevent drying of PKH-exosomes, the nonwoven fabric was pre-positioned outside the mouse skin tissue, and PKH-exosomes were injected between the nonwoven fabric and skin tissue. Then, the reaction was carried out for 30 minutes to 1 hour. In addition, the PKH-exosomes were injected between the nonwoven fabric and the skin tissue, and the microcurrent was flowed for a predetermined time, for example, 30 minutes to 1 hour. After completion of the reaction, the PKH-exosomes transferred into the skin tissue were confirmed immediately using a confocal fluorescence microscope (Leica, SP8X) or the skin tissue and the PKH-exosomal solution were further reacted for 1 hour to 6 hours , And PKH-exosomes were identified by confocal fluorescence microscopy. As a result, it was confirmed that the exosomes were more efficiently passed through the epidermis, deeply transferred into the skin tissue, and more effectively absorbed to the skin when active current was delivered (active transdermal delivery) (see Figs. 12 and 13 ).

It is expected that the exosome kits of the present invention can effectively pass through the epidermis to effectively prevent the dermatitis from being prevented, inhibited, alleviated, improved or treated when the exosome kit of the present invention is applied to the skin.

Example 8: Treatment of exosome kit against human skin

16, the exosome kit 100 according to one embodiment of the present invention includes a liquid immersion sheet mask 10 (a white sheet mask on which a composition containing a stem cell-derived exosome is applied or immersed) And a transdermal permeation promoting sheet mask (silver sheet mask) 20 which is an iontophoresis device. A stem cell-derived exosome (12) is applied or deposited on the surface or inside of the liquid immersion sheet mask (10). A battery 22 for generating a microcurrent is mounted on the percutaneous permeation enhancing sheet mask 20 and a conductive pattern 24 electrically connected to the cell 22 is attached to the percutaneous permeation enhancing sheet mask 20, So that the micro current generated from the battery 22 can be uniformly transmitted to the entirety of the percutaneous permeation enhancing sheet mask 20. [ The conductive pattern 24 is formed by printing or plating a conductive metal such as gold, silver, or copper.

The "exosome kit (100)" having the above-described structure is applied once to the face of a person having a flushing symptom and skin troubles (hereinafter referred to as "case 1") related to the dermatitis symptom, It was evaluated whether the symptom of trouble and redness was alleviated or improved. In addition, the exosome kit 100 of the present invention is applied once to the face of a person having flushing symptoms (hereinafter referred to as " case 2 ") associated with dermatitis symptoms to evaluate whether the overall skin tone and flushing symptoms are improved Respectively. A liquid immersion sheet mask 10 containing 5 × 10 ⁵ exosomes 12 was uniformly adhered to the entire faces of the cases 1 and 2 by aligning them with the eyes and mouth parts and then a microcurrent of about 0.3 to 0.4 mA An activated percutaneous permeation enhancing sheet mask 20 which was activated to flow was laminated on the liquid immersion sheet mask 10 and used for about 25 to 30 minutes.

As a result, the "exosome kit 100" composed of the liquid-immersion sheet mask 10 containing the stem cell-derived exosome 12 at a concentration of 5 × 10 ⁵ particles / mask and the percutaneous permeation- (See case 1 in FIG. 14), the overall skin tone was improved in "case 2" and the flushing was alleviated (see case 2 in FIG. 14) . Therefore, the exosomal kit (present invention) comprising the liquid immersion sheet mask 10 in which the composition containing the stem cell-derived exosome as an active ingredient is immersed and the percutaneous permeation promoting sheet mask (iontophoresis device) 100) can be said to have an effect of preventing, suppressing, alleviating or alleviating dermatitis symptoms and redness symptoms and skin troubles associated therewith.

In addition, a composition (exosome suspension) containing a stem cell-derived exosome at a concentration of 0.29 x 10 ⁸ particles / mL was applied to the affected part (hand, neck, arm, etc.) of three severe atopic patients complaining of itching Iontophoresis was carried out by using a iontophoresis device (IONZYME DF MACHINE) (purchased from Environ) to flow a micro current of 0.4 mA for 20 minutes to the affected part of the composition. The above procedure was repeated three times a week for two weeks, and the severe itching of the patients was remarkably alleviated, and the erythema symptoms were also markedly improved (FIG. 15). Patients who applied compositions containing stem cell-derived exosomes improved their symptoms to such an extent that severe itching and erythema could be relieved to stop the prescription of steroid or antihistamine preparations.

Accordingly, it can be seen that the exosome kit 100 of the present invention can be effectively used for preventing, suppressing, ameliorating, alleviating or treating dermatitis and symptoms associated therewith, as confirmed by the clinical test as described above.

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

Claims (7)

1. A first mask pack, a mask sheet or a patch which is applied or deposited with a composition containing an exosome derived from a stem cell as an active ingredient,

   And an iontophoresis device for applying a microcurrent to the skin of the mammal to which the first mask pack, the mask sheet or the patch is applied, for preventing, suppressing, alleviating, improving or treating dermatitis.
2. The method according to claim 1,

   Wherein the first mask pack, the mask sheet or the patch is contacted or adhered onto the skin of the mammal, and the iontophoresis device is placed on the first mask pack, the mask sheet or the patch, Or patches are contacted or adhered to flow micro-currents into the skin of the mammal to which said composition is applied.
3. 3. The method according to claim 1 or 2,

   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 dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A second mask pack, a mask sheet or a patch, on which the at least one battery is mounted.
4. The method of claim 3,

   Wherein the second mask pack, the mask sheet or the patch is laminated on the first mask pack, the mask sheet or the patch.
5. (a) contacting or adhering a first mask pack, a mask sheet or a patch applied or immersed with a composition comprising an exosome derived from a stem cell as an active ingredient onto the skin of a mammal,

   (b) positioning the iontophoresis device on the first mask pack, mask sheet or patch,

   (c) performing iontophoresis, wherein the first mask pack, mask sheet or patch is contacted or adhered to flow a microcurrent into the skin of the mammal to which the composition is applied;

   (d) delivering the exosomes to the interior of the mammalian skin through the microcurrent, wherein the condition is controlled by preventing, inhibiting, alleviating or ameliorating the dermatitis except for the treatment.
6. 6. The method of claim 5,

   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 dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A second mask pack, a mask sheet or a patch, on which said at least one battery is mounted.
7. The method according to claim 6,

   Wherein the step (b) is performed by laminating the second mask pack, the mask sheet or the patch on the first mask pack, the mask sheet or the patch.

Images