Preparation and application of desoxyribonucleic acid and desoxyribonucleic acid derivative transdermal delivery system

Abstract

The invention relates to preparation and application of a desoxyribonucleic acid and desoxyribonucleic acid derivative transdermal delivery system, and belongs to the technical field of biological medicine and skin care products. Desoxyribonucleic acid and derivatives thereof have the effects of resisting aging, repairing and the like, but are difficult to penetrate through a skin barrier due to large molecular weight and strong polarity. According to the invention, hydroxyethyl chitosan, desoxyribonucleic acid and derivatives thereof are combined through electrostatic interaction to form the nano-composite. The compound is small in particle size and can promote transdermal absorption through hair follicle or cuticle gaps. The preparation method is simple, does not need an auxiliary reagent, and comprises the step of mixing a hydroxyethyl chitosan solution and a deoxyribonucleic acid derivative solution. Experiments show that the compound significantly enhances the transdermal efficiency, and has the effects of promoting cell repair and proliferation and resisting inflammation. The composition can be used for skin care products, and is high in safety and wide in application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and skin care products, and specifically relates to preparation and application of a deoxyribonucleic acid and its derivative transdermal delivery system. BACKGROUND

[0002] Deoxyribonucleic acid and its derivatives include deoxyribonucleic acid (DNA), deoxyribonucleic acid sodium (DNA sodium), deoxyribonucleic acid calcium (DNA calcium), deoxyribonucleic acid magnesium (DNA magnesium), deoxyribonucleic acid zinc (DNA zinc), deoxyribonucleic acid potassium (DNA potassium), polydeoxyribonucleotide (PDRN), polynucleotide (PN), etc., which belong to biological macromolecular skin care ingredients. As a natural substance extracted from salmon sperm or plants, it mainly undertakes two major functions of repair and moisturizing in skin care products. Among them, DNA sodium can accelerate the regeneration of epidermal cells for post-laser or sunburn skin, and clinical data shows that the use of a repair cream containing 0.5% DNA sodium can shorten the wound healing time by 3-5 days. Polydeoxyribonucleotide (PDRN) is an active polynucleotide mixture with a molecular weight range of 50-1500 kDa, which has no antigenic properties and systemic toxicity. PDRN has been used in the treatment of various diseases in clinical practice, such as diabetes, soft tissue ulcers, arthritis, etc. In recent years, studies have shown that PDRN can accelerate the synthesis of DNA nucleotides and nucleosides, improve the proliferation and growth ability of fibroblasts, promote angiogenesis, improve microcirculation, and provide nutrition for skin tissue regeneration; in addition, it can also protect tissue cells from ultraviolet-induced DNA damage. Although deoxyribonucleic acid and its derivatives have been proven to have many advantages on the skin, due to their large molecular weight and strong polarity, it is difficult for them to penetrate the skin barrier and enter the inside of the skin to exert their anti-aging, repair, etc. effects.

[0003] To solve the above problems, the deoxyribonucleic acid and its derivatives are nano-processed, which is beneficial to their transdermal absorption, thereby improving their effect in the skin. For example, the prior art CN118681062A prepares PDRN and collagen into microspheres through cross-linking reaction, which has the potential to enter the skin. In addition, studies have shown that chitosan can be combined with PDRN to prepare PDRN nanoparticles after adjusting the pH, thereby achieving the skin repair and enhancement of PDRN. However, the nano-preparation of PDRN in the above studies requires auxiliary reagents or harsh preparation conditions, thereby causing potential safety risks for its application in the skin.

[0004] In a recent study, researchers proposed a method for the spatiotemporally controlled release of PDRN from a hydrogel using exogenous stimulation. Modified short-chain chitosan (SCS) was used as a macromolecular crosslinking agent, copolymerized with molybdenum disulfide nanosheets (MoS2) and N-isopropylacrylamide (NIPAAm) to form a composite hydrogel. This nanocomposite hydrogel, under long-range stimulation with near-infrared light, enables the spatiotemporally controlled release of PDRN, overcoming the limitations of drug delivery methods that are prone to degradation and difficult to deliver.

[0005] Prior art CN116725898 A discloses a self-assembled biogel, its preparation method, and its applications. The biogel comprises palmitoyl pentapeptide-4, polydeoxyribonucleotides (PDRN), and chitosan. The PDRN is encapsulated within the self-assembled structure of palmitoyl pentapeptide-4, and chitosan is coated onto the outside of the self-assembled structure via electrostatic interactions. The combination of palmitoyl pentapeptide-4, PDRN, and chitosan enhances the bioavailability and transdermal absorption of the active substances and produces synergistic anti-aging effects.

[0006] Our previously prepared hydroxyethyl deacetylated chitosan (CN116496429B) has been shown to have good transdermal absorption for proteins, peptides, and other substances. Based on our understanding of the mechanism of action of hydroxyethyl deacetylated chitosan, we believe that this material has potential applications in the nano-sizing of deoxyribonucleic acid (DNA) and its derivatives. Based on these considerations, this invention prepares DNA and its derivative nanoparticles by mixing hydroxyethyl deacetylated chitosan with DNA and its derivatives, and then verifies their effects on the skin. Summary of the Invention

[0007] To address the aforementioned technical problems in the existing technology, the applicant, based on synthesized hydroxyethyl deacetylated chitosan, utilizes its positive charge properties and electrostatic interactions to combine it with deoxyribonucleic acid (DNA) and its derivatives to prepare a hydroxyethyl deacetylated chitosan / DNA and its derivative complex, forming nano-DNA and its derivatives. Because nano-DNA and its derivatives have the advantage of small particle size, they can penetrate the skin through hair follicles, sweat glands, or gaps in the stratum corneum, thereby exerting the skincare effects of DNA and its derivatives. Therefore, this application is filed.

[0008] The present invention first provides a transdermal delivery system for deoxyribonucleic acid and its derivatives, comprising hydroxyethyl deacetylated chitosan and deoxyribonucleic acid and its derivatives, wherein the hydroxyethyl deacetylated chitosan has a deacetylated chitosan backbone and hydroxyethyl groups are linked by ether bonds, and the number of units n=6-30.

[0009] In some embodiments of the present invention, the weight ratio of the hydroxyethyl deacetylated chitosan to deoxyribonucleic acid and its derivatives is 1:0.01-10.

[0010] In some embodiments of the present invention, the weight ratio is preferably 6:1.

[0011] According to an embodiment of the present invention, the hydroxyethyl deacetylated chitosan has the following structural units. :

[0012] Wherein, A represents the hydroxyethyl group.

[0013] In some embodiments of the present invention, the deoxyribonucleic acid and its derivatives include DNA, sodium DNA, calcium DNA, magnesium DNA, zinc DNA, potassium DNA, polydeoxyribonucleic acid (PDRN), and polynucleotide (PN), etc.

[0014] According to embodiments of the present invention, the polydeoxyribonucleic acid (PDRN) sources include Bloomage Biotechnology Co., Ltd., and Regmin (Shandong) Biotechnology Co., Ltd.

[0015] According to an embodiment of the present invention, the preparation method of the hydroxyethyl deacetylated chitosan includes the following steps:

[0016] Deacetylated chitosan was reacted with a compound containing group A to obtain hydroxyethyl deacetylated chitosan grafted with group A.

[0017] A is ethylene oxide, 2-chloroethanol, etc.

[0018] In some embodiments of the present invention, the reaction temperature in the above steps is 0-95°C, preferably 40-60°C, for example 45°C, and the reaction time can be 0.5-48 hours, for example 5 hours, 12 hours, 24 hours, 48 ​​hours, etc.

[0019] In some embodiments of the present invention, during the preparation of hydroxyethyl deacetylated chitosan, the molar ratio of the deacetylated chitosan to ethylene oxide or 2-chloroethanol is 1:0.1-1.

[0020] In some embodiments of the present invention, the deacetylated chitosan is obtained by a preparation method comprising the following steps: a1, hydrolyzing chitin to obtain deacetylated chitin; a2, oxidatively degrading the deacetylated chitin to obtain the deacetylated chitosan.

[0021] In some embodiments of the invention, the hydrolysis conditions in the above reaction include dissolving chitin in an acidic solution and then adjusting the pH to alkaline (e.g., pH 8-10) by adding an alkali, such as sodium hydroxide solution. The acidic solution may be hydrochloric acid or acetic acid solution, and the pH is preferably between 0 and 4.

[0022] In some embodiments of the present invention, the preparation of the deacetylated chitosan includes: dissolving chitin in an acidic solution (hydrochloric acid, acetic acid) with a pH between 0 and 4, wherein the solid-liquid ratio of chitin to the acidic solution is 1:5-100; adding sodium hydroxide solution (10-40%) to the acidic chitin solution to adjust the pH to 8-10 to obtain precipitate 1; subjecting precipitate 1 to steps such as pressure filtration and water washing to obtain precipitate 2; adding the obtained white precipitate 2 to a reaction vessel, adding 1M sodium hydroxide solution to adjust the pH to 8-9; then adding hydrogen peroxide solution; adjusting the temperature to 0-95°C and reacting for 0.5-48 hours to obtain a suspension; subjecting the suspension to steps such as centrifugation, water washing, and centrifugation to obtain precipitate 3.

[0023] In some embodiments of the present invention, the preparation of the hydroxyethyl deacetylated chitosan includes: dissolving the deacetylated chitosan in water and adjusting the pH to 8-10; adding ethylene oxide or 2-chloroethanol to the solution, adjusting the reaction temperature to 0-95°C, reacting for 0.5-48 hours to obtain a transparent liquid; adjusting the pH of the transparent liquid to 2-4 using an acidic solution, dialyzing (MW-500Da), and freeze-drying to obtain product 1; the molar ratio of the deacetylated chitosan to ethylene oxide or 2-chloroethanol is 1:0.1-1;

[0024] In a second aspect, the present invention provides a method for preparing the deoxyribonucleic acid and its derivative composition as described above, comprising the following steps: dissolving hydroxyethyl deacetylated chitosan in water to obtain solution A; dissolving deoxyribonucleic acid and its derivative in water to obtain solution B; and mixing solution A and solution B.

[0025] According to an embodiment of the present invention, the mass concentration of the deacetylated chitosan derivative in solution A is 0.001%-50%, for example 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.

[0026] According to an embodiment of the present invention, the mass concentration of protein in solution B is 0.001%-50%, 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.

[0027] According to an embodiment of the present invention, the mixing conditions include: a stirring speed of 10-200 rpm and a stirring time of 0.1-48 h.

[0028] The deoxyribonucleic acid and its derivatives in this invention include: DNA, sodium DNA, magnesium DNA, zinc DNA, potassium DNA, PDRN, PN, etc.

[0029] In a third aspect, the present invention also provides a skin care product comprising the deoxyribonucleic acid and its derivative composition described in the first aspect of the present invention.

[0030] The skincare products mentioned can be water, lotion, cream, etc.

[0031] In some embodiments, the amount of the deoxyribonucleic acid and its derivative transdermal delivery system added to the skin care product is 0.1%-10%.

[0032] The present invention also provides a method for promoting transdermal delivery of deoxyribonucleic acid and its derivatives, using the above-mentioned transdermal delivery system for deoxyribonucleic acid and its derivatives to achieve transdermal delivery through skin hair follicles or the stratum corneum.

[0033] Finally, this invention provides the application of the above-mentioned deoxyribonucleic acid and its derivative transdermal delivery system in the preparation of skin care products for skin repair, anti-inflammation or anti-aging.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] The deoxyribonucleic acid (DNA) and its derivative delivery system provided by this invention has significant beneficial effects: First, through nano-sizing, the complex has a small particle size and a high zeta potential, enabling it to effectively penetrate the skin barrier and improve transdermal absorption efficiency. Second, the preparation process is simple and mild, requiring no cross-linking agents or harsh conditions, ensuring high safety and avoiding potential risks. Third, experimental verification shows that the complex can promote cell repair, proliferation, and anti-inflammation, as well as improve an atopic dermatitis model. These effects make this delivery system highly effective in repairing, moisturizing, and anti-aging skincare products, with broad application prospects. Attached Figure Description

[0036] Figure 1 This is the 1H NMR spectrum of the hydroxyethyl deacetylated chitosan prepared in Example 1.

[0037] Figure 2 The infrared spectrum is that of the hydroxyethyl deacetylated chitosan prepared in Example 1.

[0038] Figure 3 This is an electron micrograph of the hydroxyethyl deacetylated chitosan / PDRN prepared in Example 1.

[0039] Figure 4This is a dynamic light scattering diagram of the hydroxyethyl deacetylated chitosan / PDRN prepared in Example 1.

[0040] Figure 5 The optimal ratio of hydroxyethyl deacetylated chitosan / PDRN in Example 3 was screened. Figure 5 In the diagram, A represents the DLS hydrated particle size analysis. Figure 5 B in the diagram represents the potential analysis. Figure 5 C in the image represents an agarose gel electrophoresis result.

[0041] Figure 6 This is a fluorescence imaging image of the animal skin test in Example 5.

[0042] Figure 7 This is a statistical graph of the fluorescence imaging results of the animal skin test in Example 5.

[0043] Figure 8 This is a diagram showing the results of the cell scratch experiment in Example 6.

[0044] Figure 9 This is a statistical chart of cell scratch healing rate in Example 6.

[0045] Figure 10 This is a diagram showing the results of the cell proliferation experiment in Example 7.

[0046] Figure 11 This is a statistical graph of the cell proliferation experiment in Example 7.

[0047] Figure 12 This is a graph showing the detection results of cellular inflammatory factors in Example 8.

[0048] Figure 13 These are comparative histological sections from different treatment groups in Example 9.

[0049] Figure 14 This is a quantitative bar chart (unit: μm) of the epidermal thickness of each treatment group in Example 9.

[0050] Figure 15 This is a collection of microscopic images of tissue sections from different treatment groups in Example 9. Detailed Implementation

[0051] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0052] Example 1: Synthesis of hydroxyethyl deacetylated chitosan

[0053] Step 1: Add 20g of chitin to 500mL of 10% hydrochloric acid solution and stir to dissolve. Then add 10% NaOH aqueous solution to adjust the pH to 8.5, resulting in a white precipitate. Filter the white precipitate, wash it, and pour it into a three-necked flask. Add 50g of 30% hydrogen peroxide and 200mL of water, adjust the temperature to 60℃, and react for 6 hours to obtain a milky white liquid. After centrifugation and washing with water, obtain the white precipitate deacetylated chitosan.

[0054] Step 2: Dissolve the deacetylated chitosan prepared in Step 1 in 100 mL, adjust the pH to 8, then add 20 g of ethylene oxide, adjust the reaction temperature to 60 °C, and after reacting for 12 hours, stop the reaction, add 10% HCl solution to adjust the pH to 4, dialyze (MW=500 Da), and freeze dry to obtain 19 g of the product hydroxyethyl deacetylated chitosan.

[0055] The 1H NMR spectrum of hydroxyethyl deacetylated chitosan is as follows: Figure 1 As shown, the characteristic peaks in the spectrum (such as the hydroxyethyl proton signal at chemical shift δ 3.0-4.0 ppm) confirm that the hydroxyethyl group was successfully grafted onto the deacetylated chitosan backbone, verifying the integrity of the chemical structure of hydroxyethyl deacetylated chitosan and providing fundamental support for the formation of the complex. The infrared spectrum of hydroxyethyl deacetylated chitosan is shown below. Figure 2 As shown, characteristic absorption bands are visible in the spectrum, such as at 3400 cm⁻¹. -1 OH stretching vibration at 2900 cm -1 CH vibration at 1100 cm -1 The presence of nearby ether bonds (COC) signals indicates successful hydroxyethyl modification and highlights the functional group characteristics of hydroxyethyl deacetylated chitosan, ensuring its stability in the nanocomposite. Electron microscopy and dynamic light scattering detection, such as... Figures 3-4 As shown.

[0056] Example 2

[0057] Dissolve 6g of the hydroxyethyl deacetylated chitosan prepared in Example 1 in 50mL of water to prepare solution A; dissolve 1g of PDRN (source: Bloomage Biotechnology Co., Ltd., product name: Biomeet™-Haiyue) in 50mL of water to prepare solution B; add solution B to solution A at room temperature and with stirring at 200rpm to prepare the hydroxyethyl deacetylated chitosan / PDRN complex; after moist heat sterilization, the obtained complex can be directly used as an active ingredient in skin care products.

[0058] Example 3

[0059] 10g of the hydroxyethyl deacetylated chitosan prepared in Example 1 was dissolved in 100mL of water as solution A; 1g of PDRN was dissolved in 100mL of water as solution B; at room temperature, solutions B and A were mixed in different proportions with stirring at 200rpm to prepare hydroxyethyl deacetylated chitosan / PDRN complexes; the optimal ratio of hydroxyethyl deacetylated chitosan / PDRN was screened using the DLS method, and the results are as follows. Figure 5 As shown.

[0060] (1) Agarose gel electrophoresis pattern:

[0061] The migration rate of electrophoretic bands directly reflects the degree of complex formation: as the N / P ratio increases, the electrostatic interaction between PDRN (a nucleic acid-like substance) and hydroxyethyl deacetylated chitosan (a cationic polymer) strengthens, inhibiting the free migration of nucleic acid molecules, and the band position gradually shifts upward. This indicates that with the increase of the N / P ratio, the polymer's ability to encapsulate / complex PDRN is enhanced, and the complex transforms from a "loosely bound state" to a "closely assembled state."

[0062] (2) DLS hydrated particle size analysis:

[0063] The hydrated particle size (Dh) of the complex varies significantly under different N / P ratios. When the N / P ratio is at a certain value, the particle size of the complex reaches the minimum and the particle size distribution is highly uniform.

[0064] This result indicates that at this N / P ratio, the assembly efficiency of hydroxyethyl deacetylated chitosan and PDRN is optimal, and the aggregation behavior between nanoparticles is effectively suppressed, forming a nanocomposite with controllable particle size and good dispersibility—which is crucial for the application performance of nucleic acid delivery systems such as "endocytosis and in vivo distribution".

[0065] (3) DLS Zeta potential analysis:

[0066] The zeta potential reflects the net charge characteristics of the complex surface. It fluctuates regularly with the N / P ratio. When the N / P ratio is at the aforementioned "optimal ratio," the absolute value of the zeta potential reaches a relatively high level (or is within a stable range), indicating a charge balance on the complex surface—ensuring both the electrostatic adsorption of PDRN by the polymer (preventing dissociation) and avoiding excessive particle aggregation through moderate charge repulsion, ultimately achieving a balance between colloidal stability and nucleic acid loading efficiency.

[0067] A three-dimensional analysis using agarose gel electrophoresis (nucleic acid binding capacity), DLS particle size (nanostructure uniformity), and Zeta potential (colloidal stability) determined that a 6:1 ratio is the optimal mass ratio for the hydroxyethyl deacetylated chitosan / PDRN complex. At this ratio, the complex exhibits "complete nucleic acid binding, controllable particle size uniformity, and excellent colloidal stability," providing key process parameters to support its application in biomedical fields such as nucleic acid delivery and gene therapy.

[0068] Example 4

[0069] 1. Mix 0.1g sodium hyaluronate, 50g glycerin, 0.5g Sepimex Zen (polyacrylate crosspolymer-6, CAS No.: 111286-86-3), 1g methylparaben, and 946.4g deionized water at 85°C, and after homogenization, obtain a mixture.

[0070] 2. After the temperature drops to 45°C, add 2g of the hydroxyethyl deacetylated chitosan / PDRN complex prepared in Example 3 (i.e., 6:1 as the optimal ratio of the complex) to the above solution, stir to dissolve it completely; obtain essence a, cool to room temperature, discharge and let stand.

[0071] Control group 1:

[0072] 1. Mix 0.1g sodium hyaluronate, 50g glycerin, 0.5g Sepimex Zen, 1g methylparaben, and 946.4g deionized water at 85°C, and then homogenize to obtain a mixture.

[0073] 2. After the temperature drops to 45℃, stir to fully dissolve it; obtain essence b, cool to room temperature, discharge and let stand.

[0074] Control group 2:

[0075] 1. Heat 0.1g sodium hyaluronate, 50g glycerin, 0.5g Sepimex Zen, 1g methylparaben, and 946.4g deionized water to 85℃, mix and homogenize to obtain a mixture;

[0076] 2. After the temperature drops to 45°C, add 1.71g of the hydroxyethyl deacetylated chitosan prepared in Example 1 to the above solution (calculated based on 2g of hydroxyethyl deacetylated chitosan / PDRN complex, the hydroxyethyl deacetylated chitosan in it is about 1.71g) and stir until fully dissolved; obtain essence c, cool to room temperature, discharge and let stand.

[0077] Control group 3:

[0078] 1. Heat 0.1g sodium hyaluronate, 50g glycerin, 0.5g Sepimex Zen, 1g methylparaben, and 948.11g deionized water to 85℃, mix and homogenize to obtain a mixture;

[0079] 2. After the temperature drops to 45℃, add 0.29 g of PDRN to the above solution (calculated based on 2 g of hydroxyethyl deacetylated chitosan / PDRN complex, of which PDRN is approximately 0.29 g), stir to dissolve fully; obtain essence d, cool to room temperature, discharge and let stand.

[0080] Example 5

[0081] 1. Sample preparation

[0082] Prepare a 20 mL aqueous solution A containing 1% of the hydroxyethyl deacetylated chitosan prepared in Example 1.

[0083] 1.2 Preparation of fluorescently labeled PDRN:

[0084] Dissolve 0.14 g Cy5.0-COOH in 20 mL of water. In an ice-water bath, add 0.11 g EDC and 0.8 g NHS. After reacting for 2 hours, add 0.1 g PDRN. After reacting for 24 hours, remove unreacted Cy5.0 and byproducts by dialysis. After freeze-drying, obtain fluorescently labeled PDRN (Cy5-PDRN).

[0085] 1.3 Preparation method of hydroxyethyl deacetylated chitosan (HCS) / fluorescently labeled PDRN:

[0086] Take 20 ml of the hydroxyethyl deacetylated chitosan solution A prepared in step 1 above, add 0.1 g of fluorescently labeled PDRN (Cy5-PDRN), and stir at 1000-2000 r / min for 2 h to obtain hydroxyethyl deacetylated chitosan HCS@fluorescently labeled PDRN with the corresponding particle size.

[0087] 2. Transdermal test

[0088] Fluorescently labeled PDRN and hydroxyethyl deacetylated chitosan (HCS) / fluorescently labeled PDRN were added to a blank emulsion at a concentration of 0.1%. Live mice were used as subjects. Different samples were applied to the skin of mice, with three mice per group. Mice were euthanized by cervical dislocation at 2 and 8 hours later. Skin tissue of approximately 0.5 cm x 0.5 cm from the center of the applied area was excised, rinsed with PBS to remove residual drug, and fixed in formalin for 24 hours. The fixed skin tissue was rinsed, dehydrated, cleared, paraffin-embedded, and then sectioned to a thickness of 4–6 mm. The sections were then dewaxed, dehydrated, cleared, unstained, and unmounted. The sections were then scanned.

[0089] 3. Quantitative PCR methods

[0090] Data were analyzed using SPSS 13.0 statistical software. The results were compared using the chi-square test, with P < 0.05 considered statistically significant. The results are as follows: Figures 6-7 As shown.

[0091] 4. Results

[0092] Depend on Figures 6-7 It was found that the fluorescently labeled hydroxyethyl deacetylated chitosan (HCS) / PDRN complex produced significant fluorescence in the skin within 8 hours, indicating that the HCS / PDRN complex can penetrate the stratum corneum and enter the skin. While fluorescently labeled PDRN also entered the skin within 8 hours, the fluorescence intensity was lower, indicating a lower efficiency of PDRN entry into the skin within 8 hours. Relatively speaking, hydroxyethyl deacetylated chitosan can promote the entry of fluorescently labeled PDRN into the skin. Figure 7 It is known that hydroxyethyl deacetylated chitosan can promote the entry of PDRN into the skin within 8 hours.

[0093] Example 6: Cell Scratch Assay

[0094] 1. Cell Culture

[0095] HaCat (keratinocytes) were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. The cell culture was carried out at 37°C and 5% CO2.

[0096] 2. Cell scratch test

[0097] HaCat cells in logarithmic growth phase were seeded into 6-well plates and cultured. When the cell density reached 80%-90%, scratching was performed. Using a 0.2 mL autoclaved pipette tip, 1-3 straight lines were drawn perpendicularly to the bottom of the plate. PBS was then added to the culture dishes for washing. Hydrogen peroxide (100 g / L) was added to the culture dishes of the treatment groups. μm Serum-free culture medium containing HCS (1.5 mg / mL), HCS (0.25 mg / mL), and PDRN (0.25 mg / mL), and HCS@PDRN (based on PDRN concentration of 0.25 mg / mL) was used. Control group culture dishes were added to serum-free culture medium and placed in an incubator for further culture. Photos were taken at 0 h and 24 h at the same scratch site. The scratch area was calculated using ImageJ software, and the cell scratch healing rate was calculated using the formula. The experiment was repeated three times.

[0098] Cell scratch healing rate =

[0099] 3. Statistical methods

[0100] Data were analyzed using SPSS 13.0 statistical software. The chi-square test was used, and a p-value < 0.05 was considered statistically significant. Results are as follows: Figure 9 As shown.

[0101] 4. Results

[0102] from Figures 8-9 It can be concluded that, compared with PDRN monomer, the hydroxyethyl deacetylated chitosan of the present invention can significantly promote the repair effect of PDRN.

[0103] Example 7: Cell proliferation experiment

[0104] 1. Cell Culture

[0105] HaCat (keratinocytes) were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. The cell culture was carried out at 37°C and 5% CO2.

[0106] An oxidative damage model of HaCat was established, and cells were divided into a control group (normal HaCaT cells) and an H2O2 group (100...). μ m ol / L), H2O2 (100 μm ol / L) + HCS group (1.5 mg / mL), H2O2 (10 ... μm ol / L) + PDRN group (0.25 mg / mL), H2O2 (10 ... μm Cells were cultured for 24 hours before subsequent experiments were conducted. The cells were cultured in the HCS@PDRN group (0.25 mg / mL PDRN) + HCS@PDRN group.

[0107] 2. Detection of HaCat cell proliferation in each group using the 5-ethynyl-2'-deoxyuridine (EDU) method

[0108] After culturing HaCaT cells in each group for 24 hours, they were used with 10 μm After labeling with 1 ol / L EDU for 2 hours, EdU staining was performed using a kit. BeyoClick™ EdU-488 Cell Proliferation Assay Kit (Item No.: C0071S, Beyotime Biotechnology Co., Ltd.), and fluorescence detection was performed.

[0109] 3. Statistical methods

[0110] Data were analyzed using SPSS 13.0 statistical software. The chi-square test was used, and a p-value < 0.05 was considered statistically significant. Results are as follows: Figure 11 As shown.

[0111] 4. Results

[0112] From Figures 10-11 It can be concluded that, compared with the PDRN monomer, the hydroxyethyl chitosan of the present invention can significantly promote the effect of PDRN on cell proliferation.

[0113] Example 8, Cell Inflammatory Inhibition Experiment

[0114] 1. Cell Culture

[0115] HaCat (keratinocytes) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the cell culture was carried out at 37°C and 5% CO2.

[0116] An oxidative damage model of HaCat was established, and the cells were divided into a control group (normal HaCaT cells), a H2O2 group (100 μ m ol / L), a H2O2 (100 μm ol / L) + HCS group (1.5 mg / mL), a H2O2 (100 μm ol / L) + PDRN group (0.25 mg / mL), and a H2O2 (100 μm ol / L) + HCS@PDRN group (based on the amount of PDRN, 0.25 mg / mL). After the above cells were cultured for another 24 hours, subsequent experiments were carried out.

[0117] Detection of cell inflammatory factors (TNF-α, IL- - 6, IL-1β): The cells in step 1 were collected with 1 mL of Trizol (Tiangen), 200 μL of chloroform was added, and after shaking vigorously for 15 s, it was left standing for 10 min, centrifuged at 4°C and 12000 rpm for 10 minutes; 400 μL of the supernatant was taken into a new 1.5 mL centrifuge tube, an equal volume (400 μL) of isopropanol was added, after inversion and mixing, it was left standing at 4°C for 10 min, and then centrifuged at 4°C and 12000 rpm for 10 minutes; the supernatant was discarded, the precipitate was washed with 70% ethanol, and centrifuged at 4°C and 10000 rpm for 3 min; the supernatant was discarded, after drying the precipitate, an appropriate amount of Rnase-free water was added to dissolve it to obtain total RNA. After reverse transcription into cDNA using a reverse transcription kit (NovoScript ® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge, E047-01B), the expression of cell inflammatory factors TNF-α, IL-6, and IL-1β was detected by the method of Realtime PCR (NovoStart ® SYBR qPCRSuperMix Plus, E096-01B).

[0118] 3. Statistical methods

[0119] Data were analyzed using SPSS 13.0 statistical software. The chi-square test was used, and a p-value < 0.05 was considered statistically significant. Results are as follows: Figure 12 As shown.

[0120] 4. Results

[0121] from Figure 12 It can be concluded that, compared with PDRN monomer, the hydroxyethyl deacetylated chitosan of the present invention can significantly promote the inhibitory effect of PDRN on cellular inflammatory response.

[0122] Example 9: The repair effect of HCS / PDRN complex on the skin barrier in mice with atopic dermatitis

[0123] Grouping and processing

[0124] Forty-eight mice were randomly divided into four groups using a random number table: normal group, model group, positive drug group, HCS group (1.71 mg / mL), PDRN group (0.29 mg / mL), low-dose HCS@PDRN group (0.5 mg / mL), medium-dose HCS@PDRN group (1 mg / mL), and high-dose HCS@PDRN group (2 mg / mL), with six mice in each group.

[0125] Except for the normal group, mice in the other groups were repeatedly stimulated with DNFB to establish an AD mouse model. The modeling process is referenced in [Kim TH, et al. The inhibitory effect of naringenin on atopicdermatitis induced by DNFB in NC / Nga mice. Life Sci., 2013, 93(15): 516-524.]. Specific method: One day before DNFB sensitization, the hair on the back of the mice was removed using electric clippers and depilatory cream, covering an area of ​​approximately 2.0 cm × 2.5 cm. On days 1, 4, 8, 11, and 15 of the experiment, except for the normal group, mice in the other groups had 30 μl of 2% DNFB solution (prepared by mixing acetone, olive oil, and DNFB, with olive oil:acetone = 1:4) evenly applied to their backs. Mice in the normal group had an equal volume of matrix solution (olive oil:acetone = 1:4) applied to their backs. On days 18 and 24, sensitization stimulation was changed to 0.2% DNFB solution, while the normal group mice continued to have an equal volume of matrix solution applied. The criteria for successful AD modeling were: histopathological examination of the skin tissue revealing intercellular edema or spongiosis of the epidermal cells, intercellular edema of the spinous layer, and superficial dermal lymphocyte infiltration. On day 29 of the experiment, the sample group was intervened, the normal group and the model group were fed normally, the positive group was treated with 0.1% dexamethasone ointment, the matrix group was treated with Example 4, and the control group 1 was treated; the HCS group was treated with Example 4, and the control group 2 was treated; the PDRN group was treated with Example 4, and the control group 3 was treated; the sample group was treated with different concentrations of HCS@PDRN corresponding to Example 4. The corresponding substances were applied to the damaged sites once a day on days 4, 6, 8 and 10.

[0126] Observation indicators and detection methods: After the experiment, mice in each group were anesthetized with 2% sodium pentobarbital solution, their eyeballs were enucleated to collect blood, and they were euthanized. Skin tissue from the lesions was collected under aseptic conditions and fixed and preserved with 4% paraformaldehyde solution; other skin tissues were preserved at -80°C for indicator detection.

[0127] Skin lesion scoring: Use a digital camera to take photos and record changes in skin lesions on the back of the skin.

[0128] Mast cell staining and image analysis: Paraffin sections were routinely dewaxed and immersed in water, stained with toluidine blue, and mounted with resin. Analysis was performed using MiVnt microscopy image analysis software, and the number of cells within each field of view was counted at 10×40 magnification.

[0129] Result: From Figures 13-15It can be seen that, compared with PDRN monomer, HCS@PDRN can significantly reduce the thickness of the stratum corneum in damaged skin and reduce the number of mast cells in the skin, indicating that HCS has a significant effect on promoting the anti-inflammatory effect of PDRN. The above description is a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A transdermal delivery system for deoxyribonucleic acid and its derivatives, characterized in that, It includes hydroxyethyl deacetylated chitosan and deoxyribonucleic acid and their derivatives, wherein the hydroxyethyl deacetylated chitosan has a deacetylated chitosan backbone and hydroxyethyl groups are linked by ether bonds, and the number of units n=6-30.

2. The transdermal delivery system for deoxyribonucleic acid and its derivatives according to claim 1, characterized in that, The weight ratio of the hydroxyethyl deacetylated chitosan to deoxyribonucleic acid and its derivatives is 1:0.01-10.

3. The transdermal delivery system for deoxyribonucleic acid and its derivatives according to claim 2, characterized in that, The weight ratio is 6:

1.

4. The transdermal delivery system for deoxyribonucleic acid and its derivatives according to claim 1, characterized in that, The deoxyribonucleic acid and its derivatives are selected from the group consisting of DNA, sodium DNA, calcium DNA, magnesium DNA, zinc DNA, potassium DNA, polydeoxyribonucleotide (PDRN), and polynucleotide (PN).

5. A method for preparing a transdermal delivery system for deoxyribonucleic acid and its derivatives as described in any one of claims 1-4, characterized in that, The process includes the following steps: dissolving hydroxyethyl deacetylated chitosan in water to obtain solution A, dissolving deoxyribonucleic acid and its derivatives in water to obtain solution B, mixing solutions A and B at a stirring speed of 10-200 rpm, and reacting for 0.1-48 hours.

6. The preparation method according to claim 5, characterized in that, The mass concentration of hydroxyethyl deacetylated chitosan in solution A is 0.001%-50%, and the mass concentration of deoxyribonucleic acid and its derivatives in solution B is 0.001%-50%.

7. A skincare product, characterized in that, A transdermal delivery system comprising any one of the deoxyribonucleic acid and its derivatives as described in claims 1-4, wherein the skin care product comprises an aqueous solution, a lotion, and a cream.

8. The skincare product according to claim 7, characterized in that, The amount of the deoxyribonucleic acid and its derivative transdermal delivery system added to skin care products is 0.1%-10%.

9. A method for promoting transdermal delivery of deoxyribonucleic acid and its derivatives, characterized in that, Transdermal delivery is achieved through skin follicles or the stratum corneum using the transdermal delivery system of any one of claims 1-4.

10. The use of the transdermal delivery system of deoxyribonucleic acid and its derivatives as described in any one of claims 1-4 in the preparation of skin care products for skin repair, anti-inflammation or anti-aging.

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