Exosome containing polydeoxyribonucleotide, composition containing exosome and preparation method of exosome

Abstract

The present invention relates to a polydeoxyribonucleotide (PDRN)-containing exosome, a composition comprising the same, and a method for preparing the same, the PDRN-containing exosome of the present invention is derived from fish testis tissue, and contains low-molecular, high-purity PDRN. Moreover, the exosome containing a large amount of low-molecular PDRN can be induced through a simple ultrasonic treatment process, the low-molecular PDRN exosome can be prepared through ultrasonic treatment of the separated exosome, and the low-molecular PDRN exosome has high intracellular absorption rate and can be effectively used for tissue regeneration or preparation of anti-inflammatory compositions.

Description

Technical Field

[0001] The present invention relates to exosomes containing polydeoxyribonucleotide (PDRN), compositions containing the same, and methods for preparing the same, and particularly to PDRN exosomes derived from fish testis tissue, tissue regeneration compositions containing the same, anti-inflammatory cosmetic compositions, and methods for preparing the same. Background Art

[0002] Polydeoxyribonucleotide (PDRN) contains phosphoric acid and four bases: adenine, guanine, thymine, and cytosine. The above four bases are combined with deoxyribose and phosphoric acid as repeating units to form a high molecular weight double-stranded polymer. In addition, four bases, adenine (A), guanine (G), cytosine (C), and uracil (U), sugar, and phosphoric acid are combined as repeating units to form a single-stranded polymer.

[0003] These genetic materials, such as DNA or RNA, can be hydrolyzed and are distributed in all organisms in a state of being bound to proteins or in a free state. The free single-stranded RNA forms a hairpin structure through hydrogen bonds between complementary parts, and a part of it becomes double-stranded.

[0004] Compared with other tissues, the PDRN content in animal sperm or eggs, plant growth points, etc. is relatively high. These genetic materials have different effects and efficiencies according to the differences in low molecular weight, high molecular weight, and extraction methods. The final product PDRN is low molecularized to ensure safety, forms a complex with adenosine A2 receptor, promotes VEGF secretion, induces angiogenesis, and activates fibroblasts, osteoblasts, and chondrocytes.

[0005] PDRN (Polydeoxyribonucleotide) has the viscoelasticity of polymer characteristics and can play a role in tissue repair when injected intradermally. That is to say, it is not a simple skin filler, but the main component of a new concept tissue repair medical device that induces skin tissue regeneration by proliferating fibroblasts that make up skin tissue and increasing their activity, and promoting the secretion of extracellular matrix such as collagen. It has a special effect on tissue regeneration and inflammation alleviation in our body.

[0006] PDRN promotes the secretion of various growth factors by stimulating the A2 purinergic receptor, a skin regeneration signaling molecule. It generates capillaries through VEGF (vascular endothelial growth factor), improves blood circulation, has an anti-inflammatory effect, prevents capillary leakage, thereby promoting skin regeneration, and has no special side effects. It is effective for burns and chronic wounds, etc. In Europe, it has been approved for various pharmaceutical applications, and in Korea, it is also used for wound treatment and tissue regeneration, etc.

[0007] Meanwhile, exosomes are naturally secreted nanovesicles with a diameter of 30 - 200 nm and are known to be important nanocarriers for transporting genetic material from their source cells. However, it is currently difficult to produce exosomes in large quantities. For example, when culturing 60 million mesenchymal stem cells in 1 liter of culture medium, based on the protein content, 1 - 2 mg of exosomes can be obtained. This amount can be used for treatment experiments on several mice, while in the case of humans, for example, the amount of exosomes used for treating graft-versus-host disease (GVHD) is 0.05 - 0.6 mg per kilogram of patient body weight, based on the protein content. Therefore, the clinical and commercial applications of exosomes require a technology that can increase the yield.

[0008] Therefore, the present invention is a technology that can produce a large amount of exosomes containing PDRN by applying ultrasonic stimulation to fish testis tissue, and it is expected to exhibit the advantages of both PDRN and exosomes.

[0009] Content of the Invention

[0010] Technical Problems to be Solved by the Invention

[0011] The technical problems to be solved by the present invention are to provide exosomes containing PDRN.

[0012] Furthermore, the technical problems to be solved by the present invention are to provide a method for preparing exosomes containing PDRN.

[0013] Furthermore, the technical problems to be solved by the present invention are to provide a tissue regeneration composition and an anti-inflammatory cosmetic composition containing exosomes containing PDRN.

[0014] The technical problems to be solved by the present invention are not limited to the above technical problems, and those skilled in the art to which the present invention pertains can clearly understand other technical problems not mentioned from the following description.

[0015] Technical Solutions for Solving the Problems

[0016] To achieve the above object, an embodiment of the present invention provides an exosome isolated from fish testis tissue, and the interior of the exosome contains polydeoxyribonucleotide (PDRN) derived from fish testis tissue.

[0017] In an embodiment of the present invention, the PDRN can be low-molecularized to a length of 1 kb or less.

[0018] In an embodiment of the present invention, the PDRN can be low-molecularized to a length of 100 bp to 500 bp.

[0019] In an embodiment of the present invention, the fish can be salmon or cod.

[0020] In an embodiment of the present invention, the fish can be salmon, and the exosome can contain 500 pg to 1000 pg of PDRN.

[0021] To achieve the above object, another embodiment of the present invention provides a method for preparing an exosome containing PDRN, including the following steps: pulverizing fish testis tissue; subjecting the tissue pulverizate to ultrasonic treatment; and isolating an exosome containing PDRN from the ultrasonically treated tissue pulverizate.

[0022] In an embodiment of the present invention, the frequency of the ultrasonic treatment can be 10 KHz to 30 KHz, the intensity can be 15% to 40% of the maximum output intensity, and the treatment time can be 5 seconds to 35 seconds.

[0023] In an embodiment of the present invention, the PDRN can be low-molecularized to a length of 1 kb or less.

[0024] In an embodiment of the present invention, the step of isolating the exosome containing PDRN can include the following steps: centrifuging the ultrasonically treated tissue pulverizate to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and centrifuging and concentrating the filtrate.

[0025] In an embodiment of the present invention, the following step can further be included: subjecting the isolated exosome containing PDRN to ultrasonic treatment.

[0026] In an embodiment of the present invention, the frequency of the ultrasonic treatment of the exosome can be 10 KHz to 30 KHz, the intensity can be 15% to 40% of the maximum output intensity, and the treatment time can be 70 seconds to 120 seconds.

[0027] In an embodiment of the present invention, after the exosome is subjected to ultrasonic treatment, the PDRN can be low-molecularized to a length of 100 bp to 500 bp.

[0028] To achieve the above object, another embodiment of the present invention provides a composition for tissue regeneration, which contains the exosomes.

[0029] To achieve the above object, still another embodiment of the present invention provides an anti-inflammatory cosmetic composition, which contains the exosomes.

[0030] Effects of the Invention

[0031] The present invention relates to exosomes containing polydeoxyribonucleotide (PDRN), a composition containing the same, and a preparation method thereof. The PDRN-containing exosomes of the present invention are derived from fish testis tissue and contain low-molecular-weight and high-purity PDRN. Moreover, through a simple ultrasonic treatment process, exosomes containing a large amount of low-molecular-weight PDRN can be induced, and by subjecting the isolated exosomes to ultrasonic treatment, low-molecular-weight PDRN exosomes can be prepared, which have a high intracellular absorption rate and can be effectively used for the preparation of tissue regeneration or anti-inflammatory compositions.

[0032] The effects of the present invention are not limited to the above effects, and it should be understood to include all effects that can be inferred from the concepts of the invention described in the specification or claims of the present invention. Brief Description of the Drawings

[0033] Figure 1 Shows the results of immunofluorescence image analysis of salmon testis PDRN exosomes. The PDRN exosomes extracted from salmon testis were subjected to overnight immunofluorescence staining and DID staining (red, Vybrant TM ) with a PE-labeled anti-CD63 antibody (green, H5C6, PE, eBioscience TM DID cell labeling solution; Thermofisher). The stained PDRN exosomes were analyzed using an immunofluorescence image confocal microscope (DMi8, LEICA), and the merged images were analyzed in yellow on the uppermost side.

[0034] Figure 2 Shows the results of flow cytometry analysis of salmon testis PDRN exosomes. After labeling the ultrasound-treated salmon testis PDRN exosomes with aldehyde / sulfate-latex beads (A37304, Thermofisher), flow cytometry (FACSMelody TM , BDBiosciences) was performed using a PE-labeled CD63 (H5C6, Invitrogen) antibody and an APC-labeled CD81 (1D6, Invitrogen) antibody.

[0035] Figure 3 Show the size distribution and quantitative analysis results of salmon low molecular weight PDRN exosomes. Salmon testis PDRN exosomes were divided into two groups according to the sonication conditions, and the size and size distribution of PDRN exosomes were confirmed using Nanosight. #1: Exosomes without sonication (No Ultrasound), #2: Exosomes with sonication (Ultrasound).

[0036] Figure 4 Show the comparative analysis results of the number of PDRN exosomes isolated from salmon testis according to the sonication conditions. PDRN exosomes were isolated from salmon testis according to the sonication conditions, and the number of exosomes in each group was compared. *p≤0.05, **p≤0.01.

[0037] Figure 5 Show the DNA electrophoresis and content analysis results of low molecular weight PDRN exosomes from salmon testis according to the sonication conditions. Among them, 5A shows the analysis results after electrophoresis on a 1.3% agarose gel for 30 minutes of genomic DNA (Tissue) extracted from salmon testis (tissue lysate), DNA extracted from salmon testis PDRN exosomes without sonication (No Ultrasound), DNA extracted from salmon testis PDRN exosomes with one-time sonication (Ultrasound 1), and DNA of salmon testis PDRN exosomes (Ultrasound 2) that were sonicated a second time after sonication and isolation of exosomes (Ultrasound 1). 5B shows the results of comparing the DNA content in each exosome after isolating PDRN exosomes from salmon testis according to the sonication conditions. *p≤0.05, **p≤0.01.

[0038] Figure 6 Show the cell viability analysis results of salmon low molecular weight PDRN exosomes (sPDRN Exosome). Human skin keratinocytes were sonicated and treated with low molecular weight PDRN exosomes (sPDRN Exosome) induced from salmon testis at different concentrations, and the cell viability was analyzed. After culturing for 24 hours, it was treated with Ez-cytox (DogenBio, Korea), and after reacting for 1 hour and 30 minutes, the absorbance was measured at 450 nm. *p≤0.05.

[0039] Figure 7Shows the results of comparative analysis of cellular uptake of low-molecular-weight-induced sPDRN exosomes and PDRN by immunofluorescence assay. To confirm the cellular uptake of ultrasound-treated low-molecular-weight-induced PDRN exosomes, fluorescently labeled PDRN and low-molecular-weight-induced sPDRN exosomes stained with DID were analyzed by immunofluorescence. The stained sPDRN exosomes were analyzed using an immunofluorescence image confocal microscope (DMi8, LEICA).

[0040] Figure 8 Shows the results of comparative analysis of intracellular uptake rates of low-molecular-weight-induced sPDRN exosomes and PDRN using flow cytometry. 1×10 9 salmon low-molecular-weight-induced exosomes (sPDRN exosomes) stained with DID and PDRN were separately treated with human fibroblasts and cultured for 24 hours. The next day, measurements were performed using flow cytometry (FACSMelody TM , BDBiosciences). The results are shown in the flow cytometry analysis graph (8A), and the measured values are shown in the chart (8B).

[0041] Figure 9 Shows the results of analysis of the tissue regeneration efficacy of low-molecular-weight-induced sPDRN exosomes. The tissue regeneration efficacy of salmon PDRN exosomes induced by low-molecular-weight through ultrasound treatment was confirmed in human skin keratinocytes (HaCaT) (9A). A migration test was performed on human skin keratinocytes. After inducing an inflammatory response with TNF-α and INF-γ for 24 hours, they were treated with culture medium (Cont), low-molecular-weight-induced PDRN exosomes (sPDRN exo), and PDRN respectively. After 24 hours, the degree of cell migration was measured under a microscope (9B). The area of Figure 9 A was quantified using the Image J program. **p ≤ 0.01.

[0042] Figure 10 Shows the results of evaluation of the anti-inflammatory efficacy of low-molecular-weight-induced sPDRN exosomes in human skin keratinocytes. After simultaneously treating human skin keratinocytes with TNF-α and INF-γ to induce an inflammatory response for 24 hours, salmon testis low-molecular-weight sPDRN exosomes were treated for 24 hours under different concentrations (10A) and different ultrasound treatment conditions (10B), and the anti-inflammatory response was analyzed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

[0043] Figure 11 Shows the results of immunofluorescence image analysis of cod testis PDRN exosomes. Using a PE-labeled anti-CD63 antibody (green, H5C6, PE, eBioscienceTM ) Overnight immunofluorescence staining and DID staining (red, Vybrant TM DID cell labeling solution; Thermofisher) were performed on PDRN exosomes extracted from cod testis. The stained PDRN exosomes were analyzed using a confocal microscope for immunofluorescence images (DMi8, LEICA), and the merged images were analyzed in yellow on the uppermost side.

[0044] Figure 12 The flow cytometry analysis results of the isolated cod testis PDRN exosomes are shown. After labeling the ultrasonically treated cod testis PDRN exosomes with aldehyde / sulfate-latex beads (A37304, Thermofisher), flow cytometry (FACSMelody TM , BDBiosciences) was performed using a PE-labeled CD63 (H5C6, Invitrogen) antibody and an APC-labeled CD81 (1D6, Invitrogen) antibody.

[0045] Figure 13 The size distribution and quantity analysis results of cod low-molecular-weight PDRN exosomes are shown. The cod testis PDRN exosomes were divided into two groups according to the ultrasonic conditions, and the size and size distribution of the PDRN exosomes were confirmed using Nanosight. #1: Exosomes without ultrasonic treatment (No Ultrasound), #2: Exosomes with ultrasonic treatment (Ultrasound).

[0046] Figure 14 The comparative analysis results of the quantity of PDRN exosomes isolated from cod testis according to the ultrasonic conditions are shown. PDRN exosomes were isolated from cod testis according to the ultrasonic conditions, and the quantity of each exosome was compared. ***p ≤ 0.001.

[0047] Figure 15Shows the results of DNA electrophoresis and content analysis of low-molecular-weight PDRN exosomes from cod testes according to ultrasonic conditions. Among them, 15A is the genomic DNA (Tissue) extracted from cod testes (tissue lysate), the DNA extracted from cod testis PDRN exosomes without ultrasonic treatment (No Ultrasound), the DNA extracted from cod testis PDRN exosomes after one ultrasonic treatment (Ultrasound 1), and the analysis results after electrophoresis for 30 minutes on a 1.3% agarose gel of the DNA of cod testis PDRN exosomes (Ultrasound 2) that are secondarily ultrasonically treated after separating exosomes (Ultrasound 1) after ultrasonic treatment. 15B is the result of comparing the DNA content in each exosome after separating PDRN exosomes from cod testes according to ultrasonic conditions. ***p≤0.001.

[0048] Figure 16 Shows the results of cell viability analysis of low-molecular-weight cod PDRN exosomes (cPDRN Exosome). Human skin keratinocytes were ultrasonically treated and then treated with low-molecular-weight induced PDRN exosomes (cPDRN Exosome) derived from cod testes at different concentrations, and the cell viability was analyzed. After culturing for 24 hours, it was treated with Ez-cytox (DogenBio, Korea), and after reacting for 1 hour and 30 minutes, the absorbance was measured at 450 nm. *p≤0.05, **p≤0.01, ***p≤0.001.

[0049] Figure 17 Shows the results of confirming the cellular uptake of low-molecular-weight induced cPDRN exosomes. To confirm the cellular uptake of low-molecular-weight induced PDRN exosomes after ultrasonic treatment (Ultrasound), fluorescently labeled PDRN and low-molecular-weight induced cPDRN exosomes stained with DID were analyzed by immunofluorescence. The stained cPDRN exosomes were analyzed using an immunofluorescence image confocal microscope (DMi8, LEICA).

[0050] Figure 18 Shows the results of comparative analysis of the intracellular uptake rates of cPDRN exosomes and PDRN. 1×10 9 low-molecular-weight cod exosomes (cPDRN exosomes) and PDRN stained with DID were respectively treated with human fibroblasts and cultured for 24 hours. The next day, it was measured by flow cytometry (FACSMelody TM , BD Biosciences), and the results were shown in the flow cytometry analysis graph (18A), and the measured values were shown in the chart (18B).

[0051] Figure 19Shows the analysis results of the tissue regeneration efficacy of low-molecular-weight-induced cPDRN exosomes. The tissue regeneration efficacy of cod PDRN exosomes induced by low molecular weight through sonication was confirmed in human skin keratinocytes. A migration test (19A) was performed on human skin keratinocytes. After inducing an inflammatory response with TNF-α and INF-γ for 24 hours, the cells were treated with culture medium (Cont), low-molecular-weight-induced PDRN exosomes (cPDRN exo), and PDRN, respectively. After 24 hours, the degree of cell migration was measured under a microscope (19B). The area of Figure 19 A was quantified using the Image J program. *p≤0.05.

[0052] Figure 20 Shows the evaluation results of the anti-inflammatory efficacy of low-molecular-weight-induced cPDRN exosomes in human skin keratinocytes. After simultaneously treating human skin keratinocytes with TNF-α and INF-γ to induce an inflammatory response for 24 hours, the cod testis low-molecular-weight PDRN exosomes were treated under different concentrations (20A) and different sonication conditions (20B) for 24 hours to analyze the anti-inflammatory response. *p≤0.05, ***p≤0.001. Detailed implementation mode

[0053] The present invention will be described in detail below.

[0054] The present invention relates to an exosome isolated from fish testis tissue, and the interior of the exosome contains polydeoxyribonucleotide (PDRN) derived from fish testis tissue.

[0055] The fish refers to a gill-bearing vertebrate living in water, which can be a saltwater fish or a freshwater fish. For example, it can be one or more genera of fish belonging to the genera Salmo, Oncorhynchus, Salvelinus, Brachymystax, and Hucho in the Salmonidae family. More specifically, it can be salmon. In addition, the fish can be, but is not limited to, cod in the Gadidae family, but is not limited thereto.

[0056] The PDRN can be contained in fish testis tissue. The PDRN in the exosomes of the present invention can be low-molecular-weighted to a length of 1 kb or less, specifically, a length of 500 bp to 1 kb.

[0057] Furthermore, when the exosomes isolated after sonication according to the following method are subjected to a second sonication, the PDRN can be low-molecular-weighted to 500 bp or less, specifically, a length of 100 bp to 500 bp or 200 bp to 400 bp.

[0058] The exosomes of the present invention contain highly purified low-molecular-weight PDRN. For example, when the fish is salmon, the exosomes may contain, for example, 500 pg to 1000 pg or 700 pg to 1000 pg of PDRN. And when the fish is cod, the exosomes may contain, for example, 50 pg to 80 pg of PDRN.

[0059] The PDRN and the exosomes are derived from the testis tissue of the same fish. The PDRN is low-molecular-weighted by subjecting the fish testis tissue to ultrasonic treatment, and the cells release the low-molecular-weighted PDRN in the form of exosomes into the extracellular environment, thereby separating and obtaining the exosomes.

[0060] The present invention relates to a method for preparing exosomes containing PDRN.

[0061] The present invention includes the following steps: pulverizing the fish testis tissue; subjecting the tissue pulverizate to ultrasonic treatment; and separating exosomes containing PDRN from the tissue pulverizate after the ultrasonic treatment.

[0062] Regarding the fish, PDRN, and exosomes, they are the same as those described above.

[0063] The step of pulverizing the fish testis tissue can be carried out by using methods known in the art and under known conditions. For example, it can be carried out by grinding the fish testis in a container, but it is not limited thereto.

[0064] For example, the frequency of the ultrasonic treatment can be 10 KHz to 30 KHz or 15 KHz to 25 KHz, the intensity can be 15% to 40% or 20% to 30% of the maximum output intensity, and the treatment time can be 5 seconds to 35 seconds, 10 seconds to 25 seconds, or 10 seconds to 15 seconds, but it is not limited to the above range. If it is less than the above range, it is difficult to low-molecular-weight the PDRN. If it exceeds the above range, the PDRN may be over-low-molecular-weighted or the cells of the fish testis tissue may be completely destroyed, thus making it impossible to separate the exosomes. Therefore, in order to effectively low-molecular-weight the PDRN and at the same time easily produce exosomes containing highly purified PDRN, it is preferably carried out within the above range.

[0065] The step of separating exosomes containing PDRN can be carried out by using methods known in the art and under known conditions. For example, it may include the following steps: centrifuging the tissue pulverizate after the ultrasonic treatment to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and centrifuging and concentrating the filtrate, but it is not limited thereto.

[0066] The present invention may further include the following step: subjecting the separated exosomes containing PDRN to ultrasonic treatment.

[0067] For example, the frequency of ultrasonic treatment of the exosomes can be 10 KHz to 30 KHz or 15 KHz to 25 KHz, the intensity can be 15% to 40% or 20% to 30% of the maximum output intensity, and the treatment time can be 70 seconds to 120 seconds, 80 seconds to 100 seconds, or 90 seconds to 95 seconds. By performing additional ultrasonic treatment on the isolated exosomes containing PDRN, the PDRN in the exosomes is further low-molecularized, thereby increasing the intracellular absorption rate.

[0068] After the ultrasonic treatment of the exosomes, PDRN can be low-molecularized to 500 bp or less, specifically, with a length of 100 bp to 500 bp or 200 bp to 400 bp.

[0069] The present invention relates to a composition for tissue regeneration containing the exosomes.

[0070] Regarding the exosomes, it is the same as described above.

[0071] Compared with the exosomes containing PDRN isolated without ultrasonic treatment, the exosomes containing PDRN of the present invention exhibit excellent tissue regeneration ability by activating cell migration and proliferation in damaged tissues or cells.

[0072] The composition may contain growth factors capable of stimulating tissue regeneration, such as epithelial growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), platelet-derived growth factors (PDGF), transforming growth factors (TGF), leukocyte-derived growth factor (LDGF), insulin-like growth factors (IGF), etc. growth factors, adhesion factors including fibronectin, laminin, vitronectin, etc., and retinoid to smoothly induce the efficacy.

[0073] In addition, in addition to the above components, lubricants, wetting agents, emulsifiers, suspending agents, preservatives, etc. can also be added.

[0074] In addition, it may further contain a pharmaceutically acceptable carrier and agent, which are described in detail in "Remington's Pharmaceutical Sciences" (19th Edition, 1995).

[0075] The present invention relates to an anti-inflammatory cosmetic composition containing the exosomes.

[0076] Regarding the exosomes, it is the same as described above.

[0077] "Anti-inflammatory" can be used interchangeably with "inhibiting or improving inflammation", and can refer to any action that reduces the immune response and inhibits the production of inflammatory factors.

[0078] The cosmetic composition of the present invention can be formulated into a skin lotion, emulsion, toner, cosmetic soap, body wash, essence, cleansing lotion, serum, nourishing cream, mask, massage cream, etc., or can also be formulated into a moisturizing toner, astringent toner, nourishing toner, eye cream, eye serum, facial cleansing foam, cleansing lotion, powder cake, body lotion, body cream, body oil, body serum, makeup primer, foundation, shampoo or hair conditioner, etc., but not limited thereto.

[0079] When used as a cosmetic composition, other substances can be added according to the dosage form of a topical skin agent or cosmetic. For example but not limited to, if the preparation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide can be used as a carrier component; if the preparation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component; especially in the case of a spray, a propellant can also be additionally included, such as chlorofluorocarbon, propane / butane or dimethyl ether. In addition, if the preparation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component. Preferably, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid ester, polyethylene glycol or sorbitan fatty acid ester can be used, but not limited thereto. If the preparation is a suspension, the carrier component can include but not limited to a liquid diluent, such as water, ethanol or propylene glycol, a suspending agent, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum hydroxyoxide, bentonite, agar or tragacanth, etc. If the preparation is a surfactant-containing cleanser, the carrier component can include but not limited to fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, hydroxyethyl sulfonate, imidazoline derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester, etc.

[0080] The present invention will be described in detail below through examples to specifically explain the present invention.

[0081] 1. Development of salmon PDRN exosomes

[0082] (1) Isolation of exosomes from salmon testes

[0083] In order to induce the acquisition of salmon PDRN exosomes containing low-molecular-weight PDRN (polydeoxyribonucleotide) from salmon testes by ultrasonic treatment, the salmon testis tissue was ultrasonically treated under the ultrasonic conditions in Table 1, and then the exosomes were isolated and characterized.

[0084] First, 4 g of salmon testes were placed in a mortar soaked in liquid nitrogen and ground, 40 mL of PBS was added, and 10 mL of it was separately isolated and stored at -20 °C (tissue lysate). The salmon testis lysate containing PBS was sequentially added dropwise using 18G, 20G, and 26G syringes, and it was made smaller by moving it up and down, and 10 mL of the sample was separately isolated and stored on ice as the sample without ultrasonic treatment (No. 1, No Ultrasound). The remaining 20 mL of the salmon testis lysate sample was treated three times at 25% intensity for 5 seconds each (15 seconds in total).

[0085] The tissue lysate sample, the sample without ultrasonic treatment (No. 1, No Ultrasound), and the sample ultrasonically treated under condition 2 (No. 2, Ultrasound) were dispensed into 1.5 mL tubes (1 mL each), centrifuged at 14000 rpm and 4 °C for 20 minutes, and the supernatants of each group were separated and combined.

[0086] In order to confirm the low-molecular-weight PDRN exosomes derived from salmon testes according to whether they have been ultrasonically treated in the samples other than the tissue lysate sample among the samples prepared above, exosome isolation was carried out as follows.

[0087] Both the sample without ultrasonic treatment and the sample with ultrasonic treatment were filtered through a 0.2 μM filter. The filtered sample was put into an Ultra-15 centrifugal filtration device (UFC9100, Millipore), and centrifuged at 10000 g and 4 °C for 30 minutes to obtain the supernatant.

[0088] Table 1

[0089]

[0090] (2) Confirmatory analysis of exosomes isolated from salmon testes by ultrasonic treatment

[0091] (2-1) Immunofluorescence analysis of isolated salmon testis PDRN exosomes

[0092] Immunofluorescence analysis was performed on the exosome-specific marker 'CD63' to confirm the low-molecular-weight PDRN exosomes isolated from salmon testis tissue by ultrasound treatment.

[0093] To perform immunofluorescence staining on PDRN exosomes extracted from salmon testis, an anti-CD63 antibody labeled with a PE fluorophore (green, H5C6, PE, eBioscience TM ) was used to stain the exosomes, and DiD dye (red, Vybran TM DiD cell labeling solution; Thermofisher) was used to stain the lipid bilayer particles overnight, and then analyzed using a confocal microscope for immunofluorescence images (DMi8, LEICA) ( Figure 1 ).

[0094] The results showed that PDRN exosomes were stained with CD63 and DiD, and the two images were merged. These results indicate that the isolated PDRN exosomes are typical and pure exosomes with a lipid bilayer ( Figure 1 ). Analysis of the nanoparticles in the images using Image J showed that the ratio of PDRN exosomes to DiD-stained particles was over 94%, confirming that the extracted particles were high-purity exosomes.

[0095] (2-2) Flow cytometry analysis of isolated salmon testis PDRN exosomes

[0096] To confirm the low-molecular-weight PDRN exosomes isolated after ultrasound treatment, exosome-specific markers CD63 (H5C6, Invitrogen) and CD81 (1D6, Invitrogen) antibodies were used for flow cytometry (FACSMelody TM , BD Biosciences) analysis.

[0097] 0.5 μL of aldehyde / sulfate latex microspheres (A37304, Thermofisher) was added to 50 μL of exosomes (ultrasound), and the reaction was carried out at room temperature for 15 minutes. Then 1 mL of 0.1% BSA solution was added, mixed well, and the reaction was carried out overnight at 4°C. The next day, it was centrifuged at 2000 g for 10 minutes and the supernatant was removed, washed with 1 mL of DPBS solution, then centrifuged again at 2000 g for 10 minutes, and the supernatant was removed. The obtained pellet was resuspended in 50 μL of DPBS.

[0098] Transfer 20 μL of the resuspended pellet into an amber tube, and additionally add 80 μL of DPBS to make the total volume reach 100 μL. Add 5 μL of CD63 (H5C6, Invitrogen) and CD81 (1D6, Invitrogen) antibodies, mix well, and react at 4 °C for 1 hour. After washing with 1 mL of DPBS, centrifuge at 2000 g for 10 minutes and remove the supernatant. Resuspend in 1 mL of DPBS, and then measure by flow cytometry ( Figure 2 ).

[0099] The experimental results showed that the staining rates of exosome-specific markers CD63 and CD81 reached over 90%, confirming that the isolated PDRN exosomes were exosome particles with high purity.

[0100] (3) Optimization experiment of ultrasonic conditions for isolating PDRN exosomes from salmon testes

[0101] To isolate effective PDRN exosomes and the best low-molecular-weight PDRN exosomes containing small-sized PDRN, the characteristics of PDRN exosomes isolated according to the presence or absence of ultrasonic treatment and ultrasonic treatment conditions were analyzed.

[0102] Isolate exosomes without ultrasonic treatment (No Ultrasound) and with ultrasonic treatment (Ultrasound) from salmon testis tissue, analyze the number and size distribution of exosomes using NTA, and confirm the characteristics of the PDRN contained therein.

[0103] (3-1) Analysis of the number and distribution of salmon PDRN exosomes (NTA)

[0104] After adjusting the final exosome volume of the PDRN exosomes isolated from salmon testes to be the same, perform NTA using a Nanosight device (Nanosight NS300, Malvern Panalytical) to measure the size distribution of PDRN exosomes ( Figure 3 ).

[0105] The experimental results showed that regardless of whether ultrasonic treatment was performed, the isolated exosomes exhibited a typical exosome size of 50 - 200 nm ( Figure 3 ).

[0106] In addition, the number of salmon PDRN exosomes obtained was confirmed through multiple repeated experiments. The results showed that the number of PDRN exosomes obtained in the ultrasonic treatment group increased significantly by approximately 4.5 times compared with the non-ultrasonic treatment group ( Figure 4 ).

[0107] (3-2) Analysis of DNA in Salmon Low Molecular Weight PDRN Exosomes

[0108] To confirm the amount of DNA, DNA was extracted from salmon low molecular weight PDRN exosomes using a DNA extraction kit (iNtRON Biotechnology, Korea).

[0109] Place 50 μL of salmon PDRN exosomes in a 1.5 mL tube, add 300 μL of cell lysis buffer, and mix well. Then add 1.5 μL of RNase A solution and incubate at 37 °C for 30 minutes. Then add 100 μL of protein precipitation (PPT) buffer and mix for 20 seconds. Centrifuge at 16,000 g at 4 °C for 5 minutes, collect 400 μL of the supernatant and transfer it to another 1.5 mL tube. Add 400 μL of isopropanol, mix well, and centrifuge at 16,000 g at 4 °C for 1 minute. Discard the supernatant, add 70% ethanol, mix well, centrifuge at 16,000 g at 4 °C for 1 minute, discard the supernatant, and dry the pellet at room temperature for about 10 minutes. Finally, add 50 μL of DNA Rehydration buffer to dissolve the DNA, and then measure the DNA concentration.

[0110] To confirm the DNA size distribution in the isolated PDRN exosomes, agarose gel electrophoresis was performed on the extracted DNA. DNA samples were extracted from salmon testis tissue lysates, exosomes isolated from salmon testis without sonication (Noultrasound), exosomes isolated after sonication (Ultrasound 1), and exosomes that were sonicated a second time (20 KHz, intensity 25%, 90 seconds) after the first sonication (Ultrasound2) and were compared and analyzed ( Figure 5 A).

[0111] Prepare a 1.3% agarose gel and place it in the electrophoresis apparatus. Fill it with TAE buffer to cover the gel, and then place the gel in iBright FL1000 (Invitrogen) at 100 V and electrophorese for 30 minutes with an exposure time of 500 ms to confirm the DNA bands.

[0112] As a result of electrophoresing an equal amount of DNA in each group of samples, it was confirmed that PDRN was present in the exosomes, and it was found that the DNA size present in the exosomes was smaller than the genomic DNA obtained from the salmon testis tissue lysate.Figure 5 A, lanes 2, 3, and 4). In particular, in exosomes treated with ultrasound, PDRN was found to be depolymerized and become smaller. When isolating exosomes by performing ultrasound treatment once (Ultrasound 1), it was confirmed that the DNA size was approximately 1.5 kb or less, distributed around 500 bp. When isolating exosomes by performing a second ultrasound treatment on the exosomes isolated by ultrasound treatment (Ultrasound 2), a band with a DNA size of approximately 1 kb or less and distributed around 200 - 400 bp was confirmed ( Figure 5 A).

[0113] The DNA concentration in the depolymerized PDRN exosomes of salmon testis was confirmed. The results showed that compared with the group without ultrasound treatment (No Ultrasound), it increased significantly by approximately 23.5 - fold in the group with one - time ultrasound treatment (Ultrasound 1) and significantly by approximately 32.1 - fold in the group with two - time ultrasound treatment (Ultrasound 2) ( Figure 5 B).

[0114] In summary, compared with the group without ultrasound treatment (No Ultrasound), the exosomes obtained under ultrasound conditions in salmon testis tissue were depolymerized. The DNA in the depolymerized PDRN exosomes obtained by ultrasound treatment (Ultrasound 1) was depolymerized and existed in a small size of approximately 500 bp. The DNA in the exosomes after two - time ultrasound treatment (Ultrasound 2) was further depolymerized and existed as PDRN with a size of approximately 200 - 400 bp. In addition, it was also confirmed that the number of exosomes increased by approximately 4.5 - fold on average during the ultrasound treatment process, and it was also confirmed that the PDRN content in the obtained depolymerized PDRN exosomes increased significantly by approximately 23 - fold and 32 - fold respectively according to the ultrasound treatment.

[0115] In subsequent experiments (cytotoxicity evaluation, intracellular uptake rate analysis, and efficacy test), depolymerized sPDRN exosomes obtained by performing a second ultrasound treatment on the exosomes isolated by ultrasound treatment (Ultrasound 2) were used for the experiments.

[0116] (4) Evaluation and analysis of the cytotoxicity of depolymerized induced PDRN exosomes derived from salmon testis

[0117] To evaluate the toxicity of PDRN exosomes induced by low molecular weight from salmon testis (sPDRN Exosome) through sonication, a cell viability experiment was conducted. Human skin keratinocytes (HaCaT) were cultured in high glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and 1X10 4 cells were seeded in each well of a 96-well plate. The next day, sPDRN exosomes were treated at different concentrations (1X10 4 to 1X10 8 particles / mL). After 24 hours, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance ( Figure 6 ) was measured at 450 nm.

[0118] The experimental results showed that when treated with low molecular weight PDRN exosomes, the cell number generally showed an increasing trend, but no cytotoxicity was observed within the entire treatment concentration range. In particular, the cell number increased significantly when treated at concentrations of 1X10 7 and 1X10 8 particles / mL.

[0119] (5) Analyze the intracellular uptake rates of low molecular weight PDRN exosomes and fluorescent DNA

[0120] (5-1) Immunofluorescence analysis

[0121] To confirm the intracellular uptake rate of salmon low molecular weight-induced exosomes (sPDRN exosomes), Cy-3-labeled PDRN (AGC TGC TGC CTT GGC AGG AAC TAA TGG GGA TCC ATA ATA AAC CCC AGG AA-[Cyanine 3]) and low molecular weight-induced PDRN exosomes stained with DiD fluorescence were treated and confirmed using immunofluorescence.

[0122] After culturing HDF cells in high glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C for 3 days, 5X10 4 cells were seeded in a 24-well plate with a cover glass coated with 0.1% gelatin. According to 1X10 9Exosomes were fixed to 50 μL with PBS, 0.25 μL of DiD staining reagent was added, mixed well, and cultured at 37 °C for 30 minutes. 50.25 μL of DiD-stained exosomes and 0.025 μL of PDRN were used to treat the cells seeded in a 24-well plate, and cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. The next day, the culture medium was aspirated, washed with 1 mL of PBS, and then 1 mL of PBS was added again. Then the cover glass was taken out and transferred to a humidity chamber, 200 μL of PBS was sprayed, and only 170 μL was taken out without allowing it to dry completely. 150 μL of 4% paraformaldehyde / PBS was added to the cover glass and fixed at room temperature for 5 minutes. After removing the 4% paraformaldehyde / PBS, 150 μL of 0.3% Triton X-100 / PBS was added for permeabilization. After removing the 0.3% Triton X-100 / PBS, 3% BSA / PBS was added and blocked at room temperature for 1 hour. For β-actin, the primary antibody was diluted 1:1000 with 100 μL of 3% BSA / PBS and treated at room temperature for 2 hours. After removing the antibody, it was washed 3 times with 200 μL of 0.03% Tween20 / PBS. The secondary antibody was diluted 1:500 with 100 μL of 3% BSA / PBS and reacted at room temperature for 1 hour. After removing the antibody, it was washed 3 times with 200 μL of 0.03% Tween20 / PBS and washed 2 times with clean PBS. 5 μL of mounting solution was added to the glass slide, the cover glass was flipped and fixed, the air was blocked with nail polish after 10 minutes, and photographed under a microscope( Figure 7 ).

[0123] Experimental results: Analysis of the intracellular uptake rates of low-molecular-weight sPDRN exosomes and PDRN showed that exosomes containing low-molecular-weight PDRN (sPDRN Exosome) exhibited higher intracellular uptake rates than conventional PDRN as confirmed by confocal microscopy.

[0124] (5-2) Flow cytometry analysis

[0125] To confirm the intracellular uptake rate of salmon sperm-derived low-molecular-weight-induced PDRN exosomes (sPDRN Exosome) under ultrasonic conditions, cells were treated with Cy-3-labeled PDRN (AGC TGC TGC CTT GGC AGG AAC TAA TGG GGA TCC ATAATAAAC CCC AGG AA-[Cyanine 3]) and DiD-fluorescently stained low-molecular-weight-induced PDRN exosomes, respectively, and then analyzed by flow cytometry (FACSMelodyTM , confirmed by analysis with BD Biosciences).

[0126] Human dermal fibroblasts (HDF) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C for 3 days, and then 2×10 5 cells were seeded into a 60-mm cell culture dish. To prepare stained exosomes, 1×10 9 exosomes were made up to 50 μL with PBS, 0.25 μL of DiD staining reagent was added, mixed well, and cultured at 37 °C for 30 minutes. As a positive control for PDRN, salmon sperm PDRN (100403-24-5, Sigma) was used. 50.25 μL of DiD-stained salmon low-molecular-weight PDRN exosomes (sPDRN Exosome) and 0.025 μL of salmon sperm PDRN were treated alone or together with the cells cultured in a 60-mm cell culture dish, and cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. The next day, the culture medium was aspirated, the cells were treated with a mixture of trypsin and EDTA, harvested, then centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, the pellet was washed with 1 mL of PBS and centrifuged at 1000 rpm for 5 minutes. The resulting pellet was suspended in 1 mL of PBS, transferred to a FACS tube, and measured by flow cytometry ( Figure 8 ).

[0127] The results of the analysis of the intracellular uptake rates of low-molecular-weight-induced PDRN exosomes and PDRN showed that the uptake rate of exosomes containing low-molecular-weight PDRN was approximately 4 times higher.

[0128] (6) Analysis of the efficacy of low-molecular-weight sPDRN exosomes

[0129] (6-1) Analysis of the tissue regeneration efficacy of low-molecular-weight PDRN exosomes

[0130] To evaluate the regeneration efficacy of low-molecular-weight sPDRN exosomes, a cell migration test was performed to analyze the difference in the degree of cell proliferation into the blank area of the culture dish.

[0131] Human skin keratinocytes (HaCaT) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C, and seeded at 7×10 per well 4Cells were seeded in 24-well plates. The next day, the cells were treated with TNF-α (10 ng / mL) and INF-γ (10 ng / mL) in high-glucose DMEM medium containing 2% fetal bovine serum and 1% penicillin / streptomycin for 24 hours. After 24 hours, the bottom of the cell culture dish was scratched with a cell scraper (SPLScarScratcher TM , SPL, Korea), and the cells were washed twice with the medium.

[0132] The low-molecular-weight PDRN exosomes isolated from salmon testis by ultrasonic treatment (sPDRN exo) were treated at 1×10 7 particles / mL, and PDRN (Sigma, 31149-F) was treated at a concentration of 70 ng / mL. Then, images of the scratched area were taken under a microscope at 0 hours. After 24 hours, photos of the same area were taken under the microscope ( Figure 9 A), and the area was quantified using the Image J program and presented in a graph ( Figure 9 B).

[0133] The experimental results confirmed that the tissue regeneration ability was significantly improved under ultrasonic treatment conditions (sPDRN exo) compared with the negative control group (Cont), and showed equal or higher efficacy than traditional PDRN ( Figure 9 ).

[0134] (6-2) Anti-inflammatory evaluation of low-molecular-weight induced sPDRN exosomes

[0135] To evaluate the anti-inflammatory activity, human skin keratinocytes (HaCaT) were used to induce inflammatory conditions, and then treated with low-molecular-weight PDRN exosomes and PDRN for a cell viability experiment (formazan is generated by dehydrogenases that are only active in living cells, and the viability is measured by absorbance).

[0136] HaCaT cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the incubator temperature was 37 °C with a CO 2 content of 5%. In the experiment, 1×10 4 cells were seeded in each well of a 96-well plate.

[0137] First, to determine the optimal concentration of the anti-inflammatory efficacy of low-molecular-weight PDRN exosomes, HaCaT cells were seeded and treated with TNF-α (10 ng / ml) and INF-γ (10 ng / ml) simultaneously the next day, and cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. Subsequently, the low-molecular-weight PDRN exosomes were used at different concentrations (1×10 7 and 1×10 8Cells / mL) were treated for 24 hours. After treatment, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of the culture medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance was measured at 450 nm ( Figure 10 A).

[0138] Next, the effective concentration of low-molecular-weight-induced sPDRN exosomes obtained by sonication, 1×10 7 cells / mL, was compared with the anti-inflammatory efficacy of traditional PDRN.

[0139] The results showed that, compared with the negative control group (Cont., PBS), the anti-inflammatory efficacy of the treatment groups with different concentrations of low-molecular-weight-induced PDRN exosomes was significantly improved ( Figure 10 A).

[0140] On the second day after HaCaT cells were seeded, they were treated simultaneously with TNF-α (10 ng / mL) and INF-γ (10 ng / mL), and cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. After 24 hours, the sonicated sPDRN exosome samples were treated at 1×10 7 cells / mL respectively, and PDRN (Sigma, 31149-F) was treated at 70 ng / mL. After 24 hours, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of the culture medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance was measured at 450 nm ( Figure 10 B).

[0141] The results showed that the anti-inflammatory efficacy of the sonication-induced sPDRN exosomes (sPDRN exo) was significantly improved compared with the negative control group, and showed the same or higher anti-inflammatory efficacy as traditional PDRN ( Figure 10 B).

[0142] 2. Development of cod PDRN exosomes

[0143] (1) Isolation of exosomes from cod testes

[0144] In order to induce and obtain cod PDRN exosomes containing low-molecular-weight PDRN (polydeoxyribonucleotide) from cod testes by sonication, cod testis tissues were sonicated under the sonication conditions in Table 1, and then the exosomes were isolated and characterized.

[0145] First, place 4g of cod testis in a mortar soaked in liquid nitrogen and grind it. Add 40 mL of PBS, separately isolate 10 mL of it and store it at -20 °C (tissue lysate). Use 18G, 20G, and 26G syringes to sequentially drip the cod testis lysate containing PBS, and make it smaller by moving it up and down. Separately isolate 10 mL of the sample and store it on ice as the sample without ultrasonic treatment (No.1, No Ultrasound). Treat the remaining 20 mL of cod testis lysate sample three times at 25% intensity for 5 seconds each (15 seconds in total).

[0146] Aliquot the tissue lysate sample, the sample without ultrasonic treatment (No.1, No Ultrasound), and the sample ultrasonically treated under condition 2 (No.2, Ultrasound) into 1.5 mL tubes (1 mL each), centrifuge at 14000 rpm and 4 °C for 20 minutes, and separate and combine the supernatants of each group.

[0147] To confirm the low-molecular-weight PDRN exosomes derived from cod testis with or without ultrasonic treatment in samples other than the tissue lysate sample among the samples prepared above, exosome isolation was performed as follows.

[0148] Both the sample without ultrasonic treatment and the sample with ultrasonic treatment were filtered through a 0.2 μM filter. Place the filtered sample into an Ultra-15 centrifugal filtration device (UFC9100, Millipore), centrifuge at 10000g and 4 °C for 30 minutes, and obtain the supernatant.

[0149] Table 2

[0150]

[0151] (2) Confirm the analysis of exosomes isolated from cod testis by ultrasonic treatment

[0152] (2-1) Immunofluorescence analysis of isolated cod testis PDRN exosomes

[0153] Perform immunofluorescence analysis on the exosome-specific marker 'CD63' to confirm the low-molecular-weight PDRN exosomes isolated from cod testis tissue by ultrasonic treatment (Ultrasound).

[0154] To perform immunofluorescence staining on the PDRN exosomes extracted from cod testis, use an anti-CD63 antibody labeled with a PE fluorescent substance (green, H5C6, PE, eBioscience TM ) to stain the exosomes, and use DiD dye (red, VybranTM The lipid bilayer particles were stained overnight with a DiD cell labeling solution (Thermofisher), and then analyzed using a confocal microscope for immunofluorescence images (DMi8, LEICA).

[0155] The results showed that PDRN exosomes were stained with CD63 and DiD, and the two images were merged. These results indicated that the isolated PDRN exosomes were typical and pure exosomes with a lipid bilayer ( Figure 11 ). Analysis of the nanoparticles in the images using Image J showed that the ratio of PDRN exosomes to DiD-stained particles was over 94%, confirming that the extracted particles were highly pure exosomes.

[0156] (2-2) Flow cytometry analysis of isolated PDRN exosomes from cod testis

[0157] To confirm the isolated low-molecular-weight PDRN exosomes after sonication, exosome-specific markers CD63 (H5C6, Invitrogen) and CD81 (1D6, Invitrogen) antibodies were used for flow cytometry (FACSMelody TM , BD Biosciences) analysis.

[0158] 0.5 μL of aldehyde / sulfate latex microspheres (A37304, Thermofisher) was added to 50 μL of exosomes (sonication), and the reaction was carried out at room temperature for 15 minutes. Then 1 mL of 0.1% BSA solution was added, mixed well, and the reaction was carried out overnight at 4°C. The next day, it was centrifuged at 2000 g for 10 minutes and the supernatant was removed. The solution was washed with 1 mL of DPBS, then centrifuged again at 2000 g for 10 minutes, and the supernatant was removed. The obtained pellet was resuspended in 50 μL of DPBS.

[0159] 20 μL of the resuspended pellet was transferred to an amber tube, and another 80 μL of DPBS was added to make the total volume reach 100 μL. 5 μL of CD63 (H5C6, Invitrogen) and CD81 (1D6, Invitrogen) antibodies were added, mixed well, and reacted at 4°C for 1 hour. After washing with 1 mL of DPBS, it was centrifuged at 2000 g for 10 minutes and the supernatant was removed, resuspended in 1 mL of DPBS, and then measured by flow cytometry ( Figure 12 ).

[0160] The experimental results showed that the staining rates of exosome-specific markers CD63 and CD81 reached over 90%, confirming that the isolated PDRN exosomes were highly pure exosome particles.

[0161] (3) Experiment on optimizing ultrasonic conditions for isolating PDRN exosomes from cod testis

[0162] To isolate effective PDRN exosomes and the best low-molecular-weight PDRN exosomes containing small-sized PDRN, the characteristics of PDRN exosomes isolated with and without ultrasonic treatment and under different ultrasonic treatment conditions were analyzed.

[0163] Exosomes were isolated from cod testis tissue without ultrasonic treatment (No Ultrasound) and with ultrasonic treatment (Ultrasound). The number and size distribution of exosomes were analyzed using NTA, and the characteristics of the PDRN contained therein were confirmed.

[0164] (3-1) Analysis of the number and distribution of cod PDRN exosomes (NTA)

[0165] After adjusting the final exosome volume of PDRN exosomes isolated from cod testis to be the same, NTA was performed using a Nanosight device (Nanosight NS300, Malvern Panalytical, Malvern Panalytical) to measure the size distribution and number of PDRN exosomes ( Figure 13 ).

[0166] The experimental results showed that the isolated exosomes exhibited a typical exosome size of 50 - 200 nm regardless of whether they were treated with ultrasound ( Figure 13 ).

[0167] In addition, the number of cod PDRN exosomes obtained was confirmed through multiple repeated experiments. The results showed that the number of PDRN exosomes obtained in the ultrasonic treatment group increased significantly by approximately 2.8 times compared with the non-ultrasonic treatment group ( Figure 14 ).

[0168] (3-2) Analysis of DNA in cod low-molecular-weight PDRN exosomes

[0169] To confirm the amount of DNA, DNA was extracted from cod low-molecular-weight PDRN exosomes using a DNA extraction kit (iNtRON Biotechnology, Korea).

[0170] Place 50 μL of cod PDRN exosomes in a 1.5 mL tube, add 300 μL of cell lysis buffer, and mix well. Then add 1.5 μL of RNase A solution, incubate at 37 °C for 30 minutes, then add 100 μL of protein precipitation (PPT) buffer, mix for 20 seconds, centrifuge at 16000 g and 4 °C for 5 minutes, collect 400 μL of the supernatant and transfer it to another 1.5 mL tube. Add 400 μL of isopropanol, mix well, centrifuge at 16000 g and 4 °C for 1 minute. Discard the supernatant, add 70% ethanol, mix well, centrifuge at 16000 g and 4 °C for 1 minute, discard the supernatant, and dry the precipitate at room temperature for about 10 minutes. Finally, add 50 μL of DNA Rehydration buffer to dissolve the DNA, and then measure the DNA concentration.

[0171] To confirm the DNA size distribution in the isolated PDRN exosomes, agarose gel electrophoresis was performed on the extracted DNA. DNA was extracted from samples of cod testis tissue lysate, exosomes isolated from cod testis without ultrasound (Noultrasound), exosomes isolated after ultrasound (Ultrasound 1), and exosomes that were secondarily ultrasonically treated (20 KHz, intensity 25%, 90 seconds) after isolation (Ultrasound2) and compared and analyzed ( Figure 15 A).

[0172] Prepare a 1.3% agarose gel and place it in the electrophoresis apparatus. Fill it with TAE buffer to cover the gel, then place the gel in the iBright FL1000 (Invitrogen) under the condition of 100 V and perform electrophoresis for 30 minutes with an exposure time of 500 ms to confirm the DNA bands.

[0173] As a result of electrophoresis of an equal amount of DNA in each group of samples, it was confirmed that PDRN was present in the exosomes, and it was found that the size of the DNA present in the exosomes was smaller than the genomic DNA obtained from the cod testis tissue lysate ( Figure 5 A). In particular, in the exosomes treated with ultrasound, it was found that PDRN was low-molecularized and became smaller. When the exosomes isolated after ultrasound treatment were secondarily isolated (Ultrasound 2), it was confirmed that the DNA size was about 1 kb or less, and bands were distributed around 200 - 400 bp ( Figure 15 A).

[0174] The DNA concentration in the low-molecular-weight PDRN exosomes from cod testes was confirmed. The results showed that, compared with the group without ultrasound treatment (No Ultrasound), it increased significantly by about 3.0 times in the group with one ultrasound treatment (Ultrasound 1) and by about 3.3 times in the group with two ultrasound treatments (Ultrasound 2). Figure 15 B).

[0175] In subsequent experiments (cytotoxicity evaluation, intracellular uptake rate analysis, and efficacy testing), low-molecular-weight cPDRN exosomes obtained by subjecting the exosomes separated by ultrasound treatment to a second ultrasound treatment separation (Ultrasound 2) were used for the experiments.

[0176] (4) Evaluation and analysis of the cytotoxicity of low-molecular-weight induced PDRN exosomes derived from cod testes

[0177] To evaluate the toxicity of low-molecular-weight induced PDRN exosomes (cPDRN Exosome, cPDRN exosomes) from cod testes by ultrasound treatment, a cell viability experiment was conducted. Human skin keratinocytes (HaCaT) were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and 1×10 4 cells were seeded in each well of a 96-well plate. The next day, cPDRN exosomes were treated at different concentrations (1×10 4 - 1×10 8 particles / mL). After 24 hours, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of the culture medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance was measured at 450 nm. Figure 16 )

[0178] The experimental results showed that when treated with low-molecular-weight PDRN exosomes, the number of cells generally showed an increasing trend, but no cytotoxicity was observed within the entire treatment concentration range. In particular, the number of cells increased significantly when treated at concentrations of 1×10 7 and 1×10 9 particles / mL.

[0179] (5) Analysis of the intracellular uptake rate of low-molecular-weight PDRN exosomes and fluorescent DNA

[0180] (5-1) Immunofluorescence analysis

[0181] To confirm the intracellular uptake rate of cod low-molecular-weight induced exosomes (cPDRN exosomes), Cy-3-labeled PDRN (AGC TGC TGC CTT GGC AGG AAC TAA TGG GGA TCC ATA ATA AAC CCC AGG AA-[Cyanine 3]) and DiD fluorescently stained low-molecular-weight induced PDRN exosomes were treated and confirmed by immunofluorescence.

[0182] After HDF cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C for 3 days, 5×10 4 Cells were seeded in 24-well plates with 0.1% gelatin-coated cover glasses. 9 The exosomes were fixed to 50 μL with PBS, and 0.25 μL DiD staining reagent was added, mixed, and incubated at 37°C for 30 minutes. The cells seeded in a 24-well plate were treated with 50.25 μL DiD-stained exosomes and 0.025 μL PDRN and incubated at 37°C and 5% CO 2 Incubate in an incubator for 24 hours. The next day, aspirate the culture medium, wash with 1 mL PBS, and then add 1 mL PBS again. Then remove the coverslip and move it to a humidity chamber, spray 200 μL PBS, and only remove 170 μL without letting it dry completely. Add 150 μL of 4% paraformaldehyde / PBS to the coverslip and fix it at room temperature for 5 minutes. After removing the 4% paraformaldehyde / PBS, add 150 μL of 0.3% Triton X-100 / PBS for permeabilization. After removing the 0.3% Triton X-100 / PBS, add 3% BSA / PBS and block at room temperature for 1 hour. For b-actin, dilute the primary antibody 1:1000 with 100 μL 3% BSA / PBS and treat at room temperature for 2 hours. After removing the antibody, wash 3 times with 200μL 0.03% Tween20 / PBS. Dilute the secondary antibody with 100μL 3% BSA / PBS at 1:500 and react at room temperature for 1 hour. After removing the antibody, wash 3 times with 200μL 0.03% Tween20 / PBS and 2 times with clean PBS. Add 5μL mounting solution to the slide, flip the coverslip to fix, block the air with nail polish after 10 minutes, and take pictures under a microscope ( Figure 17 ).

[0183] Analysis of the intracellular uptake rates of low-molecular-weight cPDRN exosomes and PDRN showed that exosomes containing low-molecular-weight PDRN (cPDRN Exosome) exhibited a higher intracellular uptake rate than conventional PDRN as confirmed by confocal microscopy.

[0184] (5-2) Flow cytometry analysis

[0185] To confirm the intracellular uptake rate of cod sperm-derived low-molecular-weight-induced PDRN exosomes (cPDRN Exosome) under ultrasonic conditions, cells were treated with Cy-3-labeled PDRN (AGC TGC TGC CTT GGC AGG AAC TAA TGG GGA TCC ATAATAAAC CCC AGG AA-[Cyanine 3]) and DiD-fluorescently stained low-molecular-weight-induced PDRN exosomes, and then confirmed by flow cytometry (FACSMelody TM , BD Biosciences).

[0186] Human dermal fibroblasts (HDF) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C for 3 days, and then 2X10 5 cells were seeded into 60-mm cell culture dishes. To prepare stained exosomes, 1X10 9 exosomes plus PBS were made up to 50 μL, 0.25 μL of DiD staining reagent was added, mixed well, and cultured at 37 °C for 30 minutes. As a positive control for PDRN, cod sperm PDRN (100403-24-5, Sigma) was used. 50.25 μL of DiD-stained cod low-molecular-weight PDRN exosomes (cPDRN Exosome) and 0.025 μL of cod sperm PDRN were treated alone or together with the cells cultured in 60-mm cell culture dishes, and cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. The next day, the culture medium was aspirated, the cells were treated with a mixture of trypsin and EDTA, harvested, then centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, the pellet was washed with 1 mL of PBS and centrifuged at 1000 rpm for 5 minutes. The resulting pellet was suspended in 1 mL of PBS, transferred to a FACS tube, and measured by flow cytometry ( Figure 18 ).

[0187] The results of the analysis of the intracellular uptake rates of low-molecular-weight-induced PDRN exosomes and PDRN showed that the uptake rate of exosomes containing low-molecular-weight PDRN was approximately 3.8 times higher.

[0188] (6) Analyze the effectiveness of low-molecular-weight cPDRN exosomes

[0189] (6-1) Analyze the tissue regeneration efficacy of low-molecular-weight PDRN exosomes

[0190] To evaluate the regenerative efficacy of low-molecular-weight cPDRN exosomes, a cell migration test was conducted to analyze the difference in the degree of cell proliferation to the blank area of the culture dish.

[0191] Human skin keratinocytes (HaCaT) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C and seeded into a 24-well plate at 7X10 4 cells per well. The next day, the cells were treated with TNF-α (10 ng / mL) and INF-γ in high-glucose DMEM medium containing 2% fetal bovine serum and 1% penicillin / streptomycin for 24 hours. After 24 hours, the bottom of the cell culture dish was scratched with a scraper (SPLScarScratcher TM , SPL, Korea) and washed twice with the medium.

[0192] Low-molecular-weight PDRN exosomes isolated from cod sperm (cPDRN exo) by ultrasonic treatment were treated at 1X10 7 particles / mL, PDRN (Sigma, 31149-F) was treated at a concentration of 70 ng / mL, and then images of the scratched area were taken under a microscope at 0 hours. Photos of the same area were taken under a microscope after 24 hours( Figure 19 A), and its area was quantified using the Image J program and presented in a graph( Figure 19 B).

[0193] The experimental results confirmed that the tissue regeneration ability was significantly improved under ultrasonic treatment conditions (cPDRN exo) compared with the negative control group (Cont), and showed the same or higher efficacy as traditional PDRN( Figure 19 ).

[0194] (6-2) Anti-inflammatory evaluation of low-molecular-weight induced cPDRN exosomes

[0195] To evaluate the anti-inflammatory activity, human skin keratinocytes (HaCaT) were induced to an inflammatory condition and then treated with low-molecular-weight PDRN exosomes and PDRN for a cell viability experiment (formazan is generated by dehydrogenase that is only active in living cells, and the viability is measured by absorbance).

[0196] HaCaT cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the temperature of the incubator was 37 °C with a CO 2 content of 5%. In the experiment, 1×10 4 cells were seeded in each well of a 96-well plate.

[0197] First, to determine the optimal concentration of the anti-inflammatory effect of low-molecular-weight PDRN exosomes, HaCaT cells were seeded. The next day, they were treated simultaneously with TNF-α (10 ng / ml) and INF-γ and cultured in an incubator at 37 °C with 5% CO 2 for 24 hours. Subsequently, the low-molecular-weight PDRN exosomes were treated at different concentrations (1×10 5 and 1×10 8 particles / mL) for 24 hours. After treatment, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of the culture medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance was measured at 450 nm ( Figure 20 A).

[0198] The results showed that compared with the negative control group (Cont., PBS), the anti-inflammatory effect of the treatment groups with low-molecular-weight induced PDRN exosomes at different concentrations (1×10 6 , 1×10 7 , 1×10 8 particles / mL) was significantly improved ( Figure 20 A).

[0199] Next, the anti-inflammatory effect of the effective concentration of 1×10 7 particles / mL of low-molecular-weight induced cPDRN exosomes obtained by sonication was compared with that of traditional PDRN.

[0200] On the second day after seeding HaCaT cells, they were treated simultaneously with TNF-α (10 ng / ml) and INF-γ and cultured in an incubator at 37 °C with 5% CO 2 for 24 hours. After 24 hours, the sonicated cPDRN exosome samples were treated at 1×10 7 particles / mL respectively, and PDRN (Sigma, 31149-F) was treated at 70 ng / mL. After 24 hours, 10 μL of Ez-cytox (DoGenBio, Korea) was added to every 100 μL of the culture medium, and the reaction was carried out in the dark at 37 °C for 1 hour and 30 minutes, and then the absorbance was measured at 450 nm ( Figure 20 B).

[0201] The results showed that the anti-inflammatory efficacy of cPDRN exosomes (cPDRN exo) induced by ultrasonic treatment was significantly improved compared with the negative control group, and exhibited the same or higher anti-inflammatory efficacy as traditional PDRN. Figure 20 B).

Claims

1. An exosome isolated from fish testis tissue, characterized in that, the interior of the exosome contains polydeoxyribonucleotide PDRN derived from fish testis tissue.

2. The exosome according to claim 1, characterized in that, the PDRN is low-molecularized to a length of 1 kb or less.

3. The exosome according to claim 1, characterized in that, the PDRN is low-molecularized to a length of 100 bp to 500 bp.

4. The exosome according to claim 1, characterized in that, the fish is salmon or cod.

5. The exosome according to claim 1, characterized in that, the fish is salmon, and the exosome contains 500 pg to 1000 pg of PDRN.

6. A method for preparing an exosome containing PDRN, characterized in that, comprises the following steps: crushing fish testis tissue; subjecting the tissue crush to ultrasonic treatment; and isolating exosomes containing PDRN from the tissue crush after ultrasonic treatment.

7. The method for preparing an exosome containing PDRN according to claim 6, characterized in that, the frequency of the ultrasonic treatment is 10 KHz to 30 KHz, the intensity is 15% to 40% of the maximum output intensity, and the treatment time is 5 seconds to 35 seconds.

8. The method for preparing an exosome containing PDRN according to claim 6, characterized in that, the PDRN is low-molecularized to a length of 1 kb or less.

9. The method for preparing an exosome containing PDRN according to claim 6, characterized in that, the step of isolating exosomes containing PDRN comprises the following steps: centrifuging the tissue crush after ultrasonic treatment to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and centrifuging and concentrating the filtrate.

10. The method for preparing an exosome containing PDRN according to claim 6, characterized in that, further comprises the following steps: subjecting the isolated exosomes containing PDRN to ultrasonic treatment.

11. The method for preparing an exosome containing PDRN according to claim 10, characterized in that, the frequency of the ultrasonic treatment of the exosomes is 10 KHz to 30 KHz, the intensity is 15% to 40% of the maximum output intensity, and the treatment time is 70 seconds to 120 seconds.

12. The method for preparing an exosome containing PDRN according to claim 10, characterized in that, after the ultrasonic treatment of the exosomes, the PDRN is low-molecularized to a length of 100 bp to 500 bp.

13. A composition for tissue regeneration, characterized in that, comprises the exosome according to claim 1.

14. An anti-inflammatory cosmetic composition, characterized in that, comprises the exosome according to claim 1.

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