Cosmetic composition comprising PDRN-captured exosomes as active ingredient for skin improvement

Abstract

The present invention relates to a cosmetic composition comprising polydeoxyribonucleotide (PDRN)-captured exosomes as an active ingredient for skin improvement. It was confirmed that PDRN exhibits a skin improvement effect by regulating expression of skin-related genes and that the use of PDRN in a form captured in exosomes increases the delivery efficiency of PDRN in the body. Thus, the composition can be advantageously used for skin improvement or for prevention or treatment of skin diseases.

Description

Cosmetic composition for skin improvement containing PDRN-encapsulated exosomes as an active ingredient

[0001] The present invention relates to a cosmetic composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0002] Aging is a phenomenon in which the physical functions of an organism degenerate over time. Cellular aging manifests as the loss of the ability for cells to divide. Generally, cellular aging includes mechanisms dependent on the Rb (retinoblastoma) protein, aging caused by the loss of telomeres (which protect chromosomes), aging due to antioxidant stress, and aging caused by DNA damage. Among these, skin aging is classified into intrinsic aging and extrinsic aging. Intrinsic aging is a phenomenon that occurs naturally over time due to the influence of genetic factors, regardless of the external environment; it is difficult to control artificially, and functional changes appear rather than macroscopic or morphological alterations. On the other hand, extrinsic aging is a phenomenon induced by external environmental factors, such as chronic exposure to sunlight; it is relatively easy to control artificially, and distinct structural and physiological changes occur. Because it is primarily caused by exposure to sunlight (ultraviolet rays), it is also referred to as photoaging. In aged skin, increased drug penetration, increased susceptibility to irritant contact dermatitis, worsening of skin dryness, itching, wrinkles, and pigmentation abnormalities (age spots, melasma, etc.) occur.

[0003] Recently, aesthetic factors have come to be recognized as important social competitiveness due to improvements in living standards, increased income levels, and growing social interest in beauty. Additionally, as the desire to maintain a younger and more beautiful appearance intensifies, the demand for and interest in cosmetics continues to rise. In the case of functional cosmetics, multi-functional products exhibiting various skin improvement effects such as anti-aging, UV protection, wrinkle reduction, and whitening are widely used, and there is particularly high demand for whitening cosmetics in the Asian market.

[0004] The objective of the present invention is to provide a skin improvement composition comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0005] Another objective of the present invention is to provide a composition for preventing or treating skin diseases comprising exosomes in which PDRN is captured as an active ingredient.

[0006] Another objective of the present invention is to provide a method for manufacturing exosomes in which the PDRN is captured.

[0007] To achieve the above objective, the present invention provides a cosmetic composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0008] In addition, the present invention provides a health functional food composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0009] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0010] In addition, the present invention provides a method for producing exosomes in which PDRN is captured, comprising the steps of: extracting PDRN (Polydeoxyribonucleotide) from salmonid fish (Step 1); extracting exosomes from skin cells (Step 2); and capturing the PDRN of Step 1 within the exosomes of Step 2 (Step 3).

[0011] According to the present invention, PDRN (Polydeoxyribonucleotide) exhibits skin improvement effects such as regulating skin-related gene expression, and by confirming that using it in a form captured in exosomes increases the delivery rate of PDRN in the body, it can be usefully utilized for skin improvement; or for the prevention or treatment of skin diseases.

[0012] Figure 1 shows the results of performing electrophoresis to confirm whether PDRN DNA fragmented by ultrasonic disruption according to Experimental Example 3 below was fragmented well. In Figure 1, 1 is the result of performing electrophoresis for 3 hours, 2 for 5 hours, 3 for 7 hours, 4 for 10 hours, and 5 for 14 hours.

[0013] Figure 2 shows the results of analyzing the size and concentration of exosomes extracted in Experimental Example 3 below.

[0014] Figure 3 shows the results of analyzing the optimal conditions for the electrochemical method for capturing DNA within exosomes.

[0015] Figure 4 shows the results of analyzing the effects of DNase treatment and heat treatment on the release of DNA captured in exosomes.

[0016] Figure 5 shows the results of analyzing the optimal concentration of exosomes used when performing the electro-cleaning method.

[0017] Figure 6 is the result of analyzing the skin regeneration effect using images of cell morphology taken over time after applying physical damage to skin cells to confirm the skin regeneration effect of PDRN-encapsulated exosomes.

[0018] Figure 7 shows the results of analyzing the effect of PDRN-encapsulated exosomes on regulating gene expression related to skin regeneration.

[0019] Figure 8 shows the results of analyzing the anti-inflammatory effect of PDRN-captured exosomes.

[0020] Figure 9 shows the results of analyzing whether changes in the characteristics of exosomes occur according to the electro-cleaning method. The left side shows the characteristics of exosomes before performing the electro-cleaning method, and the right side shows the characteristics after performing the electro-cleaning method.

[0021] The present invention will be described in more detail below.

[0022]

[0023] The present invention provides a cosmetic composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0024] The above PDRN may be derived from salmonid fish, and preferably may be derived from salmon or trout.

[0025] In addition, the exosomes containing the above PDRN can promote the gene expression of procollagenase or MMP2 (matrix metalloproteinase-2).

[0026] In addition, the exosomes containing the above PDRN can inhibit the expression of one or more selected from the group consisting of TNF-α (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), and IL-1β (Interleukin-1 beta), but are not limited thereto.

[0027] In addition, the above skin improvement may be one or more selected from the group consisting of antioxidant, anti-inflammatory, skin wrinkle improvement, skin moisturization, improvement of skin cell damage, improvement of skin troubles, anti-aging, skin regeneration, and improvement of skin elasticity, but is not limited thereto.

[0028] The above exosomes may be derived from skin cells.

[0029] In addition, the above exosomes are based on 10 μl of PDRN at a concentration of 10 pg / μl, 1 × 10 5 It may be a concentration of copies / μl.

[0030] The exosomes containing the above PDRN may be processed by one selected method from the group consisting of the application method, the MTS (Micro Needling System) method, and the injection method, but are not limited thereto.

[0031] In addition to the active ingredient, the above cosmetic composition may include conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances, and a carrier. Furthermore, the above cosmetic composition may additionally include a skin absorption promoter to enhance its effect. The formulation may be prepared in any formulation conventionally manufactured in the art; for example, the cosmetic composition may be formulated into lotion, skin, toner, essence, sunscreen, makeup base, powder, pack, ointment, patch, stick, shampoo, rinse, makeup remover, and cleanser, but is not limited thereto. Additionally, if the formulation is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. In addition, when the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and especially in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether. In addition, when the formulation is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan. In addition, when the formulation is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components.

[0032]

[0033] In addition, the present invention provides a health functional food composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0034] The present invention can be generally used as a commonly used food.

[0035] The food composition of the present invention may be used as a health functional food. The term “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0036] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0037] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0038] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0039] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0040] In the present invention, the term “prevention” refers to any act of suppressing or delaying skin disease through the administration of a composition according to the present invention.

[0041] In the present invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of a skin disease through the administration of a composition according to the present invention.

[0042] In this invention, the term “improvement” refers to any act of improving a poor condition of a skin disease through the administration of a composition according to this invention.

[0043]

[0044] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

[0045] The above skin disease may be any one selected from the group consisting of acne, skin inflammation, dry skin, athlete's foot, eczema, allergic urticaria, pruritus, ringworm, psoriasis, and atopy, but is not limited thereto.

[0046] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0047] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0048] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0049] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0050] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0051] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0052] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0053] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0054]

[0055] In addition, the present invention provides a method for preventing or treating skin diseases, comprising the step of treating an individual other than a human with exosomes in which PDRN (Polydeoxyribonucleotide) has been captured.

[0056]

[0057] In addition, the present invention provides a method for producing exosomes in which PDRN is captured, comprising the steps of: extracting PDRN (Polydeoxyribonucleotide) from fish (Step 1); extracting exosomes from skin cells (Step 2); and capturing the PDRN of Step 1 within the exosomes of Step 2 (Step 3).

[0058] The above third step may be performed through electroporation, and the electroporation may be performed under conditions of a voltage of 1000V, a pulse length of 15μs, 3 pulses, and an interval of 10 seconds.

[0059] The above manufacturing method may additionally perform DNA degrading enzyme (DNase) treatment or heat treatment after performing the above third step, and the heat treatment may be performed at 100°C for 5 minutes.

[0060] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0061]

[0062] [Experimental Example 1] PDRN Extraction

[0063] A brine solution was prepared by dissolving sodium chloride (NaCl) at 8~12% (preferably 10%) relative to the specific gravity of the purified water in 40~60L (preferably 50L) of purified water, and 10kg of frozen salmon testes (obtained from BNF Global) were placed in the brine solution and heated for about 1 hour to obtain a hot water extract. Afterward, the hot water extract was refrigerated for about 24 hours and centrifuged using a centrifuge for about 15 minutes at 4℃ and 10,000~15,000 rpm (preferably 13,000 rpm). Subsequently, 10%–12% of SDS (Sodium dodecyl sulfate), 0.4%–0.6% of a proteolytic enzyme (Alcalase, Neutrase, or Protamex), and 0.2%–0.3% of sodium hydroxide (NaOH) were added to the supernatant obtained by centrifugation, and the mixture was stirred at room temperature for about 24 hours. Then, centrifugation was repeated twice under the same conditions as above to remove the precipitate and separate the supernatant. Afterward, 2M sodium hydroxide was added to the solution to adjust the pH to 12–14 (preferably 13.00), and the mixture was stirred at 4°C for 4 hours to remove impurities such as proteins. Subsequently, isopropyl ethanol and ethyl ethanol were mixed in a ratio of approximately 3:7 (mixed ethanol) and refrigerated. Then, twice the amount of mixed ethanol relative to the separated supernatant was added, and the mixture was left at room temperature for 24 hours. Afterwards, the mixture was centrifuged for about 15 minutes at 4°C and 10,000 to 15,000 rpm (preferably 13,000 rpm) to remove ethanol and the supernatant, and precipitated high-purity PDRN was obtained. Ethyl ethanol was added again to the PDRN and centrifuged under the same conditions as above to obtain precipitated PDRN again. Afterwards, the PDRN was freeze-dried for about 72 hours.

[0064]

[0065] [Experimental Example 2] PDRN segmentation by ultrasonic fragmentation

[0066] To fragment PDRN, sonication was performed. The sonication machine was set to an amplitude of 30 and sonicated for 14 hours, and the PDRN DNA was confirmed to be fragmented to less than 200 bp by electrophoresis (*1% agarose gel, 200 V, 30 min).

[0067]

[0068] [Experimental Example 3] Exosome Extraction

[0069] Human skin cell line HaCaT (obtained from ATCC) was cultured using exosome-depleted FBS. 6×10⁶ cells per 100mm plate 6 Cells were cultured in 8 plates until a cell count of approximately 8 cells / ml was reached. Once the cells were cultured, the cell medium was harvested from the 8 plates, and the supernatant was obtained by centrifuging at 2000xg for 10 minutes to remove cell debris. Subsequently, the supernatant was transferred to a new tube, Total exosome isolation reagent (Invitrogen) was added, and the mixture was incubated overnight at 4°C. Afterward, the supernatant was removed by centrifuging at 10,000xg for 1 hour at 4°C. Then, 1×PBS was added to the exosome pellet to loosen it, and the skin cell-derived exosomes obtained through the above process were stored at -20°C.

[0070]

[0071] [Experimental Example 4] Capture of PDRN in Exosomes via Electroporation

[0072] Electroporation was performed to capture PDRN within exosomes. First, an ice bucket was prepared, and 1×10 5200 μL of exosomes at a concentration of copies / μl and 10 μL of PDRN DNA at a concentration of 10 pg were mixed. Then, the mixture was placed in an ice bucket and incubated for 10 minutes, and electroporation was performed using an electroporator under the conditions of Table 1 below. Afterward, the mixture was left at room temperature for 10 minutes.

[0073] VoltagePulse lengthPulseinterval1,000V15μs310 seconds

[0074]

[0075] [Example 1] Quantitative Evaluation of Segmented PDRN

[0076] According to Experimental Example 4 above, sonication was performed to reduce the size of the PDRN gene to 200 bp or less in order to capture the PDRN gene within the exosome, and electrophoresis was performed to confirm whether the DNA was properly fragmented. Sonication was performed at an amplitude of 30 for 3, 5, 7, 10, and 14 hours, and the electrophoresis conditions were set to 2% agarose gel, 0.5X TBE buffer, 200V, 30 min, DNA size marker: 25 / 100 bp DNA Ladder (Bioneer).

[0077] As a result, as shown in Figure 1, it was confirmed that the PDRN gene was reduced to 200 bp or less after 14 hours of treatment.

[0078]

[0079] [Example 2] Analysis of Exosome Size and Concentration

[0080] To confirm the size and concentration of the extracted exosomes (Experimental Example 3), nanoparticle tracking analysis (NTA) was performed. As a result, as shown in Fig. 2, the exosome size was found to be 100–300 nm, and the concentration was 2.4 × 10⁻⁶. 9It was found to be particles / mL.

[0081]

[0082] [Example 3] Analysis of Optimal Conditions for the Electric Cleaning Method

[0083] Two types of synthetic DNA (DNA1 and DNA2) with known nucleotide sequences were prepared (DNA1 was approximately 50 bp and DNA2 was approximately 300 bp in length). Subsequently, the synthetic DNA was captured within exosomes using an electrolytic method (Experimental Example 4). Afterward, excess DNA was removed by treatment with DNase, and the samples were heated at 100°C for 5 minutes to release the synthetic DNA captured within the exosomes. Subsequently, to detect only the DNA captured within the exosomes, primers and probes complementary to the synthetic DNA were prepared, and real-time PCR was performed. To confirm the optimal conditions for PDRN capture within exosomes and the release of the captured PDRN during the above process, the following experiment was conducted.

[0084]

[0085] 3-1. Electric Cleaning Method

[0086] To determine the optimal conditions for electroporation for PDRN capture within exosomes, DNA capture efficiency was analyzed according to the experimental group. Various conditions were varied for exosome concentration, DNA (PDRN fragmented via sonication) concentration, voltage, pulse length, pulse, and interval, and measurements were taken.

[0087] As a result, as shown in Figure 3, no band was generated during electrophoresis because exosomes are large in size. Therefore, if PDRN is captured within exosomes, the band size in electrophoresis is reduced compared to the control group. In addition, it was confirmed that the most PDRN was captured within exosomes when the electrophoresis method was performed under conditions of a voltage of 1000V, a pulse length of 15μs, 3 pulses, and an interval of 10 seconds, so it was decided to apply these conditions to future experiments.

[0088]

[0089] 3-2. DNase Treatment

[0090] To determine the effect of DNase treatment on the release of DNA captured within exosomes, experimental groups were established as a DNase-treated group and a DNase-untreated group, and electroporation was performed in the same manner as described above. Relatively long DNA2 was used as the synthetic DNA. As a result, as shown in Figure 4, in the DNase-untreated group, excess DNA that was not captured within exosomes was detected, making it difficult to accurately determine the amount of DNA captured within exosomes; whereas in the DNase-treated group, excess DNA that was not captured was removed, making it easy to accurately determine the amount of DNA captured within exosomes. From the above results, it was confirmed that removing excess DNA through DNase treatment is essential for confirming captured exosomes.

[0091]

[0092] 3-3. Heat Treatment

[0093] To determine the effect of heat treatment on the release of DNA trapped within exosomes, experimental groups were established into a heated group and a non-heated group, and the electrochemical method was performed in the same manner as described above. Relatively long DNA2 was used as the synthetic DNA. As a result, as shown in Figure 4, in the non-heated group, DNA was not released from the exosomes, so not all DNA trapped within the exosomes was detected, whereas in the heated group, DNA was released and detected in stages depending on the concentration of added DNA. From the above results, it was confirmed that the process of destroying the exosome membrane through heating is essential to release DNA trapped within exosomes.

[0094]

[0095] 3-4. Exosome Concentration

[0096] To determine the optimal concentration of exosomes used during the electrocleaning method, exosomes were serially diluted tenfold from the stock solution to 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 and 1×10 1 Copies / μl concentrations were prepared, and 10 μl of DNA at a concentration of 10 pg / μl was added to each dilution to perform electrolysis in the same manner as above (DNase treatment and heat treatment were also performed). Relatively long DNA2 was used as the synthetic DNA. As a result, as shown in Fig. 5, 1×10 exosomes 5 The lowest real-time PCR Cq value was measured at this concentration, which means that the highest amount of DNA was captured within exosomes in the dilution at this concentration compared to other concentrations. From the above results, based on the standard of treating 10 μl of DNA at a concentration of 10 pg, the exosome is 1 × 10⁶ 5 It was confirmed that the DNA capture efficiency within exosomes was the best at this concentration.

[0097]

[0098] [Example 4] Analysis of Skin Regeneration Effect

[0099] 4-1. Analysis of Skin Regeneration Effects

[0100] To confirm the skin regeneration effect of PDRN-encapsulated exosomes, cultured HaCaT cells (Experimental Example 2) were seeded into a 6-well plate, and a physical wound was created by scraping the bottom of the plate to simulate an environment identical to that of damaged skin cells. Subsequently, the experimental groups were designated as PDRN-encapsulated exosomes (PDRN group) and PDRN-unencapsulated exosomes (Control group). As shown in Figure 6, photographs were taken at 6, 12, 18, and 24 hours from 0 hours after physical damage was applied to monitor the proliferation of skin cells and the progress of wound healing. PDRN / exosomes were treated under the conditions shown in Table 2 below.

[0101] PDRN throughput 1000 ng PDRN (10 ng / μL) 100 μL exosomes (1 × 10⁶ 7 copies / mL)20μL

[0102] As a result, as shown in Figure 6, the cell migration rate after 24 hours was significantly increased in the group treated with PDRN alone (16.27%) and the group treated with PDRN-captured exosomes (20.09%) compared to the control group (10.58%). In addition, the cell migration rate after 24 hours was also significantly increased in the group treated with PDRN-captured exosomes (20.09%) compared to the group treated with PDRN alone (16.27%).

[0103]

[0104] 4-2. Analysis of Gene Expression Related to Skin Regeneration

[0105] To confirm the regulatory effect of PDRN on skin regeneration-related gene expression in captured exosomes, real-time PCR analysis was performed. For the genes to be evaluated, primers and probes were designed to detect only mRNA, considering the exon and intron regions. Each primer and probe was designed based on a region with no variation compared to all Ref transcripts, and the melting points were designed to be -55–60°C for primers and -60–65°C for probes. The melting point, GC percentage, hairpin formation temperature, energy required for self-dimer formation, and amplicon size of each primer and probe were verified using Integrated DNA Technologies (IDT). For each sample, the relative expression level (2–(target gene Cq – GAPDH gene Cq)) relative to the housekeeping gene GAPDH was calculated, and the difference in the PDRN-captured exosome group compared to the control group was analyzed.

[0106] As a result, as shown in Fig. 7, the p-values ​​of the procollagenase and MMP2 (matrix metalloproteinase-2) genes in the exosome-treated group (200E+P) in which PDRN (200ng) was captured were 0.0066 and 0.0062, respectively, indicating that the expression of the two genes was significantly increased.

[0107]

[0108] [Example 5] Analysis of Anti-inflammatory Effect

[0109] To confirm the anti-inflammatory effect through the regulation of inflammation-related gene expression in exosomes captured by PDRN, real-time PCR analysis was performed. The method was the same as in 4-2. As a result, as shown in Figure 8, the expression of inflammation-related genes TNF-α, IL-6, and IL-1β was significantly reduced in the exosome-treated groups (1000E+P, 200E+P) captured by PDRN (1000ng, 200ng).

[0110]

[0111] [Example 6] Analysis of Exosome Characteristics

[0112] To determine whether changes in the characteristics of exosomes occurred due to the electroporation method, a transmission electron microscope (TEM) was used. As a result, as shown in Figure 9, no significant changes in the characteristics of exosomes were observed depending on whether or not electroporation treatment was performed. From the above results, it was confirmed that since the electroporation method does not affect the characteristics of exosomes, the application of PDRN in a form captured in exosomes is safe.

[0113]

[0114] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A cosmetic composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

2. A cosmetic composition according to claim 1, characterized in that the PDRN is derived from salmonid fish.

3. A cosmetic composition according to claim 1, characterized in that the exosomes containing the PDRN promote the gene expression of procollagenase or MMP2 (matrix metalloproteinase-2).

4. A cosmetic composition according to claim 1, characterized in that the exosomes containing the PDRN inhibit the expression of one or more selected from the group consisting of TNF-α (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), and IL-1β (Interleukin-1 beta).

5. A cosmetic composition according to claim 1, characterized in that the skin improvement is one or more selected from the group consisting of antioxidant, anti-inflammatory, skin wrinkle improvement, skin moisturization, improvement of skin cell damage, improvement of skin troubles, anti-aging of skin, skin regeneration, and improvement of skin elasticity.

6. A cosmetic composition according to claim 1, characterized in that the exosomes are derived from skin cells.

7. In claim 1, the exosomes are 1×10 based on 10μl of PDRN at a concentration of 10pg / μl. 5 A cosmetic composition characterized by a concentration of copies / μl.

8. A cosmetic composition according to claim 1, characterized in that the exosomes containing the PDRN are processed by one selected method from the group consisting of a topical method, an MTS (Micro Needling System) method, and an injection method.

9. A health functional food composition for skin improvement comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

10. A pharmaceutical composition for the prevention or treatment of skin diseases comprising exosomes containing PDRN (Polydeoxyribonucleotide) as an active ingredient.

11. A pharmaceutical composition according to claim 10, characterized in that the skin disease is any one selected from the group consisting of acne, skin inflammation, dry skin, athlete's foot, eczema, allergic urticaria, pruritus, ringworm, psoriasis, and atopy.

12. Step of extracting PDRN (Polydeoxyribonucleotide) from fish (Step 1); Step of extracting exosomes from skin cells (Step 2); and A method for producing exosomes in which PDRN is captured, comprising the step (step 3) of capturing the PDRN of the first step within the exosomes of the second step.

13. A manufacturing method according to claim 12, characterized in that the third step is performed through electroporation.

14. A manufacturing method according to claim 13, characterized in that the above-mentioned electro-cleaning method is performed under conditions of a voltage of 1000V, a pulse length of 15μs, 3 pulses, and an interval of 10 seconds.

15. A manufacturing method according to claim 12, characterized in that, after performing the third step, the manufacturing method further performs DNA degrading enzyme (DNase) treatment or heat treatment.

16. A manufacturing method according to claim 15, characterized in that the heat treatment is performed at 100℃ for 5 minutes.

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