Exosomes comprising cartilage homeostasis factor and composition comprising same for regenerating skin or cartilage

Abstract

The present invention relates to exosomes comprising a cartilage homeostasis factor, a cosmetic composition for skin regeneration comprising same as an active ingredient, or a composition for wound treatment comprising same as an active ingredient.

Description

Exosomes containing cartilage homeostasis factors and compositions for skin or cartilage regeneration containing the same

[0001] The present application relates to an exosome containing a cartilage homeostasis factor and a composition for skin or cartilage regeneration containing the same.

[0002] The skin is the body's first line of defense. It consists of the epidermis, dermis, and subcutaneous tissue. The stratum corneum, the outermost layer of the epidermis, is composed of keratin and lipids. It inhibits the penetration of harmful substances and moisture evaporation, protecting the body from bacteria and external irritants.

[0003] Meanwhile, skin damage can occur due to various factors, including stress, UV rays, and physical force. The process of skin regeneration after damage can be divided into four stages: hemostasis, inflammation, proliferation, and remodeling. Various growth factors and cytokines are involved in this process.

[0004] Fibroblasts play a crucial role in skin regeneration and wound healing. They migrate other cells to the wound site and regulate cell division at the wound margin. During proliferation and migration, fibroblasts produce cytokines and extracellular matrix, modulating the interactions between fibroblasts, keratinocytes, and the endothelium.

[0005] Meanwhile, exosomes are nano-sized vesicles naturally produced within cells, containing proteins and genetic information. They transmit various signals, including genetic information, to other cells outside the cell, and are involved in development, proliferation, differentiation, immune regulation, angiogenesis, and the progression of various diseases. Exosomes, which are biological nanovesicles, have recently attracted great attention as drug delivery vehicles due to their advantages such as avoidance of immune responses, excellent compatibility with the human body, drug loading function, targeted delivery to specific cells, and stability in the blood.

[0006] The technical challenge of the present application is to provide cell-derived exosomes containing cartilage homeostasis factors.

[0007] The technical task of the present application is to provide a cosmetic composition for skin regeneration containing the exosome.

[0008] The technical task of the present application is to provide a pharmaceutical composition for wound treatment, cartilage regeneration, and prevention or treatment of osteoarthritis, including exosomes.

[0009] The technical task of the present application is to provide a method for treating a wound or osteoarthritis, which comprises a step of administering a therapeutically effective amount of a pharmaceutically acceptable salt of the exosome to a subject.

[0010] The technical problem of the present application is to provide a use of the exosomes for wound healing or osteoarthritis treatment.

[0011] To solve the above-described problem, one embodiment of the present application provides a cell-derived exosome containing a cartilage homeostasis factor.

[0012] Cell-derived exosomes containing the aforementioned cartilage homeostasis factors can promote the growth and migration of fibroblasts when brought into contact with them, thus enabling their use for skin regeneration or wound healing. Furthermore, these exosomes promote the growth of chondrocytes, thus facilitating cartilage regeneration and the treatment of osteoarthritis.

[0013] The above exosomes can be isolated from the medium in which the cells are cultured.

[0014] The above cell culture medium may be a common medium used in the field of cell culture, and examples thereof include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, and the like. In addition, the medium may further include one or more of a carbon source, a nitrogen source, and a trace element component.

[0015] The above exosomes can be isolated and purified from the medium in which cells are cultured using a method commonly used in the art.

[0016] The above exosomes may be secreted from cells transformed by introduction of a gene encoding a cartilage homeostasis factor.

[0017] The above cartilage homeostasis factor may be one or more selected from the group consisting of Transforming growth factor β1 (TGFβ1), Transforming growth factor β2 (TGFβ2), Transforming growth factor β3 (TGFβ3), Leucine-rich repeat-containing protein 32 (LRRC32), Fibroblast growth factor 18 (FGF18), Bone morphogenetic protein 2 (BMP2), Bone morphogenetic protein 6 (BMP6), and Bone morphogenetic protein 7 (BMP7). Specifically, the gene for Transforming growth factor β3 (TGFβ3) may be included, or the gene for Bone morphogenetic protein 6 (BMP6) may be introduced. Alternatively, the gene for Transforming growth factor β3 (TGFβ3) may be introduced, and genes for one or more of the remaining cartilage homeostasis factors may be further introduced. Both TGFβ3 and BMP6 may be introduced.

[0018] Gene information encoding cartilage homeostasis factors such as the above TGFβ1, TGFβ2, TGFβ3, LRRC32, BMP2, BMP6, BMP7 and FGF18 can be selected by a person skilled in the art from a database commonly used to search for genetic information in the field, such as GenBank.

[0019] In one embodiment of the present application, the cell may be a transformed cell into which a cartilage homeostasis factor gene has been introduced.

[0020] The above transformation can be accomplished using a conventional vector used in the field of cell transformation, and may be selected from the group consisting of a lentivirus vector, a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. Vectors that can be used as the above recombinant vectors can be produced based on, but are not limited to, plasmids used in the art (e.g., pcDNA series, pLVTHM, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.), or viral vectors (e.g., adeno-associated virus (AAV) vectors, etc.).

[0021] The above recombinant vector can be produced using a genetic recombination technique well known in the art, and site-specific DNA cutting and ligation can be performed using enzymes generally known in the art.

[0022] The above recombinant vector can be introduced into stem cells by a method known in the art for introducing nucleic acid molecules into organisms, cells, tissues, or organs, and can be performed by selecting an appropriate standard technique depending on the cell type as known in the art. Such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method (e.g., Lipofectamine 2000), and lithium acetate-DMSO method.

[0023] The cell that secretes or produces the above exosome may be any one cell selected from the group consisting of stem cells, immune cells, somatic cells, and tumor cells.

[0024] The cell that secretes or produces the above exosome may be a human cell line or an animal cell line. The above cell lines are CHO cells, NS0 cells, Sp2 / 0 cells, BHK cells, C127 cells, HEK293 cells, HEK293T cells, HEK-293 STF cells, 293T / 17 cells, 293T / 17 SF cells, or HEK-2932sus cells, HT-1080 cells, PERC6 cells, NuLi-1 cells, ARPE-19 cells, VK2 / E6E7 cells, Ect1 / E6E7 cells, RWPE-2 cells, WPE-stem cells, End1 / E6E7 cells, WPMY-1 cells, NL20 cells, NL20-TA cells, WT 9-7 cells, WPE1-NB26 cells, WPEint cells, RWPE2-W99 cells, HaCaT cells, hTERT-immortalized human fibroblast cells, It may be selected from the group consisting of BJ-5ta cells and BEAS-2B cells, but is not limited thereto.

[0025] The above tumor cells may be cells derived from ovarian cancer, breast cancer, liver cancer, brain cancer, colon cancer, prostate cancer, cervical cancer, lung cancer, stomach cancer, skin cancer, pancreatic cancer, oral cancer, rectal cancer, laryngeal cancer, thyroid cancer, parathyroid cancer, colon cancer, bladder cancer, peritoneal cancer, adrenal cancer, tongue cancer, small intestine cancer, esophagus cancer, renal pelvis cancer, kidney cancer, heart cancer, duodenum cancer, ureter cancer, urethra cancer, pharynx cancer, vaginal cancer, tonsil cancer, anal cancer, pleura cancer, thymus cancer, or nasopharyngeal cancer, but are not limited thereto. Specifically, the tumor cells may be selected from the group consisting of, but are not limited to, human ovarian cancer cell lines (SKOV3, OVCAR3), human breast cancer cell lines (MCF-7, T47D, BT-474), human hepatocellular carcinoma cell lines (Hep3B, HepG2), human glioblastoma cell lines (U87MG, U251), human colon cancer cell lines (SW480, HT-29, HCT116, Caco-2), human lung cancer cell lines (A549, NCIH358, NCI-H460), human prostate cancer cell lines (22RV1), human cervical cancer cell lines (HeLa), human melanoma cell lines (A375), human embryonic kidney cell lines (HEK293T), and human gastric cancer cell lines (NCI-N87).

[0026] The cell that secretes or produces the above exosome may be a cell line that overexpresses the gasdermin protein group.

[0027] The above-mentioned gasdermin protein group overexpressing cell lines can induce exosome production and secretion when apoptosis is induced by staurosporine, etc.

[0028] The above gasdermin protein may be one or more proteins selected from the gasdermin protein group consisting of GSDMA, GSDMB, GSDMC, GSDMD, DFNA5 (GSDME) and DFNB59.

[0029] GSDMD (GasderminD) is cleaved from the N-terminal domain by caspase activation by the inflammasome, and the separated N-terminal domain oligomerizes in the cell membrane to create a pore with a diameter of 10 to 16 nm. Through this pore, the inflammatory cytokines IL-1 and IL-18 are secreted, and eventually, pyroptosis occurs with the rupture of the cell membrane, which can induce the extracellular secretion of intracellular components.

[0030] GSDME (GasderminE), expressed by the DFNA5 gene, is cleaved by caspase 3, which is activated during the apoptosis process, causing necroptosis (secondary necrosis) or pyroptosis, which can induce the extracellular secretion of intracellular components by creating a hole in the cell membrane.

[0031] The term "pyroptosis" refers to a type of cell death characterized by caspase-dependent cell death induced by one or more of the Caspases CASP 1, 3, 4, and 5, which activate the gasdermin family of proteins, resulting in cell membrane perforation.

[0032] The above stem cell may be any one cell selected from the group consisting of mesoderm stem cell, pluripotent stem cell, multipotent stem cell, and unipotent stem cell.

[0033] The above stem cells may be immortalized stem cells.

[0034] The above immune cell may be any one cell selected from the group consisting of a dendritic cell, a natural killer cell, a T cell, a B cell, a regulatory T cell (Treg cell), a natural killer T cell, an innate lymphoid cell, a macrophage, a granulocyte, a chimeric antigen receptor-T cell, a lymphokine-activated killer cell, and a cytokine-induced killer cell.

[0035] The above somatic cell may be any one cell selected from the group consisting of fibroblasts, chondrocytes, synovial cells, keratinocytes, adipocytes, osteoblasts, osteoclasts, and peripheral blood mononuclear cells.

[0036] One embodiment of the present application provides a cosmetic composition for skin regeneration comprising the cell-derived exosomes.

[0037] The above cosmetic composition may contain the cell-derived exosomes in an amount of 0.0001 to 10 wt%, or 0.001 to 5 wt%, or 0.01 to 3 wt%, or 0.1 to 2 wt%, or 0.5 to 1.5 wt% based on the total weight of the cosmetic composition.

[0038] The above cosmetic composition may be prepared in liquid or solid form using bases, adjuvants, and additives commonly used in the cosmetic field. Liquid or solid cosmetics may include, but are not limited to, toners, creams, lotions, bath products, and the like. The bases, adjuvants, and additives are not particularly limited, and examples thereof include water, alcohol, propylene glycol, stearic acid, glycerol, cetyl alcohol, and liquid paraffin.

[0039] The above cosmetic composition may include ingredients commonly used in cosmetic compositions, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

[0040] The above cosmetic composition can be manufactured into a formulation commonly manufactured in the art, and can be formulated as, 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 manufactured 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.

[0041] When the formulation of the above cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0042] When the formulation of the above cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0043] When the formulation of the above cosmetic composition is a solution or emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0044] When the formulation of the above cosmetic composition is a suspension, 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, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0045] When the formulation of the present invention is a surfactant-containing cleansing agent, a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, an imidazolinium derivative, methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, or an ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0046] The cosmetic composition of the present invention can be used alone or in combination, or in combination with other cosmetic compositions. Furthermore, the cosmetic composition of the present invention, which exhibits excellent skin regeneration effects, can be used according to conventional methods, and the frequency of use can vary depending on the user's skin condition or preference.

[0047] When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed off with water. As specific examples, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.

[0048] One embodiment of the present application provides a pharmaceutical composition for wound treatment comprising the cell-derived exosomes.

[0049] The above pharmaceutical composition may be a pharmaceutical composition for preventing or treating skin wounds. The skin wounds may be at least one selected from the group consisting of wounds, burns, abrasions, photodamage caused by ultraviolet (UV) rays, scars, or wounds caused by skin diseases.

[0050] One embodiment of the present application provides a pharmaceutical composition for cartilage regeneration or prevention or treatment of osteoarthritis, comprising the cell-derived exosome.

[0051] One embodiment of the present application provides a method for treating a wound or treating osteoarthritis, comprising administering to a subject a therapeutically effective amount of a pharmaceutically acceptable salt of the exosome.

[0052] One embodiment of the present application provides the use of said exosomes for wound healing or osteoarthritis treatment.

[0053] The above "treatment" includes an action that occurs while the subject is suffering from a particular disease, disorder or condition, reduces the severity of the disease, disorder or condition, or delays or slows the progression of the disease, disorder or condition.

[0054] The above “prevention” means inhibiting the occurrence of the condition, disease or illness by administering the composition of the present application to a subject.

[0055] In the present application, the "subject" may be a human or a non-human animal. Examples include, but are not limited to, a human (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly person)) and / or a non-human animal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus macaque), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and / or a dog. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.

[0056] The pharmaceutical composition may contain conventional excipients, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings, or carriers.

[0057] The route of administration of the pharmaceutical composition above includes oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal, and may be applied by topical application, for example. The parenteral route includes subcutaneous, intradermal, intravenous, intramuscular, or intralesional injection or infusion techniques.

[0058] The above pharmaceutical composition can be administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to promote regeneration of damaged skin or treat skin diseases caused by skin wounds at an applicable reasonable benefit / risk ratio, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0059] The above pharmaceutical composition can be administered as an individual treatment or in combination with other treatments, and can be administered sequentially or simultaneously with conventional treatments. It can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination that can be readily made by those skilled in the art.

[0060] The above pharmaceutical composition can be used not only for humans but also for non-human mammals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, fish, etc.

[0061] The above pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods.

[0062] As preparations for parenteral administration, they can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, sterile aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, syrups, suppositories, aerosols, etc., and sterile injection preparations according to conventional methods, and examples thereof include, but are not limited to, external preparations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0063] The appropriate dosage of the above pharmaceutical composition can be determined by considering factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity.

[0064] Cell-derived exosomes according to one embodiment of the present application can promote the growth and migration of fibroblasts, and thus have excellent skin regeneration or wound healing effects.

[0065] In addition, cell-derived exosomes according to one embodiment of the present application have an excellent effect on the growth of chondrocytes, and thus have an excellent effect on cartilage regeneration or prevention and / or treatment of osteoarthritis.

[0066] Figure 1 shows a Western blot confirming that the exosomes of the present invention express cartilage homeostasis factors BMP6 or TGFβ3.

[0067] Figures 2 and 3 confirm that BMP6-expressing exosomes or TGFβ3-expressing exosomes exhibit a fibroblast growth-promoting effect.

[0068] Figure 4 shows the migration ability of cells after scratching a fibroblast culture plate treated with BMP6-expressing exosomes or TGFβ3-expressing exosomes.

[0069] Figure 5 shows the results of confirming the effect of BMP6-expressing exosomes or TGFβ3-expressing exosomes on the proliferation capacity of fibroblasts using crystal violet staining.

[0070] Figure 6 shows the toxicity of BMP6-expressing exosomes or TGFβ3-expressing exosomes to fibroblasts confirmed by MTS assay.

[0071] Figures 7 and 8 confirm that BMP6-expressing exosomes or TGFβ3-expressing exosomes exhibit a chondrocyte growth-promoting effect.

[0072] Figure 9 shows the migration ability of cells after scratching a chondrocyte culture plate treated with TBMP6-expressing exosomes or TGFβ3-expressing exosomes.

[0073] Figure 10 shows the results of confirming the effect of BMP6-expressing exosomes or TGFβ3-expressing exosomes on the proliferation ability of chondrocytes using crystal violet staining.

[0074] Figure 11 shows the toxicity of BMP6-expressing exosomes or TGFβ3-expressing exosomes to chondrocytes confirmed by MTS assay.

[0075] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these examples.

[0076]

[0077] Example 1: Production of transformed stem cells into which a cartilage homeostasis factor gene has been introduced.

[0078] According to the methods of Examples 1-1 to 1-3, a cartilage homeostasis factor (BMP6 or TGFβ3) gene was introduced into stem cells that release exosomes, thereby producing transformed stem cells that express exosomes containing the cartilage homeostasis factor gene.

[0079]

[0080] 1-1. Preparation of immortalized stem cells

[0081] Immortalized stem cells (ASC52Telo, TERT overexpressing adipose-derived mesenchymal stem cells hTERT immortalized adipose-derived mesenchymal stem cells (ATCC, SCRC-4000)) were purchased from ATCC and cultured in DMEM (Dulbeccos modified Eagle's medium) at 5% CO2 and 37°C.

[0082]

[0083] 1-2. Lentiviral vector manufacturing and storage

[0084] Lentiviral vectors were used to transduce cartilage homeostasis factor genes.

[0085] Specifically, the GFP (green fluorescent protein) site of the pLVTHM vector (Addgene) was replaced with polynucleotide sequences encoding TGFβ3 (Transforming growth factor β3), LRRC32 (Leucine-rich repeat-containing protein 32), and BMP6 (Bone morphogenetic protein 6), thereby producing pLVTHM-TGFβ3, pLVTHM-LRRC32, and pLVTHM-BMP6 recombinant lentiviral vectors for use in transfection of cartilage homeostasis factors, respectively.

[0086] To concentrate the recombinant lentiviral vector, 293T cells (HEK293T cells overexpressing gasdermin proteins, see KR10-2022-0147494) were transfected with 12 μg of the recombinant lentiviral vector, 85 μg of psPax2, and 3 μg of pMD2G using Lipofectamine 2000 (Invitrogen). After 24 and 48 hours, the cells were centrifuged at 26,000 rpm for 2 hours at 4°C, the supernatant was removed, and the recombinant lentiviral pellet was resuspended in 200 μl of DMEM and stored at -80°C.

[0087]

[0088] 1-3. Lentiviral vector transfection

[0089] The immortalized stem cells prepared in Example 1-1 were 5×10 4 The cells were seeded into a 24-well plate at a density of 10 cells / well, and after 24 hours, 20 μl of the recombinant lentiviral vector prepared in Example 1-2 was added to DMEM medium, cultured for 16 hours, and then the cells were washed. After 48 hours, the above process was repeated once more to increase the transfection rate of the recombinant lentiviral vector.

[0090]

[0091] Example 2: Culturing of transformed stem cells and exosome isolation

[0092] 1×10 transformed stem cells into which the cartilage homeostasis factor gene was introduced, manufactured in Example 1 6 Cells / ㎖ were cultured in DMEM medium under 5% CO2 and 37°C conditions. To isolate exosomes from transformed stem cells, the medium was simultaneously treated with IFN-γ (10 U / ㎖) and TNF-α (50 ng / ㎖), the oxygen concentration was lowered to 3%, and cultured for 24 hours. Then, each culture medium was collected, filtered, and concentrated at low temperature to obtain a culture medium containing exosomes derived from immortalized stem cells.

[0093] Afterwards, the culture medium was centrifuged at 300×g for 10 minutes, and the supernatant was separated. The separated supernatant was centrifuged at 2,000×g for 20 minutes, and the supernatant was separated. The separated supernatant was centrifuged again at 100,000×g for 70 minutes, and the pellet from which the supernatant was removed was suspended in PBS and sonicated to isolate exosome particles.

[0094]

[0095] Example 3: Confirmation of expression of cartilage homeostasis factors in isolated exosomes.

[0096] Western blot analysis was performed to determine whether cartilage homeostasis factors were expressed from the isolated exosomes.

[0097] Specifically, the exosomes isolated in Example 2 were washed with PBS and then dissolved in 50 μL of an aqueous solution containing 12.5 μL of RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) and a phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA) and 5 μL of 1 M DiThioThreitol (DTT). The lysate was boiled at 100°C for 5 min to prepare an exosome sample for Western blotting. The sample was analyzed by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and blocked with PBS containing Tween-20 in 5% skim milk powder. Afterwards, the cells were incubated with primary antibodies against TGFβ3, BMP6, LRRC32, and GAPDH at 4°C overnight, and then reacted with secondary antibodies at room temperature for 1 hour.

[0098] Antibody-antigen complexes were detected using an enhanced chemiluminescent kit (Pierce, Rockford, IL, USA), and signal intensities were measured using a Fusion Solo 2 image analyzer (Vilber Lourmat, Paris, France).

[0099] As a result, as illustrated in Fig. 1, exosomes derived from immortalized stem cells transfected with the pLVTHM-TGFβ3, pLVTHM-BMP6, and pLVTHM-LRRC32 recombinant lentiviral vectors were confirmed to express TGFβ3, BMP6, or LRRC32, respectively. In addition, exosomes expressing both TGFβ3 and BMP6 were also confirmed.

[0100]

[0101] Example 4. Effect of exosomes containing cartilage homeostasis factors on fibroblast proliferation.

[0102] 4-1. Growth and wound healing effects of fibroblasts

[0103] Human fibroblasts (hTERT-immortalized human foreskin fibroblast cell line, BJ-5ta (ATCC® CRL-4001™)) were prepared. Fibroblasts cultured in a 150 mm plate were seeded at 8 × 10 in a 6-well plate. 4 Inoculate cells / well, 3.2 × 10 in 60 mm plates 6 Cells were inoculated per well and cultured in an incubator under 5% CO2 and 37°C conditions for 48 hours.

[0104] Exosomes filtered through 0.22 μm were dispensed in 100 μL or 500 μL amounts to 6-well plates, excluding the control group, and the difference in cell growth over time was confirmed.

[0105] After confirming cell growth, a 60 mm plate was scratched on the bottom with a sterilized tip, 100 μL or 500 μL of exosomes were dispensed, and the difference in cell growth in the scratched area was confirmed.

[0106] After obtaining cell images of 6-well plates and 60 mm plates, they were washed three times with warmed 1X PBS, and then 4% paraformaldehyde was dispensed enough to submerge the plate surface for cell fixation and fixed for 30 minutes at room temperature. After fixation, each plate was washed three times with 1X PBS, and 1 g (1%) of crystal violet was made into 100 ml of 20% ethanol, dispensed enough to submerge the plate surface, and the staining process was performed for 10 minutes at room temperature.

[0107] After removing the crystal violet, the cells were washed five times with 1XPBS to remove any dye that was not bound to the cells. The shape and distribution of the washed cells were observed under a microscope.

[0108] According to Figures 2 to 4, human fibroblasts grew faster as the amount of exosomes expressing TGFβ3 or BMP6 increased. Figures 2 and 3 confirm that BMP6-expressing exosomes or TGFβ3-expressing exosomes have a fibroblast growth-promoting effect. Figure 4 confirms the migration ability of cells after scratching a fibroblast culture plate treated with TGFβ3-expressing exosomes. In Figure 4, A shows immediately after scratching, B shows 6 hours after scratching, C shows the state before staining after overnight scratching, and D shows the state after staining after overnight scratching. As can be seen in Figures 2 to 4, the damaged area was regenerated in all experimental groups treated with exosomes expressing TGFβ3 or BMP6.

[0109] The results of the experiment suggest that exosomes containing cartilage homeostasis factors have an excellent skin regeneration effect.

[0110]

[0111] 4-2. MTS Analysis

[0112] Fibroblasts were cultured in 150 mm plates and after cell stabilization, 1 × 10 fibroblasts were seeded in 96 well plates. 4 Cells were plated at the number per well and cultured in an incubator under 5% CO2 and 37℃ conditions for 48 hours.

[0113] Excluding the control group, 2 μL or 10 μL of exosomes filtered through a 0.22 μm filter were dispensed into 96-well plates. After 48 hours, 10 μL of MTS reagent was dispensed into each well and cultured in an incubator under 5% CO2 and 37°C conditions to induce color development, which was checked every 5 minutes until the color began to change.

[0114] When the MTS reagent started to change color from yellow to purple, the absorbance was measured at a wavelength of 490 nm.

[0115] As shown in Table 1 and Figures 5 and 6, the experimental results showed that TGFβ3-expressing exosomes, BMP6-expressing exosomes, and exosomes expressing both TGFβ3 and BMP6 were non-toxic to fibroblasts and further promoted cell growth. It was confirmed that cell growth increased rapidly as the amount of exosomes increased. In addition, it was confirmed that the cell growth rate of exosomes expressing both TGFβ3 and BMP6 was greater.

[0116] Control exosomes 2 μl Exosomes 10 μl BMP6-expressing exosomes 0.258 0.32 10.44 85 TGFβ3-expressing exosomes 0.234 0.28 10.410 BMP6 and TGFβ3-expressing exosomes 0.210 0.38 50.483

[0117] According to the experimental results, the exosomes derived from transformed stem cells into which the cartilage homeostasis factor gene of the present invention has been introduced can be effectively utilized for skin regeneration and wound treatment.

[0118] According to the experimental results, the exosomes derived from transformed stem cells into which the cartilage homeostasis factor gene of the present invention has been introduced can be effectively utilized for skin regeneration and wound treatment.

[0119]

[0120] Example 5. Effect of exosomes containing cartilage homeostasis factors on chondrocyte proliferation.

[0121] 5-1. Growth of chondrocytes and wound healing effects

[0122] Chondrocytes were obtained from the orthopedic laboratory of Ajou University Hospital using rabbit chondrocytes isolated using the following method. Specifically, cartilage from the knee joint of a New Zealand White rabbit was sliced ​​thinly and isolated, and then washed three times with PBS. The cartilage was placed in DMEM containing 0.2% type II collagenase and incubated at 120 rpm for 6 hours in a shaking incubator at 37°C. After the reaction, the cell suspension was filtered through a cell strainer and centrifuged at 1600 rpm for 10 minutes. The isolated chondrocytes were washed three times with PBS and a cell suspension was prepared with medium (DMEM + 10% FBS). The chondrocytes were cultured at a density of 5 × 10 4 cells / cm 2 After placing the cells in a 150 mm plate to achieve a cell density of , they were cultured in an incubator under conditions of 5% CO2 and 37°C.

[0123] Chondrocytes cultured in 150 mm plates were seeded at 8 × 10 in 6-well plates. 4 Inoculate cells / well, 3.2 × 10 in 60 mm plates 6 Cells were inoculated per well and cultured in an incubator under 5% CO2 and 37°C conditions for 48 hours.

[0124] Exosomes filtered through 0.22 μm were dispensed in 100 μL or 500 μL amounts to 6-well plates, excluding the control group, and the difference in cell growth over time was confirmed.

[0125] After confirming cell growth, a 60 mm plate was scratched on the bottom with a sterilized tip, 100 μL or 500 μL of exosomes were dispensed, and the difference in cell growth in the scratched area was confirmed.

[0126] After obtaining cell images of 6-well plates and 60 mm plates, they were washed three times with warmed 1X PBS, and then 4% paraformaldehyde was dispensed enough to submerge the plate surface for cell fixation and fixed for 30 minutes at room temperature. After fixation, each plate was washed three times with 1X PBS, and 1 g (1%) of crystal violet was made into 100 ml of 20% ethanol, dispensed enough to submerge the plate surface, and the staining process was performed for 10 minutes at room temperature.

[0127] After removing the crystal violet, the cells were washed five times with 1XPBS to remove any dye that was not bound to the cells. The shape and distribution of the washed cells were observed under a microscope.

[0128] According to Figures 7 to 9, as the amount of exosomes expressing TGFβ3 or BMP6 increased, chondrocytes grew faster. Figures 7 and 8 confirmed that BMP6-expressing exosomes or TGFβ3-expressing exosomes exhibited a growth-promoting effect on chondrocytes. Figure 9 shows the migration ability of cells after scratching a chondrocyte culture plate treated with TGFβ3-expressing exosomes. In Figure 9, A shows immediately after scratching, B shows 6 hours after scratching, C shows the state before staining after overnight scratching, and D shows the state after staining after overnight scratching. As can be seen in Figures 7 to 9, the damaged area was regenerated in all experimental groups treated with exosomes expressing TGFβ3 or BMP6.

[0129] The results of the experiment suggest that exosomes containing cartilage homeostasis factors have an excellent cartilage regeneration effect.

[0130]

[0131] 5-2. MTS Analysis

[0132] Chondrocytes were cultured in a 150 mm plate and after cell stabilization, 1 × 10 chondrocytes were seeded in a 96-well plate. 4 Cells were plated at the number per well and cultured in an incubator under 5% CO2 and 37℃ conditions for 48 hours.

[0133] Excluding the control group, 2 μL or 10 μL of exosomes filtered through a 0.22 μm filter were dispensed into 96-well plates. After 48 hours, 10 μL of MTS reagent was dispensed into each well and cultured in an incubator under 5% CO2 and 37°C conditions to induce color development, which was checked every 5 minutes until the color began to change.

[0134] When the MTS reagent started to change color from yellow to purple, the absorbance was measured at a wavelength of 490 nm.

[0135] As shown in Table 2 and Figures 10 and 11, the experimental results showed that TGFβ3-expressing exosomes, BMP6-expressing exosomes, and exosomes expressing both TGFβ3 and BMP6 were non-toxic to chondrocytes and further promoted cell growth. It was confirmed that cell growth increased rapidly as the amount of exosomes increased. In addition, it was confirmed that the cell growth rate of exosomes expressing both TGFβ3 and BMP6 was greater.

[0136] Control exosomes 2 μl Exosomes 10 μl BMP6-expressing exosomes 0.20 3 0.26 1 0.37 TGFβ3-expressing exosomes 0.20 9 0.26 0 0.39 4 BMP6 and TGFβ3-expressing exosomes 0.20 5 0.27 5 0.415

[0137] According to the experimental results, the exosomes derived from transformed stem cells into which the cartilage homeostasis factor gene of the present invention has been introduced can be effectively used for cartilage regeneration treatment and osteoarthritis treatment.

[0138] The present invention has been described above, focusing on specific embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. Cell-derived exosomes containing cartilage homeostasis factors.

2. In paragraph 1, The above cartilage homeostasis factors are A cell-derived exosome, wherein at least one is selected from the group consisting of TGFβ1 (Transforming growth factor β1), TGFβ2 (Transforming growth factor β2), TGFβ3 (Transforming growth factor β3), LRRC32 (Leucine-rich repeat-containing protein 32), FGF18 (Fibroblast growth factor 18), BMP2 (Bone morphogenetic protein 2), BMP6 (Bone morphogenetic protein 6), and BMP7 (Bone morphogenetic protein 7).

3. In paragraph 1, The above cells are cell-derived exosomes, which are transformed cells by introducing a cartilage homeostasis factor gene.

4. In paragraph 1, A cell-derived exosome, wherein the cell is any one cell selected from the group consisting of stem cells, immune cells, and somatic cells.

5. In paragraph 5, A cell-derived exosome, wherein the stem cell is any one cell selected from the group consisting of mesoderm stem cells, pluripotent stem cells, multipotent stem cells, and unipotent stem cells.

6. In paragraph 5, The above stem cell is an immortalized stem cell, a cell-derived exosome.

7. In paragraph 5, A cell-derived exosome, wherein the immune cell is any one cell selected from the group consisting of a dendritic cell, a natural killer cell, a T cell, a B cell, a regulatory T cell (Treg cell), a natural killer T cell, an innate lymphoid cell, a macrophage, a granulocyte, a chimeric antigen receptor-T cell, a lymphokine-activated killer cell, and a cytokine-induced killer cell.

8. In paragraph 5, A cell-derived exosome, wherein the somatic cell is any one cell selected from the group consisting of fibroblasts, chondrocytes, synovial cells, keratinocytes, adipocytes, osteoblasts, osteoclasts, and peripheral blood mononuclear cells.

9. Cosmetic composition for skin regeneration containing the exosome of paragraph 1 10. A pharmaceutical composition for wound treatment, cartilage regeneration, or prevention or treatment of osteoarthritis, comprising the exosome of paragraph 1.

11. A method for treating a wound or osteoarthritis, comprising administering to a subject a therapeutically effective amount of the exosome of paragraph 1 in a pharmaceutically acceptable salt.

12. Use of the exosome of claim 1 for wound treatment or osteoarthritis treatment.

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