Use of composition comprising exosome derived from adipose-derived stem cell as effective ingredient in ameliorating dermatitis

Abstract

The present invention provides a composition for preventing, ameliorating, alleviating or treating dermatitis comprising exosomes derived from adipose-derived stem cells as an active ingredient. The composition of the present invention is able to act against dermatitis-inducing multiple cytokine targets, and thus be widely applied against dermatitis caused by various factors and effectively suppress and alleviate dermatitis.

Claims

1. A method for preventing, ameliorating, alleviating or treating atopic dermatitis mediated by IL-4, the method comprising: administering a composition to a subject in need thereof, wherein the composition comprises exosomes derived from adipose-derived stem cells as an active ingredient, wherein the composition excludes adipose-derived stem cells, and wherein the administering of the composition decreases expression and/or production of IL-4 in the subject.

2. The method of claim 1, wherein the exosomes are obtained by performing the steps of: (a) adding trehalose to a conditioned medium of adipose-derived stem cells; (b) filtering the conditioned medium having the trehalose added thereto; (c) isolating exosomes from the filtered conditioned medium by tangential flow filtration (TFF); and (d) adding trehalose to a buffer for diafiltration, and performing diafiltration on the isolated exosomes by the TFF using the buffer having the trehalose added thereto.

3. The method of claim 2, wherein the diafiltration is performed continuously or discontinuously.

4. The method of claim 2, wherein the diafiltration is performed using a buffer having at least 4 times the volume of the isolated exosomes.

5. The method of claim 2, wherein a TFF filter having a molecular weight cutoff (MWCO) of 100,000 Da, 300,000 Da, 500,000 Da or 750,000 Da, or a 0.05 μm filter is used for the TFF.

6. The method of claim 2, wherein step (c) further comprises concentrating the conditioned medium containing the exosomes to a volume of between 1/100 and 1/25 of the original volume of the conditioned medium by the TFF.

7. The method of claim 1, wherein the exosomes decrease expression levels of IL-4 and IL-31 in skin tissue or skin cells.

8. The method of claim 7, wherein the exosomes additionally decrease expression level of at least one selected from the group consisting of IL-23 and TNF-α in skin tissue or skin cells.

9. The method of claim 1, wherein the exosomes decrease the level of IgE in blood, and the number of white blood cells and eosinophils in blood.

10. The method of claim 1, wherein the exosomes decrease the number of mast cells, CD86+ cells and CD206+ cells in skin tissue.

11. The method of claim 1, wherein the subject is at least one selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

12. A method for preventing, ameliorating, alleviating or treating atopic dermatitis mediated by IL-4 in a subject in need thereof, the method comprising: (a) (a1) applying a composition to skin of the subject, wherein the composition comprises exosomes derived from adipose-derived stem cells as an active ingredient; or (a2) contacting or attaching a patch, a mask pack or a mask sheet, which has the composition applied thereto or soaked therein, to skin of the subject; or (a3) sequentially performing (a1) and (a2); and (b) decreasing expression and/or production of IL-4 in the skin tissue and/or the skin cells of the subject.

13. The method of claim 12, wherein the exosomes are contained in or mixed with at least one of hydrogel, hyaluronic acid, salt of hyaluronic acid, or hyaluronate gel.

14. The method of claim 13, wherein the hydrogel is obtained by dispersing a gelled polymer in a polyhydric alcohol.

15. The method of claim 14, wherein the gelled polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum, and locust bean gum; and the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin.

16. The method of claim 12, wherein the composition is used in at least one form selected from the group consisting of a patch, a mask pack, a mask sheet, a cream, a tonic, an ointment, a suspension, an emulsion, a paste, a lotion, a gel, an oil, a spray, an aerosol, a mist, a foundation, a powder, and an oilpaper.

17. The method of claim 16, wherein the composition is applied to or soaked in at least one surface of the patch, the mask pack, or the mask sheet.

18. The method of claim 12, further comprising performing iontophoresis by allowing a microcurrent to flow through the skin having the composition applied thereto.

19. The method of claim 18, further comprising contacting or attaching an iontophoresis device to the skin.

20. The method of claim 19, wherein the iontophoresis device comprises at least one battery selected from the group consisting of a flexible battery, a lithium-ion secondary battery, an alkaline battery, a dry cell, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery, or comprises a patch, a mask pack or a mask sheet provided with the at least one battery.

21. The method of claim 12, wherein the subject is at least one selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a flowchart illustrating a method of isolating and purifying exosomes in a method of preparing exosomes from culture media of adipose-derived stem cells according to one embodiment of the present invention.

(3) FIG. 2 shows the results of measuring the relative amount of proteins contained in a solution in each step of preparing exosomes from culture media of adipose-derived stem cells according to one embodiment of the present invention. The relative amount of proteins in each step was expressed as the relative ratio of the total amount of proteins in solution of each step to the total amount of proteins in conditioned media of stem cells. The experimental results as shown are the results obtained from two different batches, respectively.

(4) FIG. 3 shows the results of measuring the productivity and purity of exosomes obtained according to one embodiment of the present invention. The productivity of exosomes was calculated as the number of exosome particles obtained per mL of conditioned media of stem cells (CM), and the purity of exosomes was calculated as the number of exosome particles per μg of proteins contained in a final fraction. The experimental results as shown are the results obtained from five different batches, respectively.

(5) FIGS. 4A to 4E show the results of analyzing the physical properties of exosomes obtained according to one embodiment of the present invention. “FIG. 4A” shows the particle size distribution and the number of particles obtained by tunable resistive pulse sensing (TRPS) analysis. “FIG. 4B” shows the particle size distribution and the number of particles obtained by nanoparticle tracking analysis (NTA). “FIG. 4C” shows different magnifications of particle images obtained by transmitted electron microscopy (TEM) analysis. “FIG. 4D” shows the results of Western blot analysis of exosomes obtained according to one embodiment of the present invention. “FIG. 4E” shows the results of flow cytometry for CD63 and CD81 in the analysis of markers for exosomes obtained according to one embodiment of the present invention.

(6) FIGS. 5A to 5C show the results of NTA analysis of particle size distributions, which indicate that exosomes having a uniform particle size distribution and high purity are obtained by the addition of trehalose. As the amount of trehalose added increases, a particle size distribution with a single peak can be obtained.

(7) FIGS. 6A to 6C show the results of NTA analysis that indicate particle size distributions obtained depending on whether or not trehalose was added in a process of preparing exosomes according to one embodiment of the present invention. “FIG. 6A” shows the results obtained when trehalose was added throughout the preparation process; “FIG. 6B” shows the results obtained in the case that conditioned media are freeze-stored and thawed, and then trehalose was added to the thawed media; and “FIG. 6C” shows the results obtained when no trehalose was added. “FIG. 6D” shows the results of comparing the relative productivity and relative concentration of exosomes isolated by the methods of FIGS. 6A to 6C. “FIG. 6E” shows the mean size of exosomes isolated by the methods of FIGS. 6A to 6C.

(8) FIG. 7 shows experimental results indicating that the exosomes according to one embodiment of the present invention have the effect of reducing NO formation, a kind of inflammatory reaction. In FIG. 7, PBS denotes phosphate-buffered saline; DEX denotes dexamethasone; EXO denotes exosomes; CM denotes conditioned media of adipose-derived stem cells; and CM-EXO denotes exosome-depleted conditioned media of adipose-derived stem cells.

(9) FIGS. 8A to 8C show experimental results comparing the NO formation-reducing effect of exosomes isolated according to one embodiment of the present invention, with the NO formation-reducing effect of exosomes isolated by a conventional precipitation method (PPT). FIG. 8A shows the results of NTA analysis of exosomes isolated by a conventional precipitation method; FIG. 8B shows the results of NTA analysis of exosomes isolated by the method according to one embodiment of the present invention; and FIG. 8C is a graph comparing the NO formation-reducing effects. The extent of reduction in NO formation was expressed as a relative ratio (%) to the extent of reduction in NO formation by dexamethasone (Dex) as a positive control.

(10) FIGS. 9A to 9C show results indicating that when atopy-induced mice (dermatitis-induced animal model 1) were treated with exosomes according to one embodiment of the present invention, atopic symptoms were alleviated.

(11) FIGS. 10A and 10B depict graphs showing the results of real-time PCR performed to examine changes in the mRNA expression levels of inflammatory cytokines IL-4 and IL-31 in samples obtained from the skin lesion of dermatitis-induced animal model 1, after treating the mice in which atopy was induced, with exosomes according to one embodiment of the present invention.

(12) FIG. 11 shows results indicating that when atopy-induced mice (dermatitis-induced animal model 2) were treated with exosomes according to one embodiment of the present invention, atopic symptoms were alleviated in a manner of depending on the dose of the exosomes.

(13) FIGS. 12A to 12E depict graphs quantifying and comparing the results shown in FIG. 11.

(14) FIGS. 13A to 13D depict graphs showing the results of real-time PCR performed to examine changes in the mRNA expression levels of inflammatory cytokines IL-4, IL-31, TNF-α and IL-23 in samples obtained from the skin lesion of dermatitis-induced animal model 2, after treating the mice in which atopy was induced, with exosomes according to one embodiment of the present invention.

(15) FIGS. 14A to 14C depict graphs showing the results of measuring IgE (FIG. 14A), white blood cells (FIG. 14B) and eosinophils (FIG. 14C) present in blood, after treating the dermatitis-induced animal model 2 with exosomes according to one embodiment of the present invention.

(16) FIG. 15 shows the results of measuring fluorescence intensity to identify exosomes stained with PKH67.

(17) FIG. 16 depicts fluorescence microscopic images showing the extent to which fluorescently stained exosomes of the present invention were delivered into porcine skin tissue. The fluorescence microscopic images were obtained at a certain time after diluting the fluorescently stained exosomes of the present invention with buffer and applying the dilution to the porcine skin surface.

(18) FIG. 17 depicts confocal fluorescence microscopic images showing the extent to which fluorescently stained exosomes of the present invention were delivered into mouse skin tissue, and graphs comparing the total fluorescence intensity obtained by measuring the fluorescence intensity on each of the images. The top of FIG. 17 depicts confocal fluorescence microscopic images obtained after a certain time after diluting the fluorescently stained exosomes of the present invention with buffer and applying the dilution to the mouse skin surface.

(19) FIG. 18 shows results indicating that exosomes according to one embodiment of the present invention were not cytotoxic after human fibroblast HS68 cells were treated with the exosomes.

(20) FIG. 19 depicts photographs showing that erythema and the like on human skin (affected part) with severe dermatitis were remarkably ameliorated as a result of applying a composition including exosomes according to one embodiment of the present invention to human skin (affected part) and then performing iontophoresis to allow a microcurrent to flow through the human skin (affected part) to which the composition was applied.

(21) FIGS. 20A to 20F depict photographs showing that atopic symptoms were remarkably ameliorated as a result of subcutaneously injecting a composition including exosomes according to one embodiment of the present invention into Shetland Sheepdogs suffering from naturally occurring severe atopic dermatitis.

EXAMPLES

(22) Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be easily inferred by those skilled in the art from the detailed description and examples of the present invention are interpreted as falling within the scope of the present invention. References referred to in the present invention are incorporated herein by reference.

(23) Throughout the present specification, it is to be understood that, when any part is referred to as “comprising” any component, it does not exclude other components, but may further include other components, unless otherwise specified.

Example 1: Cell Culture

(24) RAW 264.7 cells (mouse macrophage cell line) were purchased from the Korean Cell Line Bank and cultured. For cell culture, cells were subcultured in DMEM (purchased from ThermoFisher Scientific) medium containing 10% fetal bovine serum (FBS; purchased from ThermoFisher Scientific) and 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific) at 37° C. under 5% CO.sub.2.

(25) Human dermal fibroblast HS68 cells purchased from ATCC were subcultured in DMEM (purchased from ThermoFisher Scientific) medium containing 10% fetal bovine serum (FBS; purchased from ThermoFisher Scientific) and 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific) at 37° C. under 5% CO.sub.2.

(26) According to a cell culture method known in the technical field to which the present invention pertains, adipose-derived stem cells were cultured at 37° C. under 5% CO.sub.2. Next, the cells were washed with phosphate-buffered saline (purchased from ThermoFisher Scientific), and then the medium was replaced with serum-free, phenol red-free medium, and the cells were cultured for 1 to 10 days. The supernatant (hereinafter, referred to as “conditioned medium”) was recovered.

(27) In order to obtain exosomes having a uniform particle size distribution and high purity in an exosome isolation process, 2 wt % of trehalose was added to the conditioned medium. After addition of trehalose, the conditioned medium was filtered through 0.22 μm filter to remove impurities, such as cell debris, waste, macroparticles and the like. From the filtered conditioned medium, exosomes were immediately isolated. In addition, the filtered conditioned medium was stored in a refrigerator (10° C. or below), and then used for exosome isolation. Furthermore, the filtered conditioned medium was freeze-stored in an ultra-low temperature freezer at −60° C. or below, thawed, and then subjected to exosome isolation. Thereafter, exosomes were isolated from the conditioned medium by TFF.

Example 2: Isolation and Purification of Exosomes by TFF Method

(28) For isolating, concentrating and diafiltrating exosomes from the conditioned medium filtered through 0.22 μm filter in Example 1, TFF method was used. The filtered conditioned medium was sonicated to loose potential aggregation of exosomes before isolating and concentrating exosomes using TFF. As a filter for TFF method, a cartridge filter (known as a hollow fiber filter; purchased from GE Healthcare) or a cassette filter (purchased from Pall, Sartorius or Merck Millipore) was used. The TFF filter may be selected with various molecular weight cutoffs (MWCOs). Using the filter having selected MWCO, exosomes were isolated and concentrated, and particles, proteins, lipids, nucleic acids, low-molecular-weight compounds, etc., were removed, which are smaller than the MWCO.

(29) To isolate and concentrate exosomes, a TFF filter having MWCO of 100,000 Da (Dalton), 300,000 Da or 500,000 Da was used. Exosomes were isolated from the conditioned medium by removing substances smaller than the MWCO and concentrating the conditioned medium to a volume of about 1/100 to 1/25 by the TFF method.

(30) The isolated and concentrated solution of exosomes was additionally subjected to diafiltration. The diafiltration was performed continuously (continuous diafiltration) or discontinuously (discontinuous diafiltration), using a buffer having at least 4 times, preferably at least 6 to 10 times, more preferably at least 12 times volume of the isolated exosomes. To obtain exosomes having a uniform particle size distribution and high purity, 2 wt % trehalose in PBS was added to the buffer. FIGS. 6A to 6E show the results that by the addition of trehalose, exosomes having a uniform particle size distribution and high purity can be obtained in high yield.

Example 3: Analysis of Characteristics of Isolated Exosomes

(31) The amounts of proteins of the isolated exosomes, the conditioned medium and the fractions of TFF isolation process were measured using BCA colorimetric assay (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence assay (purchased from Sigma). With regard to exosomes isolated and concentrated by the TFF method according to one embodiment, the extent, to which proteins, lipids, nucleic acids, low-molecular-weight compounds, etc. were removed, was monitored by the protein assays, and the results of the monitoring are shown in FIG. 2. As a result, it could be seen that proteins present in the conditioned medium were very effectively removed by the TFF method according to one embodiment.

(32) FIG. 3 shows the results of comparing the productivity and purity of exosomes in each of five independent batches when exosomes were isolated by the TFF method according to one embodiment. The results obtained from the five independent batches were analyzed, and as a result, it was confirmed that exosomes were very stably isolated by the TFF method according to one embodiment.

(33) The particle size and concentration of the isolated exosomes were measured by nanoparticle tracking analysis (NTA) instrument (purchased from Malvern) or tunable resistive pulse sensing (TRPS) instrument (purchased from Izon Science). The uniformity and size of the isolated exosomes were analyzed by transmission electron microscopy (TEM). FIGS. 4A to 4C show the results of TRPS, NTA and TEM of the exosomes isolated by the isolation method according to one embodiment of the present invention.

(34) After exosomes were isolated by the TFF method, the size distribution of the exosomes was analyzed by NTA depending on whether trehalose was added. The results of the analysis are shown in FIGS. 5A to 5C. The concentration of trehalose was increased from 0 wt % to 1 wt % and 2 wt % (from the top to the bottom in FIGS. 5A to 5C), and the experiment was repeated three times. It was confirmed that when no trehalose was used, particles having a size of 300 nm or more were observed, whereas as the amount of trehalose added was increased, the number of particles having a size of 300 nm or more decreased and the size distribution of the exosomes became uniform.

(35) The effect due to the addition of trehalose in the process of isolating exosomes by the TFF method was additionally examined. As shown in FIGS. 6A to 6C, when 2 wt % trehalose in PBS was added throughout the process of preparing exosomes, exosomes having a uniform size distribution could be obtained (FIG. 6A). However, when the conditioned medium, which had been freeze-stored without adding trehalose, was used, but the TFF process was performed with adding trehalose only in the diafiltration process, or the TFF process was performed without adding any trehalose, uneven exosomes including a large amount of large particles were obtained (FIGS. 6B and 6C).

(36) The relative productivity and concentration of the isolated exosomes were compared, and as a result, exosomes could be obtained with very high productivity when trehalose was added throughout the exosome production process. The obtained exosomes were at least 5 times concentration of the control (in which trehalose was not added throughout the exosome production process) (FIG. 6D). As shown in the NTA analysis result, it was confirmed that the mean size of the isolated exosomes was uniform (200 nm) when trehalose was added throughout the exosome production process (FIG. 6E).

(37) FIG. 4D shows the results of Western blot analysis of the exosomes isolated by the isolation method according to one embodiment of the present invention. As shown therein, the presence of CD9, CD63, CD81 and TSG101 markers was confirmed. As antibodies for each of the markers, 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, respectively.

(38) FIG. 4E shows the results of flow cytometry of the exosomes isolated by the isolation method according to one embodiment of the present invention. As shown therein, the presence of CD63 and CD81 markers was confirmed. To isolate CD63-positive exosomes, an Exosome-Human CD63 Isolation/Detection Reagent kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's instruction. The markers were stained with PE-Mouse anti-human CD63 (purchased from BD) or PE-Mouse anti-human CD81 (purchased from BD), and then analyzed using a flow cytometer (ACEA Biosciences).

(39) Taking the above results together, it could be confirmed that the isolation method according to one embodiment of the present invention could economically and efficiently isolate and purify exosomes having a uniform particle size distribution and high purity in high yield by adding trehalose in the isolation and/or purification process based on tangential flow filtration. In addition, it could be seen that the processes of the isolation method according to one embodiment of the present invention can be scaled-up and are also suitable for GMP.

Example 4: Measurement of Cytotoxicity Following Exosome Treatment

(40) In order to evaluate the cytotoxicity of exosomes, isolated by the isolation method according to one embodiment of the present invention, in human skin fibroblast HS68 cells, the cells were treated with various concentrations of the exosomes, and the proliferation rate of the cells was examined. Specifically, HS68 cells were suspended in 10% FBS-containing DMEM, and then seeded and grown to 80 to 90% confluency and cultured in an incubator at 37° C. under 5% CO.sub.2 for 24 hours. After 24 hours, the medium was removed, and the cells were treated with various concentrations of the exosomes prepared in Example 2. Then, the viability of the cells was evaluated while the cells were cultured for 24 to 72 hours. The cell viability was measured using WST-1 reagent (purchased from Takara), MTT reagent (purchased from Sigma), CellTiter-Glo reagent (purchased from Promega) or alamarBlue reagent (purchased from ThermoFisher Scientific) with a microplate reader (purchased from Molecular Devices).

(41) As a control, the cells cultured in conventional cell culture medium not treated with the exosomes was used. It was confirmed that the exosomes of the present invention showed no cytotoxicity in the concentration range used in the test (FIG. 18).

Example 5: Measurement of Inflammatory Response Using Microphage Cell Line

(42) RAW 264.7 cells were suspended in 10% FBS-containing DMEM medium, and seeded into each well of a multiwell plate resulting in 80 to 90% confluency. Next day, the cells were treated and cultured with a suitable concentration of the exosomes of the present invention (exosomes prepared in Example 2) diluted in fresh serum-free medium containing LPS for 1 to 24 hours. After completion of the culture, the culture supernatant was collected, and NO present in the culture medium were measured to examine inflammatory response. Inflammatory response in the culture medium was measured using an NO detection kit (purchased from Intronbio or Promega). As a positive control, cells were treated with dexamethasone (purchased from Sigma).

(43) As shown in FIG. 7, it was confirmed that when mouse macrophage RAW 264.7 cells were treated with exosomes of the present invention under the presence of LPS, NO production, an LPS-induced inflammatory response, decreased in a concentration-dependent manner. This result shows that the exosomes of the present invention have a functional activity useful for the prevention, amelioration, alleviation or treatment of dermatitis, that is, an activity of reducing LPS-induced inflammatory response, and that the exosomes of the present invention is useful as an active ingredient in a composition for the prevention, amelioration, alleviation or treatment of dermatitis.

Example 6: Comparison of NO Formation-Reducing Effect Between Isolation Methods

(44) To compare NO formation-reducing effect of exosomes between isolation methods, exosomes isolated by a conventional precipitation method were prepared besides the exosomes obtained by the TFF isolation and purification according to one embodiment of the present invention. The precipitation method was performed according to the protocol of the manufacturer (System Biosciences). It was confirmed that the exosomes isolated by the conventional precipitation method (see FIG. 8A) had a lower uniformity of the particle size distribution and various particle sizes as compared with the exosomes isolated and purified by the TFF method of one embodiment of the present invention (see FIG. 8B). In addition, as shown in FIG. 8C, it was confirmed that the exosomes isolated and purified by the TFF method of one embodiment of the present invention inhibited NO formation at a remarkably higher level than the exosomes obtained by the conventional precipitation method. These results show that the exosomes isolated and purified according to one embodiment of the present invention are superior to the exosomes isolated according to the conventional method, in terms of the uniformity of particle size distribution and the inhibition of NO formation.

(45) Thus, the exosomes obtained according to the isolation method of one embodiment of the present invention have excellent performance or functional activities (e.g., uniformity of particle size distribution, inhibition of NO production, reduction of inflammatory response, etc.), and the composition of the present invention, which contains, as an active ingredient, the stem cell-derived exosomes having excellent functional activities as described above, is superior to the conventional art in terms of the effect of preventing, ameliorating, alleviating or treating dermatitis.

Example 7: Dermatitis-Induced Animal Model 1

(46) Male NC/Nga mice (16 to 18 g, 5-week-old; purchased from Central Laboratory Animal Inc.) were purchased, adapted for 7 days, and then used in this experiment. The adapted mice were divided into five groups as follows after dermatitis was induced in the mice.

(47) (1) Normal: Normal control group;

(48) (2) Vehicle (dermatitis-induced group): negative control group in which dermatitis was induced by house dust mite extracts;

(49) (3) IV: a test group in which the exosomes prepared in Example 2 were intravenously (IV) injected at a dose of 2.8 μg/head three times a week for two weeks, after dermatitis was induced by house dust mite extracts;

(50) (4) SC: a test group in which the exosomes prepared in Example 2 were subcutaneously (SC) injected at a dose of 2.8 μg/head three times a week for two weeks, after dermatitis was induced by house dust mite extracts; and

(51) (5) Pred: a test group in which prednisolone was administered orally every day, after dermatitis was induced by house dust mite extracts.

(52) The auricles of each of NC/Nga mice (purchased from Central Laboratory Animal Inc.) was shaved with a razor, and then depilated by applying a suitable amount of a depilatory. After wiping off the depilatory, AD induction reagent (house dust mite extracts; purchased from BioStir Inc.) was applied uniformly to the auricles by a micropipette tip. After shaving with a razor, if necessary, 150 μL of 4% SDS aqueous solution was applied uniformly to the auricles by a micropipette tip. After the auricles were dried with cold air from a dryer and further dried naturally for about 2 to 3 hours, AD induction reagent was applied uniformly to the auricles by a micropipette tip. All the pretreatments were performed twice a week for 3 weeks, i.e. six times in total.

(53) Before starting administration of the exosomes prepared in Example 2, clinical skin score assessment was performed. According to the ranked scores, the animals were randomly grouped so that the average score of each group was distributed as uniformly as possible.

(54) FIG. 9A depicts a photograph of an atopy-induced mouse (No treatment) and photographs showing that atopic symptoms are alleviated by exosome treatment (IV and SC) according to one embodiment of the present invention. FIG. 9B is a graph showing the atopic clinical score of each of test groups (2) to (5), and as shown therein, it was confirmed that the atopic clinical score of the groups treated with the exosomes according to one embodiment of the present invention was improved. In addition, FIG. 9C is a graph showing relative changes in the ear thickness measured in each of test groups (2) to (5) as compared with the ear thickness of normal group (1), and as shown therein, it was confirmed that the ear thickness of the groups treated with the exosomes according to one embodiment of the present invention decreased.

(55) Taken together, it was confirmed through the experiment that the skin clinical score and the ear thickness decreased in the groups (both IV and SC) treated with the exosomes of the present invention.

Example 8: Dermatitis-Induced Animal Model 2

(56) To evaluate the dose-dependent effect of exosomes, mice were divided into 9 groups as follows after dermatitis was induced as described in Example 7 above.

(57) (1) Normal: normal control group (indicated by “N” in FIG. 11);

(58) (2) Control (dermatitis-induced group): a negative control group in which dermatitis was induced by house dust mite extracts (indicated by “C” in FIG. 11);

(59) (3) IV, L (exosome, low): a test group in which the exosomes prepared in Example 2 above were intravenously (IV) injected at a dose of 0.14 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts;

(60) (4) IV, M (exosome, medium): a test group in which the exosomes prepared in Example 2 above were intravenously (IV) injected at a dose of 1.4 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts;

(61) (5) IV, H (exosome, high): a test group in which the exosomes prepared in Example 2 above were intravenously (IV) injected at a dose of 10 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts;

(62) (6) SC, L (exosome, low): a test group in which the exosomes prepared in Example 2 above were subcutaneously (SC) injected at a dose of 0.14 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts;

(63) (7) SC, M (exosome, medium): a test group in which the exosomes prepared in Example 2 above were subcutaneously (SC) injected at a dose of 1.4 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts;

(64) (8) SC, H (exosome, high): a test group in which the exosomes prepared in Example 2 above were subcutaneously (SC) injected at a dose of 10 μg/head three times a week for 4 weeks, after dermatitis was induced by house dust mite extracts; and

(65) (9) Pred: a test group in which prednisolone was administered orally every day, after dermatitis was induced by house dust mite extracts (indicated by “P” in FIG. 11).

(66) Dermatitis induction was performed as described in Example 7, and an excessive amount of AD induction reagent was applied so that the mean clinical skin score at the time of administration of the exosomes was 9. Before starting administration of the exosomes prepared in Example 2, clinical skin score assessment was performed. According to the ranked scores, the animals were randomly grouped so that the average score of each group was distributed as uniformly as possible.

(67) The first and second rows from the top of FIG. 11 are photographs of atopy-induced mice (Day 0) and photographs showing that atopic symptoms are alleviated in a dose-dependent manner by treatment with the exosomes according to one embodiment of the present invention (Day 28). FIG. 12A is a graph showing relative improvement in the atopic clinical score of each of test groups (2) to (9), and as shown therein, it was confirmed that the atopic clinical score of the groups (IV and SC) treated with the exosomes according to one embodiment of the present invention was improved in a dose-dependent manner.

(68) The third and fourth rows from the top of FIG. 11 show the results of staining ear skin tissue sections with H&E and toluidine blue. The ear skin tissue of each euthanized mouse was stained with H&E, and then the thickness of the ear skin tissue was measured. FIG. 12B is a graph showing the thickness of the ear skin tissue measured in each of test groups (2) to (9) in comparison with that of normal group (1), and as shown therein, it was confirmed that in the groups (IV and SC) treated with the exosomes according to one embodiment of the present invention, the thickness of the ear skin tissue decreased in a dose-dependent manner. In addition, the ear skin tissue of each euthanized mouse was stained with toluidine blue, and then the infiltration of mast cells, a type of inflammatory cells, was measured. FIG. 12C is a graph showing the number of mast cells measured in each of test groups (2) to (9) in comparison with that in normal group (1), and as shown therein, it was confirmed that in the groups (IV and SC) treated with the exosomes according to one embodiment of the present invention, the infiltration of mast cells decreased in a dose-dependent manner.

(69) The fifth and sixth rows from the top of FIG. 11 show the results of subjecting the ear skin tissue of each euthanized mouse to immunohistochemical staining with anti-CD86 antibody and anti-CD206 antibody. It is known that inflammatory dendritic epidermal cells (IDECs), which are abundantly present in dermatitis lesions without being found in normal skin, display CD86 antigen and CD206 antigen on their surface (J Invest Dermatol. 2002; 118:327-334; Arch Dermatol Res. 2001; 293:448-454). Thus, by measuring the number of CD86+ cells and CD206+ cells in atopic dermatitis lesion, the extent of amelioration of dermatitis symptoms can be determined.

(70) The ear skin tissue section of each euthanized mouse was subjected to immunohistochemical staining with anti-CD86 antibody and anti-CD206 antibody (Abcam, Cambridge, Mass.), and then the number of CD86+ cells and the number of CD206+ cells were counted. FIGS. 12D and 12E are graphs showing the number of CD86+ cells and CD206+ cells measured in each of test groups (2) to (9) in comparison with that in normal group (1), and as shown therein, it was confirmed that in the groups (IV and SC) treated with the exosomes according to one embodiment of the present invention, the number of CD86+ cells and the number of CD206+ decreased in a dose-dependent manner. These results show that the infiltration of inflammatory dendritic epidermal cells in the atopic dermatitis lesion decreased and dermatitis symptoms were alleviated or ameliorated.

(71) Taken together, it was confirmed through the experiments that in the groups (both IV and SC) treated with the exosomes of the present invention, the skin clinical score, the thickness of the ear skin tissue, the infiltration of mast cells, and the infiltration of inflammatory dendritic epidermal cells decreased in a dose-dependent manner.

Example 9: Measurement of mRNA Expression Levels of Cytokines

(72) cDNA was prepared from the total RNA obtained by grinding tissues of skin lesions of each euthanized mouse, and changes in the mRNA expression levels of various inflammatory cytokines which are a major cause of dermatitis, were measured using a real-time PCR method. As a reference gene for normalizing IL-4, IL-31, IL-23 and TNF-α genes, GAPDH gene was used. The sequences of primers used in the real-time PCR are shown in Table 1 below.

(73) TABLE-US-00001 TABLE 1 Nucleotide sequences of primers used in real-time PCR Sequences Forward primer Reverse primer Genes (5′ .fwdarw. 3′) (5′ .fwdarw. 3′) IL-4 ACA GGA GAA GGG ACG  GAA GCC CTA CAG ACG AGC CC A T  TC A  (SEQ ID NO: 1) (SEQ ID NO: 2) IL-31  CAC ACA GGA ACA ACG  CGA TAT TGG GGC ACC GAA   AA G CC  G  (SEQ ID NO: 3) (SEQ ID NO: 4) IL-23 CAC ATG CAC CAG CGG  CTT TGC AAG CAG AAC TGG GA C AT  CTG TTG  (SEQ ID NO: 5) (SEQ ID NO: 6) TNF-α CGT CGT AGC AAA CCA  TTG AAG AGA ACC TGG GAG  CC A AG  TA G ACA  (SEQ ID NO: 7) (SEQ ID NO: 8) GAPDH CAT GGC CTT CCG TGT  CCT GCT TCA CCA CCT TCT TCC TA  TGA T  (SEQ ID NO: 9) (SEQ ID NO: 10)

(74) Through the experiment on the above animal model 1, it was confirmed that in the groups (both IV and SC) treated with the exosomes of the present invention, the mRNA expression levels of inflammation-related cytokines IL-4 and IL-31, which cause dermatitis, decreased (FIGS. 10A and 10B). In addition, through the experiment on the above animal model 2, it was confirmed that in the groups (both IV and SC) treated with the exosomes of the present invention, the mRNA expression levels of various inflammation-related cytokines (i.e., IL-4, IL-31, TNF-α and IL-23), which cause dermatitis, decreased in a dose-dependent manner (FIGS. 13A to 13D).

(75) The group treated with the exosomes of the present invention decreased the mRNA expression levels of IL-4, IL-31, TNF-α and IL-23 in a dose-dependent manner as compared with the control and the prednisolone-treated group, and these inflammation-related cytokines are major targets for the development of dermatitis-related therapeutic agents. Decreases in the expression levels of these multiple targets are related to the suppression and alleviation of dermatitis. IL-4 initiates isotype class switching to IgE and activates eosinophils. In addition, it is known that IL-31 affects isotype class switching to IgE and recruits inflammatory cells into the skin, and increased IL-31 correlates with severity of dermatitis. It is known that IL-23 induces the differentiation of ThO-type T cells into pathogenic helper T cells that produce TNF-α, and the high plasma concentration of TNF-α is correlated with the severity of dermatitis. Considering these aspects collectively, it is thought that the exosomes of the present invention regulate inflammatory response by inhibiting the expression of multiple cytokines that are major causes of dermatitis.

(76) Therefore, the exosomes of the present invention is able to act against various inflammatory cytokines (i.e., IL-4, IL-31, IL-23, and TNF-α) that cause dermatitis, and thus be widely applied against dermatitis caused by various factors and effectively suppress and alleviate dermatitis.

Example 10: Blood Assay

(77) Plasma was isolated from the blood of each euthanized mouse, and the concentration of immunoglobulin E (IgE) in the blood was measured using an ELISA kit. Through the experiment, it was confirmed that in the group treated with the exosomes prepared in Example 2, the blood IgE level decreased in a manner of depending on the dose of the exosomes (FIG. 14A).

(78) In addition, using the whole blood of each euthanized mouse, the number of blood cells was counted. A predetermined amount of whole blood cells were centrifuged using a Cytospin (purchased from ThermoFisher Scientific) at 1,000 rpm for 10 minutes, and then subjected to slide-drying, followed by Diff-Quik staining. The number of white blood cells and the number of eosinophils were measured. Through the experiment, it was confirmed that in the group treated with the exosomes prepared in Example 2, the number of white blood cells and the number of eosinophils decreased in a manner of depending on the dose of the exosomes (FIGS. 14B and 14C).

(79) From the above-described results, it can be seen that the composition of the present invention reduces the level of the inflammatory response factor IgE in blood that causes dermatitis, and also reduces the number of white blood cells and the number of eosinophils in blood. In addition, the composition of the present invention reduces the production of various inflammatory cytokines and inflammation-related factors, and inhibits the activity or involvement of inflammation-related immune cells. Therefore, the composition of the present invention is useful as a pharmaceutical composition, a skin external preparation and a cosmetic composition for the prevention, amelioration, alleviation or treatment of dermatitis.

Example 11: Test for Skin Penetration Ability of Exosomes

(80) To prepare fluorescently stained exosomes, PKH67 dye (purchased from Sigma) was used. 1 mM PKH67 was diluted in Diluent C (purchased from Sigma) to prepare 10 μM PKH67 solution. The solution was mixed with a suitable concentration of exosome solution and allowed to react at room temperature under a light-shielded condition for 10 minutes. After completion of the reaction, MW3000 spin column (purchased from ThermoFisher Scientific) was used to remove the remaining free PKH67 dye from the exosomes stained with PKH67 (hereinafter, abbreviated as “PKH-exosomes”). After removing PKH67 that did not react with the exosomes, analysis was performed using a fluorometer (purchased from Molecular Devices), and as a result, it was confirmed that fluorescence with sufficient intensity was detected in the PKH-exosomes (FIG. 15).

(81) The PKH-exosomes were dispersed in phosphate buffered saline (PBS) at a suitable concentration, for example, a concentration of 1×10.sup.5 particles/mL to 1×10.sup.9 particles/mL, and applied to the outer surface of porcine skin. The porcine skin was covered with nonwoven fabric to prevent drying of the PKH-exosome solution, and then the PKH-exosomes and the skin tissue allowed to react for a suitable time, for example 30 minutes to 1 hour, so that the PKH-exosomes reached the subcutaneous tissue of the porcine skin. After completion of the reaction, the porcine skin tissue was fixed overnight in 3.7% formaldehyde solution, and washed three times with PBS for 5 minutes each time. The washed porcine skin tissue was soaked in 30% sucrose solution, and then treated with OCT compound. Next, the tissue was washed three times with PBS for 5 minutes each time, and then sectioned using a microtome. The tissue section was placed on a slide glass. Meanwhile, preparation of the tissue section may be performed before the tissue is fixed with formaldehyde solution. The fluorescence detected from the PKH-exosomes in the tissue section was observed using a fluorescence microscope. As a result, it was confirmed that the PKH-exosomes were delivered through the epidermis of the porcine skin tissue into the subcutaneous tissue (FIG. 16). As shown in FIG. 16, the exosomes of the present invention could effectively penetrate through the skin barrier, so that it could be delivered deep into the skin tissue and effectively absorbed into the skin. Therefore, a skin external preparation or cosmetic composition containing the exosomes as an active ingredient will effectively act in the prevention, amelioration, alleviation or treatment of dermatitis.

(82) Next, the skin tissue of hairless mice was dissected and placed in the upper chamber of a Franz diffusion cell. The inside of the diffusion cell was filled with PBS. The PKH-exosomes were dispersed in PBS at a suitable concentration, for example, a concentration of 1×10.sup.5 particles/mL to 1×10.sup.9 particles/mL, and then applied to the outer surface of the mouse skin tissue. At this time, nonwoven fabric was pre-placed on the outer surface of the mouse skin tissue in order to prevent drying of the PKH-exosome solution, and the PKH-exosome solution was injected between the nonwoven fabric and the skin tissue. Then, the PKH-exosomes and the skin tissue were allowed to react for 30 minutes to 1 hour. After completion of the reaction, the PKH-exosomes delivered into the skin tissue were immediately observed with a confocal fluorescence microscope (Leica, SP8X), or the skin tissue and the PKH-exosome solution were additionally allowed to react for 1 to 6 hours, and then the PKH-exosomes were observed with a confocal fluorescence microscope. As a result, it was confirmed that the exosomes of the present invention are able to effectively penetrate through the skin barrier, so that exosomes of the present invention are able to be delivered deep into the skin tissue and effectively absorbed into the skin (FIG. 17).

(83) Therefore, a skin external preparation or cosmetic composition containing the exosomes as an active ingredient will effectively act in the prevention, amelioration, alleviation or treatment of dermatitis.

Example 12: Treatment of Human Skin with Composition Containing Exosomes as Active Ingredient

(84) The composition containing the exosomes obtained according to the isolation method of one embodiment of the present invention, that is, a suspension containing the exosomes, was applied to the affected parts (hand, neck, arm, etc.) of three severe atopic patients three times a week for 1 to 2 weeks, and then iontophoresis allowing a microcurrent to flow through the composition-applied affected part was performed using an iontophoresis device. As a result, severe pruritus in the patients was remarkably alleviated, and dermatitis-related erythema symptoms in the patients were also remarkably ameliorated (FIG. 19). In the patients to which the composition containing the exosomes of the present invention was applied, severe pruritus and dermatitis-related erythema symptoms were alleviated and ameliorated so that the prescription of steroids or anti-histamines for these patients would be stopped.

(85) Thus, it can be seen that a skin external preparation or cosmetic composition containing, as an active ingredient, the exosomes obtained by the isolation method according to one embodiment of the present invention, exhibits the effect of preventing, ameliorating, alleviating or treating dermatitis, as confirmed through the above-described clinical tests.

Example 13: Treatment of Canine with Composition Containing Exosomes as Active Ingredient

(86) An experiment was performed on a Shetland Sheepdog (body weight: 13 kg; 9 years old) suffering from naturally occurring severe atopic dermatitis. The composition containing the exosomes obtained according to the isolation method of one embodiment of the present invention (the exosomes prepared in Example 2) was subcutaneously injected into the Shetland Sheepdog suffering from naturally occurring severe atopic dermatitis, 12 times in total for 5 weeks. In one injection, the exosomes prepared in Example 2 were injected at a dose of 117 μg/head. At weeks 1 and 2, the composition was subcutaneously injected three times a week, and at weeks 3, 4 and 5, the composition was subcutaneously injected twice a week. In addition, after administration of the exosomes of the present invention, administration of other drug was stopped.

(87) FIG. 20A depicts photographs of affected parts before administration of the exosomes of the present invention, and as can be seen therein, erythema and inflammation in the abdomen were severe. It could be seen that dermatitis symptoms were remarkably ameliorated from 3 days after administration of the exosomes of the present invention (FIG. 20B), and dermatitis almost disappeared at 10 days after administration (FIG. 20C) and at 2 weeks after administration (FIG. 20D). In addition, after administration of the exosomes of the present invention, the vitality of the tested Shetland Sheepdog clearly increased, and its body weight increased to 16 kg. The increase in vitality and weight gain is believed to be due to the amelioration of dermatitis symptoms.

(88) In order to confirm whether the dermatitis treating effect of the exosome of the present invention would be sustained, the affected part of the tested Shetland sheepdog was continuously observed after the end of administration of the exosomes of the present invention. As a result, it could be confirmed that the effect of ameliorating dermatitis was maintained even at 50 days (FIG. 20E) and 93 days (FIG. 20F) after the end of administration.

(89) Therefore, as confirmed through the above-described animal test for the canine, the composition containing, as an active ingredient, the exosomes obtained according to the isolation method of one embodiment of the present invention can effectively alleviate or ameliorate dermatitis, and the dermatitis treating effect thereof can be sustained.

Example 14: Preparation of Cosmetic Composition Containing Exosomes of the Present Invention

(90) 1704 μg/mL of the exosomes prepared in Example 2 above was mixed with and suspended in the components shown in Table 2 below, thereby preparing a cosmetic composition (lotion). The content of each component is shown in Table 2 below.

(91) TABLE-US-00002 TABLE 2 Components and their contents of lotion containing exosomes of the present invention Components Contents (wt%) Exosomes prepared in Example 2 1 Glycerin 7.375 Caprylic/capric triglyceride 6 Cetyl ethylhexanoate 5 Propanediol 5 Phenyl trimethicone 3.5 Stearic acid 3 1,2-hexanediol 2 Panthenol 2 Cetearyl olivate 1.8 Sorbitan olivate 1.2 Diisostearyl malate 1 Fructan 1 Ammonium acryloyldimethyl 0.3 taurate/VP copolymer Arachidyl alcohol 0.25 Behenyl alcohol 0.15 Arachidyl glucoside 0.1 Hydrogenated lecithin 0.1 Shea butter 0.09 Xanthan gum 0.05 Lavender oil 0.02 Bergamot oil 0.02 Ceramide NP 0.02 Orange peel oil 0.02 Phytospingosine 0.015 Palmitoyl tetrapeptide-7 0.01 Palmitoyl tripeptide-1 0.01 Purified water Balance

(92) Although the present invention has been described with reference to the embodiments, the scope of the present invention is not limited to these embodiments. Any person skilled in the art will appreciate that various modifications and changes are possible without departing from the spirit and scope of the present invention and these modifications and changes also fall within the scope of the present invention.