Hybrid exosome and use thereof

Abstract

The present invention relates to a hybrid exosome formed by the fusion of a human cell-derived exosome and an artificial exosome. When an active ingredient labeled with a fluorescent material was attached or encapsulated in the hybrid exosome of the present invention and applied to cells, the active ingredient was observed in the cells. In addition, when the exosome was applied to skin tissue, fluorescence expression could also be observed in the dermal layer. Therefore, the hybrid exosome according to the present invention can be used as a novel drug delivery system.

Description

Hybrid exosomes and their uses

[0001] The present invention relates to a hybrid exosome and its use.

[0002] Extracellular vesicles (EVs) are nanoscale membrane structures that transport cellular materials, such as proteins, between cells, mediating intercellular communication. In particular, exosomes are known to be produced by the budding of late endosomes and fuse with the plasma membrane before being released into the extracellular space. Exosomes are 40 to 200 nm in size, composed of a lipid bilayer membrane rich in phosphocholine, cholesterol, and ceramide. They are secreted by virtually all cell types and exist stably in all body fluids, such as blood, lymph, and sweat. Furthermore, their small size and weak negative charge allow them to circulate for extended periods and reach the interior of organs. Exosomes are also expected to be next-generation drug delivery systems, as they can evade phagocytosis and deliver hydrophilic or hydrophobic drugs (Bunggulawa et al., J Nanobiotechnology (2018) 16:81).

[0003] However, natural exosomes are heterogeneous in size, shape, and even the composition of their active ingredients. Furthermore, the extremely small amount secreted from cells and the significant effort required for collection and concentration limit their commercialization. Therefore, the development of new technologies to overcome existing limitations is urgently needed.

[0004] Accordingly, the inventors of the present invention conducted research to develop a more superior drug delivery system, and as a result, they produced a hybrid exosome by fusing an artificial exosome containing an exosome and a lipid component of an exosome component, and confirmed that the active ingredient contained in the exosome was delivered into a cell, thereby completing the present invention.

[0005] To achieve the above purpose, one aspect of the present invention provides a hybrid exosome in which an exosome and an artificial exosome are fused.

[0006] Another aspect of the present invention provides a method for producing a hybrid exosome, comprising the steps of: i) producing an exosome or an artificial exosome; and ii) mixing the exosome and the artificial exosome to induce fusion thereof.

[0007] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, comprising the hybrid exosome as an active ingredient.

[0008] Another aspect of the present invention provides a cosmetic composition for improving skin condition, which comprises a hybrid exosome fused with an artificial exosome containing the hybrid exosome as an active ingredient.

[0009] Another aspect of the present invention provides a drug delivery composition comprising a hybrid exosome fused with an exosome and an artificial exosome containing an active ingredient.

[0010] Another aspect of the present invention provides a use of a hybrid exosome, which is a fusion of an exosome and an artificial exosome, for preventing or treating skin diseases.

[0011] Another aspect of the present invention provides a method for preventing or treating a skin disease comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

[0012] Another aspect of the present invention provides a use of hybrid exosomes, which are fused exosomes and artificial exosomes, for improving skin condition.

[0013] Another aspect of the present invention provides a method for improving skin condition comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

[0014] When an active ingredient labeled with a fluorescent substance was attached or encapsulated in a hybrid exosome according to the present invention and treated with cells, the active substance was observed within the cells. Furthermore, when the exosome was applied to skin tissue, fluorescence expression was observed even in the dermal layer. The hybrid exosome is manufactured by fusing an exosome and an artificial exosome loaded with an active substance, and thus can simultaneously exhibit the pharmacological activity of the existing exosome itself and the pharmacological activity of the loaded active substance. Therefore, the hybrid exosome according to the present invention can be used not only as a novel drug delivery vehicle but also as a therapeutic agent for various diseases.

[0015] Figure 1 is a diagram showing the function of the hybrid exosome of the present invention.

[0016] Figure 2 is a drawing showing the characteristics of the hybrid exosome of the present invention.

[0017] Figure 3 schematically illustrates a method for manufacturing artificial exosomes.

[0018] FIG. 4 is a drawing showing a specific example of a lipid bilayer component used in the production of a hybrid exosome, which is one specific example of the present invention.

[0019] Figure 5 is a drawing showing the results of confirming the ability of a hybrid exosome, which is an example of the present invention, to carry hydrophobic or hydrophilic substances.

[0020] Figure 6 is a drawing showing the results of FACS analysis of each exosome after preparing a hybrid exosome, which is an embodiment of the present invention, by mixing green fluorescently labeled mesenchymal stem cell-derived exosomes (MSC exosomes) and red fluorescently labeled artificial exosomes. Green: MSC exosomes, red: artificial exosomes, yellow: hybrid exosomes.

[0021] Figure 7 is a drawing showing the results of checking skin permeability using the Franz diffusion cell test after loading a red fluorescently labeled peptide (palmitoyl-WKYMVm_TAMRA) (SEQ ID NO: 1) into a hybrid exosome, which is an example of the present invention. Blue: nuclei of cells in skin tissue (DAPI), red: palmitoyl-WKYMVm_TAMRA peptide loaded into the hybrid exosome.

[0022] Figure 8 is a diagram showing the results of confirming the intracellular uptake of MSC exosomes labeled with green fluorescence in mesenchymal stem cells. Blue: DAPI (nucleus), green: MSC exosomes.

[0023] Figure 9 is a diagram showing the results of confirming the intracellular uptake of artificial exosomes labeled with red fluorescence in mesenchymal stem cells. Blue: nucleus (DAPI), red: artificial exosomes.

[0024] Figure 10 is a diagram showing the results of confirming the intracellular uptake of hybrid exosomes (yellow), which are one specific example of the present invention, in mesenchymal stem cells. Green: MSC exosome components in hybrid exosomes, red: artificial exosome components in hybrid exosomes, blue: nuclei (DAPI), yellow: MSC exosome components and artificial exosome components in hybrid exosomes.

[0025] Figure 11 is a diagram and graph showing the results of examining the degree of infiltration into skin cells after treating keratinocytes (HaCaT cells) with fluorescently labeled exosomes or liposomes, respectively. Blue: nuclei (DAPI), green: MSC exosomes, red: artificial exosomes or liposomes, blue / red: hybrid exosomes.

[0026] Figure 12 is a diagram and graph showing the results of confirming the skin permeability of artificial exosomes labeled with red fluorescence using a Franz Diffusion Cell. Blue: nucleus (DAPI), red: artificial exosomes.

[0027] Figure 13 is a diagram and graph showing the results of confirming the drug delivery capacity of artificial exosomes loaded with red fluorescent dye using a Franz Diffusion Cell. Blue: nucleus (DAPI), red: artificial exosomes.

[0028] Hybrid exosomes

[0029] One aspect of the present invention provides a hybrid exosome in which an exosome and an artificial exosome are fused.

[0030] The term "exosome" used in the present invention may refer to a nano-sized particle that is naturally secreted by living cells, packaged in a lipid bilayer, and serves as a role in transmitting information between cells. The size of exosomes is known to be approximately 30 nm to 250 nm in diameter. Although there are some differences depending on the type of cell of origin, it is known that the exosome membrane contains surface proteins (surface markers) such as CD9, CD63, and CD81, and the inside of the exosome contains proteins such as TSG101 and ALIX that can prove that it is of endosomal origin. In addition, it contains proteins including growth factors and cytokines with various functions, as well as nucleic acids such as mRNA and miRNA, and has components and effects that reflect the characteristics of the cell of origin. In particular, stem cell-derived exosomes are known to have effects such as regulating stem cell differentiation, promoting regeneration and growth, and inducing specific immune responses.

[0031] In the present invention, the exosome may be an exosome derived from a human or plant, but exosomes of various origins that are used or may be used in the art in the future may be used without limitation as long as they do not cause adverse effects on the human body.

[0032] Specifically, the human-derived exosomes may be derived from human blood, tissue or cells.

[0033] More specifically, it may be isolated from human blood, or a culture of tissues or cells. At this time, the cells may be stem cells, immune cells, blood cells, somatic cells, or germ cells. The stem cells may be mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, or neural stem cells, but are not limited thereto. In addition, the mesenchymal stem cells may be derived from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerves, skin, amniotic fluid, or amniotic membrane, but are not limited thereto.

[0034] Specifically, the plant-derived exosomes may be isolated from a plant culture, a plant extract, or a biological solution equivalent thereto. In this case, the plant culture, extract, or biological solution equivalent thereto may be derived from the entire plant, or may be derived from plant tissue, callus, or cells.

[0035] In one specific example, the culture or extract of the plant may be a culture or extract of cells, tissues or callus derived from any one selected from the group consisting of flowers, leaves, stems, branches, fruits, fruit peels, roots, seeds and combinations thereof of the plant.

[0036] In one embodiment of the present invention, the exosome may be an exosome isolated from a culture medium of human mesenchymal stem cells.

[0037] In the present invention, the diameter of the exosome may be from about 50 nm to about 400 nm. Specifically, it may be from about 50 nm to about 400 nm, from about 80 nm to about 350 nm, from about 100 nm to about 300 nm, or from about 150 nm to about 250 nm, but is not limited thereto.

[0038] Additionally, the exosome may have a zeta potential of negative charge (<0 mV). Specifically, the zeta potential of the exosome may be, but is not limited to, about -100 mV to -1 mV, about -80 mV to -3 mV, about -50 mV to about -6 mV, about -40 mV to about -9 mV, about -30 mV to about -12 mV, or about -30 mV to about -15 mV.

[0039] As used herein, the term "artificial exosome" refers to a structure composed of a lipid membrane surrounding an aqueous internal compartment. The membrane of an artificial exosome is mainly composed of phospholipids and their derivatives. When phospholipids and their derivatives are dispersed in an aqueous solution, etc., vesicles of a single layer or lipid bilayer are spontaneously formed. Artificial exosomes can contain water-soluble active ingredients in the aqueous internal space and can also contain hydrophobic active ingredients within the lipid bilayer, and thus can be utilized as a delivery vehicle for various drugs.

[0040] In the present invention, the artificial exosome may include any one selected from the group consisting of lecithin, phosphatidylcholine (PC), ceramide, cholesterol, phytosterol or a derivative thereof, phosphatidylethanolamine (PE), phosphatidylserine (PS), and a combination thereof.

[0041] The term "lecithin" as used herein refers to a phospholipid containing glycerol phosphate. Lecithin is a major component of biological membranes and is abundant in animal brains, spinal cords, blood cells, egg yolks, and plant seeds, yeast, and molds. An example of lecithin is phosphatidylcholine (PC), which is characterized by having hydrophilic components such as phosphoric acid and choline bound to one side of glycerol, and a hydrophobic acyl group bound to the other side.

[0042] In the present invention, the lecithin may be, without limitation, hydrogenated lecithin, unsaturated lecithin, lyso lecithin, etc. In addition, extracted lecithin (egg yolk lecithin, soybean lecithin, etc.), synthetic lecithin, or a combination thereof may be used, but is not limited thereto.

[0043] The term "ceramide" used herein refers to one of the sphingolipids having a structure in which a fatty acid is linked to sphingosine or phytosphingosine. The ceramide accounts for more than about 40% of the intercellular lipids that constitute the stratum corneum of the skin, and is a component of the double-layer cell membrane and the skin lipid membrane. In addition, ceramide is an essential component for the formation of the structure or function of the stratum corneum, and ceramides existing in the human body are classified into various types according to the degree of polarity. Ceramide is an important element of the skin barrier, and has the function of acting as a lipid barrier that suppresses the evaporation of moisture and maintaining the orderly structure of the stratum corneum, and therefore is attracting attention as a raw material for moisturizing cosmetics.

[0044] In the present invention, the ceramide may be one or more selected from the group consisting of ceramide NS, ceramide AS, ceramide EOS, ceramide NDS, ceramide ADS, ceramide EODS, ceramide NP, ceramide AP, ceramide EOP, ceramide NH, ceramide AH, and ceramide EOH, but is not limited thereto. In one embodiment of the present invention, the ceramide may be ceramide NP.

[0045] As used herein, the term "cholesterol" refers to a type of sterol (a combination of a steroid and an alcohol), a lipid found in the cell membranes of all animal cells. Cholesterol is transported through the bloodstream. In the present invention, the cholesterol is included in the artificial exosome to increase the membrane strength of the lipid bilayer, thereby enhancing the stability of the artificial exosome.

[0046] As used herein, the term "sphingolipid" refers to a key epithelial lipid involved in maintaining the skin's barrier function. Sphingolipids, such as sphingosine, sphinganine, sphingomyelin, and phytosphingosine, generally contain a long sphingoid base as a central group or "backbone," with amide-linked long-chain fatty acids and a head group. There are hundreds of known classes of sphingolipids, each with a different head group (e.g., choline phosphate, glucose, galactose, polysaccharides) and a different fatty acid and sphingoid base. Phytosphingosine is a ceramide precursor concentrated in healthy stratum corneum, where it is enzymatically converted to sphingolipids in the skin. In one embodiment, the sphingolipid in the present invention may be sphingomyelin.

[0047] As used herein, the term "phytosterol" refers to a plant-derived lipid containing a sterol backbone. This includes plant sterols and stanols, and more than 250 sterols and related compounds are currently known. Phytosterols can synchronize the metabolic cycle of epithelial cells in the skin and can be used as structural components of artificial exosomes or other biological membranes to enhance the strength of their bilayer membrane structures.

[0048] The phytosterol may include, for example, sitosterol, β-sitosterol, stigmasterol, campesterol, chalinosterol, clionasterol, brassicasterol, alpha-spinasterol, Delta5-avenasterol, luphenol, danchosterol, desmosterol, poriferasterol, stigmasterol, campestanol, cycloartenol, etc. In one embodiment of the present invention, the phytosterol may be β-sitosterol.

[0049] As used herein, the term "phosphatidylethanolamine (PE)" is a type of phospholipid found in biological membranes, known to account for approximately 25% of all phospholipids. Phosphatidylethanolamine is synthesized by adding cytidine diphosphate-ethanolamine to diglycerides, and S-adenosylmethionine can subsequently methylate the amine of phosphatidylethanolamine to produce phosphatidylcholine.

[0050] The term "phosphatidylserine (PS)" used in this specification is a membrane lipid that constitutes a cell membrane, and is known to account for about 13% to about 15% of the phospholipids of the human cerebral cortex.

[0051] Specifically, the artificial exosome may include any one selected from the group consisting of lecithin and ceramide, phytosterol and combinations thereof.

[0052] In one specific example, the artificial exosome may comprise lecithin. In one specific example, the artificial exosome may comprise lecithin and ceramide. In one specific example, the artificial exosome may comprise lecithin and phytosterol. In one specific example, the artificial exosome may comprise lecithin, ceramide, and phytosterol.

[0053] Preferably, the artificial exosome may include lecithin, ceramide, and phytosterol. At this time, the lecithin, ceramide, and phytosterol may be included in a ratio of about 1:0.1:0.01 to about 1:0.8:0.4 based on 1 part by weight of lecithin. Specifically, the ratio may be about 1:0.1:0.01 to about 1:0.8:0.4, about 1:0.2:0.03 to about 1:0.7:0.3, about 1:0.3:0.03 to about 1:0.6:0.2, or about 1:0.4:0.03 to about 1:0.5:0.3. In one embodiment, the lecithin, ceramide, and phytosterol may be included in a ratio of about 1:0.45:0.03 to about 1:0.45:0.2 based on 1 part by weight of lecithin.

[0054] In the present invention, the artificial exosome may have a diameter of about 50 nm to about 300 nm. Specifically, the artificial exosome may have a diameter of about 50 nm to about 300 nm, about 70 nm to about 250 nm, about 90 nm to about 200 nm, or about 110 nm to about 150 nm.

[0055] Additionally, the zeta potential of the artificial exosome may be from about -30 mV to about +20 mV. Specifically, it may be from about -30 mV to about +20 mV, from about -25 mV to about +10 mV, from about -20 mV to +1 mV, or from about -15 mV to about -5 mV, but is not limited thereto.

[0056] In the present invention, the artificial exosome may additionally contain an active ingredient. The active ingredient is not limited to a specific type, and any ingredient capable of being encapsulated in the artificial exosome and exhibiting the desired effect as determined by those skilled in the art may be used. Furthermore, the active ingredient may be hydrophobic or hydrophilic.

[0057] The above active ingredient may be any one selected from the group consisting of amino acids, minerals, sugars, vitamins (or precursors or derivatives thereof) or salts thereof, energy sources of cells, peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds or salts thereof, natural products (including extracts) or salts thereof, or glycosides, semi-synthetic compounds, toxins, and combinations thereof, but is not limited thereto. In this case, the active ingredient may be loaded onto any one site selected from the group consisting of the surface, interior, between lipid bilayers, and combinations thereof of the artificial exosome.

[0058] In one specific example, the active ingredient is glycine, glutamic acid, leucine, alanine, phenylalanine, valine, isoleucine, methionine, cysteine, proline, sodium pyruvate, calcium chloride, magnesium chloride, zinc oxide, calcium pantothenate, glucose, inositol, pyridoxine or a salt thereof (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or a salt thereof (HCl or nitrate), nicotinamide. Adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), adenosine triphosphate (ATP), niacinamide, arbutin (α or β), ethyl ascorbyl ether, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, (-)-α-bisabolol, paper mulberry extract, soluble licorice extract, glabridin, hydroquinone, kojic acid, ascorbic acid,Dipotassium glycyrrhizate, tranexamic acid, phloretin, ergothioneine, retinol, retinyl palmitate, polyethoxylated retinamide, bakuchiol, adenosine, ubiquinone, thioctic acid, peptides, Trp-Lys-Tyr-Met-Val-D-Met (WKYMV or WKYMVm) (SEQ ID NO: 1), glutathione, palmitoyl tritapeptide-1, palmitoyl pentapeptide-4, acetyl Acetyl hexapeptide-8, myristoyl pentapeptide-17, copper peptide, acetyl hydroxyproline, hydroxypropyl tetrahydropyrantriol, collagen, elastin, trehalose, ceramide, glycerin, chitosan, hyaluronic acid, shea butter, luteolin, apigenin, astaxanthin, protocatechuic acid (PCA), urolithin, panthenol, tocopherol, resveratrol, Lycopene, beta-carotene, thiotic acid, curcumin,It may be any one selected from the group consisting of ubiquinone, catechins, flavonoids, polyphenols, salicylic acid, allantoin, aloe vera extract, chamomile flower extract, green tea extract, tea tree leaf oil, centella asiatica extract, Houttuynia cordata extract, azelaic acid, caffeine, dexpantenol, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, Argania spinosa kernel oil, and combinations thereof, but is not limited thereto.

[0059] In the present invention, the hybrid exosome may be a fusion of the exosome and the artificial exosome at a ratio of about 1:1 to about 1:10000. Specifically, it may be fused in a number ratio of about 1:1 to about 1:10000, about 1:1 to about 1:1000, about 1:1 to about 1:500, about 1:1 to about 1:100, about 1:1 to about 1:90, about 1:1 to about 1:80, about 1:1 to about 1:70, about 1:1 to about 1:60, about 1:1 to about 1:50, about 1:1 to about 1:40, about 1:1 to about 1:30, about 1:1 to about 1:20, about 1:1 to about 1:10, about 1:1 to about 1:5, or about 1:1 to about 1:3. Preferably, it may be fused in a number ratio of about 1:1, but is not limited thereto.

[0060] The diameter of the hybrid exosome may be about 50 nm to about 300 nm. Specifically, the hybrid exosome may have a diameter of about 50 nm to about 300 nm, about 70 nm to about 250 nm, about 90 nm to about 200 nm, or about 110 nm to about 150 nm.

[0061] Additionally, the zeta potential of the hybrid exosome may be from about -30 mV to about +20 mV. Specifically, it may be from about -30 mV to about +20 mV, from about -25 mV to about +10 mV, from about -20 mV to +1 mV, or from about -15 mV to about -10 mV, but is not limited thereto.

[0062] In the present invention, the hybrid exosome obtained by fusion of the exosome and the artificial exosome may be a single structure containing both the exosome-derived component and the artificial exosome-derived component. The exosome and the artificial exosome are the same as described above.

[0063] Accordingly, it may include both the lipid bilayer component of the exosome and the lipid bilayer component of the artificial exosome. The lipid bilayer of the hybrid exosome may include any one selected from the group consisting of lecithin, ceramide, cholesterol, sphingolipid, phytosterol or derivatives thereof and combinations thereof. More specifically, it may include any one selected from the group consisting of lecithin, ceramide, phytosterol and combinations thereof. In one embodiment, the lipid bilayer of the hybrid exosome may include lecithin, ceramide and phytosterol, and these components may be included in a ratio of about 1:0.45:0.03 to about 1:0.45:0.2 based on 1 part by weight of lecithin.

[0064] In addition, when an active ingredient is loaded into the artificial exosome, a hybrid exosome obtained by fusing the exosome and the artificial exosome loaded with the active ingredient may contain the same active ingredient as that loaded into the artificial exosome. The active ingredient may be hydrophobic or hydrophilic. At this time, the active ingredient may be included in any one compartment selected from the group consisting of the surface of the hybrid exosome, between lipid bilayers, inside the exosome, and combinations thereof. Therefore, the hybrid exosome loaded with the active ingredient according to the present invention can simultaneously exhibit the pharmacological effect of the active ingredient contained in the exosome and the pharmacological effect of the active ingredient loaded into the artificial exosome. The active ingredient is the same as described above.

[0065] The encapsulation rate of the above-mentioned active ingredient in the hybrid exosome may be about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, from more than about 80% to less than about 99%, from more than about 80% to less than about 98%, from more than about 80% to less than about 97%, from more than about 80% to less than about 96%, or from more than about 80% to less than about 95%.

[0066] Here, the above "encapsulation" means encapsulating to surround and efficiently introduce the delivery substance into the body, and the drug encapsulation rate (encapsulation efficiency) means the content of the drug encapsulated in the hybrid exosome with respect to the total drug content used in manufacturing.

[0067] Method for preparing hybrid exosomes

[0068] Another aspect of the present invention provides a method for producing a hybrid exosome, comprising the steps of: i) producing an exosome or an artificial exosome; and ii) mixing the artificial exosome and the exosome to induce fusion thereof.

[0069] At this time, the exosomes, artificial exosomes and hybrid exosomes are the same as described above.

[0070] Specifically, the method for producing the hybrid exosome may include the following steps.

[0071] First, it may include a step of manufacturing exosomes or artificial exosomes.

[0072] a. Exosome production

[0073] The above exosomes can be obtained from humans or plants. Specifically, they can be isolated and obtained from human blood, tissue, or cell cultures. Alternatively, they can be isolated and obtained from plant cultures or extracts.

[0074] The term "cultivation" used in the present invention refers to growing tissues, cells, or calli under appropriately controlled environmental conditions, and the culturing process of the present invention can be performed according to appropriate media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected tissue, cell, or calli. The medium refers to a known medium used in the culturing of tissues, cells, or calli, and includes all known cell culture media or modified media thereof.

[0075] The human-derived tissue or cell can be cultured in a culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, about 18 hours to about 7 days, or about 1 day to about 3 days.

[0076] The plant-derived tissue, cell or callus can be cultured in a culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, about 18 hours to about 7 days or about 1 day to about 3 days.

[0077] In one specific example of the present invention, human-derived mesenchymal stem cells (MSCs) can be cultured in a culture medium for about 1 hour to about 4 weeks, about 6 hours to about 3 weeks, about 12 hours to about 2 weeks, about 18 hours to about 7 days, or about 1 day to about 3 days.

[0078] The exosomes of the present invention can be separated from the culture medium by a separation method that is used or may be used in the future in the art, such as ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, or polymer-based precipitation, but is not limited thereto.

[0079] In one specific example, it can be separated by the ultracentrifugation method of the present invention.

[0080] The above ultracentrifugation may include low-speed centrifugation and / or high-speed centrifugation. Specifically, it may be performed by including (a) a process of obtaining a supernatant by centrifugation for 10 to 30 minutes under centrifugation conditions of about 1,000×g to about 3,000×g, (b) a process of obtaining a supernatant by centrifugation for 10 minutes to 2 hours under high-speed centrifugation conditions of about 10,000×g to about 5,000×g, and (c) a process of obtaining a pellet containing exosomes by ultracentrifugation for 60 minutes to 2 hours under ultracentrifugation conditions of about 100,000×g to about 150,000×g.

[0081] At this time, after the above process (a), a process of filtering the culture solution may be additionally performed. At this time, the filtration may be performed under the condition that the filter size is about 0.1 μm to about 20 μm.

[0082] In addition, the process of (c) above can be performed repeatedly. Specifically, the ultracentrifugation process can be performed by repeating it once, twice, or three times. In addition, a process of filtering the exosomes after the ultracentrifugation can be additionally performed. Through this process, small-sized impurities present in the exosomes can be filtered out and large-sized exosomes can be separated. The filtration can be performed under the condition that the filter size is about 0.1 μm to about 20 μm.

[0083] b. Manufacture of artificial exosomes

[0084] Artificial exosomes can be manufactured by any manufacturing method currently used or available in the art, but are not limited thereto.

[0085] In one specific example, the artificial exosome may be prepared using an ethanol injection method. The ethanol injection method may include: (a) preparing a lipid solution by dissolving lecithin, phytosterol, and ceramide in alcohol; (b) preparing an artificial exosome dispersion solution by adding the lipid solution dropwise to sterilized water; and (C) performing extrusion molding by passing the lipid solution through an extruder.

[0086] At this time, the mixing ratio of lecithin, phytosterol, and ceramide is the same as described above. The alcohol may be ethanol, isopropyl alcohol, or propyl alcohol. Specifically, it may be ethanol.

[0087] In the step (b), the lipid solution and sterilized water may be mixed in a volume ratio of about 1:1 to about 1:20. Specifically, the lipid solution may be dispersed by dropping it into the sterilized water in a ratio of about 1:5 to about 1:50, about 1:5 to about 1:40, about 1:5 to about 1:30, about 1:5 to about 1:20, or about 1:5 to about 1:10. At this time, the dropping speed may be performed at a speed of about 400 μl / min or less.

[0088] Additionally, steps (a) and (b) may be performed at room temperature. Specifically, the temperature may be from about 15°C to about 30°C, from about 20°C to about 30°C, or from about 25°C to about 30°C. Additionally, the steps may be performed under stirring to ensure even dispersion of the components within the solution. The stirring speed and time are not particularly limited. In one embodiment, the stirring may be performed at a speed of about 500 rpm for 60 minutes.

[0089] In the above step (c), extrusion molding can be performed through a high-pressure disperser.

[0090] At this time, the extrusion pressure can be adjusted as needed, but can be from about 300 bar to about 3000 bar. Specifically, extrusion can be performed at a pressure of from about 300 bar to about 3000 bar, from about 500 bar to about 2500 bar, or from about 1000 bar to about 2000 bar. In addition, the extrusion molding can be repeated several times. Specifically, it can be performed repeatedly from 1 time to 10 times. The extrusion molding can be performed once, twice, three times, four times, six times, six times, seven times, eight times, nine times, or ten times, but is not limited thereto.

[0091] In order to produce a small amount of artificial exosomes or hybrid exosomes in the step (c) above, a mini extruder (mini extruder; Avanti Pola Lipid) can be used instead of a high-pressure disperser. The mini extruder uses a syringe to extrude a solution through a membrane filter to break down particles into small pieces. In the present invention, the extrusion molding can be performed by gradually changing the diameter of the holes in the used filter to a smaller one, or by overlapping multiple filters so that the filter diameter decreases along the extrusion progress direction. At this time, the filter used is preferably about 1000 nm to about 100 nm in diameter. In addition, during the extrusion molding, a process of increasing the extrusion pressure can be additionally performed if necessary.

[0092] Additionally, the artificial exosomes manufactured by the above method may include a process of finally obtaining a pellet containing exosomes by ultracentrifugation. The ultracentrifugation conditions are the same as those described above.

[0093] Second, it may include a step of mixing the above exosomes and artificial exosomes to induce their fusion.

[0094] Specifically, the fusion of the exosome and the artificial exosome may include (a) a step of dissolving the exosome and the artificial exosome in an alcohol solution and mixing them; (b) a step of loading the mixture into sterilized water to prepare a hybrid exosome dispersion solution; and (c) a step of performing extrusion molding by passing the hybrid exosome dispersion solution through an extruder.

[0095] The mixing ratio of alcohol, exosomes and artificial exosomes in step (a) and the extrusion molding in step (c) are the same as described above.

[0096] In the step (b), the hybrid exosome dispersion solution and sterile water may be mixed in a volume ratio of about 1:1 to about 1:20. Specifically, the lipid solution may be dispersed by dropping into the sterile water at a ratio of about 1:5 to about 1:50, about 1:5 to about 1:40, about 1:5 to about 1:30, about 1:5 to about 1:20, or about 1:5 to about 1:10. At this time, the dropping speed may be performed at a speed of about 400 μl / min or less.

[0097] Additionally, steps (a) and (b) may be performed at room temperature and may be performed under stirring to ensure even dispersion of the components within the solution. At this time, the temperature, stirring speed, and time are the same as those described above.

[0098] Additionally, the hybrid exosomes manufactured by the above method may include a process of finally obtaining a pellet containing exosomes by ultracentrifugation. The ultracentrifugation conditions are the same as those described above.

[0099] Third, the above manufacturing method may additionally include a step of encapsulating an active ingredient into the hybrid exosome of ii).

[0100] The above active ingredient can be encapsulated into hybrid exosomes by mixing them together in the exosome and artificial exosome mixing step of ii). At this time, if the active ingredient is hydrophobic, it can be encapsulated into hybrid exosomes by dissolving it in ethanol and mixing it with the exosomes and artificial exosomes. Additionally, if the active ingredient is hydrophilic, it can be encapsulated into hybrid exosomes by dissolving it in sterilized water and mixing it with the exosome and artificial exosome mixture.

[0101] The above alcohol is the same as described above.

[0102] Pharmaceutical composition

[0103] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, comprising a hybrid exosome, a fusion of an exosome and an artificial exosome, as an active ingredient. The hybrid exosome may contain an active ingredient. The exosome, the artificial exosome, the hybrid exosome, and the active ingredient are the same as those described above.

[0104] Specifically, the active ingredient may be used alone or in combination with a component that promotes cell activity and energy supply or production, or a functional ingredient that is effective in preventing or treating skin diseases.

[0105] More specifically, the component that promotes the activity of the cell and the supply or production of energy within the cell may be selected from the group consisting of amino acids, minerals, sugars, vitamins, intracellular energy sources, and combinations thereof.

[0106] More specifically, the amino acid may be selected from the group consisting of, but is not limited to, glycine, glutamic acid, leucine, alanine, phenylalanine, valine, isoleucine, methionine, cysteine, proline, and combinations thereof.

[0107] The above mineral may be selected from the group consisting of, but not limited to, sodium pyruvate, calcium chloride, magnesium chloride, zinc oxide, calcium pantothenate, and combinations thereof.

[0108] The above sugar may be glucose, inositol or a combination thereof.

[0109] The above vitamins may include salts, precursors and derivatives thereof, and may be selected from the group consisting of, but not limited to, pyridoxine or a salt thereof (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or a salt thereof (HCl or nitrate), and combinations thereof.

[0110] The component that promotes the activity of the cell and the supply or production of energy within the cell may be selected from the group consisting of nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), adenosine triphosphate (ATP), and combinations thereof, but is not limited thereto.

[0111] More specifically, the functional ingredient effective in preventing or treating the above skin disease may be an ingredient exhibiting any one function selected from the group consisting of whitening, wrinkle improvement, skin elasticity improvement, moisturizing, antioxidant function, anti-inflammation or acne improvement, hair loss improvement or hair growth promotion, and combinations thereof.

[0112] More specifically, the whitening functional ingredients include glutathione, niacinamide, arbutin (α or β), ethyl ascorbyl ether, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, (-)-α-bisabolol, paper mulberry extract, oil-soluble licorice extract, glabridin, hydroquinone, kojic acid, ascorbic acid, dipotassium glycyrrhizate, tranexamic acid, phloretin, may be selected from the group consisting of, but not limited to, ergothioneine and combinations thereof.

[0113] The above wrinkle-improving functional ingredient may be selected from the group consisting of retinol, retinyl palmitate, polyethoxylated retinamide, bakuchiol, adenosine, ubiquinone, glutathione, thioctic acid, peptides, palmitoyl tritapeptide-1 (Pal-GHK), palmitoyl pentapeptide-4, acetyl hexapeptide-8, and combinations thereof, but is not limited thereto.

[0114] The above skin elasticity improvement functional ingredient may be selected from the group consisting of acetyl hydroxyproline, hydroxypropyl tetrahydropyrantriol, collagen, elastin, and combinations thereof, but is not limited thereto.

[0115] The above moisturizing functional ingredient may be selected from the group consisting of trehalose, ceramide, glycerin, chitosan, hyaluronic acid, shea butter, and combinations thereof, but is not limited thereto.

[0116] The above antioxidant functional ingredient may be selected from the group consisting of palmitoyl tripeptide-1, copper peptide, luteolin, apigenin, astaxanthin, protocatechuic acid (PCA), urolithin, panthenol, ascorbic acid, tocopherol, resveratrol, bakuchiol, lycopene, β-carotene, thiotic acid, curcumin, ubiquinone, catechins, flavonoids, polyphenols, and combinations thereof, but is not limited thereto.

[0117] The above anti-inflammatory or acne-improving functional ingredient may be selected from the group consisting of sialic acid, panthenol, allantoin, aloe vera extract, chamomile flower extract, green tea extract, tea tree leaf oil, centella asiatica extract, miltiorrhiza uralensis extract (Houttuynia cordata extract), and combinations thereof, but is not limited thereto.

[0118] The above hair loss improvement or hair growth promotion functional ingredient may be selected from the group consisting of Trp-Lys-Tyr-Met-Val-D-Met (WKYMV or WKYMVm) (SEQ ID NO: 1), palmitoyl tripeptide-1, myristoyl pentapeptide-17, azelaic acid, caffeine, niacinamide, dexpanthenol, sialic acid, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, Argania spinosa kernel oil, and combinations thereof, but is not limited thereto.

[0119] The above "skin disease" may be selected from the group consisting of, but is not limited to, dermatitis, acne, wounds, skin wrinkles, skin aging, loss of skin elasticity, dry skin, sensitive skin, hair loss, and skin pigmentation and combinations thereof.

[0120] The term "prevention" as used herein can comprehensively mean preventing a disease in advance or reducing the likelihood or frequency of occurrence by administering the pharmaceutical composition in a pharmaceutically effective amount. For example, it can mean reducing the probability of occurrence or the probability of recurrence in a patient who is likely to develop a skin disease or a patient who has previously developed the disease. The "pharmaceutically effective amount" has the same meaning as "therapeutically effective amount," and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, administration route, administration method, number of administrations, treatment period, combination, or concurrently used drugs.

[0121] The term "treatment" used herein may comprehensively mean improving a disease by administering the pharmaceutical composition in a pharmaceutically effective amount, may provide relief or cure of the symptoms of the disease in a shorter period of time compared to natural healing, and may improve one or most of the symptoms caused by the disease. The pharmaceutically effective amount is the same as described above. The pharmaceutical composition of the present invention may be a composition for treating a skin disease on its own, or may be administered together with other pharmacological ingredients and used as a therapeutic adjuvant for the disease. Accordingly, the term "treatment" includes the meaning of "treatment assistance."

[0122] The above “included as an effective ingredient” means that the hybrid exosome according to the present invention is added to an extent that it can exhibit the above-mentioned effect, and may include formulation in various forms by adding various components as auxiliary components for delivery and stabilization, etc.

[0123] The pharmaceutical composition of the present invention may include the hybrid exosome in an amount of about 0.001 wt% to about 30 wt% based on the total weight of the composition.

[0124] Specifically, the composition may comprise about 1 μg to about 1000 μg, about 5 μg to about 500 μg, about 10 μg to about 200 μg, about 15 μg to about 100 μg, or about 20 μg to about 50 μg of hybrid exosomes.

[0125] Meanwhile, the pharmaceutical composition of the present invention can be administered to a subject in a "therapeutically effective amount." The therapeutically effective amount is the same as described above.

[0126] The term "administration" as used herein means introducing a given substance into an individual in an appropriate manner, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto. In addition, the pharmaceutical composition of one embodiment of the present invention may be administered by any device that allows the active substance to travel to the target tissue or cell. Specifically, it may be administered parenterally, and more specifically, it may be administered subcutaneously or transdermally. In addition, the pharmaceutical composition may be directly applied to the skin. When applying the pharmaceutical composition to the skin, it may include directly applying the pharmaceutical composition according to the present invention to the skin or spraying it, depending on its form.

[0127] The subject to which the pharmaceutical composition can be administered here may be a mammal, and specifically a human.

[0128] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition according to the present invention may be administered in one to several divided doses at a dose of about 0.001 mg / kg to about 100 mg / kg for adults. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0129] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exhibit toxicity beyond what the subject of application (prescription) can tolerate. The carrier may be included in an amount of about 1 wt% to about 99.99 wt%, preferably about 70 wt% to about 99.99 wt%, based on the total weight of the pharmaceutical composition of the present invention. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition, but are not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington's Pharmaceutical Sciences (19th ed., 1995)".

[0130] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration agents, nasal inhalants, and suppositories according to methods known in the art together with a suitable carrier. In one specific example, the pharmaceutical composition of the present invention can be prepared as an injection. The injection may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection that is dissolved or suspended, but is not limited thereto. Depending on the type of the injection, the injection may contain at least one of distilled water for injection, vegetable oil (e.g., peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, estradiol benzoate, bismuth subsalicylate, sodium arsenobenzol, or streptomycin sulfate, and may optionally contain a stabilizer or preservative.

[0131] In one specific example, the pharmaceutical composition of the present invention may be prepared as a topical skin preparation. When the pharmaceutical composition is prepared as a topical skin preparation, it may be formulated in the form of an ointment, a solution, a cream, a spray, a patch, etc. At this time, as long as the effects of the present invention are not impaired, ingredients commonly used in cosmetics or topical skin preparations, such as moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powdered ingredients, coloring agents, aqueous ingredients, water, various skin nutrients, etc., may be appropriately blended as needed.

[0132] In addition, the pharmaceutical composition may additionally contain or be used in combination with a known substance that exhibits a preventive or therapeutic effect against the disease.

[0133] Another aspect of the present invention provides a use of the hybrid exosome or a pharmaceutical composition comprising the hybrid exosome as an active ingredient for preventing or treating skin diseases. Another aspect of the present invention provides a method for preventing or treating skin diseases, comprising administering the hybrid exosome or a pharmaceutical composition comprising the hybrid exosome as an active ingredient to a subject.

[0134] Here, the hybrid exosome, pharmaceutical composition, object, administration, skin disease, prevention and treatment are the same as described above.

[0135] Cosmetic composition

[0136] Another aspect of the present invention provides a cosmetic composition for improving skin condition, comprising a hybrid exosome, a fusion of an exosome and an artificial exosome, as an active ingredient. The hybrid exosome may include an active ingredient. The exosome, the artificial exosome, the hybrid exosome, and the active ingredient are the same as those described above.

[0137] Specifically, the active ingredient may be used alone or in combination with a component that promotes cell activity and energy supply or production, or a functional ingredient effective in preventing or treating skin diseases. Here, the component that promotes cell activity and energy supply or production, or a functional ingredient effective in preventing or treating skin diseases, are the same as those described above.

[0138] The above skin condition improvement may be selected from, but is not limited to, the group consisting of dermatitis improvement, acne improvement, wrinkle suppression, skin aging inhibition, skin elasticity improvement, whitening, moisturizing, hair loss suppression, and combinations thereof. Furthermore, the improvement may be characterized by protecting the skin from skin cell function decline or loss, improving skin condition, or preventing or improving skin diseases.

[0139] The cosmetic composition of the present invention may include the hybrid exosome in an amount of about 0.001 wt% to about 30 wt% based on the total weight of the composition.

[0140] Specifically, the cosmetic composition may comprise about 1 μg to about 1000 μg, about 5 μg to about 500 μg, about 10 μg to about 200 μg, about 15 μg to about 100 μg, or about 20 μg to about 50 μg of hybrid exosomes.

[0141] The above cosmetic composition can be formulated into a cosmetic formulation commonly manufactured in the art. The cosmetic composition can be formulated into, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. More specifically, the cosmetic composition can be formulated into a formulation of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0142] When the formulation of the cosmetic composition according to the present invention is a paste, cream or gel, it may include a carrier component selected from the group consisting of animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and mixtures thereof.

[0143] The formulation of the cosmetic composition according to the present invention may include a carrier component selected from the group consisting of a solvent, a solvating agent, an emulsifying agent, and mixtures thereof, which are solutions or emulsions. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol, sorbitan fatty acid esters, and mixtures thereof.

[0144] When the formulation of the cosmetic composition according to the present invention is a suspension, it may include a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and a carrier component selected from the group consisting of microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth and mixtures thereof.

[0145] The above cosmetic composition may additionally contain various known additives in addition to the carrier depending on the formulation.

[0146] The above additives include emulsifiers, moisturizers, surfactants, chelating agents, antioxidants, bactericides, and stabilizers.

[0147] The emulsifier may include liquid paraffin, cetyl thanoate, stearic acid, etc. The moisturizer may include a polyol selected from the group consisting of glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, sorbitol, and combinations thereof.

[0148] The above chelating agent may include sodium ethylenediaminetetraacetate (EDTA), α-hydroxy fatty acids, lactoferrin, α-hydroxy acids, citric acid, lactic acid, malic acid, bilirubin, biliverdin, and the like.

[0149] The antioxidant may include butylhydroxyanisole, dibutylhydroxytoluene, or propyl gallate. In addition, ingredients that can be mixed into the cosmetic composition or external skin preparation include fat components, emollients, organic and inorganic pigments, organic powders, ultraviolet absorbers, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, vitamins, and the like.

[0150] Another aspect of the present invention provides a cosmetic method for improving skin condition using the hybrid exosome or a cosmetic composition containing the hybrid exosome as an active ingredient. The cosmetic method may include applying the cosmetic composition to the skin of a subject. Here, the hybrid exosome, the cosmetic composition, the skin condition improvement, and the subject are the same as described above.

[0151] The step of applying to the skin may include directly applying or spraying the cosmetic composition according to the present invention to the skin, depending on its form. At this time, the amount of application and the number of times per day the cosmetic composition is used may be appropriately set depending on the user's age, sex, purpose, severity of symptoms, etc. For example, an appropriate amount of the cosmetic composition may be applied to the skin 1 to 6 times per day.

[0152] Drug delivery vehicle composition

[0153] Another aspect of the present invention provides a drug delivery composition comprising a hybrid exosome fused with an exosome and an artificial exosome containing an active ingredient.

[0154] The above exosomes, active ingredients, artificial exosomes and hybrid exosomes are the same as described above.

[0155] In the present invention, the hybrid exosome obtained by fusion of the exosome and the artificial exosome may be a single structure including both the exosome-derived component and the artificial exosome-derived component. Accordingly, it may include both the double lipid layer component of the exosome and the double lipid layer component of the artificial exosome. In addition, the hybrid exosome may include the same active ingredient as that loaded in the artificial exosome. The active ingredient may be hydrophobic or hydrophilic. In this case, the active ingredient may be included in any one compartment selected from the group consisting of the surface of the hybrid exosome, between the lipid bilayers, the interior of the exosome, and combinations thereof. In this case, the active ingredient is the same as described above.

[0156] In one embodiment of the present invention, the hybrid exosome was able to carry both a hydrophobic substance (palmitate-conjugated WKYMVm (SEQ ID NO: 1)) and a hydrophilic substance (trypan blue) (Fig. 5).

[0157] For therapeutic and remedial purposes

[0158] Another aspect of the present invention provides a use of a hybrid exosome, which is a fusion of an exosome and an artificial exosome, for preventing or treating skin diseases.

[0159] The above exosomes, artificial exosomes, hybrid exosomes, skin diseases, prevention and treatment are the same as described above.

[0160] Another aspect of the present invention provides a method for preventing or treating a skin disease comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

[0161] The above exosomes, artificial exosomes, hybrid exosomes, administration, skin diseases, prevention and treatment are the same.

[0162] The above-mentioned entity refers to a subject suffering from or likely to suffer from a skin disease. The above-mentioned entity may be a mammal, preferably a human. The dosage may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and responsiveness.

[0163] Another aspect of the present invention provides a use of hybrid exosomes, which are fused exosomes and artificial exosomes, for improving skin condition.

[0164] The above exosomes, artificial exosomes, hybrid exosomes and skin condition improvement are the same as described above.

[0165] Another aspect of the present invention provides a method for improving skin condition comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

[0166] The above exosomes, artificial exosomes, hybrid exosomes, objects, administration and skin condition improvement are the same as described above.

[0167]

[0168] The present invention is described in more detail through the following examples. However, the following examples are provided to exemplify the present invention, and the scope of the present invention is not limited to these examples.

[0169] Example 1. Hybrid exosome production

[0170] Example 1.1, Isolation of exosomes derived from human mesenchymal stem cells

[0171] Human mesenchymal stem cell-derived exosomes (MSC exosomes) were isolated through the following process.

[0172] First, human tonsil tissue was treated with 1% collagenase enzyme to dissociate into single cells, and then human mesenchymal stem cells were isolated according to a known method and cultured in an incubator at 37°C using alpha-MEM medium containing 10% FBS. When the mesenchymal stem cells proliferated to 90% confluency in the culture dish, they were washed twice with HBSS (Hank's balanced salt solution) to remove the remaining FBS and replaced with alpha-MEM medium without FBS. Then, the mesenchymal stem cell culture medium was obtained by culturing for 48 hours in an incubator at 37°C.

[0173] The obtained culture medium was filtered using a 0.2 μm filter to remove impurities, and then centrifuged at 10,000 × g for 30 minutes at 4°C to collect only the supernatant. The supernatant was centrifuged at 100,000 × g for 70 minutes at 4°C in an ultracentrifuge. Thereafter, the supernatant was removed, and the precipitated exosomes were resuspended in HBSS and centrifuged again under the same conditions to obtain exosomes.

[0174] The exosomes obtained through the above process were resuspended in HBSS, and the size and number of exosomes were measured using a Nanosight device, and then used in the following experiments.

[0175] Example 1.2. Preparation of artificial exosomes

[0176] Artificial exosomes were manufactured through the following process. At this time, the lipid composition and ratio were improved to produce large quantities of exosomes (Table 1 and Figure 4).

[0177] A lipid solution was prepared by dissolving 0.45 mg of phytosterol, 1 mg of lecithin, and 0.2 mg of ceramide in 200 μl of 100% ethanol. The lipid solution (200 μl) was slowly dropped into 2 ml of sterile water at a rate of 400 μl / min or less and stirred at 500 rpm for 60 minutes to prepare an artificial exosome dispersion solution. In this case, when the artificial exosome carries a substance, the hydrophobic substance was dissolved in 100% ethanol, and the hydrophilic substance was dissolved in sterile water and mixed to prepare an artificial exosome dispersion solution.

[0178] After installing membranes with pore sizes of 1000 nm, 400 nm, 200 nm, and 100 nm in a mini extruder, the artificial exosome dispersion solution was sequentially extruded 10 times per membrane using a syringe to extrude artificial exosomes into small sizes.

[0179] The artificial exosomes obtained through the above process were collected by centrifugation at 100,000Хg at 4°C for 70 minutes in an ultracentrifuge.

[0180] Lipid component (original) ratio (mass ratio) Lipid component (improved) ratio (mass ratio) lecithin 1 lecithin 1 cholesterol 0.9 phytosterol 0.45 sphingomyelin 0.4--ceramide 0.03 DS-ceramide Y30 (ceramide NP) 0.03~0.2

[0181] Example 1.3. Hybrid exosome production

[0182] Hybrid exosomes, which are one specific example of the present invention, were prepared using the same method as in Example 1.2. At this time, the MSC exosomes of Example 1.1 were mixed with the artificial exosome dispersion solution of Example 1.2, and then prepared using the sequential extrusion method using the small extruder described in Example 1.2. At this time, the MSC exosomes and artificial exosomes were mixed so that the number ratio was 1:1.

[0183] Example 2. Evaluation of the material loading capacity of hybrid exosomes

[0184] Hybrid exosomes have an internal space that can contain active substances and can contain hydrophobic or hydrophilic substances.

[0185] To confirm the material loading capacity of hybrid exosomes, palmitoyl-WKYMVm (SEQ ID NO: 1) labeled with the fluorescent material TAMRA was used as a hydrophobic material, and trypan blue was used as a hydrophilic material. At this time, palmitoyl-WKYMVm was dissolved in 100% ethanol and mixed so that it would be 1% mass ratio of lipids, and trypan blue was dissolved in sterile water, and hybrid exosomes were prepared in the same manner as in Example 1.3.

[0186] The generated exosomes were captured using ultracentrifugation, and the absorbance of the supernatant and the base solution was measured, and the loading efficiency was calculated through the difference in the absorbance ratio (Fig. 5).

[0187] As a result, it was confirmed that hydrophobic and hydrophilic substances in exosomes were loaded at more than 90% and 82.5%, respectively.

[0188] Example 3. Characterization of hybrid exosomes

[0189] The results of measuring the size and zeta potential of each particle using the Malvern Zetasizer nano-zs instrument are shown in Table 2. Compared to mesenchymal stem cell-derived exosomes (MSC exosomes), hybrid exosomes showed significantly reduced size and heterogeneity. Furthermore, compared to artificial exosomes, the zeta potential shifted toward negative charge. Furthermore, it was confirmed that the polydispersity index (PDI) value of hybrid exosomes was lower than that of MSC exosomes. These results indicate that the uniformity of hybrid exosomes was increased compared to that of MSC exosomes.

[0190] Size (nm) PDI Zeta potential (mV) MSC exosomes 240.1 0.354-20.1 Artificial exosomes 133.9 0.072-8.05 Artificial exosomes + peptide 148.8 0.089-5.69 Hybrid exosomes 149.8 0.079-14.4

[0191] Example 4. Synthesis and efficiency analysis of hybrid exosomes combining mesenchymal stem cell-derived exosomes and artificial exosomes.

[0192] MSC exosomes and artificial exosomes were reprocessed to produce hybrid exosomes using the same method as in Example 1.3. At this time, MSC exosomes were stained with a green fluorescent dye (PKH67GL) and artificial exosomes were stained with a red fluorescent dye (rhodamine PE) and FACS analysis was performed.

[0193] As a result, as shown in Fig. 6, in the case of hybrid exosomes, it was confirmed that the hybrid exosomes existed in a section that simultaneously displayed green and red fluorescence, and the synthesis efficiency was 52% or higher.

[0194] Example 5. Evaluation of skin permeability of hybrid exosomes

[0195] The skin permeability of hybrid exosomes prepared using the same method as in Example 1.3 was evaluated. At this time, the experimental group was divided into three groups: a control group, a group treated with peptide_TAMRA alone (peptide), and a group in which hybrid exosomes loaded with the active ingredient peptide_TAMRA (hybrid exosome+peptide) were evaluated. Skin permeability was verified to determine the degree of exosome infiltration into the skin based on the Franz diffusion cell experiment.

[0196] Specifically, to measure skin penetration, skin tissue was collected from the ear of a pig, and then subcutaneous fat tissue was removed to secure skin tissue consisting of the epithelium and dermis. The skin tissue was attached to a Franz diffusion cell device with the epithelium facing upward, and hybrid exosomes loaded with peptide-TAMRA alone or peptide-TAMRA were dissolved in a PBS solution and injected into the upper chamber of the Franz diffusion cell device where the epithelium was in contact, and reacted in a constant temperature and humidity chamber for 24 hours. PBS solution was used as a control. At this time, the PBS solution was injected into the lower chamber of the Franz diffusion cell device with care to prevent air from being trapped, and the solution was stirred using a magnetic stirrer. In addition, the temperature of the constant temperature and humidity chamber was maintained at 32℃, which is the temperature of the skin surface.

[0197] After 24 hours of reaction in a constant temperature and humidity chamber, the Franz diffusion cell device was disassembled, and skin tissue was isolated and analyzed using confocal microscopy. Epithelial and dermal tissues were identified through DAPI staining.

[0198] As a result, as shown in Fig. 7, in the peptide_TAMRA only treatment group, fluorescent markers were observed only around the stratum corneum, whereas in the hybrid exosome + peptide treatment group, fluorescent markers were observed in the dermis layer as well as the epidermis. Through the above results, it was confirmed that hybrid exosomes loaded with peptides can be used as carriers mediating transdermal delivery of peptides.

[0199] Example 6. Confirmation of intracellular uptake of exosomes derived from mesenchymal stem cells.

[0200] MSC exosomes prepared using the same method as in Example 1.1 were stained with a green fluorescent dye (PKH67GL). The MSC exosomes were treated with mesenchymal stem cells and allowed to react for 14 hours, after which fluorescence was observed using a confocal microscope. At this time, the nuclei were observed by staining them with DAPI.

[0201] As a result, as shown in Figure 8, MSC exosomes stained with green fluorescence were identified in the nuclear periphery of DAPI-stained mesenchymal stem cells. The results confirmed that MSC exosomes enter the cells.

[0202] Example 7. Confirmation of intracellular uptake of artificial exosomes

[0203] Artificial exosomes were prepared using the same method as in Example 1.2, mixing rhodamine PE at a ratio of 0.1 mol% relative to lipid content. The artificial exosomes prepared as described above were treated with mesenchymal stem cells, and after reacting for 14 hours, fluorescence was observed using a confocal microscope. At this time, the nucleus was stained with DAPI and observed.

[0204] As a result, as shown in Figure 9, artificial exosomes stained with red fluorescence were identified in the nuclear periphery of DPAI-stained mesenchymal stem cells. The results confirmed that artificial exosomes entered the cells.

[0205] Example 8. Confirmation of intracellular uptake of hybrid exosomes

[0206] Hybrid exosomes prepared in the same manner as in Example 1.3 were treated with mesenchymal stem cells and reacted for 14 hours, after which fluorescence was observed using a confocal microscope. At this time, the nuclei were observed by staining them with DAPI.

[0207] As a result, as shown in Fig. 10, MSC exosomes with green fluorescence, artificial exosomes stained red, and hybrid exosomes with both green and red fluorescence, resulting in a yellow color, were observed around the nucleus of mesenchymal stem cells. The results confirmed that hybrid exosomes entered the cells.

[0208] Example 9. Analysis of hybrid exosome uptake into skin cells

[0209] To analyze the uptake of hybrid exosomes produced by the method of Example 1.3 into skin cells, artificial exosomes, liposomes, MSC exosomes (natural exosomes), and hybrid exosomes were each treated to HaCaT cells, which are skin keratinocytes, and the uptake of exosomes into skin cells was observed using a laser fluorescence microscope. At this time, the nuclei were observed by staining them with DAPI.

[0210] At this time, the double membrane of exosomes and liposomes was stained using rhodamine PE, a red fluorescent dye, and MSC exosomes (natural exosomes) were stained with a green fluorescent dye (PKH67GL). Afterwards, the dyed artificial exosomes and MSC exosomes were fused in the same manner as in Example 1.3 to prepare hybrid exosomes (Table 3). At this time, the liposomes used as a control group were prepared by dissolving lecithin in 100% ethanol in the same manner as in Example 1.2 (ethanol injection method and sequential extrusion method).

[0211] Experimental group MSC exosomes (PKH67GL labeled) Vehicle (Rhodamine PE labeled) MSC exosomes (natural exosomes) O-artificial exosomes-O-liposomes-O-hybrid exosomes OO

[0212] The hybrid exosomes, MSC exosomes, artificial exosomes, and liposomes manufactured as described above were each treated to HaCaT cells and reacted for 30 minutes. After the reaction, each of the cells was fixed, and the degree of intracellular uptake of the exosomes and liposomes dyed with each fluorescent dye was confirmed through fluorescence expression. At this time, the nucleus was observed by staining it with DAPI. As a result, as shown in Fig. 11, when artificial exosomes or hybrid exosomes were treated, the degree of fluorescence expression in skin cells increased. The degree of fluorescence expression was similar to that of the MSC exosome (natural exosome) treatment group and was higher than that of the liposome treatment group. Through the above results, it was confirmed that the hybrid exosomes of the present invention had a high degree of intracellular uptake.

[0213] Example 10. Analysis of skin permeability and drug delivery capacity of exosomes.

[0214] To analyze the permeability of artificial exosomes into skin tissue, skin permeability analysis was performed using a Franz Diffusion Cell.

[0215] Specifically, artificial exosomes were stained with the red fluorescent dye rhodamine PE and then applied to pig skin using a Franz Diffusion Cell. After 24 hours, the skin tissue to which the exosomes had been applied was fixed and histologically analyzed for fluorescence expression within the skin tissue. Nuclei were observed by staining with DAPI.

[0216] As a result, as shown in Fig. 12, fluorescence expression was confirmed in the skin dermis layer. Through the above results, it was confirmed that the artificial exosome has skin permeability.

[0217] In addition, to confirm the drug delivery ability of the artificial exosome, rhodamine B dye dissolved in a hydrophilic or hydrophobic solvent was encapsulated in the exosome, and then applied to the skin.

[0218] As a result, as shown in Fig. 13, rhodamine fluorescence expression was confirmed up to the skin dermis layer. Through the above results, the skin permeability and drug delivery ability of the artificial exosome according to the present invention could be confirmed.

Claims

1. Hybrid exosomes fused with exosomes and artificial exosomes.

2. In paragraph 1, The above exosome is a hybrid exosome, which is of human or plant origin.

3. In paragraph 2, The above human-derived exosomes are hybrid exosomes derived from human blood, tissue or cells.

4. In paragraph 3, The above cell is a hybrid exosome, wherein the cell is a stem cell, an immune cell, a blood cell, a somatic cell or a germ cell.

5. In paragraph 4, Hybrid exosome, wherein the stem cell is a mesenchymal stem cell, an adult stem cell, an induced pluripotent stem cell, an embryonic stem cell, a hematopoietic stem cell, or a neural stem cell.

6. In paragraph 5, The above mesenchymal stem cells are hybrid exosomes derived from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerve, skin, amniotic fluid or amniotic membrane.

7. In paragraph 2, The above plant-derived exosome is a hybrid exosome isolated from a plant culture.

8. In paragraph 7, A hybrid exosome, wherein the culture of the above plant is a cell, tissue or callus culture derived from any one selected from the group consisting of a flower, leaf, stem, branch, fruit, fruit peel, root, seed and combinations thereof of the plant.

9. In paragraph 1, The above artificial exosome is a hybrid exosome comprising any one selected from the group consisting of lecithin, ceramide, cholesterol, sphingolipid, phytosterol or a derivative thereof, phosphatidylethanolamine (PE), phosphatidylserine (PS), and a combination thereof.

10. In paragraph 9, The above artificial exosome is a hybrid exosome containing lecithin, ceramide and phytosterol.

11. In paragraph 10, The above artificial exosome is a hybrid exosome containing lecithin, ceramide, and phytosterol in a mass ratio of 1:0.1:0.01 to 1:0.8:0.

4.

12. In paragraph 1, The above artificial exosome is a hybrid exosome that additionally contains an active ingredient.

13. In paragraph 12, A hybrid exosome, wherein the active ingredient is any one selected from the group consisting of amino acids, minerals, sugars, vitamins (or precursors or derivatives thereof) or salts thereof, energy sources of cells, peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds or salts thereof, natural products (including extracts) or salts or glycosides thereof, semi-synthetic drugs, toxins and combinations thereof.

14. In paragraph 13, The above active ingredients are glycine, glutamic acid, leucine, alanine, phenylalanine, valine, isoleucine, methionine, cysteine, proline, sodium pyruvate, calcium chloride, magnesium chloride, zinc oxide, calcium pantothenate, glucose, inositol, pyridoxine or a salt thereof (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or a salt thereof (HCl or nitrate), nicotinamide adenine Nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), adenosine triphosphate (ATP), niacinamide, arbutin (α or β), ethyl ascorbyl ether, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, (-)-α-bisabolol, paper mulberry extract, soluble licorice extract, glabridin, hydroquinone, kojic acid, ascorbic acid,Dipotassium glycyrrhizate, tranexamic acid, phloretin, ergothioneine, retinol, retinyl palmitate, polyethoxylated retinamide, bakuchiol, adenosine, ubiquinone, thioctic acid, peptides, Trp-Lys-Tyr-Met-Val-D-Met (WKYMV or WKYMVm) (SEQ ID NO: 1), glutathione, palmitoyl tritapeptide-1, palmitoyl pentapeptide-4, acetyl Acetyl hexapeptide-8, myristoyl pentapeptide-17, copper peptide, acetyl hydroxyproline, hydroxypropyl tetrahydropyrantriol, collagen, elastin, trehalose, ceramide, glycerin, chitosan, hyaluronic acid, shea butter, luteolin, apigenin, astaxanthin, protocatechuic acid (PCA), urolithin, panthenol, tocopherol, resveratrol, Lycopene, beta-carotene, thiotic acid, curcumin,A hybrid exosome, wherein the hybrid exosome is any one selected from the group consisting of ubiquinone, catechin, flavonoid, polyphenol, salicylic acid, allantoin, aloe vera extract, chamomile flower extract, green tea extract, tea tree leaf oil, centella asiatica extract, Houttuynia cordata extract, azelaic acid, caffeine, dexpantenol, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, Argania spinosa kernel oil, and combinations thereof.

15. In paragraph 1, A hybrid exosome in which the above exosomes and artificial exosomes are fused at a ratio of 1:1 to 1:10000.

16. In paragraph 1, A hybrid exosome, wherein the diameter of the hybrid exosome is 50 nm to 300 nm.

17. In paragraph 1, A hybrid exosome, wherein the zeta potential of the hybrid exosome is -30 mV to +20 mV.

18. In paragraph 1, A hybrid exosome, wherein the hybrid exosome further comprises an active ingredient.

19. In paragraph 18, A hybrid exosome, wherein the active ingredient is contained in any one compartment selected from the group consisting of the surface of the hybrid exosome, between lipid bilayers, internal compartments, and combinations thereof. 20.i) a step of preparing exosomes or artificial exosomes; and ii) A method for producing a hybrid exosome, comprising the step of mixing the above exosomes and artificial exosomes to induce their fusion.

21. In paragraph 20, A method for producing a hybrid exosome, wherein the above manufacturing method further comprises the step of iii) encapsulating an active ingredient into the hybrid exosome of ii).

22. A pharmaceutical composition for preventing or treating skin diseases, comprising a hybrid exosome in which an exosome and an artificial exosome are fused as an active ingredient.

23. In paragraph 22, A pharmaceutical composition for preventing or treating skin diseases, wherein the hybrid exosome comprises an active ingredient.

24. In paragraph 23, The above active ingredients are glycine, glutamic acid, leucine, alanine, phenylalanine, valine, isoleucine, methionine, cysteine, proline, sodium pyruvate, calcium chloride, magnesium chloride, zinc oxide, calcium pantothenate, glucose, inositol, pyridoxine or a salt thereof (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or a salt thereof (HCl or nitrate), nicotinamide adenine Nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), adenosine triphosphate (ATP), niacinamide, arbutin (α or β), ethyl ascorbyl ether, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, (-)-α-bisabolol, paper mulberry extract, soluble licorice extract, glabridin, hydroquinone, kojic acid, ascorbic acid,Dipotassium glycyrrhizate, tranexamic acid, phloretin, ergothioneine, retinol, retinyl palmitate, polyethoxylated retinamide, bakuchiol, adenosine, ubiquinone, thioctic acid, peptides, Trp-Lys-Tyr-Met-Val-D-Met (WKYMV or WKYMVm) (SEQ ID NO: 1), glutathione, palmitoyl tritapeptide-1, palmitoyl pentapeptide-4, acetyl Acetyl hexapeptide-8, myristoyl pentapeptide-17, copper peptide, acetyl hydroxyproline, hydroxypropyl tetrahydropyrantriol, collagen, elastin, trehalose, ceramide, glycerin, chitosan, hyaluronic acid, shea butter, luteolin, apigenin, astaxanthin, protocatechuic acid (PCA), urolithin, panthenol, tocopherol, resveratrol, Lycopene, beta-carotene, thiotic acid, curcumin,A pharmaceutical composition for preventing or treating skin diseases, wherein the pharmaceutical composition comprises at least one selected from the group consisting of ubiquinone, catechins, flavonoids, polyphenols, salicylic acid, allantoin, aloe vera extract, chamomile flower extract, green tea extract, tea tree leaf oil, centella asiatica extract, Houttuynia cordata extract, azelaic acid, caffeine, dexpantenol, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, Argania spinosa kernel oil, and combinations thereof.

25. In paragraph 22, A composition for preventing or treating a skin disease, wherein the skin disease is selected from the group consisting of dermatitis, acne, wounds, skin wrinkles, skin aging, loss of skin elasticity, dry skin, sensitive skin, acne, hair loss, and skin pigmentation and combinations thereof.

26. A cosmetic composition for improving skin condition, comprising a hybrid exosome in which exosomes and artificial exosomes are fused as an active ingredient.

27. In paragraph 26, A cosmetic composition for improving skin condition, wherein the hybrid exosome comprises an active ingredient.

28. In paragraph 26, A cosmetic composition for improving skin condition, wherein the improvement in skin condition is selected from the group consisting of improvement in dermatitis, improvement in acne, inhibition of wrinkles, inhibition of skin aging, improvement in skin elasticity, whitening, moisturizing, inhibition of hair loss, and combinations thereof.

29. A drug delivery composition comprising a hybrid exosome fused with an exosome and an artificial exosome containing an active ingredient.

30. Use of hybrid exosomes fused with exosomes and artificial exosomes for preventing or treating skin diseases.

31. A method for preventing or treating a skin disease, comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

32. Use of hybrid exosomes fused with exosomes and artificial exosomes for improving skin condition.

33. A method for improving skin condition, comprising administering to a subject a hybrid exosome in which an exosome and an artificial exosome are fused.

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