Hybrid exosomes and their applications

Abstract

This invention relates to hybrid exosomes formed by the fusion of human cell-derived exosomes and artificial exosomes. When an active ingredient labeled with a fluorescent substance was attached to or encapsulated in the hybrid exosomes according to the present invention and then treated with cells, the active ingredient was observed within the cells. Furthermore, when the exosomes were applied to skin tissue, fluorescence expression could be observed in the dermis layer as well. Therefore, the hybrid exosomes according to the present invention can be used as a novel drug delivery system.

Description

Technical Field

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

Background Art

[0002] Extracellular vesicles are nano-sized membrane structures that transmit cellular substances such as proteins between cells and mediate intercellular communication. In particular, exosomes are known to be produced from the budding of late endosomes and fuse with the plasma membrane before being released into the extracellular space. Exosomes are small vesicles sized 40 - 200 nm composed of a lipid bilayer membrane rich in phosphocholine, cholesterol, and ceramide, are secreted from almost all cell types, and stably exist in all kinds of body fluids such as blood, lymph, and sweat. Also, exosomes have the advantages of a long in vivo circulation time and reaching the inside of organs due to their small size and weak negative charge. Exosomes can also avoid phagocytosis and transmit hydrophilic or hydrophobic drugs, and are expected to be the next generation of drug delivery carriers (Bunggulawa et al., J Nanobiotechnology (2018) 16:81).

[0003] However, natural exosomes have not only non-uniform sizes and shapes but also non-uniform active ingredients inside the exosomes. Also, the amount secreted from cells is extremely small, and considerable effort is required for collection and concentration, so there is a limit to commercialization. Therefore, there is an urgent need to develop new technologies to overcome existing limitations.

Summary of the Invention

Problems to be Solved by the Invention

[0004] As a result of research conducted to develop a superior drug delivery system, the inventors manufactured a hybrid exosome by fusing an exosome with an artificial exosome containing lipid components of exosomes, and confirmed that the active ingredient contained in the exosome was delivered into the cell, thereby completing the present invention. [Means for solving the problem]

[0005] To achieve the aforementioned objective, one aspect of the present invention provides a hybrid exosome in which exosomes and artificial exosomes are fused.

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

[0007] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of 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 contains a hybrid exosome fused with an artificial exosome containing the aforementioned hybrid exosome as an active ingredient.

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

[0010] Another aspect of the present invention provides applications for the prevention or treatment of skin diseases of hybrid exosomes, which are fused with exosomes and artificial exosomes.

[0011] A further aspect of the present invention provides a method for preventing or treating a skin disease, comprising the step of administering a hybrid exosome, which is a fusion of an exosome and an artificial exosome, to an individual.

[0012] Another aspect of the present invention provides applications for improving skin conditions using hybrid exosomes, which are fused with exosomes and artificial exosomes.

[0013] Another aspect of the present invention provides a method for improving skin condition, which includes the step of administering a hybrid exosome, in which exosomes and artificial exosomes are fused, to an individual. [Effects of the Invention]

[0014] When a fluorescently labeled active ingredient was attached to or encapsulated in the hybrid exosome according to the present invention and then treated with cells, the active ingredient was observed within the cells. Furthermore, when the exosome was applied to skin tissue, fluorescence expression could be observed in the dermis layer as well. The hybrid exosome is manufactured by fusing an exosome with an artificial exosome carrying an active ingredient, thereby simultaneously exhibiting the pharmacological activity of the existing exosome itself and the pharmacological activity of the carried active ingredient. Therefore, the hybrid exosome according to the present invention can be used not only as a novel drug delivery system but also as a therapeutic agent for a variety of diseases. [Brief explanation of the drawing]

[0015] [Figure 1] This is a diagram illustrating the function of the hybrid exosome of the present invention. [Figure 2] This is a diagram illustrating the characteristics of the hybrid exosome of the present invention. [Figure 3] This is a schematic representation of the method for producing artificial exosomes. [Figure 4] This is a diagram showing a specific example of a lipid bilayer component used in the production of hybrid exosomes, which is one specific example of the present invention. [Figure 5] This figure shows the results of confirming the hydrophobic or hydrophilic material carrying capacity of hybrid exosomes, which are one specific example of the present invention. [Figure 6]This diagram shows the results of a hybrid exosome, one specific example of the present invention, produced by mixing mesenchymal stem cell-derived exosomes (MSC exosomes) labeled with green fluorescence and artificial exosomes labeled with red fluorescence, and then confirming each exosome through FACS analysis. Green: MSC exosomes, Red: Artificial exosomes, Yellow: Hybrid exosomes. [Figure 7] This figure shows the results of confirming skin permeability using the Franz diffusion cell test after loading a red fluorescently labeled peptide (palmitoyl-WKYMVm_TAMRA) (SEQ ID NO: 1) onto a hybrid exosome, which is one specific example of the present invention. Blue: Cell nuclei (DAPI) in skin tissue; Red: Palmitoyl-WKYMVm_TAMRA peptide loaded within the hybrid exosome. [Figure 8] This diagram shows the results of confirming the influx (uptake) of green-fluorescent-labeled MSC exosomes into mesenchymal stem cells. Blue: DAPI (nucleus), Green: MSC exosome. [Figure 9] This diagram shows the results of confirming the influx of red-fluorescently labeled artificial exosomes into mesenchymal stem cells. Blue: nucleus (DAPI), red: artificial exosome. [Figure 10] This diagram shows the results of confirming the influx of hybrid exosomes (yellow), a specific example of the present invention, into mesenchymal stem cells. Green: Components of MSC exosomes within hybrid exosomes, Red: Components of artificial exosomes within hybrid exosomes, Blue: Nucleus (DAPI), Yellow: Components of MSC exosomes and artificial exosomes within hybrid exosomes. [Figure 11] These diagrams and graphs show the results of examining the degree of influx into skin cells after treating keratinocytes (HaCaT cells) with fluorescently labeled exosomes or liposomes, respectively. Blue: nucleus (DAPI), Green: MSC exosomes, Red: artificial exosomes or liposomes, Blue / Red: hybrid exosomes. [Figure 12] The drawings and graphs show the results of confirming the skin permeability of artificially labeled exosomes with red fluorescence using a Franz Diffusion Cell. Blue: nucleus (DAPI), red: artificially labeled exosomes. [Figure 13] The drawings and graphs show the results of confirming the drug delivery ability of artificially labeled exosomes encapsulated with a red fluorescent dye using a Franz Diffusion Cell. Blue: nucleus (DAPI), red: artificially labeled exosomes.

Embodiments for Carrying Out the Invention

[0016] <Hybrid Exosomes> One aspect of the present invention provides hybrid exosomes in which exosomes and artificially labeled exosomes are fused.

[0017] The term "exosome" used in the present invention can mean nano-sized particles that are naturally secreted by living cells and encapsulated by a lipid bilayer, and play a role in information transmission between cells. The size of exosomes is known to be approximately 30 to 250 nm in diameter. Although there are some differences depending on the type of origin cell, the exosome membrane contains surface proteins (surface markers) such as CD9, CD63, and CD81, and it is known that proteins such as TSG101 and ALIX, which can prove that they are of endosomal origin, are contained inside the exosomes. In addition, it contains proteins such as growth factors and cytokines having various functions, as well as nucleic acids such as mRNA and miRNA, and has components and effects that reflect the characteristics of the origin cell. In particular, exosomes derived from stem cells are known to have functions such as regulating the differentiation of stem cells, promoting regeneration and growth, and inducing specific immune reactions.

[0018] In the present invention, the exosomes are exosomes derived from humans or plants, but any exosomes of various origins that are used in the art or will be used in the future can be used without limitation as long as they do not cause adverse effects on the human body.

[0019] Specifically, the human-derived exosomes are also derived from human blood, tissues or cells.

[0020] More specifically, they are also isolated from human blood, or tissue or cell culture media. At this time, the cells are also stem cells, immune cells, blood cells, somatic cells or germ cells. The stem cells are 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. Further, the mesenchymal stem cells are derived from umbilical cord blood, umbilical cord, bone marrow, adipose tissue, muscle, nerve, skin, amniotic fluid or amnion, but are not limited thereto.

[0021] Specifically, the plant-derived exosomes are also isolated from plant cultures, plant juices, or biological solutions of plants equivalent thereto. At this time, the plant cultures, juices or biological solutions equivalent thereto are derived from the whole plant or from plant tissues, callus or cells.

[0022] As a specific example, the plant culture or juice is also a culture or juice of cells, tissues or callus derived from any one selected from the group consisting of plant flowers, leaves, stems, branches, fruits, fruit skins, roots, seeds, and combinations thereof.

[0023] In one embodiment of the present invention, the exosomes are also exosomes isolated from the culture medium of human mesenchymal stem cells.

[0024] In the present invention, the diameter of the exosomes is also about 50 - 400 nm. Specifically, it is about 50 - 400 nm, about 80 - 350 nm, about 100 - 300 nm or about 150 - 250 nm, but is not limited thereto.

[0025] Furthermore, the exosomes also have a negative zeta potential (<0mV). Specifically, the zeta potentials of the exosomes are approximately -100 to -1mV, approximately -80 to -3mV, approximately -50 to -6mV, approximately -40 to -9mV, approximately -30 to -12mV, or approximately -30 to -15mV, but are not limited to these values.

[0026] 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 mainly consists of phospholipids and their derivatives. When phospholipids and their derivatives are dispersed in an aqueous solution, a single-layer or lipid bilayer endoplasmic reticulum is spontaneously formed. Artificial exosomes can carry water-soluble active ingredients in their aqueous internal space, and hydrophobic active ingredients in their lipid bilayer, making them useful as drug delivery systems for a variety of drugs.

[0027] In the present invention, the artificial exosome may contain any one selected from the group consisting of lecithin, phosphatidylcholine (PC), ceramide, cholesterol, phytosterol or its derivatives, phosphatidylethanolamine (PE), phosphatidylserine (PS), and combinations thereof.

[0028] As used herein, the term "lecithin" 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, etc., as well as in plant seeds, yeasts, and fungi. An example of lecithin is phosphatidylcholine (PC), which is characterized by hydrophilic components such as phosphate and choline being bonded to one side of glycerol, and a hydrophobic acyl group being bonded to the other side.

[0029] In the present invention, the lecithin can be hydrogenated lecithin, unsaturated lecithin, lyso lecithin, etc., without limitation. Extracted lecithin (egg yolk lecithin, soy lecithin, etc.), synthetic lecithin, or combinations thereof can also be used, but are not limited to these.

[0030] As used herein, the term "ceramide" refers to one of the sphingolipids having a structure in which a fatty acid is linked to sphingosine or pitosphingosine. Ceramide accounts for more than 40% of the intercellular lipids that make up the stratum corneum of the skin, and is a component of the bilayer cell membrane and skin lipid membrane. Ceramide is also an essential component for the formation and function of the stratum corneum, and ceramide present in the human body is classified into various types depending on its degree of polarity. Ceramide is an important element of the skin barrier and has the role of a lipid barrier that suppresses water evaporation and the function of maintaining the orderly structure of the stratum corneum, and is therefore attracting attention as a raw material for moisturizing cosmetics.

[0031] In the present invention, the ceramide is 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 to these. In one embodiment of the present invention, the ceramide is also ceramide NP.

[0032] As used herein, the term "cholesterol" refers to a type of sterol (a combination of a steroid and an alcohol) lipid found in the cell membranes of all animal cells. Cholesterol is transported through the blood. In the present invention, the cholesterol can be incorporated into artificial exosomes to enhance the stability of the artificial exosomes by increasing the membrane strength of the lipid bilayer.

[0033] As used herein, the term "sphingolipid" refers to core epithelial lipids associated with maintaining the skin's barrier function. Sphingolipids such as sphingosine, sphinganin, sphingomyelin, and phytosphingosine generally constitute a central group or "backbone" comprising long sphingoid bases, amide-linked long-chain fatty acids, and a head group. Hundreds of known sphingolipid classes exist, each with different head groups (e.g., choline phosphate, glucose, galactose, polysaccharides) and different fatty acids and sphingoid bases. Phytosphingosine is a ceramide precursor concentrated in the healthy stratum corneum, which is converted to sphingolipids by enzymes in the skin. In one specific example, in the present invention, the sphingolipid is also sphingomyelin.

[0034] As used herein, the term "phytosterol" refers to plant-derived lipids that have a sterol backbone. This includes plant sterols and stanols, and currently, more than 250 sterols and related compounds are 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 improve the strength of septa with bilayer membrane structures.

[0035] The phytosterols mentioned above may include, for example, sitosterol, β-sitosterol, stigmasterol, campesterol, charinosterol, clionasterol, brassicasterol, α-spinasterol, Delta 5-avennasterol, lupeol, dancosterol, desmosterol, polyferasterol, stigmathanol, campestanol, cycloartenol, and the like. In one embodiment of the present invention, the phytosterol is also β-sitosterol.

[0036] As used herein, the term "phosphatidylethanolamine (PE)" refers to a type of phospholipid found in biological membranes, known to account for approximately 25% of the total phospholipids. Phosphatidylethanolamine is synthesized by adding cytidine diphosphate-ethanolamine to a diglyceride, and S-adenosylmethionine can subsequently methylate the amine of phosphatidylethanolamine to produce phosphatidylcholine.

[0037] As used herein, the term "phosphatidylserine (PS)" refers to a phospholipid that constitutes the cell membrane, and is known to make up approximately 13-15% of the phospholipids in the human cerebral cortex.

[0038] Specifically, the artificial exosome may contain lecithin and any one selected from the group consisting of ceramide, phytosterol, and combinations thereof.

[0039] As a specific example, the artificial exosome may contain lecithin. As a specific example, the artificial exosome may contain lecithin and ceramide. As a specific example, the artificial exosome may contain lecithin and phytosterol. As a specific example, the artificial exosome may contain lecithin, ceramide, and phytosterol.

[0040] Preferably, the artificial exosome may contain lecithin, ceramide, and phytosterols. In this case, the lecithin, ceramide, and phytosterols are included in a ratio of approximately 1:0.1:0.01 to 1:0.8:0.4 based on 1 part by weight of lecithin. Specifically, they are included in a ratio of approximately 1:0.1:0.01 to 1:0.8:0.4, approximately 1:0.2:0.03 to 1:0.7:0.3, approximately 1:0.3:0.03 to 1:0.6:0.2, or approximately 1:0.4:0.03 to 1:0.5:0.3. In one example, the lecithin, ceramide, and phytosterols are included in a ratio of approximately 1:0.45:0.03 to 1:0.45:0.2 based on 1 part by weight of lecithin.

[0041] In the present invention, the artificial exosome is also about 50 to 300 nm in size. Specifically, the artificial exosome may have a diameter of about 50 nm to about 300 nm, about 70 to 250 nm, about 90 to 200 nm, or about 110 to 150 nm.

[0042] Furthermore, the zeta potential of the artificial exosome is approximately -30 to +20 mV. Specifically, it is approximately -30 to +20 mV, approximately -25 to +10 mV, approximately -20 to +1 mV, or approximately -15 to -5 mV, but is not limited to these ranges.

[0043] In the present invention, the artificial exosome may further contain an active ingredient. The active ingredient is not limited to a specific type and is applicable as long as it exerts the desired effect as a person skilled in the art and is supported on the artificial exosome. Furthermore, the active ingredient may be hydrophobic or hydrophilic.

[0044] The active ingredient is one selected from the group consisting of amino acids, minerals, sugars, vitamins (or their precursors or derivatives) or their salts, cellular energy sources, peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds or their salts, natural products (including extracts) or their salts or glycosides, semi-synthetic compounds, toxins, and combinations thereof, but is not limited to these. In this case, the active ingredient is supported on any one site selected from the group consisting of the surface, interior, between the lipid bilayers, and combinations thereof of the artificial exosome.

[0045] In one specific example, the active ingredients include 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 its salt (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or its salt (HCl or nitrate), and nicotinamide adenine dinucleotide. (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, oil-soluble licorice extract, glabridin, hydroquinone, kojic acid, ascorbic acid, dipotassium glycyrrhizinateglycyrrhizate), 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 tripeptide-1, palmitoyl pentapeptide-4, acetyl hexapeptide-8 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 acid, PCA), urolithin, panthenol, tocopherol, resveratrol, lycopene, β-carotene, thiotic acidThe following are selected from the group consisting of, but are not limited to, acid, curcumin, 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.

[0046] In the present invention, hybrid exosomes are also formed by fusing the aforementioned exosomes and the artificial exosomes in a number ratio of approximately 1:1 to 1:10,000. Specifically, they are also formed by fusing in number ratios of approximately 1:1 to 1:10,000, approximately 1:1 to 1:1000, approximately 1:1 to 1:500, approximately 1:1 to 1:100, approximately 1:1 to 1:90, approximately 1:1 to 1:80, approximately 1:1 to 1:70, approximately 1:1 to 1:60, approximately 1:1 to 1:50, approximately 1:1 to 1:40, approximately 1:1 to 1:30, approximately 1:1 to 1:20, approximately 1:1 to 1:10, approximately 1:1 to 1:5, or approximately 1:1 to 1:3. Preferably, they are fused in a number ratio of approximately 1:1, but are not limited to these.

[0047] The diameter of the aforementioned hybrid exosome is also approximately 50-300 nm. Specifically, the hybrid exosome may have diameters of approximately 50 nm, approximately 300 nm, approximately 70-250 nm, approximately 90-200 nm, or approximately 110-150 nm.

[0048] Furthermore, the zeta potential of the aforementioned hybrid exosome is approximately -30 to +20 mV. Specifically, it is approximately -30 to +20 mV, approximately -25 to +10 mV, approximately -20 to +1 mV, or approximately -15 mV to -10 mV, but is not limited to these ranges.

[0049] In the present invention, the hybrid exosome obtained by fusing the exosome and the artificial exosome is a single structure containing both the component derived from the exosome and the component derived from the artificial exosome. The exosome and the artificial exosome are the same as those described above.

[0050] Therefore, the bilipid layer of the hybrid exosome may contain both the components of the bilipid layer of the exosome and the components of the bilipid layer of the artificial exosome. The bilipid layer of the hybrid exosome may contain any one selected from the group consisting of lecithin, ceramide, cholesterol, sphingolipids, phytosterols or their derivatives, and combinations thereof. More specifically, it may contain any one selected from the group consisting of lecithin, ceramide, phytosterols, and combinations thereof. In one example, the lipid bilayer of the hybrid exosome may contain lecithin, ceramide, and phytosterols, and these components are included in a ratio of about 1:0.45:0.03 to 1:0.45:0.2 based on 1 part by weight of lecithin.

[0051] Furthermore, when an active ingredient is supported on the artificial exosome, the hybrid exosome obtained by fusing the exosome and the artificial exosome supporting the active ingredient may contain the same active ingredient as that supported on the artificial exosome. The active ingredient may be hydrophobic or hydrophilic. In this case, the active ingredient may be contained in any one compartment selected from the group consisting of the surface of the hybrid exosome, between the lipid bilayers, inside the exosome, and combinations thereof. Therefore, the hybrid exosome supporting the active ingredient according to the present invention can simultaneously exhibit the pharmacological effects of the active ingredient contained in the exosome and the pharmacological effects of the active ingredient supported on the artificial exosome. The active ingredient is the same as described above.

[0052] The encapsulation rate of the supported active ingredient within the hybrid exosome is approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately over 80% to 99% or less, approximately over 80% to 98% or less, approximately over 80% to 97% or less, approximately over 80% to 96% or less, or approximately over 80% to 95% or less.

[0053] In this context, "encapsulation" refers to the process of surrounding a signaling molecule to enable efficient invagination into the body through encapsulation, while the drug encapsulation rate (encapsulation efficiency) refers to the content of the drug encapsulated within the hybrid exosome relative to the total drug content used in the manufacturing process.

[0054] <Method for producing hybrid exosomes> Another aspect of the present invention provides a method for producing hybrid exosomes, comprising the steps of i) producing exosomes or artificial exosomes; and ii) mixing the artificial exosomes and exosomes to induce their fusion.

[0055] In this case, the exosomes, artificial exosomes, and hybrid exosomes are the same as those described above.

[0056] Specifically, the method for producing the hybrid exosome may include the following steps:

[0057] Firstly, the process may include the step of producing exosomes or artificial exosomes. a. Exosome production The exosomes can be obtained from humans or plants. Specifically, they can be isolated and obtained from cultures of human blood, tissue, or cells, or from plant cultures or juices.

[0058] As used in this invention, the term "culture" means growing tissue, cells, or callus under appropriately controlled environmental conditions, and the culture process of this invention is carried out using appropriate culture media and culture conditions known to those skilled in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected tissue, cells, or callus. The culture media means known culture media used when culturing tissue, cells, or callus, and includes either known cell culture media or modified versions thereof.

[0059] The aforementioned human-derived tissues or cells are cultured in culture medium for approximately 1 hour to 4 weeks, approximately 6 hours to 3 weeks, approximately 12 hours to 2 weeks, approximately 18 hours to 7 days, or approximately 1 to 3 days.

[0060] The plant-derived tissues, cells, or callus are cultured in culture medium for approximately 1 hour to 4 weeks, approximately 6 hours to 3 weeks, approximately 12 hours to 2 weeks, approximately 18 hours to 7 days, or approximately 1 to 3 days.

[0061] As a specific example of the present invention, human-derived mesenchymal stem cells (MSCs) are cultured in culture medium for approximately 1 hour to 4 weeks, approximately 6 hours to 3 weeks, approximately 12 hours to 2 weeks, approximately 18 hours to 7 days, or approximately 1 to 3 days.

[0062] The exosomes of the present invention are separated in the culture medium by separation methods currently used or to be used in the industry, such as, but not limited to, ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, or polymer-based precipitation.

[0063] In one specific example, separation is performed by the ultracentrifugation method of the present invention.

[0064] The aforementioned ultracentrifugation may include low-speed centrifugation and / or high-speed centrifugation. Specifically, it may include (a) a process of obtaining a supernatant after centrifugation for 10 to 30 minutes under conditions of centrifugation speed of approximately 1,000 to 3,000 × g, (b) a process of obtaining a supernatant after centrifugation for 10 minutes to 2 hours under conditions of high-speed centrifugation speed of approximately 10,000 to 5,000 × g, and (c) a process of obtaining a pellet containing exosomes by ultracentrifugation for 60 minutes to 2 hours under conditions of ultracentrifugation speed of approximately 100,000 to 150,000 × g.

[0065] In this case, after the process described in (a) above, a further step of filtering the culture medium may be performed. In this case, the filtration can be carried out under conditions where the filter size is approximately 0.1 to 20 μm.

[0066] Furthermore, the process described in (c) above is repeated. Specifically, the ultracentrifugation process is repeated once, twice, or three times. After ultracentrifugation, a further process of filtering the exosomes can be carried out. Through this process, small-sized impurities present in the exosomes can be filtered out and larger-sized exosomes can be separated. The filtration can be carried out under conditions where the filter size is approximately 0.1 to 20 μm.

[0067] b. Production of artificial exosomes Artificial exosomes can be manufactured using, but are not limited to, methods currently used or to be used by those skilled in the art.

[0068] In one specific example, the artificial exosomes can be produced by an ethanol injection method. The ethanol injection method may include the steps of (a) dissolving lecithin, phytosterols, and ceramides in alcohol to produce a lipid solution; (b) dropping the lipid solution into sterile water to produce an artificial exosome dispersion solution; and (c) passing the lipid solution through an extruder to perform extrusion molding.

[0069] In this case, the mixing ratio of lecithin, phytosterol, and ceramide is the same as described above. The alcohol is ethanol, isopropyl alcohol, or propyl alcohol. Specifically, it is ethanol.

[0070] In step (b) above, the lipid solution and sterile water are mixed in a volume ratio of approximately 1:1 to 1:20. Specifically, the lipid solution can be dispersed in sterile water by dropping it into the sterile water at ratios of approximately 1:5 to 1:50, approximately 1:5 to 1:40, approximately 1:5 to 1:30, approximately 1:5 to 1:20, or approximately 1:5 to 1:10. In this case, the dropping rate is approximately 400 μL / min or less.

[0071] Furthermore, steps (a) and (b) are carried out at room temperature. Specifically, this can be approximately 15°C to 30°C, approximately 20°C to 30°C, or approximately 25°C to 30°C. The process may also be carried out under stirring to ensure uniform dispersion of the components in the solution. The stirring speed and duration are not particularly limited. In one embodiment, the stirring may be carried out at a speed of approximately 500 rpm for 60 minutes.

[0072] In step (c) above, the extrusion molding can be carried out through a high-pressure disperser.

[0073] In this process, the extrusion pressure is adjusted as needed, but is typically around 300 to 3000 bar. Specifically, extrusion can be performed at pressures of approximately 300 to 3000 bar, 500 to 2500 bar, or 1000 to 2000 bar. The extrusion process is repeated several times, specifically 1 to 10 times. The extrusion process may be performed 1, 2, 3, 4, 6, 6, 7, 8, 9, or 10 times, but is not limited to these.

[0074] In step (c) above, to produce a small amount of artificial or hybrid exosomes, a mini extruder (product of Avanti Pola Lipid) can be used instead of a high-pressure disperser. The mini extruder breaks down the solution into smaller particles by extruding it through a membrane filter using a syringe. In this invention, the extrusion is performed by gradually replacing the filters with smaller diameter holes, or by stacking multiple filters so that the filter diameter decreases along the direction of extrusion. In this case, the filters used should preferably have a diameter of approximately 1000 to 100 nm. Furthermore, during the extrusion process, the extrusion pressure may be increased as needed.

[0075] Furthermore, the artificial exosomes produced by the above method may include a final step of ultracentrifugation to obtain a pellet containing the exosomes. The conditions for the ultracentrifugation are the same as those described above.

[0076] Secondly, the procedure may include a step of mixing the exosomes and artificial exosomes to induce their fusion.

[0077] Specifically, the fusion of the exosomes and artificial exosomes may include: (a) a step of dissolving and mixing the exosomes and artificial exosomes in an alcohol solution; (b) a step of loading the mixture into sterile water to produce a hybrid exosome dispersion solution; and (c) a step of passing the hybrid exosome dispersion solution through an extruder to perform extrusion molding.

[0078] 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.

[0079] In step (b) above, the hybrid exosome dispersion and sterile water are mixed in a volume ratio of approximately 1:1 to 1:20. Specifically, the lipid solution is added dropwise to the sterile water and dispersed in ratios of approximately 1:5 to 1:50, 1:5 to 1:40, 1:5 to 1:30, 1:5 to 1:20, or 1:5 to 1:10. The rate of addition is approximately 400 μL / min or less.

[0080] Furthermore, steps (a) and (b) above may be carried out at room temperature and may be performed under stirring to ensure uniform dispersion of the components in the solution. In this case, the temperature, stirring speed, and time are the same as described above.

[0081] Furthermore, the hybrid exosomes produced by the above method may include a final step of ultracentrifugation to obtain a pellet containing the exosomes. The conditions for the ultracentrifugation are the same as those described above.

[0082] Thirdly, the manufacturing method may further include the step of encapsulating the active ingredient in the hybrid exosomes of ii) above.

[0083] The active ingredient is mixed with the exosomes and artificial exosomes in the exosome and artificial exosome mixing step described in ii) above and encapsulated in the hybrid exosome. If the active ingredient is hydrophobic, it is dissolved in ethanol together with the exosomes and artificial exosomes and mixed and encapsulated in the hybrid exosome. If the active ingredient is hydrophilic, it is dissolved in sterile water and mixed with the exosome and artificial exosome mixture and encapsulated in the hybrid exosome.

[0084] The aforementioned alcohol is the same as described above.

[0085] <Pharmaceutical Compositions> Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases, comprising a hybrid exosome formed by the fusion of exosomes and artificial exosomes as an active ingredient. In this case, the hybrid exosome contains an active ingredient. The exosomes, artificial exosomes, hybrid exosomes, and active ingredient are the same as those described above.

[0086] Specifically, the active ingredient may be a component that promotes cell activity and energy supply or production, or a functional ingredient that is effective in preventing or treating skin diseases, either alone or in combination.

[0087] More specifically, the components that promote the activity of the cells and the supply or production of energy within the cells are selected from the group consisting of amino acids, minerals, sugars, vitamins, intracellular energy sources, and combinations thereof.

[0088] More specifically, the amino acids are selected from, but are not limited to, the group consisting of glycine, glutamic acid, leucine, alanine, phenylalanine, valine, isoleucine, methionine, cysteine, proline, and combinations thereof.

[0089] The aforementioned minerals are selected from, but are not limited to, the group consisting of sodium pyruvate, calcium chloride, magnesium chloride, zinc oxide, calcium pantothenate, and combinations thereof.

[0090] The aforementioned sugars are glucose, inositol, or a combination thereof.

[0091] The aforementioned vitamins may include their salts, precursors, and derivatives, and are selected from, but are not limited to, the group consisting of pyridoxine or its salt (HCl), cyanocobalamine, folic acid, riboflavin, thiamine or its salt (HCl or nitrate), and combinations thereof.

[0092] The components that promote the activity of the cells and the supply or production of energy within the cells are selected from, but are not limited to, the group consisting of nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), adenosine triphosphate (ATP), and combinations thereof.

[0093] More specifically, functional ingredients effective in preventing or treating the aforementioned skin diseases are also ingredients that exhibit any one of the following functions selected from the group consisting of skin whitening, wrinkle improvement, skin elasticity improvement, moisturizing, antioxidant function, anti-inflammatory or acne improvement, hair loss improvement or hair growth promotion, and combinations thereof.

[0094] More specifically, the aforementioned whitening functional ingredients are 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, and tranexamic acid. The following are selected, but are not limited to, the group consisting of acid, phloretin, ergothioneine, and combinations thereof.

[0095] The aforementioned wrinkle-improving functional ingredients are selected from, but are not limited to, the group consisting of retinol, retinyl palmitate, polyethoxylated retinamide, bakuchiol, adenosine, ubiquinone, glutathione, thioctic acid, peptides, palmitoyl tripeptide-1 (Pal-GHK), palmitoyl pentapeptide-4, acetyl hexapeptide-8, and combinations thereof.

[0096] The aforementioned skin elasticity-improving functional ingredient is selected from the group consisting of acetyl hydroxyproline, hydroxypropyl tetrahydropyrantriol, collagen, elastin, and combinations thereof, but is not limited to these.

[0097] The aforementioned moisturizing functional ingredients are selected from, but are not limited to, the group consisting of trehalose, ceramide, glycerin, chitosan, hyaluronic acid, shea butter, and combinations thereof.

[0098] The aforementioned antioxidant functional components can be selected from, but are not limited to, 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.

[0099] The aforementioned anti-inflammatory or acne-improving functional ingredient is selected from, but is not limited to, the group consisting of sialic acid, panthenol, allantoin, aloe vera extract, chamomile flower extract, green tea extract, tea tree leaf oil, centella asiatica extract, Houttuynia cordata extract (fish-salt

[0100] The aforementioned hair loss improving or hair growth promoting functional ingredients are selected from, but are not limited to, 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, dexpantenol, sialic acid, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, argania spinosa kernel oil, and combinations thereof.

[0101] The aforementioned "skin disease" is selected from, but is not limited to, the group consisting of dermatitis, acne, wounds, wrinkles, skin aging, weakened skin elasticity, dry skin, sensitive skin, hair loss, and skin discoloration, and combinations thereof.

[0102] As used herein, the term "prevention" comprehensively means preventing a disease in advance or reducing its likelihood or frequency of occurrence by administering the pharmaceutical composition in a pharmaceutically effective dose. For example, it also means reducing the probability of developing or recurring a skin disease in a patient who is at risk of developing it or who has previously developed it. The term "pharmaceutically effective dose" is synonymous with "therapeutic effective dose" and is readily determined by a skilled technician based on factors well known in the pharmaceutical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of doses, the duration of treatment, the combination, or any other drugs used concurrently.

[0103] As used herein, the term "treatment" can comprehensively mean improving a disease by administering the pharmaceutical composition in a pharmaceutically effective amount, providing relief or cure of the symptoms of the disease in a shorter time than natural healing, and also improving one or most of the symptoms of the disease. The pharmaceutically effective amount is the same as described above. The pharmaceutical composition of the present invention may be a composition for the treatment of skin diseases on its own, or it may be administered together with other pharmacological components and applied as a therapeutic adjunct to the disease. Thus, "treatment" includes the meaning of "therapeutic adjunct."

[0104] The phrase "included as an active ingredient" means that the hybrid exosome according to the present invention is added to an extent that allows it to exhibit the effects described above, and includes the addition of various components as auxiliary components for purposes such as transmission and stabilization, and formulation into various forms.

[0105] The pharmaceutical composition of the present invention may contain the hybrid exosomes in an amount of about 0.001 to 30% by weight relative to the total weight of the composition.

[0106] Specifically, the composition may contain hybrid exosomes in amounts of approximately 1 to 1000 μg, approximately 5 to 500 μg, approximately 10 to 200 μg, approximately 15 to 100 μg, or approximately 20 to 50 μg.

[0107] On the other hand, the pharmaceutical composition of the present invention can be administered to an individual in a "therapeutably effective amount." The therapeutically effective amount is the same as described above.

[0108] As used herein, "administration" means introducing a given substance into an individual in an appropriate manner, and the administration route of the composition is administered through a given general route, insofar as it can reach the target tissue. This includes, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, topical, intranasal, intrapulmonary, and rectal administration. Furthermore, a specific example of the pharmaceutical composition of the present invention is administered by any device that can move the active substance to target tissue or cells. Specifically, it is administered parenterally, and more specifically, subcutaneously or transdermally. The pharmaceutical composition is also applied directly to the skin. When the pharmaceutical composition is applied to the skin, this may include applying or spraying the pharmaceutical composition according to the present invention directly to the skin in its form.

[0109] Here, the individual to whom the pharmaceutical composition is administered is a mammal, and specifically, a human.

[0110] The appropriate dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition according to the present invention can be administered in one to several divided doses at a dose of approximately 0.001 to 100 mg / kg based on adult standards. Such dosages should not be construed as limiting the scope of the present invention in any way.

[0111] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have a toxicity that exceeds the suitability of the target of application (prescription). The carrier is included in about 1 to 99.99% by weight, preferably about 70 to 99.99% by weight, based on the total weight of the pharmaceutical composition of the present invention. The pharmaceutically acceptable carrier can be any non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inactive solids are included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) are also included in the pharmaceutical composition, but are not limited to these. Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington's Pharmaceutical Sciences (19th ed, 1995)".

[0112] When the pharmaceutical composition is manufactured in parenteral dosage form, it is formulated with a suitable carrier by methods known in the art to be in the form of an injectable, transdermal, nasal inhalant, or suppository. As a specific example, the pharmaceutical composition of the present invention can be manufactured as an injectable. The injectable is, but is not limited to, an aqueous injectable, a non-aqueous injectable, an aqueous suspension injectable, a non-aqueous suspension injectable, or a solid injectable used after dissolution or suspension. Depending on the type, the injectable may contain at least one of the following: distilled water for injection, vegetable oil (e.g., peanut oil, sesame oil, camellia oil, etc.), monoglycerides, diglycerides, propylene glycol, camphor, estradiol benzoate, bismuth subsalicylate, sodium arsenobenzol, or streptomycin sulfate, and may selectively contain stabilizers and preservatives.

[0113] As a specific example, the pharmaceutical composition of the present invention can be manufactured as a topical skin preparation. When the pharmaceutical composition is manufactured as a topical skin preparation, it is formulated in the form of an ointment, liquid, cream, spray, patch, etc. In this case, ingredients commonly used in cosmetics and topical skin preparations, such as moisturizers, antioxidants, oily components, UV absorbers, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, and various skin nutrients, can be appropriately added as needed, within the limits that do not impair the effects of the present invention.

[0114] Furthermore, the pharmaceutical composition may further contain, or be used in combination with, known substances that exhibit preventive or therapeutic effects against the disease.

[0115] A further aspect of the present invention provides applications for the prevention or treatment of skin diseases of the hybrid exosome or a pharmaceutical composition containing the same as an active ingredient. A further aspect of the present invention provides a method for the prevention or treatment of skin diseases, comprising the step of administering the hybrid exosome or a pharmaceutical composition containing the same as an active ingredient to an individual.

[0116] Here, the hybrid exosome, pharmaceutical composition, individual, administration, skin disease, prevention, and treatment are the same as described above.

[0117] <Cosmetic composition> Another aspect of the present invention provides a cosmetic composition for improving skin condition, comprising a hybrid exosome, which is a fusion of exosomes and artificial exosomes, as an active ingredient. In this case, the hybrid exosome may contain an active ingredient. The exosomes, artificial exosomes, hybrid exosomes, and active ingredient are the same as those described above.

[0118] Specifically, the active ingredient may be a component that promotes cell activity and energy supply or production, or a functional ingredient effective in preventing or treating skin diseases, either alone or in combination. Here, the component that promotes cell activity and energy supply or production, or the functional ingredient effective in preventing or treating skin diseases, is the same as described above.

[0119] The aforementioned improvement of skin condition is selected from, but is not limited to, the group consisting of improvement of dermatitis, improvement of acne, suppression of wrinkle formation, suppression of skin aging, improvement of skin elasticity, whitening, moisturizing, suppression of hair loss, and combinations thereof. Furthermore, it is characterized by protecting the skin from functional decline or loss of skin cells, improving skin condition, or preventing or improving skin diseases.

[0120] The cosmetic composition of the present invention may contain the hybrid exosomes in an amount of about 0.001 to 30% by weight relative to the total weight of the composition.

[0121] Specifically, the cosmetic composition may contain hybrid exosomes in amounts of approximately 1 to 1000 μg, approximately 5 to 500 μg, approximately 10 to 200 μg, approximately 15 to 100 μg, or approximately 20 to 50 μg.

[0122] The cosmetic composition is formulated into cosmetic dosage forms commonly manufactured in the industry. The cosmetic composition is formulated into, but is not limited to, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays. More specifically, it is formulated into softening lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, or powders.

[0123] When the dosage form of the cosmetic composition according to the present invention is a paste, cream, or gel, it may contain a carrier component selected from the group consisting of animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, and mixtures thereof.

[0124] The dosage form of the cosmetic composition according to the present invention may include a carrier component selected from the group consisting of solvents, solvating agents, emulsifying agents, and mixtures thereof, in which the composition is in solution or emulsion form. Examples include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, sorbitan fatty acid esters, and mixtures thereof.

[0125] When the dosage form of the cosmetic composition according to the present invention is a suspension, it may contain water, a liquid diluent such as 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 methhydroxyl, bentonite, agar, tragacanth, and mixtures thereof.

[0126] The cosmetic composition may further contain various known additives in addition to the carrier, depending on the dosage form.

[0127] The aforementioned additives include emulsifiers, humectants, surfactants, chelating agents, antioxidants, disinfectants, and stabilizers.

[0128] The emulsifier may include liquid paraffin, cetyl octanoate, stearic acid, and the like. The humectant may include polyols selected from the group consisting of glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, sorbitol, and combinations thereof.

[0129] The chelating agent may include sodium ethylenediaminetetraacetate (EDTA), α-hydroxy fatty acids, lactoferrin, α-hydroxy acids, citric acid, lactic acid, malic acid, bilirubin, and biliverdin.

[0130] The aforementioned antioxidants may include butylhydroxyanisole, dibutylhydroxytoluene, or propyl gallate. Other ingredients that can be incorporated into the cosmetic composition or topical skin preparation include oils and fats, emollients, organic and inorganic pigments, organic powders, UV absorbers, pH adjusters, alcohols, dyes, fragrances, blood circulation promoters, cooling agents, antiperspirants, and vitamins.

[0131] A further aspect of the present invention provides a method of cosmetic treatment through improvement of skin condition using the hybrid exosome or a cosmetic composition containing the same as an active ingredient. The method of cosmetic treatment may include the step of applying the cosmetic composition to the skin of an individual. Here, the hybrid exosome, cosmetic composition, skin condition improvement, and individual are the same as described above.

[0132] The step of applying to the skin may include directly applying or spraying the cosmetic composition according to the present invention onto the skin in its form. In this case, the amount of the cosmetic composition to be applied and the number of times it is used per day are appropriately set according to the user's age, gender, intended use, the severity of symptoms, etc. For example, an appropriate amount of the cosmetic composition can be applied to the skin at a frequency of 1 to 6 times per day.

[0133] <Drug delivery system composition> Another aspect of the present invention provides a drug delivery system composition comprising exosomes; and hybrid exosomes fused with artificial exosomes containing an active ingredient.

[0134] The exosomes, active ingredients, artificial exosomes, and hybrid exosomes are the same as those described above.

[0135] In the present invention, the hybrid exosome obtained by fusing the exosome and the artificial exosome is a single structure containing both components derived from the exosome and components derived from the artificial exosome. Therefore, it may contain both components of the bilipid layer of the exosome and components of the bilipid layer of the artificial exosome. Furthermore, the hybrid exosome may contain the same active ingredient as that supported on the artificial exosome. The active ingredient can be hydrophobic or hydrophilic. In this case, the active ingredient may be contained in any one compartment selected from the group consisting of the surface of the hybrid exosome, between the lipid bilayers, inside the exosome, and combinations thereof. In this case, the active ingredient is the same as described above.

[0136] In one embodiment of the present invention, the hybrid exosome was able to support either a hydrophobic substance (WKYMVm with palmitate attached, SEQ ID NO: 1) or a hydrophilic substance (trypan blue) (Figure 5).

[0137] <Therapeutic and ameliorative uses> Another aspect of the present invention provides applications for the prevention or treatment of skin diseases of hybrid exosomes, which are fused with exosomes and artificial exosomes.

[0138] The exosomes, artificial exosomes, hybrid exosomes, skin diseases, prevention, and treatment described above are the same as those described previously.

[0139] A further aspect of the present invention provides a method for preventing or treating a skin disease, comprising the step of administering a hybrid exosome, which is a fusion of an exosome and an artificial exosome, to an individual.

[0140] The aforementioned exosomes, artificial exosomes, hybrid exosomes, administration, skin diseases, prevention, and treatment are all the same.

[0141] The aforementioned individual refers to a subject that is suffering from or is likely to suffer from a skin disease. The aforementioned individual is a mammal, and preferably a human. The aforementioned dosage is prescribed in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, pathological condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0142] Another aspect of the present invention provides applications for improving skin conditions using hybrid exosomes, which are fused with exosomes and artificial exosomes.

[0143] The exosomes, artificial exosomes, hybrid exosomes, and improvement of skin condition described above are the same as those described previously.

[0144] Another aspect of the present invention provides a method for improving skin condition, which includes the step of administering a hybrid exosome, in which exosomes and artificial exosomes are fused, to an individual.

[0145] The exosomes, artificial exosomes, hybrid exosomes, individuals, administration, and improvement of skin condition are the same as described above.

[0146] The present invention will be described in more detail through the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. [Examples]

[0147] <Example 1. Production of Hybrid Exosomes> [Example 1.1. Isolation of exosomes derived from human mesenchymal stem cells] Human mesenchymal stem cell-derived exosomes (MSC exosomes) were isolated through the following process.

[0148] First, human tonsil tissue was treated with 1% collagenase enzyme to isolate single cells. Then, human mesenchymal stem cells were isolated using a known method and cultured in an alpha-MEM medium containing 10% FBS in a 37°C constant temperature and humidity chamber. When the mesenchymal stem cells had grown to 90% of the culture dish's density, the medium was washed twice with HBSS (Hank's balanced salt solution) to remove any remaining FBS, and then replaced with alpha-MEM medium without FBS. Finally, the medium was cultured in a 37°C constant temperature and humidity chamber for 48 hours to obtain the mesenchymal stem cell culture medium.

[0149] 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 then centrifuged in an ultracentrifuge at 100,000 × g for 70 minutes at 4°C. Next, the supernatant was removed, the precipitated exosomes were resuspended in HBSS, and then recentrifuged under the same conditions to obtain the exosomes.

[0150] After the exosomes obtained in the above process were resuspended in HBSS, their size and number were measured using a Nanosight instrument, and then they were used in the following experiments.

[0151] [Example 1.2. Production of Artificial Exosomes] Artificial exosomes were produced through the following process. During this process, the lipid composition and ratios were improved to facilitate large-scale exosome production (Table 1 and Figure 4).

[0152] 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 mixed with 2 mL of sterile water while stirring at 500 rpm for 60 minutes, gradually reducing the mixing rate to 400 μL / min or less, to prepare an artificial exosome dispersion solution. In this case, if the artificial exosomes carried a substance, hydrophobic substances were dissolved in 100% ethanol, and hydrophilic substances were dissolved in sterile water and mixed to prepare the artificial exosome dispersion solution.

[0153] After setting 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 and mold the artificial exosomes into small sizes.

[0154] The artificial exosomes obtained through the above process were collected by centrifugation at 100,000 × g for 70 minutes at 4°C using an ultracentrifuge.

[0155] [Table 1]

[0156] [Example 1.3. Production of Hybrid Exosomes] A specific example of the present invention, a hybrid exosome, was produced by the same method as in Example 1.2. In this case, the MSC exosomes from Example 1.1 were mixed with the artificial exosome dispersion solution from Example 1.2, and then produced by sequential extrusion using a small extruder as described in Example 1.2. In this process, the MSC exosomes and artificial exosomes were mixed in a 1:1 ratio.

[0157] <Example 2. Evaluation of the material carrying capacity of hybrid exosomes> Hybrid exosomes have an internal space that can support active substances and can support either hydrophobic or hydrophilic substances.

[0158] To confirm the material-carrying capacity of the hybrid exosomes, palmitoyl-WKYMVm (SEQ ID NO: 1) labeled with the fluorescent substance TAMRA was used as the hydrophobic substance, and trypan blue was used as the hydrophilic substance. Palmitoyl-WKYMVm was dissolved in 100% ethanol and mixed to a ratio of 1% by mass of lipids, and trypan blue was dissolved in sterile water. Hybrid exosomes were then prepared in the same manner as in Example 1.3.

[0159] The generated exosomes were collected by ultracentrifugation, and the absorbance of the supernatant and the base solution was measured. The loading efficiency was calculated from the difference in the ratio of each absorbance (Figure 5).

[0160] As a result, it was confirmed that hydrophobic and hydrophilic substances were supported at a rate of 90% and 82.5% or higher, respectively, within the exosomes.

[0161] <Example 3. Characterization of Hybrid Exosomes>

[0162] Table 2 shows the results of measuring the size and zeta potential of each particle using the Malvern Zetasizer nano-zs instrument. Hybrid exosomes showed significantly reduced size and heterogeneity compared to mesenchymal stem cell-derived exosomes (MSC exosomes). Furthermore, the zeta potential shifted to the negative charge side compared to artificial exosomes. In addition, it was confirmed that the polydispersity index (PDI) value of hybrid exosomes was lower than that of MSC exosomes. These results indicate increased homogeneity of hybrid exosomes compared to MSC exosomes.

[0163] [Table 2]

[0164] <Example 4. Synthesis and efficiency analysis of hybrid exosomes combining mesenchymal stem cell-derived exosomes and artificial exosomes> Hybrid exosomes were produced by reprocessing MSC exosomes and artificial exosomes using the same method as in Example 1.3. In this process, 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.

[0165] As a result, as shown in Figure 6, in the case of hybrid exosomes, the region exhibiting both green and red fluorescence simultaneously was found to be present, and the synthesis efficiency was confirmed to be 52% or higher.

[0166] <Example 5. Evaluation of skin permeability of hybrid exosomes> The skin permeability of hybrid exosomes produced by the same method as in Example 1.3 was evaluated. The experimental group was divided into three groups: a control group, a group treated with peptide_TAMRA alone (peptide), and a group in which the active ingredient peptide_TAMRA was loaded onto hydrated exosomes (hybrid exosome + peptide). Skin permeability was determined by verifying the degree of exosome infiltration into the skin based on the Franz diffusion cell method.

[0167] Specifically, to measure skin infiltration, skin tissue was collected from pig ears, and then subcutaneous adipose tissue was removed to secure skin tissue consisting of epithelial and dermal layers. The skin tissue was then attached to a Franz diffusion cell instrument with the epithelial layer facing upwards. Peptide-TAMRA alone or hybrid exosomes carrying peptide-TAMRA were dissolved in PBS solution and injected into the upper chamber of the Franz diffusion cell instrument in contact with the epithelial layer, and the mixture was reacted in a constant temperature and humidity chamber for 24 hours. PBS solution was used as a control group. During this process, care was taken to avoid collecting air layers when injecting PBS solution into the lower chamber of the Franz diffusion cell instrument, and the solution was stirred using a magnetic stirrer. The temperature of the constant temperature and humidity chamber was maintained at 32°C, the temperature of the skin surface.

[0168] After a 24-hour reaction in a constant temperature and humidity chamber, the Franz diffusion cell apparatus was disassembled to separate the skin tissue, which was then analyzed using a confocal microscope. Epithelial and dermal tissues were identified by DAPI staining.

[0169] As a result, as shown in Figure 7, in the peptide_TAMRA-only treatment group, the fluorescent labeling factor was observed only around the stratum corneum, while in the hybrid exosome + peptide treatment group, the fluorescent labeling factor was observed not only in the epidermis but also in the dermis layer. Through these results, it was confirmed that hybrid exosomes carrying peptides can be used as carriers for transdermal delivery of peptides.

[0170] <Example 6. Confirmation of intracellular influx of mesenchymal stem cell-derived exosomes> MSC exosomes prepared by the same method as in Example 1.1 were stained with a green fluorescent dye (PKH67GL). After treating mesenchymal stem cells with the MSC exosomes, the cells were allowed to react for 14 hours, and then fluorescence was observed using a confocal microscope. The nuclei were stained with DAPI and observed.

[0171] As a result, as shown in Figure 8, MSC exosomes stained with green fluorescence were found in the periphery of the nucleus of mesenchymal stem cells stained with DAPI. Through these results, we were able to confirm that MSC exosomes enter the cell interior.

[0172] <Example 7. Confirmation of intracellular influx of artificial exosomes> Artificial exosomes were prepared by mixing rhodamine PE at a ratio of 0.1 mol% relative to the lipid content, using the same method as in Example 1.2. After treating mesenchymal stem cells with the artificial exosomes prepared as described above, the cells were allowed to react for 14 hours, and fluorescence was confirmed using a confocal microscope. The nuclei were stained with DAPI and observed.

[0173] As a result, as shown in Figure 9, artificial exosomes stained with red fluorescence were observed in the periphery of the nucleus of mesenchymal stem cells stained with DPAI. Through these results, we were able to confirm that artificial exosomes can enter the cell interior.

[0174] <Example 8. Confirmation of intracellular influx of hybrid exosomes> After treating mesenchymal stem cells with hybrid exosomes prepared by the same method as in Example 1.3, the cells were allowed to react for 14 hours, and fluorescence was observed using a confocal microscope. The nuclei were stained with DAPI for observation.

[0175] As a result, as shown in Figure 10, MSC exosomes stained green, artificial exosomes stained red, and hybrid exosomes that exhibited both green and red colors simultaneously, resulting in a yellow appearance, were observed in the periphery of the mesenchymal stem cell nucleus. Through these results, it was confirmed that hybrid exosomes enter the cell interior.

[0176] <Example 9. Analysis of hybrid exosome influx into skin cells> To analyze the influx of hybrid exosomes into skin cells produced by the method described in Example 1.3, artificial exosomes, liposomes, MSC exosomes (natural exosomes), and hybrid exosomes were treated with HaCaT cells (skin keratinocytes), respectively, and the influx of exosomes into skin cells was observed using a laser fluorescence microscope. The nuclei were stained with DAPI for observation.

[0177] In this process, the bilayer membranes of exosomes and liposomes were stained with rhodamine PE, a red fluorescent dye, and MSC exosomes (natural exosomes) were stained with a green fluorescent dye (PKH67GL). Subsequently, the stained artificial exosomes and MSC exosomes were fused in the same manner as in Example 1.3 to produce hydrated exosomes (Table 3). In this process, liposomes used as a control group were prepared by dissolving lecithin in 100% ethanol and using the same method as in Example 1.2 (ethanol injection method and sequential extrusion method).

[0178] [Table 3]

[0179] The hybrid exosomes, MSC exosomes, artificial exosomes, and liposomes produced as described above were used to treat HaCaT cells, and the cells were allowed to react for 30 minutes. After the reaction, the cells were fixed, and the degree of intracellular influx of exosomes and liposomes stained with each fluorescent dye was confirmed by fluorescence expression. The nuclei were stained with DAPI and observed. As shown in Figure 11, the degree of fluorescence expression in skin cells increased when treated with artificial exosomes or hybrid exosomes. The degree of fluorescence expression was similar to that of the MSC exosome (natural exosome) treated group and higher than that of the liposome treated group. Through these results, it was confirmed that hybrid exosomes have a high degree of intracellular influx in the present invention.

[0180] <Example 10. Analysis of exosome skin permeability and drug delivery capacity> To analyze the permeability of artificial exosomes into skin tissue, we used Franz Diffusion Cells to analyze skin permeability.

[0181] Specifically, artificial exosomes were stained with rhodamine PE, a red fluorescent dye, and then coated onto pig skin using Franz Diffusion Cells. After 24 hours, the skin tissue coated with exosomes was fixed, and fluorescence expression within the skin tissue was analyzed by histological analysis. The nuclei were observed after staining with DAPI.

[0182] As a result, fluorescence expression was confirmed in the dermis layer of the skin, as shown in Figure 12. Through these results, it was confirmed that the artificial exosomes have skin permeability.

[0183] Furthermore, to confirm the drug delivery ability of the artificial exosomes, rhodamine B dye dissolved in a hydrophilic or hydrophobic solvent was encapsulated in the exosomes, and then applied to the skin.

[0184] As a result, as shown in Figure 13, rhodamine fluorescence expression was confirmed up to the dermis layer of the skin. Through these results, we were able to confirm the skin permeability and drug delivery ability of the artificial exosomes according to the present invention.

Claims

1. A hybrid exosome, a fusion of exosomes and artificial exosomes.

2. The hybrid exosome according to claim 1, wherein the exosome is of human or plant origin.

3. The hybrid exosome according to claim 2, wherein the human-derived exosome is derived from human blood, tissue, or cells.

4. The hydrated exosome according to claim 3, wherein the cells are stem cells, immune cells, blood cells, somatic cells, or germ cells.

5. The hybrid exosome according to claim 4, wherein the stem cells are mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, or neural stem cells.

6. The hybrid exosome according to claim 5, wherein the mesenchymal stem cells are derived from umbilical cord blood, umbilical cord, bone marrow, fat, muscle, nerve, skin, amniotic fluid, or amniotic membrane.

7. The plant-derived exosome is isolated from a plant culture, and is a hybrid exosome according to claim 2.

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

9. The hybrid exosome according to claim 1, wherein the artificial exosome comprises any one selected from the group consisting of lecithin, ceramide, cholesterol, sphingolipids, phytosterols or their derivatives, phosphatidylethanolamine (PE), phosphatidylserine (PS), and combinations thereof.

10. The hybrid exosome according to claim 9, wherein the artificial exosome comprises lecithin, ceramide, and phytosterol.

11. The hybrid exosome according to claim 10, wherein the artificial exosome contains lecithin, ceramide, and phytosterol in a mass ratio of 1:0.1:0.01 to 1:0.8:0.

4.

12. The hybrid exosome according to claim 1, wherein the artificial exosome further comprises an active ingredient.

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

14. The 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 its salt (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or its salt (HCl or nitrate), nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), and adenosine triphosphate (ATP). 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, 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 Tripeptide-1, Palmitoyl Pentapeptide-4, 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 One of the following selected from the group consisting of ol, tocopherol, resveratrol, lycopene, β-carotene, thioctic acid, curcumin, 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 (fish-saltwood extract), azelaic acid, caffeine, dexpanthenol, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, argania spinosa kernel oil, and combinations thereof.The hybrid exosome according to claim 13.

15. The hybrid exosome according to claim 1, wherein the exosomes and artificial exosomes are fused in a number ratio of 1:1 to 1:10000.

16. The hybrid exosome according to claim 1, wherein the diameter of the hybrid exosome is 50 to 300 nm.

17. The hybrid exosome according to claim 1, wherein the zeta potential of the hybrid exosome is -30 to +20 mV.

18. The hybrid exosome according to claim 1, further comprising an active ingredient.

19. The hybrid exosome according to claim 18, wherein the active ingredient is contained in any one compartment selected from the group consisting of the surface of the hybrid exosome, between the lipid bilayers, internal compartments, and combinations thereof.

20. i) The step of manufacturing exosomes or artificial exosomes, ii) A step of mixing the exosomes and artificial exosomes to induce their fusion, A method for producing hybrid exosomes, including

21. The method for producing hybrid exosomes according to claim 20, further comprising the step of iii) encapsulating an active ingredient in the hybrid exosomes of ii).

22. A pharmaceutical composition for the prevention or treatment of skin diseases, comprising a hybrid exosome formed by the fusion of exosomes and artificial exosomes as an active ingredient.

23. The aforementioned hybrid exosome comprises an active ingredient, wherein the pharmaceutical composition for the prevention or treatment of skin diseases is as described in claim 22.

24. The 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 its salt (HCl), cyanocobalamin, folic acid, riboflavin, thiamine or its salt (HCl or nitrate), nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), and adenosine triphosphate (ATP). 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, 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 Tripeptide-1, Palmitoyl Pentapeptide-4, 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 One of the following selected from the group consisting of ol, tocopherol, resveratrol, lycopene, β-carotene, thioctic acid, curcumin, 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 (fish-saltwood extract), azelaic acid, caffeine, dexpanthenol, L-menthol, biotin, zinc pyrithione, minoxidil, keratin, argania spinosa kernel oil, and combinations thereof.A pharmaceutical composition for the prevention or treatment of skin diseases as described in claim 23.

25. The composition for preventing or treating a skin disease according to claim 22, wherein the skin disease is selected from the group consisting of dermatitis, acne, wounds, wrinkles, skin aging, weakening of skin elasticity, dry skin, sensitive skin, hair loss and skin discoloration, and combinations thereof.

26. A cosmetic composition for improving skin condition, containing hybrid exosomes, which are a fusion of exosomes and artificial exosomes, as an active ingredient.

27. The aforementioned hybrid exosome is a cosmetic composition for improving skin condition according to claim 26, comprising an active ingredient.

28. The skin condition improvement described above is selected from the group consisting of improvement of dermatitis, improvement of acne, suppression of wrinkle formation, suppression of skin aging, improvement of skin elasticity, whitening, moisturizing, suppression of hair loss, and combinations thereof, as described in claim 26.

29. A drug delivery system composition comprising exosomes; and hybrid exosomes fused with artificial exosomes containing an active ingredient.

30. Applications of hybrid exosomes, which are fused with exosomes and artificial exosomes, for the prevention or treatment of skin diseases.

31. A method for preventing or treating a skin disease, comprising the step of administering a hybrid exosome, which is a fusion of an exosome and an artificial exosome, to an individual.

32. Applications of hybrid exosomes, which are a fusion of exosomes and artificial exosomes, for improving skin condition.

33. A method for improving skin condition, comprising the step of administering a hybrid exosome, which is a fusion of exosomes and artificial exosomes, to an individual.

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