A kind of oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion and preparation method thereof

Abstract

The present invention belongs to the technical field of cosmetic raw materials, and specifically relates to a kind of oxidized hyaluronic acid-PDRN-peptide nanoemulsion and its preparation method. The nanoemulsion comprises oxidized hyaluronic acid, PDRN and polypeptide, and the nanoemulsion is formed by oxidized hyaluronic acid, PDRN and polypeptide through layer-by-layer self-assembly technology. Specifically: positively charged polypeptide is used to wrap negatively charged PDRN to form positively charged composite particles, or negatively charged PDRN is used to wrap positively charged polypeptide to form electrostatic composite particles, and then, the positively charged composite particles or the electrostatic composite particles are further wrapped with oxidized hyaluronic acid as a packaging material, and finally oxidized hyaluronic acid-PDRN-peptide nanoemulsion is formed. The nanoemulsion of the present invention can improve the stability of polypeptides, improve the problems that polypeptide molecules are difficult to penetrate the skin and have low bioavailability, and effectively improve the transdermal rate and stability of PDRN, give play to the tissue repair function of PDRN, further amplify the anti-inflammatory effects of the two, and improve the bioavailability of active ingredients.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to an oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion and a preparation method thereof. Background Art

[0002] PDRN, also known as "polydeoxyribonucleotide", is a regenerative substance extracted from salmon testes and optimized by DNA. It has multiple activities such as tissue repair, anti-ischemia and anti-inflammatory. Studies have shown that PDRN can effectively relieve wrinkles caused by skin aging by stimulating the proliferation of collagen and elastic fibers, help improve pigmentation, and help accelerate wound healing and relieve skin problems such as acne pits and acne marks. PDRN has a high compatibility of ingredients and can be compatible with any cosmetic formula. Its application in hydration and whitening is widely recognized by people from all walks of life in the cosmetics industry.

[0003] Peptides are protein fragments, widely found in animals and plants, can regulate the body's balance, and have extremely strong activity and diversity. Natural active peptides such as carnosine and glutathione can effectively remove and inhibit the accumulation of free radicals in the body, effectively slow down skin aging, and have good effects in sunscreen, anti-wrinkle and whitening skin care products. Studies have reported that small molecule peptides can enhance the body's immunity, improve and repair skin damage by regulating the expression levels of cell growth factors and inflammatory factors. For example, low concentrations of bee venom peptides can directly bind to protein p50, inhibit LPS-induced p50 translocation to the nucleus, reduce inflammatory gene transcription, lead to NF-KB target inactivation, and produce a wide range of anti-inflammatory effects. At present, peptides are used as core ingredients in many high-end skin care products and have broad application prospects, but their application is limited to a certain extent due to the problems of difficulty in transdermal penetration and low bioavailability of peptide ingredients. To solve the above problems, common application methods such as peptide encapsulation technology and peptide ingredient compounding are currently used, but they are all optimized and combined based on peptide raw materials, and the difficulties of peptide application technology are not solved from the root. Summary of the invention

[0004] In view of the above-mentioned technical problems, the present invention provides an oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion and a preparation method thereof. The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion prepared by the present invention has a milky white translucent appearance, a uniform and stable state, and no suspended matter is generated. The nanoemulsion can improve the stability of polypeptides, improve the problems such as the difficulty of polypeptide molecules to penetrate the skin and low bioavailability, and effectively improve the transdermal rate and stability of PDRN, give play to the PDRN tissue repair function, further amplify the anti-inflammatory effects of the two, and improve the bioavailability of active ingredients.

[0005] The technical solution of the present invention is as follows:

[0006] An oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion, the nanoemulsion comprises oxidized hyaluronic acid, PDRN and polypeptide, and the nanoemulsion is formed by the oxidized hyaluronic acid, PDRN and polypeptide through a layer-by-layer self-assembly technology.

[0007] Specifically, the layer-by-layer self-assembly technology refers to using a positively charged polypeptide to wrap a negatively charged PDRN to form a positively charged composite particle, or using a negatively charged PDRN to wrap a positively charged polypeptide to form a negatively charged composite particle, and then further wrapping the positively charged composite particles or the negatively charged composite particles with oxidized hyaluronic acid as a packaging material, finally forming an oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion.

[0008] The first option is:

[0009] The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion is an oxidized hyaluronic acid-PDRN-melittin nanoemulsion.

[0010] Further, the preparation method of the oxidized hyaluronic acid-PDRN-melittin nanoemulsion is:

[0011] (1) Weigh PDRN into a beaker and add deionized water to dissolve it to prepare a 0.5 mg / ml PDRN solution;

[0012] (2) Weigh melittin into a beaker and add deionized water to dissolve it to prepare a 1 mg / ml melittin solution;

[0013] (3) Weigh oxidized hyaluronic acid into a beaker, add deionized water to dissolve it, and prepare a 0.05% oxidized hyaluronic acid solution by mass percentage;

[0014] (4) slowly dripping the PDRN solution of step (1) into the melittin solution of step (2), stirring at a constant temperature to form a colorless and transparent PDRN-melittin solution for later use;

[0015] (5) The PDRN-melittin solution obtained in step (4) is slowly dripped into the oxidized hyaluronic acid solution, and stirred continuously at a constant temperature to form a milky white and translucent oxidized hyaluronic acid-PDRN-melittin nanoemulsion.

[0016] Preferably, the stirring time in step (4) and step (5) is 0.5 to 3 hours; the temperature is 20 to 60° C.; and the final pH of the nanoemulsion is 4 to 6.

[0017] Furthermore, in the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: bee venom peptide solution = 100-300: 2.5-9: 7-30.

[0018] The second technical solution provided is:

[0019] The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion is an oxidized hyaluronic acid-PDRN-polylysine nanoemulsion.

[0020] Further, the preparation method of the oxidized hyaluronic acid-PDRN-polylysine nanoemulsion is:

[0021] (1) Weigh PDRN into a beaker and add deionized water to dissolve it to prepare a 0.2 mg / ml PDRN solution;

[0022] (2) Weigh polylysine into a beaker and add deionized water to dissolve it to prepare a 5 mg / ml polylysine solution;

[0023] (3) Weigh oxidized hyaluronic acid into a beaker, add deionized water to dissolve it, and prepare a 0.05% oxidized hyaluronic acid solution by mass percentage;

[0024] (4) slowly dripping the polylysine solution of step (2) into the PDRN solution of step (1), stirring at a constant temperature to form a colorless and transparent PDRN-polylysine solution for standby use;

[0025] (5) The PDRN-polylysine solution obtained in step (4) is slowly dripped into the oxidized hyaluronic acid solution, and stirred continuously at a constant temperature to form a milky white and translucent oxidized hyaluronic acid-PDRN-polylysine nanoemulsion.

[0026] Preferably, the stirring time in step (4) and step (5) is 0.5 to 3 hours; the temperature is 20 to 60° C.; and the final pH of the nanoemulsion is 4 to 6.

[0027] Furthermore, in the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: polylysine solution = 100-200: 18-40: 0.3-1.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion provided by the present invention is mainly prepared by a layer-by-layer self-assembly method, that is, the positively charged bee venom peptide is electrostatically combined with the negatively charged PDRN to form positively charged composite particles, and then the oxidized hyaluronic acid is used as a packaging material to further wrap the positively charged composite particles to form a nanoemulsion;

[0030] The reaction mechanism is that after bee venom peptide wraps PDRN, positively charged composite particles are formed. The positively charged composite particles combine with oxidized hyaluronic acid through two reactions: the first is that the positively charged composite particles combine with the negatively charged oxidized hyaluronic acid electrostatically; the second is that after bee venom peptide wraps PDRN, bee venom peptide is in the outer layer, and bee venom peptide reacts with oxidized hyaluronic acid through Schiff base reaction. The oxidized hyaluronic acid then wraps the bee venom peptide again, layer by layer, and finally the nanoemulsion we need is prepared.

[0031] Compared with free peptides, it can effectively improve the stability of peptides, reduce the particle size, increase the transdermal penetration and bioavailability of peptide molecules, and at the same time, it is also beneficial to improve the transdermal penetration rate and stability of PDRN, exert the tissue repair function of PDRN, and further amplify the antibacterial and anti-inflammatory repair effects of the two.

[0032] 2. The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion provided by the present invention has a milky white and translucent appearance, a uniform and stable emulsion state, and no suspended matter is produced; at the same time, the raw materials for preparation are easily available, the preparation method is simple, and it is easy to industrialize and produce, and has great application potential in the field of cosmetic technology.

[0033] Mechanism of oxidized hyaluronic acid-PDRN-polylysine nanoemulsion: First, the negative charge of PDRN combines with the positively charged polylysine to form negatively charged composite particles, and then the polylysine is wrapped with PDRN. The hydrogen bond between PDRN and oxidized hyaluronic acid is used to form a nanoemulsion containing oxidized hyaluronic acid-PDRN-polylysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0035] Figure 1 It is the cell proliferation effect of the oxidized hyaluronic acid-PDRN-polylysine nanoemulsion of the present invention;

[0036] Figure 2 It is the result of cell scratch microscopy observation of the oxidized hyaluronic acid-PDRN-polylysine nanoemulsion of the present invention;

[0037] Figure 3 The cell migration rate of the oxidized hyaluronic acid-PDRN-polylysine nanoemulsion described in the present invention. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0039] The layer-by-layer self-assembly technology of the present invention is to alternately adsorb polyelectrolyte solutions with opposite charges by electrostatic action, and form a multilayer polymer film on the surface of the carrier. Compared with other traditional technologies, the biggest advantage of layer-by-layer self-assembly technology is that the physical properties of the multilayer film can be regulated by controlling the number of adsorbed molecular layers, and the structure and chemical properties of the assembled multilayer film can also be regulated by changing the polyelectrolyte or other charged species. Based on this, the design principle of this patent is to use the electrostatic binding of positively charged polypeptides to form positively charged composite particles, and then use oxidized hyaluronic acid as a packaging material to further wrap the above-mentioned electrostatic particles through electrostatic and Schiff base to form a nanoemulsion, so as to achieve the improvement of polypeptide stability, improve the problems such as polypeptide molecules are difficult to penetrate the skin and low bioavailability, and at the same time improve PDRN transdermal absorption rate and stability, effectively play the PDRN tissue repair function, and further amplify the purpose of the anti-inflammatory and antibacterial effects of the two, which has excellent application prospects in the field of cosmetics.

[0040] It should be noted that the synthesis of the oxidized hyaluronic acid (OHA) of the present invention refers to the literature [Preparation and Properties of Aldehyde-modified Hyaluronic Acid / Hydroxypropyl Chitosan Self-Healing Hydrogel [J / OL]. Journal of Composite Materials: 1-9 [2023-05-23]; the principle is to use sodium periodate to oxidize the hydroxyl groups on hyaluronic acid (HA) to aldehyde groups to synthesize aldehyde-modified hyaluronic acid (OHA).

[0041] The melittin of the present invention is from application number 202210112859.6, and the patent name is: a gold-melittin nanohybrid and its application. The amino acid sequence of the melittin is as shown in SEQ ID No. 1: Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2. The melittin adopts Fmoc chemical solid phase peptide synthesis (SPPS) or the HBTU / HOBt-based Fmoc method optimized HBTU activation / DIEA in situ neutralization scheme provided by ABI, and all peptides are synthesized on MBHA resin in the peptide synthesis reactor; after cleavage and deprotection in a reagent mixture containing 88% TFA, 5% phenol, 5% H2O and 2% TIPS (Fmoc chemistry), the crude product is precipitated with cold ether and purified to homogeneity by a preparative C18 reverse phase HPLC method, the molecular weight is determined by electrospray ionization mass spectrometry (ESI-MS), and freeze-dried on a freeze dryer to obtain the target product.

[0042] Example 1

[0043] A method for preparing an oxidized hyaluronic acid-PDRN-melittin nanoemulsion comprises the following steps:

[0044] Weigh 0.5 mg PDRN and dissolve it in 1 mL deionized water, and prepare a 0.5 mg / ml PDRN solution by ultrasonic dissolution for later use; weigh 1 mg bee venom peptide and dissolve it in 1 mL deionized water to prepare a 1 mg / ml bee venom peptide solution for later use; weigh 5 mg oxidized hyaluronic acid and dissolve it in 10 mL deionized water to prepare a 0.05% oxidized hyaluronic acid solution by mass fraction for later use;

[0045] 0.34 mL of 0.5 mg / ml PDRN was slowly dripped into 1 mL of 1 mg / mL bee venom peptide solution, stirred at room temperature for 30 to 60 min, and then 1 mL of the above mixture was slowly dripped into 10 mL of 0.05% oxidized hyaluronic acid solution, stirred at 20 to 60 ° C for 3 h to obtain oxidized hyaluronic acid-PDRN-bee venom peptide nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: bee venom peptide solution = 100: 2.5: 7.4.

[0046] Example 2

[0047] A method for preparing an oxidized hyaluronic acid-PDRN-melittin nanoemulsion comprises the following steps:

[0048] Weigh 0.5 mg PDRN and dissolve it in 1 mL deionized water, and prepare a 0.5 mg / ml PDRN solution by ultrasonic dissolution for use; weigh 2 mg bee venom peptide and dissolve it in 2 mL deionized water to prepare a 1 mg / ml bee venom peptide solution for use; weigh 10 mg oxidized hyaluronic acid and dissolve it in 20 mL deionized water to prepare a 0.05% oxidized hyaluronic acid solution by mass fraction for use;

[0049] 0.68 mL of 0.5 mg / ml PDRN solution was slowly dripped into 2 mL of 1 mg / mL bee venom peptide solution, stirred and reacted at 20-60° C. for 30-60 min to obtain PDRN-bee venom peptide solution, and then 2 mL of the above PDRN-bee venom peptide solution was slowly dripped into 20 mL of 0.05% oxidized hyaluronic acid solution, stirred and reacted at 20-60° C. for 3 h to obtain oxidized hyaluronic acid-PDRN-bee venom peptide nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: bee venom peptide solution = 200: 5.07: 14.9.

[0050] Example 3

[0051] A method for preparing an oxidized hyaluronic acid-PDRN-melittin nanoemulsion comprises the following steps:

[0052] Weigh 1 mg of PDRN and dissolve it in 2 mL of deionized water, and prepare a 0.5 mg / ml PDRN solution by ultrasonic dissolution for later use; weigh 4 mg of melittin and dissolve it in 4 mL of deionized water to prepare a 1 mg / ml melittin solution for later use; weigh 15 mg of oxidized hyaluronic acid and dissolve it in 30 mL of deionized water to prepare a 0.05% oxidized hyaluronic acid solution by mass fraction for later use;

[0053] 1.3 mL of 0.5 mg / ml PDRN was slowly dripped into 4 mL of 1 mg / mL bee venom peptide solution, stirred at room temperature for 30 to 60 min, and then 3.5 mL of the above mixture was slowly dripped into 30 mL of 0.05% oxidized hyaluronic acid solution, stirred at 20 to 60 ° C for 3 h to obtain oxidized hyaluronic acid-PDRN-bee venom peptide nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: bee venom peptide solution = 300: 8.50: 26.4.

[0054] Example 4

[0055] A method for preparing an oxidized hyaluronic acid-PDRN-polylysine nanoemulsion comprises the following steps:

[0056] Weigh 2 mg of PDRN and dissolve it in 10 mL of deionized water, and ultrasonically dissolve a 0.2 mg / mL PDRN solution for later use; weigh 5 mg of polylysine and dissolve it in 1 mL of deionized water to prepare a 5 mg / ml polylysine solution for later use; weigh 5 mg of oxidized hyaluronic acid and dissolve it in 10 mL of deionized water, and prepare a 0.05% oxidized hyaluronic acid solution by mass for later use;

[0057] Take 0.2mL of 5mg / ml polylysine and slowly drip it into 10mL of 0.2mg / mL PDRN solution, stir and react at room temperature for 30-60min, then take 2mL of the above mixture and slowly drip it into 10mL of 0.05% oxidized hyaluronic acid solution, stir and react at 20-60℃ for 3h to obtain oxidized hyaluronic acid-PDRN-polylysine nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: polylysine solution = 100: 19.6: 0.39.

[0058] Example 5

[0059] A method for preparing an oxidized hyaluronic acid-PDRN-polylysine nanoemulsion comprises the following steps:

[0060] Weigh 4 mg of PDRN and dissolve it in 20 mL of deionized water, and ultrasonically dissolve a 0.2 mg / mL PDRN solution for later use; weigh 5 mg of polylysine and dissolve it in 1 mL of deionized water to prepare a 5 mg / ml polylysine solution for later use; weigh 5 mg of oxidized hyaluronic acid and dissolve it in 10 mL of deionized water to prepare a 0.05% oxidized hyaluronic acid solution by mass for later use;

[0061] Take 0.45mL of 5mg / ml polylysine and slowly drip it into 20mL of 0.2mg / mL PDRN solution, stir and react at room temperature for 30-60min, then take 2mL of the above mixture and slowly drip it into 10mL of 0.05% oxidized hyaluronic acid solution, stir and react at 20-60℃ for 3h to obtain oxidized hyaluronic acid-PDRN-polylysine nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: polylysine solution = 100: 19.5: 0.44.

[0062] Example 6

[0063] A method for preparing an oxidized hyaluronic acid-PDRN-polylysine nanoemulsion comprises the following steps:

[0064] Weigh 4 mg of PDRN and dissolve it in 20 mL of deionized water, and ultrasonically dissolve a 0.2 mg / mL PDRN solution for later use; weigh 5 mg of polylysine and dissolve it in 1 mL of deionized water to prepare a 5 mg / ml polylysine solution for later use; weigh 10 mg of oxidized hyaluronic acid and dissolve it in 20 mL of deionized water to prepare a 0.05% oxidized hyaluronic acid solution by mass for later use;

[0065] Take 0.45mL of 5mg / ml polylysine and slowly drip it into 20mL of 0.2mg / mL PDRN solution, stir and react at room temperature for 30-60min, then take 4mL of the above mixture and slowly drip it into 20mL of 0.05% oxidized hyaluronic acid solution, stir and react at 20-60℃ for 3h to obtain oxidized hyaluronic acid-PDRN-polylysine nanoemulsion. In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: polylysine solution = 200: 39.1: 0.88.

[0066] Application and verification experiments:

[0067] A kind of oxidized hyaluronic acid-PDRN-polylysine nanoemulsion ( Figure 1 and Figure 2Both are represented by sample groups), and pure PDRN, combined with blank control and positive control groups, respectively, based on human skin fibroblasts to measure cell proliferation rate and cell migration rate to evaluate its efficacy in anti-aging and repair. The test methods are as follows:

[0068] Main materials and reagents:

[0069] The cells used in this test are human skin fibroblasts (HFB), which were obtained by Guangdong Boxi Biotechnology Co., Ltd. through primary and subculture. The cell generation used in the experiment is P3. DMEM culture medium (Gibco), fetal bovine serum (Sijiqing), PBS (Bosder), CCK-8 detection solution (Mi Mouse), pancreatin (Gibco), DMEM (Gibco), fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.), PBS (Solebo), pancreatin (Gibco).

[0070] 1. Cell proliferation rate determination:

[0071] (1) Cell recovery, passaging and inoculation: HFB cells with a passage number of P3 were taken from the liquid nitrogen tank, recovered and inoculated into culture flasks, and cultured in a 37°C, 5% CO2 incubator until the cells grew to 70%-80%, then digested and passaged. The digested cells were resuspended in culture medium and counted for later use; then 5000 cells / well were inoculated into a 96-well plate, incubated overnight in an incubator (37°C, 5% CO2), and the inoculation volume per well was 200uL.

[0072] (2) Experimental grouping: When the cell plating rate reaches 30%-40%, the culture plates are taken out and marked in time periods as 0h, 24h, and 48h. A control group, a positive control group, a PDRN-treated group, and a sample group are set up in each time period, and three replicate wells are set up in each group.

[0073] (3) Control test: Discard the supernatant from all wells during the 0h period, add 100uL PBS to each well for washing, discard the PBS, add 100uL CCK-8 treatment solution to each well, and incubate in a 37°C, 5% CO2 incubator in the dark. After 2h, aspirate 90uL of the wells in a new 96-well plate and read the OD value using a microplate reader adjusted to 450nm.

[0074] (4) Sample determination: discard the supernatant from all remaining wells, add 100uL PBS to each well for washing, discard the PBS and add samples in groups, with a sample volume of 200uL per well. Add DMEM medium containing 2% FBS to the control group, add DMEM medium containing 10% FBS to the positive control group, and add DMEM medium containing 2% FBS containing samples of corresponding concentrations to the sample group. After the sample addition is completed, place the culture plate in a 37°C, 5% CO2 incubator and continue to culture for 24h and 48h respectively, then discard the supernatant, add 100uL PBS to each well for washing, discard the PBS and add 100uCCK-8 treatment solution to each well, incubate in a 37°C, 5% CO2 incubator in the dark, and after 2h, aspirate 90uL from each well in turn and place it in a new 96-well plate. Adjust the microplate reader to 450nm to read the OD value.

[0075] (5) Calculation of cell viability: Taking the 0 h well of each group as the control well, the relative cell viability value of each group was calculated.

[0076] Figure 1 It is the effect diagram of cell proliferation activity in different treatment groups. Figure 1 The results showed that compared with the control group, the cell viability of the positive control group increased significantly after 48 hours of treatment, indicating that this experiment was effective; after 24 hours of administration at a concentration of 10% in the PDRN treatment group, the cell viability increased compared with the control group, but after 48 hours of administration, there was no significant change in cell viability compared with the control group; and after 24 hours of administration at a concentration of 10% in the sample group, the cell viability increased significantly compared with the control group; after 48 hours of administration, the cell viability was significantly higher than that of the control group. It shows that PDRN can effectively induce and improve HFB cell viability, promote cell proliferation rate, and promote cell regeneration and tissue repair, but this advantage is gradually lost as the administration time is prolonged, which may be related to the partial degradation of PDRN after entering the human body. The sample group treatment with the same concentration showed that as the administration time was prolonged, the HFB cell viability gradually increased, and was significantly higher than the control group and the PDRN treatment group, indicating that the oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion described in the present invention can effectively improve the stability of PDRN, enhance cell proliferation activity, and help to play the tissue repair function of PDRN.

[0077] 2. Cell migration rate determination:

[0078] (1) Cell recovery, passaging and inoculation: HFB cells with a passage number of P3 were taken from the liquid nitrogen tank, recovered and inoculated into culture bottles, and cultured in an incubator (37°C, 5% CO2) until the cells grew to 70%-80%, then digested and passaged. The digested cells were resuspended in culture medium and counted for later use; then the cells were counted at 3.5*10 5 The cells were inoculated into 6-well plates at a volume of 1000 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ). The inoculation volume per well was 2 mL.

[0079] (2) Experimental grouping: When the cell plating rate reaches 30%-40%, the culture plates are taken out and marked in time periods as 0h, 24h, and 48h. A control group, a positive control group, a PDRN-treated group, and a sample group are set up in each time period, and three replicate wells are set up in each group.

[0080] (3) Scratch treatment: When the cell plating rate in the 6-well plate reaches 90%, the cells are scratched. After the scratches are completed, the supernatant is discarded, 1 mL of PBS is added to each well, and photos are taken under a 5x microscope. The scratch morphology at 0 h is recorded, and the photo position is marked.

[0081] (4) Sample determination: After discarding PBS, add samples in groups, with a sample volume of 2 mL per well, and 3 replicates per group. Add DMEM culture medium to each well of the blank control group; add DMEM culture medium containing 10% FBS to each well of the positive control group; add DMEM culture medium containing samples of corresponding concentrations to each well of the sample group. After adding samples, place the 6-well plate in an incubator (37°C, 5% CO2) for incubation; observe the cells 18 hours after scratching and take pictures under a 5x microscope. Note: Try to keep the same scratch position when taking pictures at different time periods for each experimental well.

[0082] (5) Calculation of cell migration rate: The scratch area of ​​each photo at each time was calculated for each group of samples. The scratch area at different times was subtracted from the scratch area at 0 h of each group of samples to obtain the cell migration area. The migration rate of the BC group was assumed to be 100%. The t-test method was used for statistical analysis to calculate the relative migration rate of each group of samples.

[0083] Figure 2 The results of scratch microscopy observation of cells in different treatment groups are shown in Figure 2. Figure 3 is the cell migration rate of HFB cells treated with different treatments after 18 hours of culture. The results showed that compared with the control group, the cell migration rate of the PDRN treatment group and the sample group was significantly increased, and the HFB cell migration rate of the sample group after 18 hours of culture was higher than that of the PDRN treatment group. This shows that the oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion of the present invention can actively promote skin anti-aging repair.

[0084] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. An oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion, characterized in that: The nanoemulsion comprises oxidized hyaluronic acid, PDRN and a polypeptide, and the nanoemulsion is formed by oxidized hyaluronic acid, PDRN and the polypeptide through a layer-by-layer self-assembly technology; The oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion is an oxidized hyaluronic acid-PDRN-polylysine nanoemulsion; The preparation method of the oxidized hyaluronic acid-PDRN-polylysine nanoemulsion is: (1) Weigh PDRN into a beaker and add deionized water to dissolve it to prepare a 0.2 mg / ml PDRN solution; (2) Weigh polylysine into a beaker and add deionized water to dissolve it to prepare a 5 mg / ml polylysine solution; (3) Weigh oxidized hyaluronic acid into a beaker, add deionized water to dissolve it, and prepare a 0.05% oxidized hyaluronic acid solution by mass percentage; (4) slowly dripping the polylysine solution of step (2) into the PDRN solution of step (1), stirring at a constant temperature to form a colorless and transparent PDRN-polylysine solution for standby use; (5) slowly dripping the PDRN-polylysine solution obtained in step (4) into the oxidized hyaluronic acid solution, stirring continuously at a constant temperature to form a milky white and translucent oxidized hyaluronic acid-PDRN-polylysine nanoemulsion; The stirring time in step (4) and step (5) is 0.5 to 3 hours; the temperature is 20 to 60° C.; the final pH of the nanoemulsion is 4 to 6; In the final reaction system, the volume ratio of oxidized hyaluronic acid solution: PDRN solution: polylysine solution = 100-200: 18-40: 0.3-1.

2. An oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion according to claim 1, characterized in that, The layer-by-layer self-assembly technology refers to using a positively charged polypeptide to wrap a negatively charged PDRN to form a positively charged composite particle, or using a negatively charged PDRN to wrap a positively charged polypeptide to form a negatively charged composite particle, and then further wrapping the positively charged composite particles or the negatively charged composite particles with oxidized hyaluronic acid as a packaging material, and finally forming an oxidized hyaluronic acid-PDRN-polypeptide nanoemulsion.

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