KR102415344B1 - Skin wrinkle improvement composition comprising fragmented DNA mixtures with increased skin permeability - Google Patents

Abstract

The present invention relates to a skin-permeable composition containing a DNA fragment mixture and a method for preparing the same.

Description

Skin wrinkle improvement composition comprising fragmented DNA mixtures with increased skin permeability

The present invention relates to a skin-permeable composition containing a DNA fragment mixture and a method for preparing the same.

Nucleic acids are DNA and RNA present in the nucleus of a cell, and include purine bases (adenine, A; guanine, G) or pyrimidine bases (cytosine, C; thymine, T; uracil, U), a polymer of nucleotides composed of a pentose and phosphoric acid. Such nucleic acids have been recognized as materials for storage and transmission of genetic information, but recently, nucleic acids are broken down into small fragments outside the cell and selectively bind to receptors on the cell surface to activate signals inside the cell and small fragments of nucleic acids. As they are known to play a role in inducing DNA synthesis quickly with little energy by moving into cells, new functions and technologies using them are being developed.

Polydeoxyribonucleotide, a kind of mixture of DNA fragments with specific specifications extracted from animals or plants, stimulates adenosine A2 receptors to achieve anti-inflammation and cell proliferation. , is known to have a tissue regeneration effect (Kim, Y.H., et al., The wound healing effect of PDRN (polydeoxyribonucleotide) material on full thickness skin defect in the mouse, J. Korean Soc. Plast. Reconstr. Surg., 37(3), 220-226, 2010.).

Accordingly, there is a need to increase skin permeability for rapid absorption of the DNA fragment mixture for cell regeneration or cell growth.

It is an object of the present invention to provide a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive.

Another object of the present invention is to provide a composition for preventing skin aging or skin regeneration comprising the composition as an active ingredient.

Another object of the present invention is to provide a quasi-drug or cosmetic composition comprising the composition as an active ingredient.

Another object of the present invention is to provide a method for preparing a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

One aspect of the present invention provides a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive.

The composition may include the DNA fragment mixture in an amount of more than 1 part by weight based on 1 part by weight of the cationic additive.

The DNA fragment mixture in the composition may be present in the form of monodisperse particles.

The present invention surprisingly provides the composition of the present invention by confirming that the composition comprising the DNA fragment mixture and the cationic additive in a specific content includes the DNA fragment mixture in the form of monodisperse particles and increasing the skin permeability.

In the present invention, the term "DNA fragment mixture" refers to an extracted form of DNA corresponding to a biopolymer composed of phosphoric acid, four types of bases, and deoxyribose, which is mixed in the form of DNA fragments having various molecular weights. do.

In one embodiment, the DNA fragment mixture may be in a hydrolyzed form.

In one embodiment, the DNA fragment mixture may be at least one selected from the group consisting of polydeoxyribonucleotides (PDRN) and polynucleotides.

As used herein, the term "PDRN" refers to a polydeoxyribonucleotide, and is a mixture of short deoxyribonucleotides. PDRN is a kind of low molecular weight DNA complex made by fractionating DNA chains into a certain size.

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are linked in a chain form by covalent bonds. For example, the polynucleotide may have a relatively long nucleic acid length or a higher molecular weight than polydeoxyribonucleotides, but is not limited thereto.

In any one of the above embodiments, the DNA fragment mixture of the present invention may have a molecular weight of about 1 to 100,000 kDa, specifically, about 5 to 50,000 kDa, and more specifically, 50 to 10,000 kDa. .

In any one of the above embodiments, the DNA fragment mixture of the present invention may be a DNA fragment mixture obtained by extraction from testis or semen of fish. Specifically, the fish may be salmonid fish. More specifically, it may be salmon or trout, but is not limited thereto.

In one embodiment, the cationic additive of the present invention may be at least one selected from the group consisting of cationic peptides, cationic lipids and cationic polymers.

Specifically, the cationic peptide may be one or more selected from the group consisting of polylysine, protamine, and KALA peptide (cationic fusogenic peptide).

Specifically, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (N-[1-(2,3-dioleoyloxy)propyl ]-N,N,N-trimethylammonium chloride), 1,2-dioleoyloxy-3-(trimethylammonio)propane (1,2-dioleoyloxy-3-(trimethylammonio)propane), 1,2-dioleo yl-3-(4'-trimethylammonio)butanoyl-sn-glycerol (1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol), 1,2-diacyl-3-dimethyl Ammonium-propane (1,2-diacyl-3-dimethylammonium-propane), 1,2-diacyl-3-trimethylammonium-propane (1,2-diacyl-3-trimethylammonium-propane), 1,2- Diacyl-sn-glycerol-3-ethylphosphocholine (1,2-diacyl-sn-glycerol-3-ethylphosphocholine), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] It may be at least one selected from the group consisting of cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, dimethyloctadexylammonium bromide, and copolymers thereof.

Specifically, the cationic polymer may be at least one selected from the group consisting of chitosan, spermine, putrescine, cadaverine and spermidine. .

In any one of the above-described embodiments, the cationic additive of the present invention is at least one selected from spermine, spermidine, or dimethyloctadecylammonium bromide. it could be

In any one of the above-described embodiments, the cationic additive of the present invention may include spermine, but is not limited thereto.

The composition of the present invention may include the cationic additive and the DNA fragment mixture in a specific content ratio, so that the DNA fragment mixture is present in the form of monodisperse particles.

In one embodiment, the composition of the present invention may contain the DNA fragment mixture in an amount of more than 2.5 parts by weight based on 1 part by weight of the cationic additive. Specifically, based on 1 part by weight of the cationic additive, the DNA fragment mixture may be included in an amount of about 3 parts by weight or more, about 4 parts by weight or more, about 4.5 parts by weight or more, or about 5 parts by weight or more, but is not limited thereto. does not

The term “about” includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values within a range equal to or similar to the value following the term about, but not limited

The upper limit of the content of the DNA fragment mixture based on the content of the cationic additive is not particularly limited as long as the DNA fragment mixture is included in the form of monodisperse particles. For example, the content of the DNA fragment mixture based on 1 part by weight of the cationic additive may be about 200 parts by weight or less. In another example, the content of the DNA fragment mixture based on 1 part by weight of the cationic additive is about 100 parts by weight or less, about 50 parts by weight or less, about 40 parts by weight or less, about 35 parts by weight or less, about 30 parts by weight or less, about 25 parts by weight or less. or less, or about 20 parts by weight or less, but is not limited thereto.

In any one of the above-described embodiments, the composition of the present invention may include 0.01 wt% to 5.0 wt% of the DNA fragment mixture based on the total composition.

In any one of the above-described embodiments, the composition of the present invention may include 0.0005 wt% to 2.0 wt% of the cationic additive based on the total composition.

In the present invention, the term "monodisperse" refers to the uniformity of size and shape. As an example, particles having a narrow size distribution in a size distribution mode, such as a distribution average of sizes, may be referred to as “monodisperse”. As an example, a series of polymer molecules having similar molecular weights may be referred to as “monodisperse”.

In any one of the above-described embodiments, the monodisperse particles may have a condensed form rather than an amorphous dispersed form, for example, a sphere.

In the present invention, "spherical" in the shape of a particle means that it is observed in a circular or spherical shape at a specific magnification on an electron microscope, and can be oval, toroid, irregularly shaped, etc. It is not meant to exclude other forms. That is, to express the shape of a specific particle in the present invention as "spherical" does not necessarily mean that the particle has a shape having a sphericity of 1. " can be used as a term to refer to particles having a sphericity (equivalent diameter/maximum diameter) of 0.7 to 1.0. In one embodiment, the spherical particles of the present invention may be solid or hollow particles. However, it is not limited thereto.

In any one of the above embodiments, the surface potential of the monodisperse particles may be about 0 mV to -60 mV. Specifically, it may be about 0mV to -30mV, but is not limited thereto.

In any one of the above-described embodiments, the monodisperse particles may have a structure in which a mixture of DNA fragments is condensed around a cationic additive forming a core. However, it is not limited thereto.

The composition of the present invention may have increased skin permeability.

The composition of the present invention may have increased skin permeability compared to a DNA fragment mixture that does not include a cationic additive. The mixture of DNA fragments not containing the cationic additive may be a mixture of hydrolyzed DNA fragments, and may not form monodisperse particles or spherical shapes by being dispersed in various sizes.

In the present invention, the term "skin permeability" refers to the ability or property to penetrate the skin and penetrate into the skin, and is used in the same sense as "skin permeability" and "skin permeability".

As an example, a criterion for comparing the level of increase in skin permeability of the composition of the present invention may be a composition containing no cationic additive, or a composition containing less than 1 part by weight of the DNA fragment mixture based on 1 part by weight of the cationic additive. have. However, the present invention is not limited thereto, and the level of increase in skin permeability may be evaluated based on a composition different from the content of the DNA fragment mixture and cationic additive included in the composition of the present invention.

The composition of the present invention may be formulated in various forms.

In one embodiment, the composition of the present invention may be used as an injection, liquid, cream, ointment, gel, lotion, internal solution, external solution, transdermal absorbent, and the like.

In any one of the above embodiments, the composition of the present invention may further comprise a physiologically active substance.

The physiologically active substance is, for example, an antibody, protein, peptide, miRNA (microRNA), siRNA (small interfering RNA), aptamer (aptamer), synthetic drug (chemical drug), etc. may be, but is not limited thereto, and may further include an appropriate material according to the intended use.

In any one of the above-described embodiments, the composition of the present invention may further include a medical device raw material.

The medical device raw material may be, for example, hyaluronic acid, carboxylmethyl cellulose, collagen, alginate, poloxamer, bone graft materials, etc. However, it is not limited thereto, and may further include an appropriate material depending on the intended use.

In any one of the above embodiments, the composition of the present invention may further comprise at least one of a carrier, an excipient and a diluent.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, unknown. vaginal cellulose, polyvinyl pyrrolidone, purified water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. For example, when the composition of the present invention is formulated and used, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant may be included in the composition of the present invention, but is not limited thereto.

Another aspect of the present invention provides a composition for preventing skin aging, comprising a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive provided in the present invention.

Another aspect of the present invention provides a composition for skin regeneration, comprising a composition having increased skin permeability comprising the DNA fragment mixture and the cationic additive provided in the present invention.

As used herein, the term "skin aging" refers to symptoms such as reduced skin elasticity, reduced gloss, wrinkle generation, weakened regeneration, or severe dryness. The skin aging may be caused by the passage of time or an external environment.

In the present invention, the term “skin regeneration” refers to a process of restoring skin tissue against damage caused by external and internal causes of the skin. Examples of the damage caused by the external cause include ultraviolet rays, external pollutants, wounds, trauma, and the like, and examples of the damage caused by the internal cause include stress, but are not limited thereto.

In one embodiment, skin aging inhibition and/or skin regeneration is one or more proteins of collagen I, collagen II, tropoelastin, MMP-1, MMP-9, and procollagen type I and elastase Or it can be evaluated by checking the expression level of the gene encoding it.

In any one of the preceding embodiments, skin aging inhibition and/or skin regeneration is

(i) increased expression of one or more of procollagen type I, collagen I, collagen II, tropoelastin, and elastase; and

(ii) a decrease in the expression level of one or more of MMP-1 and MMP-9, but is not limited thereto.

In one embodiment of the present invention, the composition of the present invention having increased skin permeability was administered to confirm the effect of inhibiting skin aging and regenerating the skin, so the composition of the present invention can be usefully used for preventing skin aging and regenerating the skin.

Another aspect of the present invention provides a quasi-drug composition comprising a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive provided in the present invention.

In the present invention, the term "quasi-drug" refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act of Korea Quasi-drugs exclude products used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body.

In one embodiment, the quasi-drug composition of the present invention may be prepared in a form selected from the group consisting of body cleanser, foam, soap, mask, ointment, cream, lotion, essence and spray, but is not limited thereto.

Another aspect of the present invention provides a cosmetic composition comprising a composition with increased skin permeability comprising a DNA fragment mixture and a cationic additive provided in the present invention.

In one embodiment, the cosmetic composition of the present invention is a product with professional functionality emphasizing physiologically active efficacy and effect, and is a product that helps skin whitening, a product that helps improve skin wrinkles, burns the skin finely, Among the products that help protect the skin from UV rays, it may be included in cosmetics prescribed by Ordinance of the Ministry of Health and Welfare. However, it is not limited thereto.

In one embodiment, the cosmetic composition of the present invention is a solution, external ointment, cream, foam, nutrient lotion, soft lotion, pack, soft water, emulsion, makeup base, essence, soap, liquid detergent, bath agent, sunscreen cream, sunscreen It can be prepared in a formulation selected from the group consisting of oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays. , but not limited thereto.

Another aspect of the present invention comprises mixing a cationic additive and a DNA fragment mixture, and condensing the DNA fragment mixture into monodisperse particles. Preparation of a composition comprising a DNA fragment mixture and a cationic additive provide a way

The preparation method may include mixing the DNA fragment mixture in an amount of more than 1 part by weight based on 1 part by weight of the cationic additive.

In one embodiment, the preparation method of the present invention contains more than about 2.5 parts by weight, about 3 parts by weight or more, about 4 parts by weight or more, about 4.5 parts by weight or more, or about 5 parts by weight of the DNA fragment mixture based on 1 part by weight of the cationic additive. It may be mixed in an amount greater than or equal to parts. It is not limited thereto.

In any one of the above-described embodiments, other optional components may be additionally used in the step of mixing the cationic additive and the DNA fragment mixture. For example, but not limited to, isotonic agents, solvents, and appropriate salt ingredients may be used.

In any one of the above-described embodiments, the preparation method of the present invention may further include the step of dissolving the DNA fragment mixture by heating.

In any one of the above-described embodiments, the preparation method of the present invention may further include the step of dissolving the cationic additive by heating.

In any one of the above-described embodiments, the dissolving by heating may be at about 50° C. to 90° C., specifically, at about 60° C. to 80° C., but is not limited thereto.

In any one of the above embodiments, the dissolving by heating may be performed before the mixing of the cationic additive and the DNA fragment mixture, but is not limited thereto.

In any one of the above-described embodiments, the preparation method of the present invention may further include sterilizing the composition.

The composition of the present invention has increased skin permeability, and can be usefully used in various fields such as quasi-drugs, cosmetics, and raw materials for medical devices.

1A and 1B show the results of analyzing the size distribution of a DNA fragment mixture using DLS.
Figure 2 shows the result of confirming the shape of the DNA fragment mixture using TEM.
3 schematically shows a skin absorption test method.
4 shows a mixture of DNA fragments that have penetrated the skin layer using an HPLC instrument.
5 and 6 compare the skin permeability of the DNA fragment mixture.
7 confirms the evaluation results of cytotoxicity and proliferation of the DNA fragment mixture.
FIG. 8 shows changes in the mRNA expression levels of collagen I, collagen III, tropoelastin, MMP-1, and MMP-9 according to the DNA fragment mixture treatment after UV-B irradiation in NHDF cells.
Figure 9 shows the changes in the protein expression levels of Procollagen type I, MMP-1, and Elastase according to the DNA fragment mixture treatment after UV-B irradiation to NHDF cells.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: Preparation of a composition comprising a mixture of condensed DNA fragments and confirmation of particle morphology of the mixture of DNA fragments

Hydrolyzed DNA (manufactured by Pharma Research, Inc.) was used for the DNA fragment mixture, and spermine-4HCl as a cationic additive was used.

Purified water was added to the components having the contents shown in Table 1 below to prepare a 1ml composition.

content (wt%) Experimental example control hydrolyzed DNA 0.5 0.5 0.5 0.5 0.5 NaCl 0.9 0.9 0.9 0.9 0.9 Spermine-4HCl 0.01 0.05 0.1 0 0.2

Particle size distribution was confirmed by dynamic light scattering using DLS, a particle size analyzer. Specifically, the size of the sample in the Brownian motion state having irregular motion in the colloidal state was measured by the diffusion coefficient, and the particle size was calculated by the following equation.

D=kT/6πηR

(D = diffusion coefficient, k = Boltzmann constant, T = temperature, η = solution viscosity, R = particle diameter)

Here, the particle distribution was confirmed by applying a temperature of 25°C and a scattering angle of 90°. For comparative particle size comparison, as a control, a mixture of DNA fragments without cationic additives (hereinafter referred to as Hydrolyzed DNA) and a mixture of DNA fragments prepared to contain a relatively high content (0.2%) of cationic additives were used. did. The results of analyzing the size distribution of the DNA fragment mixture using DLS are shown in FIGS. 1A and 1B .

DNA fragment mixtures generally have non-uniform base lengths in most cases. Consistent with this, it was confirmed that hydrolyzed DNA is polydisperse (FIG. 1a). However, when a cationic additive was used, it was confirmed that the content of the experimental example was monodisperse (FIG. 1b).

In addition, as a result of confirming the shape of the DNA through TEM (FIG. 2), when the cationic additive was not used, the DNA had an inconsistent shape, whereas when the cationic additive was used as the content of the experimental example, the DNA was condensed. could check

The composition containing the condensed DNA fragment prepared in this Example (DNA fragment mixture 0.5%, spermine salt 0.05%, NaCl 0.9% content; hereinafter referred to as condensed hydrolyzed DNA) was used in the experiments of the following examples.

Example 2: Confirmation of skin absorption and penetration level of a composition comprising a condensed DNA fragment mixture (1)

The skin delivery efficiency of the composition comprising the condensed DNA fragment mixture of the present invention was evaluated at an in vitro level.

As an evaluation method for skin delivery efficiency, Franz diffusion cell was used according to the Franz method, a representative in vitro skin absorption test method, and the test method was carried out according to the OECD guideline (2004). The test method performed is schematically shown in FIG. 3, and the specific test method is as follows.

During the test period, the skin absorbent device and skin were maintained at normal skin temperature (32 ± 1℃) and humidity (30~70%), and the skin type was Pig skin and the thickness was 400㎛.

The skin was placed on the receptor chamber with the stratum corneum facing upward, and PBS Buffer was used for the receptor fluid in the lower part of the cell. It was applied so that the sample concentration was 10 mg on the upper part of the stratum corneum. The test was repeated 3 times for each test substance, and after 30 minutes, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24 hours had elapsed, the sample was permeated through the sample port at a certain concentration. 0.5ml each of the tested substance solution was collected, and 0.5ml of the same amount of receptor fluid was added again.

If the test exceeds 24 hours, the skin condition deteriorates, so the sample collection time should not exceed 24 hours. Analysis was carried out for content analysis using HPLC. The experimental results are shown in FIG. 4 .

As a result of quantification of Hydrolyzed DNA that has penetrated the skin layer using HPLC equipment, Hydrolyzed DNA was 7.35 μg/cm2 and 44.97 μg/cm2 at 12H and 24H, respectively, and for condensed Hydrolyzed DNA, 411.5 μg/cm2 at 12H and 24H, respectively. , 1063.52 μg/cm 2 　 drug permeation was confirmed. This is a result of the hydrolyzed DNA condensed with the addition of cationic additives showing a transmittance that is about 11 times higher than that of the hydrolyzed DNA.

Based on this, it was confirmed that the condensed DNA fragment mixture of the present invention is a material capable of improving skin permeability, and it was confirmed that it can be used as an active ingredient in pharmaceuticals and cosmetics.

Example 3: Confirmation of skin absorption and permeation level of a composition comprising a condensed DNA fragment mixture (2)

The skin delivery efficiency of the composition comprising the condensed DNA fragment mixture of Example 1 was evaluated through an in vivo experiment. The specific experimental method is as follows.

For 5 female testers, 500 μl of each of Hydrolyzed DNA and Condensed Hydrolyzed DNA were applied to cotton at the measurement site (2x3 ㎠), let it absorb for 20 minutes, and then remove the remaining sample with a tissue. Before and after application of the product, stay in a constant temperature and humidity room. When measuring the device, do not move the test site and do not apply external physical stimuli to the test site in the middle of the test.

After washing the test site with tap water, the test subjects rested their skin for 30 minutes in a space where there is no movement of air or direct sunlight and constant temperature and humidity conditions (temperature 22±2℃, humidity 50±10%) are maintained, then get involved

Test sites are selected as test sites 1, 2, and 3 by randomizing the sites 5 cm and 10 cm away from the left and right anterior medial flexion sites, and apply Hydrolyzed DNA and Condensed Hydrolyzed DNA according to the designated test method for each site. Measurements are performed before, immediately after (20 minutes of absorption), and 1 hour after use of Hydrolyzed DNA and Condensed Hydrolyzed DNA.

The test site was measured using Raman spectroscopy (Skin composition analyzer Model 3510 SCA (River Diagnostics, The Netherlands)), a device that can non-destructively check the percutaneous penetration and absorption of substances without biopsy, and the amount of permeation Raman fingerprint spectra (785 nm, 400 -1800/cm) to measure the skin penetration of Hydrolyzed DNA and Condensed Hydrolyzed DNA 5 times from the outermost layer of the epidermis to a total depth of 24 μm in 2 μm steps. The measured data is analyzed through Skin Tools 2.0, the equipment software, and the analyzed data is expressed in arbitrary units (a.u.).

When comparing the skin penetration depth of Hydrolyzed DNA and Condensed Hydrolyzed DNA, the test results are shown in [Fig. 5] and [Fig. 6]. Immediately after use, hydrolyzed DNA increased significantly to a skin depth of 4 μm, and condensed hydrolyzed DNA up to 16 μm, compared to the measured values before application ( p<0.05 ).

After 1 hour, the hydrolyzed DNA increased significantly to a skin depth of 4 μm and the condensed hydrolyzed DNA up to 10 μm compared to the measured values before application ( p<0.05 ).

The maximum depth of skin penetration of Hydrolyzed DNA and Condensed Hydrolyzed DNA was 4㎛ for Hydrolyzed DNA and 16㎛ for Condensed Hydrolyzed DNA.

Through this, it was confirmed that the skin permeability of the condensed DNA fragment of the present invention was effectively increased even in vivo.

Example 4: Confirmation of cell proliferation ability of a composition comprising a condensed DNA fragment mixture

An experiment was performed to evaluate the cell proliferation ability of the composition comprising the condensed DNA fragment mixture of the present invention.

The specific experimental method is as follows. NHDFs were adhered to 96-well plates at a density of 5×10 ³ cells/well, and toxicity and proliferation were measured using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). Cells were treated and serum-starved for 24 h, then treated with various concentrations of condensed Hydrolyzed DNA (1, 2.5, 5, 10, 12.5, 25, 50 and 100 ppm) for 24 h, 48 h, and 72 h. . CCK-8 solution (10 μl) was added to cells in 1 ml DMEM (Thermo Fisher Scientific, Inc.) and incubated at 37° C. for 2 hours. Absorbance was measured at 450 nm using a microplate reader (SpectraMax 340; Molecular Devices, Inc.).

When the condensed Hydrolyzed DNA was treated for 24 hours, 48 hours, and 72 hours, the results of evaluation of cytotoxicity and proliferation are shown in FIG. 7 .

After treatment with condensed Hydrolyzed DNA, it showed no cytotoxicity at any concentration up to 72 hours and 24 hours; concentrations of 12.5, 25, 50 and 100, after 48 hours; At concentrations of 10, 12.5, 25, 50, and 100, cell proliferation was shown at all treatment concentrations after 72 hours.

Through this, it was confirmed that the composition comprising the condensed DNA of the present invention does not exhibit cytotoxicity and can be safely used.

Example 5: Identification of factors related to skin aging inhibition and skin regeneration of a composition comprising a condensed DNA fragment mixture

An experiment was performed to evaluate the skin aging inhibition and skin regeneration ability of the composition comprising the condensed DNA fragment mixture of the present invention.

NHDF was cultured in microtiter plates (96 wells) at a density of 1x10 ⁵ cells per well. After pretreatment of cells and depletion of serum for 24 hours, Hydrolyzed DNA condensed after UV-B irradiation (50mJ /cm2) using Biospectra (Vilber Lourmat) was treated at a concentration of 10, 100 ppm and harvested after 24 hours. did Prior to UV-B irradiation, the culture medium was rinsed with PBS and irradiated with a specified intensity in PBS to prevent absorption by phenol-red present in the culture medium. All cell treatment groups were equally incubated in PBS at room temperature during the experimental procedure to ensure identical treatment conditions. Based on this, changes in the expression levels of mRNA and protein involved in skin aging inhibition and skin regeneration were measured.

Example 5-1. Confirmation of changes in mRNA expression level

Total RNA was extracted from cultured cells and isolated using Trizol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA synthesis was performed according to the protocol provided by Takara PrimeScript™ RT Master Mix (Takara Bio, Inc.). Reverse transcription PCR was performed at 37 °C for 15 minutes and at 85 °C for 5 seconds. For quantitative PCR, TB Green® Premix Ex Taq ™ II (Takara Bio, Inc.) and CFX96™ Real-Time System (BioRad Laboratories, Inc.) were used, and all reactions were repeated three times under the following conditions. 40 cycles of denaturation at 95°C for 30 seconds, 5 seconds at 95°C, and 30 seconds at 60°C were performed. Relative gene expression values were determined using the 2- ΔΔ Cq method.

24 hours after treatment with the condensed Hydrolyzed DNA, Collagen I, Collagen III, Tropoelastin, MMP-1, and MMP-9 expressed by the treatment with the condensed Hydrolyzed DNA are shown in FIG. 8 .

After UV-B irradiation, increase and decrease in mRNA expression level significantly occurred by UV-B irradiation compared to the control group. When 100ppm of condensed Hydrolyzed DNA was treated, the mRNA expression levels of Collagen I, Collagen III, and Tropoelastin, which were decreased by UV-B irradiation, increased, and the mRNA expression levels of MMP-1 and MMP-9, which were increased by UV-B irradiation, decreased. confirmed that

Example 5-2. Confirmation of protein expression level change

Type 1 collagen secretion was assessed using a procollagen type 1 C-peptide (PIP) enzyme immunoassay (MK101; Takara Bio Inc.). After the incubation period, 100 μl of antibody-peroxidase conjugate solution was added to each well followed by addition of 20 μl of diluent or standard solution. After incubation at 37°C for 3 hours, the well contents were removed and all wells were washed 4 times with 400 μl PBS. Then, 100 μl substrate solution was added to each well and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μl stop solution. Absorbance was measured at 450 nm using an ELISA reader (SpectraMax 340; Molecular Devices, LLC).

MMP-1 activity was confirmed using the MMP-1 immunoassay kit (DY901; R&D Systems, Inc.). The activity of MMP-1 in the cell supernatant was confirmed using the method described in the MMP-1 Assay Kit.

Elastase activity was assayed in the supernatant using the Fastin Elastin Assay kit (Biocolor Ltd.). The Fastin Elastin Assay uses dye reagents that bind to 'basic' and 'non-polar' amino acid sequences found in mammalian elastin. The dye-bound elastin and standards recovered from each sample were detected by reading the absorption at 513 nm. The measured amount of each elastin protein was normalized to the corresponding cell number.

Changes in procollagen type I reduced by UV-B and increased MMP-1 and elastase activity after 48 hours of treatment with condensed hydrolyzed DNA were confirmed. Changes in procollagen type I, MMP-1, and Elastase are shown in FIG. 9 .

After UV-B irradiation, the increase and decrease in protein expression level significantly occurred by UV-B irradiation compared to the control group. It was confirmed that when 100 ppm of condensed Hydrolyzed DNA was treated, the synthesis of Procollagen type I, which was reduced by UV-B irradiation, was increased. In addition, it was confirmed that the protein expression levels of MMP-1 and Elastase, which were increased by UV-B irradiation, were decreased.

From the results of Examples 5-1 and 5-2 described above, it was confirmed that the composition including the condensed DNA fragment mixture of the present invention was excellent in skin aging inhibition and skin regeneration effects.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (16)

A composition for skin regeneration with increased skin permeability comprising a DNA fragment mixture having a molecular weight of 50 to 10,000 kDa and a cationic additive, wherein the composition contains more than 5 parts by weight of the DNA fragment mixture based on 1 part by weight of the cationic additive and not more than 50 parts by weight As a composition comprising, the DNA fragment mixture in the composition is present in the form of monodisperse particles, wherein the cationic additive is spermine, spermidine, or dimethyldioctadecyl The composition of one or more selected from ammonium bromide (dimethyloctadexylammonium bromide).
The composition of claim 1, wherein the particle has a surface potential of 0 mV to -30 mV.
The composition of claim 1 , wherein the particles are in a condensed form.
The composition of claim 1, wherein the content of the DNA fragment mixture of the composition is 0.01 wt% to 5.0 wt% based on the total composition.
The composition of claim 1, wherein the content of the cationic additive in the composition is 0.0005 wt% to 2.0 wt%, based on the total composition.
The composition of claim 1, wherein the DNA fragment mixture is at least one selected from the group consisting of polydeoxyribonucleotides and polynucleotides.
The composition of claim 1, wherein the DNA fragment mixture is a polydeoxyribonucleotide having a molecular weight of 50 to 2,000 kDa.
The composition of claim 1, wherein the DNA fragment mixture is isolated from testis or semen of fish.
According to claim 8, wherein the fish is characterized in that the salmon family, the composition.
delete The composition according to any one of claims 1 to 9, wherein the composition is a formulation selected from injections, liquids, creams, ointments, gels, lotions, internal solutions, external solutions, and transdermal absorption agents. .
A composition for preventing skin aging, comprising the composition of any one of claims 1 to 9 as an active ingredient.
The composition of claim 1, wherein the skin regeneration is for wrinkle improvement.
A quasi-drug composition comprising the composition of any one of claims 1 to 9 as an active ingredient.
A cosmetic composition comprising the composition of any one of claims 1 to 9 as an active ingredient.
Condensing the DNA fragment mixture into monodisperse particles by mixing the DNA fragment mixture having a molecular weight of 50 to 10,000 kDa based on 1 part by weight of the cationic additive in an amount of more than 5 parts by weight and not more than 50 parts by weight, A method for preparing a composition for skin regeneration comprising a DNA fragment mixture and a cationic additive, wherein the cationic additive is spermine, spermidine, or dimethyloctadexylammonium bromide. One or more selected from, the manufacturing method.

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