KR20190049474A - Exosome kit and method for improving transdermal delivery of exosomes using the same - Google Patents

Abstract

The present invention provides an exosome kit for promoting transdermal permeation of an exosome, which comprises a mask pack, a mask sheet or a patch having a composition containing an exosome as an active ingredient applied or deposed thereto, and an iontophoresis device. The present invention facilitates skin application of exosome having functions of functional cosmetics or quasi-drugs, and allows the exosome to effectively penetrate skin barriers while penetrating deeply into the skin. Accordingly, the present invention is applied to skin care which improves a bad skin state or restores the skin to a normal state to have an excellent effect, and provides an excellent effect in terms of skin disease treatments for alleviating, improving deteriorated skin state or restoring the same to a normal state.

Description

Exosome kit and method for enhancing transdermal permeation of exosome using the same -

The present invention relates to an exosomal kit for the percutaneous permeation enhancement of exosomes comprising a mask pack, a mask sheet or a patch applied or immersed with a composition comprising an exosome as an active ingredient, and an iontophoresis device will be.

The present invention also relates to a method for enhancing transdermal permeability of exosome and its application, which enables the exosome to pass through the skin barrier effectively and to be deeply transferred into the skin using the exosome kit.

The skin has a three-layer structure of epidermis, dermis, and subcutaneous fat. The stratum corneum of the epidermis, which is located at the outermost part of the skin, suppresses moisture evaporation of the skin and acts as a skin barrier. However, the skin barrier of the stratum corneum has a problem of impairing the penetration of the active ingredient when the functional cosmetic or the medicinal product is applied to the skin, thereby deteriorating the efficacy of the functional cosmetic or the medicinal product.

A transdermal delivery system is an effective ingredient applied to the surface of the skin to easily pass through the skin barrier and deliver it deeply into the skin. Typically known transdermal delivery systems include liposomes. Liposomes are composed of lipid bilayers that are structurally similar to cell membranes or intercellular lipids of the stratum corneum, so they can be fused with cell membranes, effectively transferring the active ingredients inside the liposomes into the skin. Artificially synthesized liposomes, however, have limited stability and limited skin penetration.

An attempt has been made to create an elastomer that is flexible and easy to deform so as to increase the skin absorption rate and to penetrate the horny layer of the skin when the skin penetrates, for example an etosome. Ethosomes are made by dissolving phospholipids in ethanol known as skin permeation enhancers. Ethanol ethanol weakens the intercellular lipid membranes of the skin's horny layer, increasing the skin's absorption rate of the active ingredient and making the membrane of the endoplasmic reticulum itself flexible. This feature allows the etosome to deliver effective ingredients to the deeper and deeper part of the active ingredient in the skin. However, ethosomes also have problems such as particle aggregation due to curing of nanoparticles and layer separation phenomenon.

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. Particularly, development of application technology related to improvement of skin penetration ability of exosome is insufficient.

The inventors of the present invention have developed an exosomic kit for transcutaneous permeation enhancement of exosomes which greatly promotes transdermal permeation of exosomes while repeatedly studying the new application fields of exosomes and medical or cosmetic techniques Thus completing 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 "

Korean Registered Patent No. 10-1443180 (2014.09.22)

Kor. J. Aesthet. Cosmetol., Vol. 12 No. 5, 597-605 (October 2014)

It is an object of the present invention to provide an exosome kit for enhancing transcutaneous permeation of exosomes comprising a mask pack, a mask sheet or a patch applied or immersed with a composition containing an exosome as an active ingredient, and an iontophoresis device, .

It is another object of the present invention to provide a method for enhancing transdermal permeability of exosomes, which enables the exosome to pass through the skin barrier effectively and deeply into the skin using the exosome kit, and its application.

 It is still another object of the present invention to provide a cosmetic method or a skin disease treatment method for controlling the condition of mammalian skin except the therapeutic use using the exosomatic 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 accomplish the above object, the present invention provides a method for treating a transplant of an exosomes comprising an iontophoresis device and a mask pack, a mask sheet or a patch, on which a composition containing an exosome as an active ingredient is applied or immersed, Thereby providing an exosome kit for enhancing permeability.

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 " biological solution " means a liquid solution of biogenic origin, in which the exosome is dispersed, suspended, precipitated, suspended or mixed, and includes, for example, a cell culture solution, a cell culture supernatant, Cell culture supernatant, whole blood, serum, cord blood, plasma, multiple fluid, brain and cerebrospinal fluid, placenta extract, and bone marrow aspirate. However, the present invention is not limited thereto, and it is needless to say that it does not exclude various biological origin solutions such as various animals, plants, bacteria, fungi, algae and the like. A " biological solution " may be cultured or incubated under conditions that release and / or secrete exosomes, and may be frozen and thawed.

As used herein, the term " exosome " is intended to mean an exosome that is secreted from cells of various animals, plants, bacteria, fungi, algae and the like, preferably stem cells, (E. G., Exosome-like vesicles) having a nano-sized bezacl structure and a composition similar to exosome.

However, it is needless to say that the exosome used in the present invention can be applied to skin and various exosomes which are used in the art or can be used in the future can be used as long as they do not cause adverse effects on the human body. 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.

As used herein, the term " improvement of skin condition " is a positive change visually and / or tactually perceptible to the appearance and / or feel of the skin, including skin tissue regeneration, Improving the skin condition, preventing aging of the skin, or restoring the skin condition to a mild condition, an improvement condition or a normal condition. As an example that does not limit the present invention, the term " improvement of skin condition " refers to alleviation, improvement or elimination of skin defects, skin wrinkles, reduced skin elasticity, wrinkles, wrinkles, rough and deep wrinkles, cracks, bumps, ; Skin thickening (e.g., removal of the epidermis, dermis, subcutaneous layer, nail and stratum corneum of the skin); Prevention of skin or hair elasticity loss due to loss, damage and / or inactivation of functional skin elastin; Reduction of cellulite; Discoloration of pale skin, spots, freckles, dullness, skin, hair or nails, such as blackness, dark circles under eyes, erythema, dots, café aureus, becker spots, osteoblast, pigmentary nevus, epidermal nevus, melanin pigment spot Alleviating, improving or eliminating pigmentation, capillary vasodilation or discoloration caused by spider blood vessels; Alleviating or preventing skin dryness and brittleness.

As used herein, the term " skin aesthetic " is intended to reduce, alleviate, or ameliorate skin aging such as skin sagging, dented cheeks, depressed eyes, wrinkles, reduced skin elasticity, Restoring the skin condition to a normal state; Skin, skin regeneration, skin whitening, freckles, dark circles, spots, tattoos, acne, elastin layer marks, stretch marks, scarring, enlarged pores, pale, spots, black circles, dark circles, erythema, Removal or correction of various skin defects such as spots, osteoblasts, pigmented nevi, epidermal nevus, melanin pigment spots, and pigmentation; Hair loss enhancement, scalp management, cellulite reduction, facial reduction, contour correction, soft tissue defect correction, tissue enlargement, volume-up, skin swelling, skin moisturizing and soothing, redness and blood vessels Reduction or improvement of diabetes mellitus. However, in the present invention, the term " skin beauty " is not limited to the above, and includes, for example, removing or correcting various skin imperfections.

As used herein, the term " treatment of skin diseases " refers to the treatment of facial flushing, spots, freckles, dullness, dark circles, dark circles, erythema, dots, café au lait, becker dots, Or ameliorating or alleviating pigmented skin lesions such as pigmentation, or restoring skin conditions that are deteriorated by such pigmented skin lesions to a normal state; Relieving, alleviating or ameliorating vascular skin lesions such as flame nevus, capillary vasodilatation, injection, hemangiomas, vesicular blebs, etc., or to restore normalized skin conditions deteriorated by such vascular skin lesions; Such as contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria, atopic dermatitis, seborrheic dermatitis, self-sensitizing dermatitis, autoimmune progesterone dermatitis, dermatitis dermatitis, acne or eczema , Improving or restoring normal state; Itching rash, itching rash, hairy wall, nervous scraping wound, behavioral disorders involving the skin, and parasitic warts, itching, itchy itchiness, itching in the winter, itching in the face, itching in the scrotum, itching in the scrotum, watery itching, scalp itching, nasal itching, Alleviating, ameliorating, or restoring a variety of itching, such as mental dermatological conditions, such as diarrhea; Alleviating, ameliorating or restoring a variety of fibrotic skin diseases, including skin sclerosis, to a normal state, and the like. However, in the present invention, " treatment of skin diseases " is not limited to the above-mentioned, and it is of course that the pathological condition of various skin diseases is alleviated, ameliorated or restored to a normal state.

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.

An exosome kit for transcutaneous permeation enhancement of exosome according to 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 as an active ingredient, And an iontophoresis device for applying a microcurrent to the skin of the skin.

For example, an exosome kit for transcutaneous permeation enhancement of an exosome according to an 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, By placing the iontophoresis device on a mask sheet or patch, the first mask pack, mask sheet or patch can be contacted or attached to be used in such a way that a microcurrent is applied 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 skin improving agent and / or moisturizer to the extent that the action thereof (improvement of skin condition, treatment of skin condition, skin disease, etc.) 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.

In one embodiment of the present invention, the iontophoresis device for enhancing transdermal permeation of exosome comprises a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium A battery, and a reverse electrodialysis cell, or a second mask pack, a mask sheet, or a patch having the at least one battery mounted thereon.

In the exosome kit for transcutaneous permeation enhancement of exosome in 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 one embodiment of the present invention, at least one surface of the first mask pack, mask sheet or patch is coated with a hydrogel, hyaluronic acid, hyaluronate (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. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

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

The transdermal permeation enhancement method of exosome in one embodiment of the present invention can be carried out using the exosome kit as described above.

For example, in one embodiment of the present invention, a method for enhancing transdermal permeability of an exosome comprises the steps of: (a) applying a first mask pack, a mask sheet or patch on which a composition comprising an exosome as an active ingredient is applied or immersed, (B) placing 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 first mask pack, (D) delivering the exosomes through the microcurrent to the interior of the mammalian skin. The method of claim 1, wherein the mammal is a human.

In one embodiment of the present invention, at least one surface of the first mask pack, mask sheet or patch is coated with a 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. The gelled polymer and the polyhydric alcohol may be exemplified in the above description.

In the transcutaneous permeation enhancement method of exosome according to one embodiment of the present invention, the iontophoresis device may be a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel- An electrodialysis cell, or a second mask pack, a mask sheet, or a patch with 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.

As one illustrative example of the invention, the exosome can be obtained by performing the following steps: (a) adding trehalose to the biological solution, (b) adding the trehalose (C) separating the exosomes from the filtered biological solution using TFF (Tangential Flow Filtration), and (d) removing the buffer used for desalting and buffer exchange (diafiltration). , Adding trehalose to the solution, and performing desalting and buffer exchange (diafiltration) on the separated exosome using TFF (Tangential Flow Filtration) using the buffer solution to which the trehalose is added .

On the other hand, when trehalose is added to the buffer solution used for desalting and diafiltration in step (d), exosomes having a uniform particle size distribution and high purity can be effectively obtained (see FIG. 6 ). By using trehalose in the pre-filtration step (step (b) before exocase separation by TFF) and desalting by TFF and buffer exchange step (step (d)) after separation of exosome, a high purity particle size distribution Uniform exosome can be obtained with high yield. Trehalose, on the other hand, provides the ability to efficiently isolate exosomes for impurities such as cellular debris, waste products, proteins and macromolecules.

The desalting and buffer exchange can be performed continuously or intermittently. Desalting and buffer exchange can be carried out using a buffer solution having a volume of at least 4 times, preferably 6 times to 10 times, more preferably 12 times the starting volume. For TFF, a molecular weight cutoff (MWCO) of 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da TFF filter or 0.05 占 퐉 TFF filter can be used. The step (c) may further include a step of concentrating the solution to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration).

As one example that does not limit the present invention, the biological solution may be a stem cell culture solution. The type of the stem cell is not limited, but may be a mesenchymal stem cell, for example, a fat, a bone marrow, an umbilical cord or a cord blood-derived stem cell, more preferably a fat-derived stem cell. 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 exosome used in the present invention is not limited to the exosome obtained according to the separation method described above, and it is needless to say that various exosomes which are used in the art or can be used in the future can be used. It is to be understood that the exosome isolated according to the above separation method should be understood as an example of exosome that can be used in the composition of the present invention, and the present invention is not limited thereto.

Another embodiment of the present invention is a method for improving the skin condition, comprising: (a) performing a transcutaneous permeation enhancement method of exosome as described above; and (b) Lt; RTI ID = 0.0 > mammalian < / RTI > 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, skin condition improvement may include skin regeneration, wrinkle removal or improvement, whitening, scar removal, skin texture improvement, redness reduction, moistening or smoothing of the condition of the skin, Rubbing can be an improvement of the deteriorated skin condition.

In another embodiment of the present invention, there is provided a method for enhancing transdermal permeation of an exosome comprising the steps of: (a) performing a transcutaneous permeation enhancing method of exosome as described above; and (b) The method comprising the steps of:

The present invention has the effect of facilitating skin application of exosomes that can function as functional cosmetics or medicines, and penetrating deeply into the skin while allowing the exosomes to pass the skin barrier effectively. Therefore, the present invention can be applied to a skin beauty which improves a poor skin condition or restores to a normal state, and can exhibit an excellent effect. In addition, the present invention provides an effect of treating a skin disease which alleviates, It is possible to exhibit an excellent effect.

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

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart illustrating a process for separating and purifying exosomes in a method for producing exosomes from a biological solution according to one embodiment of the present invention.
2 is a graph showing a result of measuring a relative amount of protein contained in a solution for each step of preparing a biological solution, for example, a stem cell culture solution, according to an embodiment of the present invention . The ratio of the total amount of protein in each step was expressed by the relative ratio of total protein amount to the total biological solution. The experimental results show the results obtained in two different batches, respectively.
FIG. 3 shows the results of measuring the productivity and purity of exosomes obtained according to one embodiment of the present invention. The productivity of exosomes was calculated as "the number of particles of exosome obtained per mL of biological solution, eg, a stem cell culture (CM)," and the purity of exosome was calculated as "the number of exosomes per microgram of protein contained in the final fraction Quot; number of particles " The experimental results show the results obtained in five different batches.
FIG. 4 shows the results of physical property analysis of exosomes obtained according to one embodiment of the present invention. 4A shows particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis. &Quot; Figure 4B " shows particle size distribution and number of particles by NTA (nanoparticle tracking analysis) analysis. 4C " shows particle images by transmission electron microscopy (TEM) according to magnification. 4D " shows the Western blot results of exosomes obtained according to one embodiment of the present invention. &Quot; Figure 4E " shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.
Figure 5 shows the results of NTA analysis on particle size distribution showing that a uniform and high-purity exosome is obtained with trehalose addition. As the amount of trehalose added increases, a particle size distribution having a single peak can be obtained.
FIG. 6 shows the results of NTA analysis showing the particle size distribution according to whether trehalose was added during the preparation of exosome according to one embodiment of the present invention. 6A shows a case where trehalose is added throughout the production process, " Fig. 6B " shows a case where trehalose is added after the cell culture solution is stored in a frozen state, and " The results are shown without adding os. 6D shows the results of comparing relative productivity and relative concentration of exosomes isolated by the methods of FIGS. 6A to 6C. 6E shows the mean size of exosomes isolated by the methods of FIGS. 6A to 6C.
FIG. 7 shows the result of confirming the absence of cytotoxicity after treatment of exosome according to one embodiment of the present invention with HS68 cells of human skin fibroblasts.
FIG. 8 shows fluorescence intensity measurement results of exosomes stained with PKH67.
Fig. 9 is an image of a fluorescence microscope confirming the degree of transfer of fluorescent stained exosomes into pig skin tissue.
FIG. 10 is a confocal fluorescence microscope image showing the degree of fluorescence-stained exosomes transferred into the mouse skin tissue.
11 is a graph showing a comparison of the total fluorescence intensity obtained by measuring the fluorescence intensity on each image in Fig.
12 is a perspective view showing a configuration of the exosomes kit 100 according to one embodiment of the present invention.
FIG. 13 is a photograph showing the skin condition before and after treatment of human skin with the exosomal kit of the present invention. FIG.
FIG. 14 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 erythema of the skin (lesion) is remarkably improved.
FIG. 15 is a photograph showing the skin condition before and after the treatment of the exosome kit of the present invention on the face of a person suffering from acne;
16A to 16I show the results of improving skin wrinkles, improving skin elasticity, skin moisturizing and dermal density when the exosome kit of the present invention is applied to human skin.

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

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. 6 shows the results of confirming the effect of obtaining an exosome with high purity and homogeneous particle size distribution at a high yield by trehalose treatment.

Example 3: Characterization of isolated exosomes

The amount of protein in the fractions of the isolated exosome, culture medium, and TFF separation process was measured using BCA colorimetry (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence (purchased from Sigma). The extent to which the exosome was isolated and concentrated by the TFF method of the present invention and the removal of proteins, lipids, nucleic acids, and low-molecular compounds was monitored by a protein determination method, and the results are shown in FIG. As a result, it was found that the protein present in the culture solution was very effectively removed by the TFF method of one embodiment of the present invention.

FIG. 3 shows the results of comparing the productivity and purity in five independent batches when isolating exosomes by the TFF method of one embodiment of the present invention. As a result of analyzing the results obtained from the five independent batches, it was confirmed that the exosome can be separated very stably by the TFF method of one embodiment of the present invention.

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 the TRPS, NTA and TEM analyzes of the separated exosomes according to one embodiment of the present invention are shown in Figs. 4A to 4C.

FIG. 5 shows the result of NTA analysis of the size distribution of exosomes according to whether or not trehalose was added after the exosome was separated by the TFF method. The concentration of trehalose was increased to 0% by weight, 1% by weight and 2% by weight (from top to bottom in FIG. 5) and repeated three times. In the absence of trehalose, particles having a size of 300 nm or more were identified, while particles having a size of 300 nm or more were reduced by increasing the amount of trehalose, and the size distribution of the exosome was uniformized .

The effect of adding trehalose was further investigated in the process of isolating exosome by the TFF method. 6, when 2 wt% trehalose dissolved in PBS was added to the whole process of exosome production, exosomes having a uniform size distribution were obtained (Fig. 6A). On the other hand, in the case of using the culture solution free of trehalose, which was stored in freeze, only TFF process was performed by addition of trehalose only in desalting and buffer exchange process, or TFF process was performed without adding trehalose On the other hand, uneven exosomes containing large particles were obtained (Figs. 6B and 6C).

As a result of comparing the relative productivity and concentration of isolated exosomes, it was found that when trehalose was added to the whole process of exosomes, exosome was obtained with high productivity and the concentration of exosome obtained was 5 times higher (Fig. 6D). As shown in the results of NTA analysis, the average size of the separated exosomes was uniformly observed at 200 nm when trehalose was added to the entire process of exosome (Fig. 6E).

FIG. 4D 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. 4E shows the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer 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 exosome used in the present invention is not limited to the exosome of the above-mentioned embodiments, and it is needless to say that various exosomes which are used in the art or can be used in the future can be used. It should be understood that the exosome isolated according to the above embodiments is an example of exosome that 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 no cytosoxicity by exosome was observed within the tested concentration range (FIG. 7).

Example 5: Preparation of fluorescent dyed exosomes

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 analyzer (purchased from Molecular Devices), it was confirmed that sufficient intensity of fluorescence was detected in the PKH-exosome after removing PKH67 not reacted with exosomes (FIG. 8).

Example 6: Skin permeability test of exosome used in the exosome kit of the present invention

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, deeply transferred into the skin tissue, and more effectively absorbed to the skin when active current was delivered (active transdermal delivery) (see FIG. 9). As shown in Fig. 9, the exosomes used in the exosome kit of the present invention can effectively pass through the epidermis and be deeply transferred into the skin tissue, and can be effectively absorbed to the skin.

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 and deeply transferred into the skin tissue when the microcurrent was passed (active transdermal delivery), and more effectively absorbed to the skin (Figs. 10 and 11) .

Example 7: Treatment of exosome kit against human skin

12, the exosome kit 100 according to one embodiment of the present invention includes a liquid immersion sheet mask 10 (a white sheet mask coated or immersed with a composition containing the exosome prepared in Example 2) And a percutaneous permeation promoting sheet mask (silver sheet mask) 20 which is an iontophoresis device. Exosome 12 is coated or deposited on the surface or inside of 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 a severe skin trouble (hereinafter referred to as "case 1") to alleviate or alleviate skin troubles and flushing symptoms . In addition, the exosome kit 100 of the present invention was applied once to the face of a person with flushing symptom (hereinafter referred to as " case 2 ") to evaluate whether the overall skin tone and flushing symptom were improved. 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, one set of the "exosome kit 100" composed of the liquid-immersion sheet mask 10 containing the exosomes 12 at a concentration of 5 × 10 ⁵ particles / mask and the percutaneous permeation- In case 1, flushing and skin troubles were remarkably improved in case 1 (see case 1 in FIG. 13) and overall skin tone was improved in case 2, and flushing was alleviated (see case 2 in FIG. 13). Therefore, the exosomatic kit 100 of the present invention, which comprises the liquid immersion sheet mask 10 on which the composition containing the exosome as an active ingredient is immersed and the percutaneous permeation promoting sheet mask (iontophoresis device) 20, It can be said that there is a skin-beauty effect or a skin disease treatment effect that prevents, suppresses, alleviates or improves symptoms and skin troubles.

In addition, a composition (exosome suspension) containing the exosome prepared in Example 2 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 An iontophoresis was performed 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. As a result of performing the above treatment three times a week for two weeks, the severe itching of patients was remarkably alleviated and the erythema symptoms were also remarkably improved (FIG. 14). Patients who applied the composition containing exosomes improved their symptoms to such an extent that severe itching and erythema could be relieved to stop the prescription of steroids or antihistamines.

Therefore, the exosome kit 100 of the present invention can be said to have a skin-beauty effect or a skin disease-treating effect for preventing, suppressing, alleviating or alleviating itching and erythema of the skin as confirmed by the clinical test as described above.

Example 8: Treatment of exosome kit on facial surface of subjects with acne symptoms

The "exosome kit" described in Example 7 was applied to the face of a person suffering from acne symptomatically once every four days to evaluate whether acne symptoms and related skin troubles were alleviated or improved. A liquid immersion sheet mask 10 containing 5 × 10 ⁵ exosomes 12 was applied to the entire face of a person suffering from acne symptomatically by aligning them with the eyes and the mouth and then a micro current of about 0.3 to 0.4 mA An activated percutaneous permeation enhancing sheet mask 20 is laminated on the liquid immersion sheet mask 10 and used for about 25 to 30 minutes.

As a result, the use of the exosome kit 100 of the present invention disappeared and the skin was soft and smooth, and the skin tone was clear (see FIG. 15).

Accordingly, the exosome kit 100 of the present invention can be said to have a skin-beauty effect or a skin disease treatment effect for preventing, suppressing, alleviating or alleviating acne symptoms and skin troubles associated therewith.

Example 9: Test for improving wrinkles, improving elasticity, moisturizing effect, etc. on human skin

For 20 healthy women (aged 35 to 59 years old) without wasting or chronic physical diseases including skin diseases as wrinkled women, the exosomes prepared in Example 2 were mixed at a concentration of 1 x 10 < ⁷ > (Translucent oil-in-water) and the exosome kit 100 of the present invention were used once every three days in the evening to test skin cosmetic effects such as skin wrinkles, skin elasticity improvement, moisturizing and skin regeneration effects.

After the facial cleansing, the contents of the ampoule were evenly applied to the face, the liquid immersion sheet mask 10 was brought into close contact with the eyes and the mouth, and the entire surface of the face was evenly adhered. Then, an activated percutaneous permeation- The mask 20 was laminated on the liquid immersion sheet mask 10 and used for about 25 to 30 minutes.

After the ampoule and the exosome kit 100 of the present invention were used, after 2 weeks of use, and after 4 weeks of use, improvement of the wrinkles of the eyes, wrinkles of the wrinkles and improvement of the wrinkles of the mouths were evaluated by using Primos Premium and Primos Light (Primos Lite) (GFMesstechnik GmbH, Germany) and Image Pro Ra (Average Roughness) value analysis. Ra means average roughness of the wrinkle profile, and as the value decreases, the wrinkle improves.

2 weeks after use of the ampoule and the exosome kit 100 of the present invention and after 4 weeks of use, the skin moisturizing effect was measured by a moisture meter (Corneometer CM825) (Courage-Khazaka eletronic GmbH, Germany) And skin elasticity was evaluated by measuring the sound pressure with CM580 (Cutometer CM580) (Courage + Khazaka electronic GmbH, Germany). The improvement of the eye tail lifting, the improvement of the ball lifting, and the improvement of the eyelid lifting were evaluated through the F-ray contour photographing and angular analysis before the use of the ampoule and the exosome kit 100 of the present invention, , Dermal density improvement was assessed by analyzing the density (%) after the skin scanner ultrasonography at the eye.

As a result of evaluating each of the test items before and after the use of the exosomous kit 100 of the present invention, significant improvement effects were obtained in all the test items after 2 weeks of use and after 4 weeks of use as compared with before use 16I). In addition, significant clinical improvement effects in the use of the exosome kit 100 of the present invention as a result of preparing for the average results of other treatments for amelioration of aging, skin moisturization and / or lifting, Were found to be very good, and wrinkles, skin elasticity, eye tail lifting and ball lifting were found to be excellent (see Table 1 below)

Contrast with the average results of other clinical procedures Test Items After 4 weeks (%) Average(%) Average contrast Eye wrinkles -5.647 -7 to -5 Average Wrinkles -7.415 -5 good Wrinkles -6.694 -7 Average Skin moisture content 21.383 10 Very good Skin elasticity 9.291 5-6 good Eyebrow lifting 16.342 10 good Ball lifting -12.053 -10 good Lifting a mouthpiece 22.851 10 Very good Dermal density 19.715 15-20 Average

After the test, a Global Assessment of Efficacy was conducted. The test subjects were tested for the test items after the use of the exosomatic kit 100 of the present invention in five steps of excellent (4), good (3), normal (2), poor (1) and very poor (0) And responded to the questionnaire. In the questionnaire survey, the mean value and the standard deviation of the questionnaire and the percentage of the subjects to the answer were obtained for each test item. As a result of the questionnaire survey on the use of the exosomatic kit 100 of the present invention, 100% of the test subjects were evaluated as above all the test items. After completion of the test, the subjects were asked to answer the questionnaire about feeling of use of the exosome kit (100) of the present invention. (4), good (3), normal (2), poor (1), and very poor (0) for the skin moisturizing, smoothness, spreading, absorbency, Respectively. The mean value and the standard deviation of the questionnaire value and the percentage of the subjects to the answer were obtained for each evaluation item. As a result of the questionnaire evaluation on the use of the exosomatic kit 100 of the present invention, 100% of the test subjects were rated above the average for all evaluation items.

On the other hand, there was no report of a specific skin adverse reaction during the period of using the exosomic kit 100 of the present invention, and no abnormality was observed on a physical examination by a dermatologist.

Therefore, the exosome kit 100 of the present invention is excellent in skin wrinkles, skin elasticity, skin regeneration and skin moisturizing effects, as confirmed by the above-mentioned clinical tests.

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.

100: Exosome kit
10: liquid immersion sheet Mask 12: exosome
20: transdermal permeation enhancing sheet mask 22:
24: conductive pattern

Claims (9)

A first mask pack, a mask sheet or a patch which is applied or deposited with a composition containing an exosome as an active ingredient,
An exosomatic kit for enhancing transcutaneous permeation of exosomes, comprising an iontophoresis device for delivering a microcurrent to the skin of a mammal to which said composition is applied. 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 through the skin of a mammal to which said composition is applied. ≪ RTI ID = 0.0 > 8. < / RTI > 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 at least one kind of battery is mounted, the exosome kit for enhancing transcutaneous permeation of exosomes. 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, and is used for the transcutaneous permeation enhancement of the exosome. (a) contacting or adhering a first mask pack, a mask sheet or a patch, onto which a composition comprising an exosome as an active ingredient is applied or deposited, onto the skin of the 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 exosome through the microcurrent to the interior of the mammalian skin. 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 the at least one kind of battery is mounted. 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. (a) performing the transdermal patch enhancement method of exosome according to any one of claims 5 to 7, and (b) improving the skin condition through the step (a). A cosmetic method for regulating the condition of a mammal skin except a dragon. (a) performing the transdermal permeation enhancing method of exosome according to any one of claims 5 to 7;
(b) treating the skin disease by relaxing, ameliorating or restoring the pathological condition of the skin disease through the step (a).

Images