A nanoemulsion containing ceramide & RGD polypeptide and its preparation method and application

Abstract

The present invention belongs to the field of cosmetic technology, specifically, a nanoemulsion containing ceramide & RGD polypeptide, and a preparation method and application thereof. The nanoemulsion containing ceramide & RGD polypeptide of the present invention comprises the following raw materials in weight percentage: ceramide & RGD polypeptide 0.06%-0.1% and deionized water. The nanoemulsion containing ceramide & RGD polypeptide has a milky white and translucent appearance. The nanoemulsion containing ceramide & RGD polypeptide can effectively enhance the water solubility of ceramide, improve its skin permeability and absorption rate, and is conducive to exerting a moisturizing effect in cosmetics, improving skin conditions, and can be compatible with cosmetic formulas under normal temperature conditions; and the preparation method of the present invention is simple and has good repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a nanoemulsion containing ceramide & RGD polypeptide, and a preparation method and application thereof. Background Art

[0002] Ceramide, also known as brain amide, is a type of lipid molecule formed by the covalent bonding of a sphingol chain and a fatty chain. It is mainly found in the gray matter and bone marrow of animal brains. It is an important component of human stratum corneum lipids. Because its structure is similar to the skin stratum corneum protein and hair fiber, and it has the characteristics of good absorption, high activity and strong efficacy, it is often used in combination with linolenic acid, hyaluronic acid and vitamins for conditioning cosmetics. It mainly promotes the self-repair of the skin barrier and regulates skin cells, improves the symptoms of dry skin and desquamation, and reduces the occurrence of skin inflammation, thereby improving skin tolerance and enhancing skin barrier function. Therefore, it has an outstanding moisturizing effect and is known as the "king of hydration". Other studies have shown that lower concentrations of ceramide can stimulate fibroblast proliferation and inhibit the expression of metalloproteinases, and have a certain anti-aging effect.

[0003] As people age, the ceramide content in the skin gradually decreases, causing dryness, desquamation and cracking of the skin. Supplementing the skin with an appropriate amount of ceramide can effectively enhance the adhesion between keratinocytes, reduce water loss in the skin, and has a moisturizing and emollient effect. This may be related to ceramide promoting the expression levels of silk polymer protein, mRNA and protein. In addition, the use of ceramide-containing skin care products or oral ceramide glycolipids can effectively inhibit the invasion of harmful substances and microorganisms by thickening the skin stratum corneum or inhibiting the formation of melanin, enhance the skin's ability to adapt to the external environment, and have a significant whitening effect, which makes ceramides of great value and wide application in the field of cosmetics. However, since ceramides are highly crystalline substances, they have problems such as poor solubility, poor water dispersion and easy crystallization when used in cosmetics, which limits their application in this field. Effectively improving the solubility and dispersibility of ceramides is of great significance to the application of ceramides in the field of cosmetics. Summary of the invention

[0004] In view of the above-mentioned technical deficiencies, the present invention provides a nanoemulsion containing ceramide & RGD polypeptide, and a preparation method and application thereof. The nanoemulsion containing ceramide & RGD polypeptide of the present invention has a milky white and translucent appearance, and the nanoemulsion containing ceramide & RGD polypeptide can effectively enhance the water solubility of ceramide, improve its skin permeability and absorption rate, is beneficial to exert a moisturizing effect in cosmetics, improves skin condition, and can be compatible with cosmetic formulas at room temperature.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A nanoemulsion containing ceramide & RGD polypeptide, comprising the following raw materials in weight percentage: 0.06%-0.1% ceramide & RGD polypeptide and deionized water, the sum of the weight percentages of the raw materials being 100%;

[0007] The ceramide & RGD polypeptide is made of the following raw materials: ceramide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine, and RGD polypeptide;

[0008] Among them, the mass ratio of ceramide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine and RGD polypeptide is 5-15:1-2:1-2:2-10.

[0009] Preferably, the RGD polypeptide is a polypeptide capable of improving skin; RGD is a sequence polypeptide consisting of three amino acids, Arg-Gly-Asp, a linear peptide, and the RGD polypeptide is used as a preferred sequence for binding to cell surface proteins.

[0010] Preferably, the ceramide is ceramide 3.

[0011] Preferably, the ceramide & RGD polypeptide is prepared according to the following steps:

[0012] S1. Weigh raw materials ceramide, RGD polypeptide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine for later use;

[0013] S2. Dissolve the RGD polypeptide in anhydrous dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine in sequence and stir, then add ceramide and stir, react at 25-50°C for 24-72h, dialyze and freeze-dry to obtain ceramide & RGD polypeptide.

[0014] Preferably, the dialysis method in step S2 is: first dialyze with dimethyl sulfoxide for 1 day, and then dialyze with ultrapure water for 3 days.

[0015] Preferably, it is made of the following raw materials in weight percentage: ceramide & RGD polypeptide 0.06%-0.1%, cholesterol 0-0.1%, preservative 0.15%-1.8% and deionized water, and the sum of the weight percentages of the raw materials is 100%.

[0016] Preferably, the preservative consists of 1,2-pentanediol, 1,2-hexanediol, and phenoxyethanol;

[0017] Among them, the volume ratio of 1,2-pentanediol, 1,2-hexanediol and phenoxyethanol is 300-540:75-150:45-100.

[0018] The present invention also protects a method for preparing a nanoemulsion containing ceramide & RGD polypeptide, comprising the following steps:

[0019] (1) Weigh the raw materials: ceramide & RGD polypeptide and deionized water according to weight percentage and set aside;

[0020] (2) After dissolving the ceramide & RGD polypeptide in ethanol, the solution was added dropwise to water at 25-45° C., and then stirred at 25-45° C. for 30-60 min, and then stirred at room temperature for 3-6 h to obtain a nanoemulsion containing the ceramide & RGD polypeptide.

[0021] Preferably, the preparation method of the nanoemulsion containing ceramide & RGD polypeptide comprises the following steps:

[0022] (1) Weigh the raw materials according to weight percentage: ceramide & RGD polypeptide, preservative, cholesterol, and deionized water for later use;

[0023] (2) dissolving the preservative in deionized water and maintaining the temperature at 25-45° C. to obtain a preservative solution;

[0024] After dissolving ceramide & RGD polypeptide and cholesterol in ethanol, the mixture is added dropwise to a preservative solution at 25-45°C, and then stirred at 25-45°C for 30-60 minutes, and then stirred at room temperature for 3-6 hours to obtain a nanoemulsion containing ceramide & RGD polypeptide.

[0025] The present invention also protects the use of the nanoemulsion containing ceramide & RGD polypeptide in the preparation of cosmetics.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. As a water-soluble RGD polypeptide molecule, the main amino acids are arginine, glycine and aspartic acid, which are widely present in various extracellular matrices such as fibronectin and laminin. It can mediate and enhance the adhesion of cells to extracellular matrices by specifically binding to integrins on cells, has a signal transduction effect, and plays an important role in regulating cell growth. It is currently widely used in medicine, but less in the field of cosmetics. The present invention mainly uses water as a solvent, integrates ceramide and RGD polypeptide molecules, and prepares water-in-oil nanoemulsions to enhance the solubility and water dispersibility of ceramide, improve the permeability of ceramide in the skin, improve the dry and desquamated state of the skin, improve skin tolerance, and enhance the moisturizing effect of the emulsion.

[0028] 2. The present invention uses RGD polypeptide to modify ceramide 3, so that it has certain hydrophilic properties (amphiphilic, RGD is easily soluble in water and Cer3 is difficult to dissolve in water); then it is used as the main raw material to prepare nanoparticles, thereby increasing the effective content of ceramide and making it easy to add and utilize in the formula; RGD peptide has cell adhesion function, increases the adhesion of ceramide in the keratinocyte layer, and resides for a longer time.

[0029] 3. The results show that the ceramide & RGD peptide nanoemulsion (Cer-RGD) has good stability, water solubility, dispersibility and biocompatibility. More importantly, the ceramide & RGD nanoemulsion effectively increases the ceramide content in the emulsion, increases the skin's absorption rate of ceramide, has a strong moisturizing effect on the skin, effectively improves the problem of dry skin and desquamation, and improves the skin's tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The infrared spectra of ceramide & RG polypeptide, ceramide 3 and RGD polypeptide prepared in Example 1 of the present invention;

[0031] Figure 2 The transmission electron micrographs of the nanoemulsion containing ceramide & RGD polypeptide prepared in Example 1 of the present invention and the transmission electron micrographs thereof for verifying its stability in a cosmetic formula, wherein (a) is a transmission electron micrograph of the nanoemulsion containing ceramide & RGD polypeptide, and (b) is a transmission electron micrograph for verifying the stability of the nanoemulsion containing ceramide & RGD polypeptide in a cosmetic formula;

[0032] Figure 3 This is a HE slice image of the 3D skin model tissue morphology for the repair efficacy detection of the nanoemulsion containing ceramide & RGD polypeptide prepared in Example 1 of the present invention;

[0033] Figure 4 This is a 3D skin model diagram of the repair efficacy test of the nanoemulsion containing ceramide & RGD polypeptide prepared in Example 1 of the present invention. Figure 4 The upper middle figure is an immunofluorescence detection diagram of the FLG content; the lower figure is a bar graph of the integrated optical density (IOD) value of the FLG content;

[0034] Figure 5 This is a bar graph of the results of the transepidermal water loss (TEWL) test of the repair efficacy of the nanoemulsion containing ceramide & RGD polypeptide prepared in Example 1 of the present invention;

[0035] Figure 6 This is the fluorescence distribution diagram of the permeability of the nanoemulsion containing ceramide & RGD polypeptide prepared in Example 1 of the present invention - the fluorescent labeling substance in pig skin. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] A method for preparing a nanoemulsion containing ceramide & RGD polypeptide comprises the following steps:

[0039] (1) Preparation of ceramide & RGD polypeptide:

[0040] 68 mg of RGD polypeptide was weighed according to a preset ratio and dissolved in 10 mL of anhydrous dimethyl sulfoxide. After dissolution, 18 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 13 mg of 4-dimethylaminopyridine (DMAP) were added to the solution in sequence for activation treatment. 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine were used as catalysts, and 100 mg of ceramide 3 was added thereto. The mixture was incubated at 45° C. overnight, dialyzed (first with DMSO for 1 day, then with ultrapure water for 3 days), and freeze-dried to obtain ceramide & RGD polypeptide for later use.

[0041] (2) Preparation of Nanoemulsion Containing Ceramide & RGD Peptide:

[0042] Weigh 20 mg of ceramide & RGD polypeptide and dissolve it in 2 mL of ethanol, then slowly drip it into 30 mL of deionized water at 45°C, stir it at 45°C for 30 minutes, cool it to room temperature and react for 3 hours to obtain a nanoemulsion containing ceramide & RGD.

[0043] Example 2

[0044] A method for preparing a nanoemulsion containing ceramide & RGD polypeptide comprises the following steps:

[0045] (1) Preparation of ceramide & RGD polypeptide:

[0046] Weigh 26 mg of RGD polypeptide according to a preset ratio and dissolve it in 5 mL of anhydrous dimethyl sulfoxide. After dissolution, add 13 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 13 mg of 4-dimethylaminopyridine (DMAP) to the solution in sequence for activation treatment, then add 65 mg of ceramide 3, react at 25°C for 72 hours, dialyze (dialyze with DMSO for 1 day, then dialyze with ultrapure water for 3 days), and lyophilize to obtain ceramide & RGD polypeptide for later use;

[0047] (2) Preparation of Nanoemulsion Containing Ceramide & RGD Peptide:

[0048] Weigh 24.2 mg of ceramide & RGD polypeptide and dissolve it in 2 mL of ethanol. Slowly drip it into 30 mL of deionized water at 35°C, stir it at 35°C for 45 minutes, and react it at room temperature for 5 hours to obtain a nanoemulsion containing ceramide & RGD.

[0049] Example 3

[0050] A method for preparing a nanoemulsion containing ceramide & RGD polypeptide comprises the following steps:

[0051] (1) Preparation of ceramide & RGD polypeptide:

[0052] 130 mg of RGD polypeptide was weighed and dissolved in 20 mL of anhydrous dimethyl sulfoxide according to a preset ratio. After dissolution, 26 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 26 mg of 4-dimethylaminopyridine (DMAP) were added to the solution in sequence for activation treatment. Then, 195 mg of ceramide 3 was added thereto. The mixture was reacted at 35° C. for 48 h. After dialysis (first with DMSO for 1 day, then with ultrapure water for 3 days), and freeze-dried, ceramide & RGD polypeptide was obtained for later use.

[0053] (2) Preparation of Nanoemulsion Containing Ceramide & RGD Peptide:

[0054] 30 mg of ceramide & RGD polypeptide was weighed and dissolved in 2 mL of ethanol, and then slowly dripped into 30 mL of deionized water at 25°C, and stirred at 25°C for 60 min, and then reacted at room temperature for 6 h to obtain a nanoemulsion containing ceramide & RGD.

[0055] Example 4

[0056] The preparation steps are the same as those in Example 1, except that the nanoemulsion containing ceramide & RGD polypeptide is prepared from ceramide & RGD polypeptide, preservative and water. The preparation method of the nanoemulsion containing ceramide & RGD polypeptide in step (2) is:

[0057] Add a preservative consisting of 540 μL of 1,2-pentanediol, 150 μL of 1,2-hexanediol, and 45 μL of phenoxyethanol to 30 mL of deionized water, and heat to 45° C. to obtain a preservative solution;

[0058] Weigh 20 mg of ceramide & RGD polypeptide and dissolve it in 2 mL of ethanol. Slowly drip it into 30 mL of a preservative solution at 45°C, stir it at 45°C for 30 minutes, cool it to room temperature and react for 3 hours to obtain a nanoemulsion containing ceramide & RGD.

[0059] Example 5

[0060] The preparation steps are the same as those in Example 1, except that the nanoemulsion containing ceramide & RGD polypeptide is made of ceramide & RGD polypeptide, preservative, cholesterol and water. The preparation method of the nanoemulsion containing ceramide & RGD polypeptide in step (2) is:

[0061] A preservative is added to 30 mL of deionized water, wherein the preservative consists of 300 μL of 1,2-pentanediol, 75 μL of 1,2-hexanediol, and 100 μL of phenoxyethanol to obtain a preservative solution;

[0062] Weigh 20 mg of ceramide & RGD polypeptide and 30 mg of cholesterol, dissolve in 2 mL of ethanol, slowly drip into 30 mL of 25°C preservative solution, stir at 25°C for 60 min, and react at room temperature for 6 h to obtain a nanoemulsion containing ceramide & RGD.

[0063] Examples 1-5 of the present invention all prepared nanoemulsions containing ceramide & RGD polypeptides with a milky white translucent appearance, excellent water solubility, and compatibility with cosmetic formulas at room temperature, and the effects are similar. The following study is conducted using the nanoemulsion containing ceramide & RGD polypeptides prepared in Example 1 as an example:

[0064] Results and Discussion:

[0065] 1. Test method:

[0066] 3D epidermal skin model used in the test Batch number: ES220607, provided by Guangdong Boxi Biotechnology Co., Ltd.; the working solution is 0.2% SLS, and the working solution preparation method is: absorb 1mL of a solution with a volume fraction of 0.4% SLS, add it to 1mL of PBS solution, and prepare a 0.2% SLS working solution. SLS can damage the skin barrier;

[0067] Model drug administration:

[0068] 1) According to the test plan, transfer the model to a 6-well plate (add 0.9 mL of EpiGrowth culture medium in advance);

[0069] 2) Add 12.5 μL of 0.2% SLS solution and 12.5 μL of PBS to the surface of the sample group, add the sample to the culture medium at the specified concentration, and incubate in a CO2 incubator (37°C, 5% CO2) for 24 h;

[0070] 3) After the incubation, the test substance remaining on the surface of the model was cleaned with sterile PBS solution, and the residual liquid inside and outside the model was wiped off with a sterile cotton swab, and the test group number was marked on the 6-well plate.

[0071] Histomorphological test: The model used for histomorphological detection was removed by ring cutting and fixed with 4% paraformaldehyde. After fixation for 24 hours, H&E staining was performed, and photos were taken under a microscope for observation, collection and analysis.

[0072] Immunofluorescence test: The model to be tested was fixed with 4% paraformaldehyde by mass. After fixation for 24 hours, immunofluorescence detection of the content of filaggrin (FLG) was performed, and photos were taken under a microscope for observation, collection and analysis.

[0073] Transepidermal water loss (TEWL) test:

[0074] 1) Preliminary preparation: After cleaning, prepare a 24-well plate according to the number of models and mark them accordingly. Add 0.3 mL of EpiGrowth culture medium to each well. Place the 24-well plate with the model in the clean bench, open the lid of the 24-well plate, and let it stand for 30 minutes before measuring.

[0075] 2) Instrument debugging: Connect the TW24 transepidermal water loss tester to the MPA6 adapter, open the instrument measurement software, calibrate the probe to zero, set the measurement time and parameters, and select the measurement parameter as transepidermal water loss (TEWL) with a measurement time of 2.5 minutes;

[0076] 3) Measurement: Clean the outer surface of the tester, put it in the clean bench, and place it on the 24-well plate where the model is placed. Each probe of the instrument corresponds to one model. Then operate the test software to measure the transepidermal water loss (TEWL). Save the data after the measurement.

[0077] 4) Data analysis: The data fluctuations will be relatively large within the first 30 seconds of measurement. When performing data analysis, the measurement values ​​of the first 30 seconds are excluded, and the test data of the last 2 minutes are used. The average value is taken as the transepidermal water loss (TEWL) measurement value of each model.

[0078] Result analysis:

[0079] Figure 1 The infrared spectra of ceramide & RG polypeptide (Cer3-RGD), ceramide 3 (Cer3), and RGD polypeptide (RGD) prepared in Example 1 of the present invention, ceramide 3 and RGD polypeptide are the raw materials used in Examples 1-5; the results show that the infrared spectrum of ceramide & RGD polypeptide is at 1725cm -1 There is an absorption peak at 1725cm -1 The absorption peak at is the absorption peak of ester, which can prove that the hydroxyl group of ceramide and the carboxyl group of RGD polypeptide undergo esterification reaction to generate ester group, verifying the successful synthesis of ceramide & RGD polypeptide.

[0080] Figure 2 (a) The results showed that ceramide & RGD polypeptide formed particles with a size of about 200 nm in water. Figure 2 The formula in (b) is composed of the following raw materials in weight percentage: 7% glycol, 1% amino acid moisturizer, 0.05% hydrolyzed sodium hyaluronate, 0.4% parahydroxyacetophenone, and the balance is water, and the sum of the weight percentages of each raw material is 100%; the formula is a simulated cosmetic, and the stability of the nanoemulsion containing ceramide & RGD in the cosmetic is studied by blending the nanoemulsion containing ceramide & RGD with the formula; Figure 2 In (b), the nanoemulsion (stock solution) containing ceramide & RGD and the formula are blended, the mass percentage of the nanoemulsion (stock solution) containing ceramide & RGD is 5%, and the mass percentage of the formula is 95%. After standing at room temperature for 10 days, the transmission electron microscopy image is tested. Figure 2 (b) The results show that the nanoemulsion containing ceramide & RGD still has good stability, water solubility and dispersibility in cosmetic formulations.

[0081] GraphPad Prism was used for graphing, and the results were expressed as Mean±SD. The groups were compared using t-test statistical analysis, and all statistical analyses were two-tailed; P<0.05 was considered to be significantly different, and P<0.01 was considered to be extremely significantly different. Figure 3 The blank control group is a normal skin tissue without adding samples or SLS treatment; the negative control group is a normal skin tissue without adding test samples and only adding SLS treatment; Model, the reference product is ceramide (Cer), and the measurement index is HE section; the results show that compared with the blank control group, the stratum corneum of the negative control group is loose and thickened, the living cell layer is damaged, and vacuoles appear, indicating that the stimulation conditions of this test are effective. Compared with the blank control group, the number of living cells in the basal layer of the nanoemulsion (Cer-RGD) sample containing ceramide & RGD and the ceramide (Cer) sample increased, the cells were closely arranged, and the vacuoles were improved.

[0082] Figure 4 The results show that based on The model was tested with ceramide (Cer) as the reference substance and the measurement index: FLG barrier protein content. The results showed that compared with the blank control group, the FLG protein content of the negative control group decreased significantly, indicating that the stimulation conditions of this test were effective. Compared with the negative control group, the FLG content of the nanoemulsion (Cer-RGD) sample containing ceramide & RGD and the ceramide (Cer) sample increased significantly, with the increase rates of 88.89% and 44.44%, respectively. Compared with the Cer group, the FLG protein content of Cer-RGD increased significantly.

[0083] Figure 5 The results show that based on The model was tested with ceramide (Cer) as the reference product and the determination index: transepidermal water loss rate TEWL. The results showed that the TEWL value of the negative control group increased significantly compared with the blank control group, indicating that the stimulation conditions of this test were effective. Compared with the negative control group, the TEWL values ​​of the nanoemulsion (Cer-RGD) sample containing ceramide & RGD and the ceramide (Cer) sample decreased significantly, with inhibition rates of 45.31% and 29.64%, respectively; compared with the Cer group, the transepidermal water loss rate of Cer-RGD decreased.

[0084] The skin penetration behavior is tested as follows:

[0085] This test is based on the suckling pig skin-Franzcell system. The distribution of fluorescent labeled substances in pig skin at different time points is observed by fluorescence microscopy to evaluate the skin penetration behavior of the samples. The equipment used includes a transdermal absorption diffusion meter (TK-12D), Franzcell, and Leica biological microscope (DM2500LED).

[0086] Preparation of ex vivo suckling pig skin: The back skin of one-month-old suckling pig stored at -20°C was thawed with deionized water at room temperature and repeatedly rinsed with PBS buffer.

[0087] Transdermal absorption capacity determination: 1) Add receiving solution to the receiving chamber: use a pipette to draw 7.0 mL of receiving solution (PBS) into the receiving chamber, and place the matching magnetic stirring bar in the receiving chamber;

[0088] 2) Assembly and fixation of the test model: Fix the skin between the supply chamber and the receiving chamber of the Franzcell, with the stratum corneum facing the supply chamber and the dermis facing the receiving chamber; after fixing the skin, add 1.5 mL of receiving solution (PBS) to the sampling tube using a pipette according to the liquid height of the sampling tube, so that the dermis layer of the skin is in close contact with the receiving solution, and the total volume of the receiving solution is 8.5 mL;

[0089] 3) Fix the Franzcell in the transdermal absorption diffusion instrument, turn on the electromagnetic stirrer and stir at a speed of 300 rpm, maintain a constant temperature water bath of (32±1)℃, and ensure that there are no bubbles in the water bath interlayer;

[0090] 4) Sample loading: After the water bath temperature of the diffusion instrument is constant, sample loading is performed. Three replicates are set for each sample at each time point. 300 μL of sample is drawn with a pipette and added to the skin surface. The sample is spread evenly from the center of the skin to the edge using the pipette tip, and the supply chamber is sealed with a sealing film.

[0091] 5) Sample collection: Skin samples were collected at 15 min, 30 min, 1 h, 2 h, 6 h, and 24 h, respectively. The skin surface was washed with PBS 5 times, and the residual liquid on the surface was wiped dry with a cotton swab. The skin was cut circularly with a blade, immersed in a 4% paraformaldehyde solution for fixation (fixation time ≥ 24 h), and frozen and sectioned for later use.

[0092] GraphPad Prism was used for graphing, and the results were expressed as Mean±SD. t-test was used for statistical analysis for comparison among groups, and all statistical analyses were two-tailed; p < 0.05 was considered to be significantly different, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, and the smaller the p value, the more significant it is.

[0093] The emulsified formula of the standard product ceramide (Cer) (water and glycerin as phase A are heated to 85°C, caprylic capric triglyceride, cetearyl alcohol, cetearyl glucoside are heated to 85°C as phase B, then B is added to A, homogenized at 5000-6000rpm for 3-5 minutes, and ceramide is added after cooling), detection index: fluorescence intensity; Figure 6 The results showed that both the nanoemulsion (Cer-RGD) sample fluorescent marker formula containing ceramide & RGD and the ceramide (Cer) sample fluorescent marker formula underwent penetration behavior; the cumulative penetration amount of the Cer-RGD fluorescent marker formula was significantly higher than that of the Cer fluorescent marker formula at 4h and 24h.

[0094] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and variations. The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and their protection scope is not limited thereto. Equivalent substitutions or changes made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention, and the protection scope of the present invention shall be subject to the claims.

Claims

1. A nanoemulsion containing ceramide & RGD polypeptide, characterized in that: It includes ceramide & RGD polypeptide and deionized water, wherein the mass proportion of the ceramide & RGD polypeptide in the nanoemulsion containing the ceramide & RGD polypeptide is 0.06%-0.1%; The ceramide & RGD polypeptide is prepared from the following raw materials: ceramide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine and RGD polypeptide; Among them, the mass ratio of ceramide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine, and RGD polypeptide is 5-15:1-2:1-2:2-10; The ceramide & RGD polypeptide is prepared according to the following steps: S1. Weigh raw materials ceramide, RGD polypeptide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine for later use; S2, dissolving the RGD polypeptide in anhydrous dimethyl sulfoxide, then sequentially adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine thereto and stirring, then adding ceramide thereto and stirring, reacting at 25-50° C. for 24-72 hours, dialyzing, and freeze-drying to obtain ceramide & RGD polypeptide; The RGD polypeptide is a polypeptide capable of improving skin, the RGD polypeptide is a sequence polypeptide consisting of three amino acids: Arg-Gly-Asp, and the RGD polypeptide is a linear peptide, and the ceramide is ceramide 3.

2. The nanoemulsion containing ceramide & RGD polypeptide according to claim 1, characterized in that: The dialysis method in step S2 is: first dialyze with dimethyl sulfoxide for 1 day, and then dialyze with ultrapure water for 3 days.

3. The nanoemulsion containing ceramide & RGD polypeptide according to claim 1, characterized in that: The invention is prepared from the following raw materials in weight percentage: 0.06%-0.1% ceramide & RGD polypeptide, 0-0.1% cholesterol, 0.15%-1.8% preservative and deionized water, and the sum of the weight percentages of the raw materials is 100%.

4. The nanoemulsion containing ceramide & RGD polypeptide according to claim 3, characterized in that: The preservative consists of 1,2-pentanediol, 1,2-hexanediol and phenoxyethanol; Among them, the volume ratio of 1,2-pentanediol, 1,2-hexanediol and phenoxyethanol is 300-540:75-150:45-100.

5. A method for preparing the nanoemulsion containing ceramide & RGD polypeptide according to claim 1, characterized in that: The steps include: (1) Weigh the raw materials: ceramide & RGD polypeptide and deionized water according to weight percentage and set aside; (2) After dissolving the ceramide & RGD polypeptide in ethanol, add it dropwise into water at 25-45°C, then stir and react at 25-45°C for 30-60 minutes, and then stir at room temperature for 3-6 hours to obtain a nanoemulsion containing ceramide & RGD polypeptide.

6. The method for preparing the nanoemulsion containing ceramide & RGD polypeptide according to claim 3, characterized in that: The steps include: (1) Weigh the raw materials according to weight percentage: ceramide & RGD polypeptide, preservative, cholesterol and deionized water, and set aside; (2) dissolving the preservative in deionized water and maintaining the temperature at 25-45° C. to obtain a preservative solution; After dissolving ceramide & RGD polypeptide and cholesterol in ethanol, the mixture is added dropwise to a preservative solution at 25-45°C, and then stirred at 25-45°C for 30-60 minutes, and then stirred at room temperature for 3-6 hours to obtain a nanoemulsion containing ceramide & RGD polypeptide.

7. Use of the nanoemulsion containing ceramide & RGD polypeptide according to claim 1 or 4 in the preparation of cosmetics.

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