A fine-line anti-wrinkle composite peptide flexible liposome and a preparation method thereof

Abstract

The application discloses a fine-line anti-wrinkle composite peptide flexible liposome and a preparation method thereof. The fine-line anti-wrinkle composite peptide flexible liposome is composed of the following components: acetylcholine blocking polypeptide, liposome lipid material, liposome membrane softener, solvent (dissolving lipid material), antioxidant, stabilizer, preservative and water. The flexible liposome has good deformability, so that the wrapped active ingredients can penetrate the skin to reach the target area. The palmitic acid modified polypeptide hydrophobic long chain and the carboxyl and amino hydrophilic groups on the side chain are used to form a three-dimensional network hydrogel through intermolecular and intramolecular hydrogen bonds, so that the stability is improved. The propylene glycol can more effectively dissolve phospholipids, so that the cost is reduced, and the moisturizing and transdermal penetration enhancer effects are achieved. The application can be mixed with water in any proportion, is convenient to mix, and can be widely used in facial, neck and whole body skin care products, such as essence water, essence liquid, essence milk, essence cream and jelly, and has significant fine-line anti-wrinkle effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a flexible nanoliposome of an immediate and long-lasting anti-wrinkle complex peptide and its preparation method. Background Technology

[0002] Most facial wrinkles are caused by the frequent movement of facial muscles, leading to repeated stretching and folding of the facial skin. Some fine wrinkles are caused by natural aging, muscle contraction, ultraviolet radiation, insufficient rest and skincare, and unhealthy lifestyle habits such as smoking and drinking. Facial dynamic wrinkles are closely related to the repetitive movement of facial muscles. When nerve impulses reach nerve endings, they release the neurotransmitter acetylcholine. After release, acetylcholine binds to the receptor nmAchR, releasing sodium ions to form a muscle action potential, causing muscle contraction. Repeated muscle contractions create dynamic wrinkles.

[0003] In recent years, functional cosmetics have become increasingly popular, and their functional ingredients, especially peptides, are playing an increasingly important role due to their safety and effectiveness. However, it is worth considering a common problem with peptide ingredients:

[0004] ① Because the peptide bonds in the structure of active polypeptides are easily broken, they are not only unstable in aqueous solutions and need to be freeze-dried and stored in a refrigerator in powder form, but also have a short-lasting effect when added to a formula. Furthermore, they are easily affected by proteolytic enzymes in the skin and become ineffective.

[0005] ② Because active peptides have high molecular weight, are hydrophilic (in most cases), and have ionizable groups in their structure, they are difficult to penetrate the lipophilic skin barrier, resulting in a significant reduction in efficacy.

[0006] To address the aforementioned issues, our company encapsulates the active ingredient complex peptides within flexible liposome hydrophilic vesicles. This solves the stability problem, and the cell membrane-like lipid bilayer structure, combined with the high deformability of flexible liposomes, enhances transdermal absorption, perfectly resolving existing issues. Furthermore, the combination with lactic acid bacteria fermentation product lysate filtrate achieves the effects of repairing and rebuilding the skin's natural barrier, moisturizing, anti-aging, and wrinkle reduction.

[0007] This invention utilizes palmitic acid, a component of the intercellular lamellar lipid matrix of the human stratum corneum. By modifying peptides with palmitic acid (C16), the hydrophobicity of the peptides can be increased, thereby improving their transdermal permeability and stability. For example, palmitoyl pentapeptide-4, which has palmitic acid attached to the amino terminus of the pentapeptide (L-lysyl-L-threonyl-L-threonyl-L-lysyl-L-serine), has both a 16-carbon hydrophobic long chain and hydrophilic groups such as amino, carboxyl, and hydroxyl groups in its structure. Therefore, it can easily self-assemble in aqueous solution in a non-covalent form to form a three-dimensional network hydrogel.

[0008] Liposomes are closed bilayer vesicles formed by the spontaneous aggregation of phospholipids in water, typically in the form of ultraspheres. Liposomes can encapsulate active ingredients (including hydrophilic and hydrophobic active ingredients) within vesicles or between bilayer structures, thereby increasing the solubility, stability, skin permeability, and improving skin feel of the active ingredients.

[0009] Flexible liposomes are self-aggregating vesicles formulated as an improvement on ordinary liposomes. Amphiphilic surfactants such as sodium cholate, sodium deoxycholate, polysorbate 80, or their derivatives are added to the phospholipid component of the liposome, giving its lipid membrane high deformability. Because their particle size is smaller than ordinary liposomes, they can pass through pores with a diameter 1 / 10 to 1 / 5 of their own, with a permeation rate and volume almost equivalent to pure water. Flexible liposomes promote the transdermal penetration of water-soluble macromolecular drugs.

[0010] In addition, traditional liposomes are usually used in the biopharmaceutical field. The preparation methods usually adopt thin film dispersion, ethanol or ether injection methods, which are only suitable for small-batch production in the laboratory. Moreover, the phospholipids used are pharmaceutical grade for injection, which are too expensive and have a very low cost-effectiveness when used in cosmetics. Furthermore, in industrial mass production, the organic solvent ethanol must be removed by vacuum evaporation, which makes the process complicated and too expensive. In addition, conventional liposomes are prone to instability problems such as aggregation, flocculation and leakage during storage, and often need to be freeze-dried for preservation, which brings inconvenience to storage and transportation.

[0011] To address the aforementioned issues, one strategy is to alter the peptide structure through chemical modification or complexation to generate new compounds; another strategy is to utilize nanodelivery systems for efficient delivery. This invention first encapsulates small-molecule active peptides within flexible liposome hydrophilic vesicles, solving the stability problem and enhancing transdermal absorption due to the cell membrane-like lipid bilayer structure and the high deformability of the flexible liposomes. Furthermore, palmitic acid (hexadecanoic acid) modifies the long hydrophobic chain of the peptide, and the carboxyl and amino hydrophilic groups on the side chains form a three-dimensional network hydrogel through intermolecular and intramolecular hydrogen bonds. This is then combined with a lactic acid bacteria fermentation product lysate filtrate containing lactic acid and over 20 amino acids, perfectly solving existing problems and achieving synergistic effects such as adjusting the skin's microecology, repairing and rebuilding the skin's natural barrier, and moisturizing, anti-aging, and wrinkle-reducing properties. Summary of the Invention

[0012] Objective of the Invention: Addressing the aforementioned problems and shortcomings of existing products, the objective of this invention is to provide a flexible liposome containing a wrinkle-reducing complex peptide and its preparation method. This flexible liposome exhibits excellent deformability, allowing the encapsulated active ingredients to penetrate transdermally to the target area. Furthermore, palmitic acid-modified hydrophobic long chains of the polypeptide and the carboxyl and amino hydrophilic groups on the side chains self-assemble through intermolecular and intramolecular hydrogen bonds to form a three-dimensional network hydrogel, increasing stability. The use of propylene glycol more effectively dissolves phospholipids, reducing costs while simultaneously acting as a moisturizing and transdermal penetration enhancer. This invention is miscible with water in any proportion, making formulation convenient. It can be widely used in facial / neck / body skincare products, such as essence water / serum / lotion / cream / gel, exhibiting significant wrinkle-reducing effects.

[0013] Technical Solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: a wrinkle-reducing and anti-wrinkle complex peptide flexible liposome, composed of the following components by mass percentage:

[0014] Acetylcholine-blocking peptides: 0.01–0.2%

[0015] Liposome lipid materials 1~5%

[0016] Liposome membrane softener 0.1~0.5%

[0017] Solvent 5-10%

[0018] Antioxidant 0.05~0.1%

[0019] Stabilizer 1~10%

[0020] Preservative 0.5~1.0%

[0021] and water 80-95%

[0022] The sum of the contents of each component is 100%.

[0023] Preferably, the acetylcholine blocking polypeptide comprises one or more combinations of arginine / lysine polypeptide (conopeptide), acetyl hexapeptide-8 (hexapeptide), and dipeptide diaminobutyryl benzylamide diacetate (snake venom peptide).

[0024] Preferably, the liposome lipid material is composed of phospholipids and cholesterol, and the mass ratio of phospholipids to cholesterol is 4:1 to 10:1.

[0025] Preferably, the phospholipids include one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and distearate phosphatidylcholine; the cholesterol includes one or more of animal-derived cholesterol and plant-derived cholesterol such as sitosterol, campesterol, and stigmasterol.

[0026] Preferably, the phospholipid is hydrogenated lecithin; and the cholesterol is sitosterol.

[0027] Preferably, the solvent for dissolving the lipid material is a polyol, including propylene glycol, glycerol, butylene glycol, or pentanediol.

[0028] Preferably, the liposome membrane softener is sodium deoxycholate or polysorbate 80, wherein the mass ratio of the liposome membrane softener to the liposome lipid material is 1:5-10.

[0029] Preferably, the antioxidant is one or more of vitamin E, BHT and BHA, and the amount added is 0.05 to 0.1% of the total mass of the composition.

[0030] Preferably, the stabilizer is a mixture of palmitic acid-modified polypeptide and lactic acid bacteria fermentation product lysate filtrate; the palmitic acid-modified polypeptide includes one or more of palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4 and palmitoyl hexapeptide-12, and the amount added is 0.05~0.1% of the total weight, and the amount added of lactic acid bacteria fermentation product lysate filtrate is 5~10% of the total weight.

[0031] Preferably, the preservative is one or more of sodium benzoate and phenoxyethanol.

[0032] This invention also provides a method for preparing the above-mentioned wrinkle-reducing and anti-wrinkle complex peptide flexible liposomes, comprising the following steps:

[0033] Step S1, Preparation of lactic acid bacteria fermentation product lysate filtrate:

[0034] Take frozen lactic acid bacteria glycerol and inoculate it onto MRS medium to activate it. Incubate at 30-42℃ for 20-30 h to obtain seed culture medium.

[0035] Take the activated seed culture medium and inoculate it into MRS culture medium at a ratio of 1%-5%, and incubate at 30-42℃ for 20-30 hours to obtain lactic acid bacteria fermentation products;

[0036] Take the lactic acid bacteria fermentation product, homogenize it once with a microfluidic high-pressure homogenizer at 18,000-20,000 psi, then increase the homogenization pressure to 23,000-25,000 psi and homogenize it twice more, while controlling the temperature at 0-10℃, to obtain the lactic acid bacteria fermentation product lysate; finally, centrifuge it in a refrigerated centrifuge at 12,000 rpm for 15 min, and take the supernatant as the lactic acid bacteria fermentation product lysate filtrate.

[0037] Step S2, Preparation of W / O Colostrum:

[0038] Weigh out phospholipids, cholesterol, membrane softener, and antioxidant and place them in a reaction vessel. Add propylene glycol and stir in a water bath at 80-90°C until completely dissolved. Cool to room temperature to obtain a lipid oil phase solution.

[0039] Take purified water at a water phase to oil phase mass ratio of 1:2-5, add acetylcholine blocking peptides and stir until completely dissolved. Add the solution to the lipid oil phase under stirring to obtain W / O colostrum for later use.

[0040] Step S3, preparation of liposomes using the W / O / W double emulsion method:

[0041] Take about 80% purified water and add W / O colostrum to it under continuous stirring. Continue stirring for 30 minutes to obtain a milky white crude liposome suspension. Homogenize it 3-5 times using a high-pressure microfluidic homogenizer at a homogenization pressure of 15000-20000 Psi. Then add lactic acid bacteria fermentation product lysate filtrate and palmitic acid modified active peptides. Stir until completely dissolved and continue stirring for 1-2 hours. Finally, add preservatives and pure water to the total volume. Filter using a 0.1-0.45μm polycarbonate membrane to obtain transparent to semi-transparent flexible nanoliposomes.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0043] (1) Effective effects of the flexible nanoliposomes of the present invention: ① The particle size is smaller than that of ordinary liposomes and they are deformable, which can quickly penetrate the stratum corneum of the skin and enter the epidermis and dermis to form a "reservoir"; ② The palmitic acid modified active peptides free or adsorbed on the surface of the liposomes take effect immediately, while the active peptides encapsulated in the flexible liposomes can be slowly released to achieve both immediate and long-lasting effects. ③ They can carry the encapsulated active peptides and act on different targets, respectively, to acetylcholine receptors, inhibit acetyl release, "downregulate" the activity of nerve cells, and adjust the release of acetylcholine, thus having a synergistic effect.

[0044] (2) Active ingredient flexible liposome encapsulation technology: The present invention encapsulates a complex active polypeptide of water-soluble arginine / lysine polypeptide, acetyl hexapeptide-8 and dipeptide diaminobutyryl benzylamide diacetate in flexible liposomes. Due to the encapsulation effect of liposomes, the opportunity for the active ingredient to come into contact with many uncertain external factors is reduced, so that the active ingredient is not degraded during the storage process, thereby improving the stability of the active ingredient in the product.

[0045] (3) The present invention designs flexible liposomes, which can control the slow release of active ingredients to exert a long-lasting effect; and have good deformability, so that the active ingredients can penetrate through the skin to reach the target area to exert their effect.

[0046] (4) Transdermal absorption technology increases penetration: Functional ingredients in cosmetics must pass through the stratum corneum to reach the corresponding sites of action in order to improve skin condition. However, the stratum corneum of human skin has a very strong barrier function, making it difficult for hydrophilic functional ingredients to penetrate. In this invention, arginine / lysine peptides, acetyl hexapeptide-8, and dipeptide diaminobutyryl benzylamide diacetate are encapsulated in flexible liposomes. Due to the similarity between the structure of liposomes and biological membranes, and the soft deformability of the liposome membrane, it is easier to penetrate the skin barrier, and the transdermal permeation is significantly increased. The self-assembly of palmitic acid-modified peptide molecules and amino acid-rich lysates from lactic acid bacteria fermentation products to form a three-dimensional network hydrogel morphology increases the stability of the flexible liposomes and synergistically enhances their efficacy.

[0047] (5) Long-lasting moisturizing: Liposomes, when applied to the skin in cosmetic form, readily associate with proteins, carbohydrates, and lipids on or inside the skin, reducing moisture loss from the skin surface. This increases skin hydration and promotes the effective penetration of active ingredients through the skin barrier to the basal layer where they exert their effects.

[0048] (6) The present invention improves the conventional ethanol injection method by adopting a modified double emulsion method-high pressure homogenization method and using propylene glycol to efficiently dissolve ordinary soybean lecithin. While avoiding the subsequent reduced pressure evaporation process of anhydrous ethanol organic solvent, the use of conventional ordinary soybean lecithin maximizes the reduction of raw material costs and production costs. Moreover, propylene glycol acts as a solvent, as well as a moisturizer and transdermal penetration enhancer, synergistically increasing the transdermal penetration of liposomes.

[0049] (7) In view of the fact that palmitic acid is a component of the intercellular lamellar lipid matrix of the human stratum corneum, and since both palmitic acid and small molecule polypeptides have linear structures, the palmitic acid-modified polypeptides are retained in the lamellar structure of the stratum corneum after transdermal penetration to exert their effects. Furthermore, the presence of a large number of carboxyl and amino groups in the compound active polypeptide molecules, as well as the large amount of lactic acid and amino acids in the lactic acid bacteria fermentation product lysate, further facilitates the self-assembly of polypeptide molecules into a three-dimensional network hydrogel, preventing problems such as lipid body aggregation, rupture, and sedimentation, thereby increasing the stability of the product.

[0050] This invention utilizes the presence of carboxyl and amino groups in the above-mentioned compound active polypeptide molecules and modifies them with palmitic acid to increase the hydrophobic chains on the polypeptide molecular chains, thereby making it more conducive to the self-assembly of polypeptide molecules within and between molecules to form a three-dimensional network hydrogel, preventing problems such as liposome aggregation, rupture, and sedimentation, thereby increasing the stability of the product and skin permeability.

[0051] (8) Simpler process, wider application, and easier compatibility: The modified double emulsion method-high pressure homogenization method is used to prepare liposomes, which has a high encapsulation rate and a simple preparation process. It is more suitable for large-scale industrial production. The resulting liposome products are transparent to semi-transparent opalescent liquids that can be miscible with water in any proportion. They are easy to compatibility and can be widely used in facial / neck / body skin care products, such as essence water / essence liquid / essence lotion / essence cream / gel, etc. Attached Figure Description

[0052] Figure 1 Transmission electron microscopy images of flexible liposomes prepared in the embodiments of the present invention, wherein: A is the flexible liposome prepared in Example 1; B is the flexible liposome prepared in Example 7;

[0053] Figure 2 This is a particle size distribution diagram of the flexible liposomes prepared in Example 1 of the present invention;

[0054] Figure 3 This is a particle size distribution diagram of the ordinary liposomes prepared in Example 7 of the present invention;

[0055] Figure 4 This is the HPLC chromatogram of the arginine / lysine polypeptide reference solution described in this invention;

[0056] Figure 5 This is the HPLC chromatogram of the acetyl hexapeptide-8 reference solution described in this invention;

[0057] Figure 6 This is the HPLC chromatogram of the dipeptide diaminobutyryl benzylamide diacetate reference solution described in this invention;

[0058] Figure 7 This is a typical HPLC chromatogram of Example 1 of the present invention;

[0059] Figure 8 This is a schematic diagram of the in vitro release test described in a specific embodiment of the present invention;

[0060] Figure 9 Example 7 of this invention: Transdermal permeation map of ordinary liposomes and aqueous solution;

[0061] Figure 10 Example 1 of the present invention: Transdermal permeation map of flexible liposomes and aqueous solution;

[0062] Figure 11 A comparison diagram of the results of flexible liposome patch assays prepared in Example 1 of this invention for typical subjects;

[0063] Figure 12The results of the flexible liposome anti-wrinkle test of typical subjects in Example 1 of this invention are shown in the bar graphs of wrinkle volume, wrinkle area, wrinkle area ratio and skin elasticity changes at different time points (*p<0.05, **p<0.01, ***p<0.001 vs D0).

[0064] Figure 13 This is a comparison image of the facial and local anti-wrinkle effects of a typical recipient after undergoing the flexible liposome anti-wrinkle test of Example 1 of this invention. Detailed Implementation

[0065] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0066] Unless otherwise specified, the equipment used in this embodiment and comparative example are all conventional experimental equipment, and the materials and reagents used are all commercially available unless otherwise specified. The experimental methods are also conventional experimental methods unless otherwise specified.

[0067] Experiment description:

[0068] Experimental materials: Active peptides: arginine / lysine peptide, acetyl hexapeptide-8 and dipeptide diaminobutyryl benzylamide diacetate, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4 and palmitoyl hexapeptide-12 (Nanjing Laimao Biotechnology Co., Ltd., content ≥98%); soybean lecithin (PC95%, injection grade) (Shanghai Aivito); soybean powder lecithin (LECIGRAN) ® 1000P, PC 60-72% (Beijing Houjia Biotechnology Co., Ltd.); Soy lecithin (Xi'an Baichuan Biotechnology Co., Ltd.); Hydrogenated lecithin (Jiangxi Tuorun); Cholesterol and sodium deoxycholate (Beijing Aoboxing Biotechnology Co., Ltd.); Phytosterols (Xi'an Guobang Industry); Polysorbate 80 (Merck, USA).

[0069] Experimental instruments: magnetic stirrer, rotary evaporator, microfluidic high-pressure homogenizer, RYJ-12B transdermal diffusion apparatus, ZEN3600 Malvern laser particle size analyzer (Malvern, UK), high performance liquid chromatograph (Shimadzu, Japan), transmission electron microscope (Hitachi, Japan).

[0070] Preparation method: The wrinkle-reducing and anti-wrinkle complex peptide flexible liposomes of the present invention are prepared by a modified double emulsion method-high pressure homogenization method. The specific preparation method includes the following steps:

[0071] Step S1, Preparation of lactic acid bacteria fermentation product lysate filtrate:

[0072] Take frozen lactic acid bacteria glycerol and inoculate it onto MRS medium to activate it. Incubate at 30-42℃ for 20-30 h to obtain seed culture medium.

[0073] Take the activated seed culture medium and inoculate it into MRS culture medium at a ratio of 1%-5%, and incubate at 30-42℃ for 20-30 hours to obtain lactic acid bacteria fermentation products;

[0074] Take the lactic acid bacteria fermentation product, homogenize it once with a microfluidic high-pressure homogenizer at 18,000-20,000 psi, then increase the homogenization pressure to 23,000-25,000 psi and homogenize it twice more, while controlling the temperature at 0-10℃, to obtain the lactic acid bacteria fermentation product lysate; finally, centrifuge it in a refrigerated centrifuge at 12,000 rpm for 15 min, and take the supernatant as the lactic acid bacteria fermentation product lysate filtrate.

[0075] Step S2, Preparation of W / O Colostrum:

[0076] Weigh out phospholipids, cholesterol, membrane softener, and antioxidant and place them in a reaction vessel. Add propylene glycol and stir in a water bath at 80-90°C until completely dissolved. Cool to room temperature to obtain a lipid oil phase solution.

[0077] Take purified water at a water-to-oil phase mass ratio of 1:2-5, add acetylcholine-blocking polypeptides and stir until completely dissolved. Add the solution to the lipid oil phase under stirring to obtain a W / O colostrum for later use.

[0078] Step S3, preparation of liposomes from W / O / W double emulsion:

[0079] Take about 80% purified water and add W / O colostrum to it under continuous stirring. Continue stirring for 30 minutes to obtain a milky white crude liposome suspension. Homogenize it 3-5 times using a high-pressure microfluidic homogenizer at a homogenization pressure of 15000-20000 Psi. Then add lactic acid bacteria fermentation product lysate filtrate and palmitic acid modified active peptides. Stir until completely dissolved and continue stirring for 1-2 hours. Finally, add preservatives and pure water to the total volume. Filter using a 0.1-0.45μm polycarbonate membrane to obtain transparent to semi-transparent flexible nanoliposomes.

[0080] Example 1: Weigh 1g of hydrogenated lecithin, 0.2g of cholesterol, 0.1g of polysorbate 80, and 0.05g of vitamin E into a flask, add 8g of propylene glycol, and stir at 80-90℃ until completely dissolved. Cool to room temperature to obtain a lipid solution. Separately, take 2g of purified water, add 0.01g of arginine / lysine peptide, 0.05g of acetyl hexapeptide-8, and 0.05g of dipeptide diaminobutyryl benzylamide diacetate, stir at room temperature until completely dissolved, and slowly add dropwise to the lipid solution while stirring continuously for 30 minutes to obtain a white, w / o colostrum. Take another 70g of purified water and slowly add the above W / O colostrum to it under continuous stirring for secondary emulsification. Continue stirring for 30 minutes, then homogenize three times in a high-pressure microfluidic homogenizer at a pressure of 15000-20000 Psi. Then add 0.05g of palmitoyl tripeptide-1 and 0.05g of palmitoyl hexapeptide-12 in sequence and stir at room temperature until completely dissolved. Then add 10g of lactic acid bacteria fermentation product lysate filtrate, mix well, add 0.8g of sodium benzoate, stir until completely dissolved, and continue stirring for 1-2 hours. Add purified water to a final volume of 100g, filter through a 0.45μm aqueous membrane, and package to obtain the drug-loaded flexible liposome product.

[0081] Examples 2-6 differ from Example 1 in that they use different phospholipids and cholesterol according to the formulation in Table 1 to prepare flexible liposomes. The results showed that in Examples 2-4, using soybean phosphatidylcholine with high, medium, and low phosphatidylcholine content and cholesterol as lipid materials, all three could be successfully prepared into translucent flexible liposomes. As the phosphatidylcholine (PC) content decreased, the appearance, color, and transparency all decreased. Because the soybean phosphatidylcholine used in Example 2 was pharmaceutical-grade for injection, it was expensive and had the best appearance. Examples 3 and 4 used ordinary cosmetic-grade soybean phosphatidylcholine, resulting in slight differences in appearance. Considering that hydrogenated lecithin is a saturated fatty acid, stable and not easily oxidized, it is more suitable for cosmetics; therefore, hydrogenated lecithin was preferred as the phospholipid lipid material. Examples 4-6 used soybean phosphatidylcholine (PC 60%) combined with different sterols as lipid materials. The results showed no significant difference in appearance among the three, but among phytosterols, sitosterol had a relatively low melting point, dispersed and dissolved quickly, and was easier to prepare; therefore, sitosterol was preferred.

[0082] Table 1. Prescriptions with different phospholipids and sterols

[0083]

[0084] Example 7: Preparation of ordinary liposomes

[0085] Unlike Example 1, no membrane softener polysorbate 80 was added to the lipid material, and no palmitic acid-modified peptides or lactic acid bacteria fermentation product lysate filtrate were added to the aqueous phase during secondary emulsification; only purified water was used.

[0086] Example 8: Preparation of ordinary flexible liposomes

[0087] Unlike Example 1, no palmitic acid-modified peptides or lactic acid bacteria fermentation product lysate filtrate were added to the aqueous phase of the secondary emulsification; only purified water was used.

[0088] The results showed that conventional flexible liposomes had better transparency than conventional liposomes.

[0089] Example 9: Preparation of ordinary flexible liposomes containing lactic acid bacteria

[0090] Unlike Example 1, palmitic acid-modified peptides were not added to the aqueous phase of the secondary emulsification; only lactic acid bacteria fermentation product lysate filtrate and purified water were added.

[0091] Examples 10-12: Effect of soybean lecithin / cholesterol ratio on liposome quality

[0092] Soybean phospholipids (PC60%) and cholesterol were selected in weight ratios of 4:1, 7:1, and 10:1, respectively, and ordinary flexible liposomes were prepared according to Example 8. They were stored at 4°C for later use, and their appearance, particle size, and encapsulation efficiency were compared. The test results are shown in Table 2.

[0093] Table 2 Results of quality index testing for flexible liposomes

[0094]

[0095] The results showed that as the mass ratio of soybean phospholipids to cholesterol increased, the liposomes obtained became more transparent, the particle size decreased, and the encapsulation efficiency increased. When the ratio was above 7:1, ideal liposomes could be obtained.

[0096] Examples 13-15: Effect of phospholipid / active peptide ratio on liposome quality

[0097] Flexible liposomes were prepared according to Example 8 with a fixed soybean phospholipid:cholesterol weight ratio of 10:1, phospholipid / active peptide ratios of 5:1, 10:1, and 20:1, and stored at 4°C for later use. Their appearance, particle size, and encapsulation efficiency were compared, and the test results are shown in Table 3.

[0098] Table 3 Results of quality index testing for flexible liposomes

[0099]

[0100] The results showed that as the concentration of active peptides in liposomes increased, the appearance of the prepared liposomes did not differ significantly; they were all translucent, opalescent liquids. However, the particle size increased, and the encapsulation efficiency decreased accordingly. The overall quality evaluation of liposomes was optimal when the phospholipid / active peptide ratio reached 10:1 or higher.

[0101] Examples 16-18: Effect of Homogenization Pressure on Liposome Quality

[0102] Flexible liposomes were prepared according to Example 8 with a fixed soybean phospholipid:cholesterol weight ratio of 10:1 and a phospholipid / active peptide ratio of 10:1. They were stored at 4°C for later use, and their appearance, particle size and encapsulation efficiency were compared. The test results are shown in Table 4.

[0103] Table 4. Results of three homogenization cycles at different homogenization pressures on the quality indicators of flexible liposomes.

[0104]

[0105] Experimental results show that as the homogenization pressure increases, the appearance transparency of the prepared liposomes improves, the particle size decreases, and the encapsulation initially increases but then remains relatively unchanged. From the perspective of the feasibility of the preparation process, a homogenization pressure of 15,000-20,000 Psi is optimal.

[0106] Examples 19-21: Effects of different types and amounts of membrane softeners on the quality of flexible liposomes

[0107] Sodium cholate and polysorbate 80 were selected as membrane softeners, and flexible liposomes were prepared according to the dosages in Table 5 in Example 8. They were stored at 4°C for later use, and their appearance, particle size and encapsulation efficiency were compared. The test results are shown in Table 5.

[0108] Table 5. Effects of different membrane softeners and dosages on liposome quality.

[0109]

[0110] The results showed that the appearance and particle size of the flexible liposomes prepared by adding different membrane softeners, sodium cholate and polysorbate 80, were not significantly different, but the encapsulation efficiency was slightly different. Adding more polysorbate 80 resulted in a better encapsulation efficiency. In Example 21, as the amount of membrane softener increased, the particle size decreased, but the encapsulation efficiency also decreased. This may be because the membrane softener is a surfactant, which has a certain solubilizing effect on phospholipids, affecting the encapsulation of active ingredients by phospholipids. Considering that polysorbate 80 is more widely used in cosmetics, polysorbate 80 was selected as the membrane softener, with an amount of 0.1%.

[0111] Comparative Examples 1-4: Preparation of ordinary flexible liposomes using the ethanol injection method

[0112] Take 1g of soybean lecithin (PC 95%), soybean powder lecithin (PC 60%), soybean powder lecithin (PC 30%), and hydrogenated lecithin, respectively, and add 0.2g of cholesterol, 0.1g of polysorbate 80, and 0.05g of vitamin E to each. Place them in a container, add 10g of anhydrous ethanol, and stir at 50-60℃ until completely dissolved. Under stirring conditions, slowly inject the solution into 80g of pure water using a syringe, and continue stirring to ensure thorough dispersion, obtaining a milky white suspension. Transfer to a rotary evaporator and evaporate under reduced pressure at 50°C to remove anhydrous ethanol. Remove the liposomes, add water to bring the total weight to 80g, and homogenize three times in a high-pressure microfluidic homogenizer at a pressure of 15000-20000 Psi to obtain semi-transparent blank liposomes. Then add 0.01g of arginine / lysine peptide, 0.05g of acetyl hexapeptide-8, and 0.05g of dipeptide diaminobutyryl benzylamide diacetate. Stir slowly at room temperature for 30min. Add 0.8g of sodium benzoate and stir until completely dissolved. Add pure water to bring the total weight to 100g. Finally, filter through a 0.22-0.45μm aqueous membrane to obtain the final product.

[0113] The results showed that only the pharmaceutical-grade soybean phospholipid with high PC content in Comparative Example 1 could be completely dissolved in anhydrous ethanol. The other three phospholipids could not be dissolved in anhydrous ethanol, even with increased amounts of anhydrous ethanol or higher temperatures. Therefore, it is difficult to prepare liposomes using the commonly used ethanol injection method with phospholipids that have low PC content and are commonly used in cosmetics.

[0114] Performance Characterization of Each Embodiment

[0115] I. Characterization of Liposomes

[0116] 1. Morphological observation:

[0117] Take samples of flexible liposomes from Example 1 and ordinary liposomes from Example 7, respectively, and dilute them 10-fold. Then, take 1 mL of the diluted solution, add two drops of 0.02% glycerol, and vortex to mix. Add one drop to a supported copper mesh using a pipette and let it stand for 2-3 minutes. Stain with 2% phosphotungstic acid for 2-3 minutes, then blot away excess liquid with filter paper and allow to air dry. Finally, observe the morphology of the samples under a transmission electron microscope. The results show that the prepared liposomes are all spherical and have good morphology. The flexible liposomes of Example 1 have smaller and more uniform particle size than the ordinary liposomes of Example 7 (see Example 7). Figure 1 ).

[0118] 2. Particle size and potential: Appropriate amounts of samples from Examples 1 and 7 were taken, diluted with pure water, and placed in a sample cell. The particle size, PDI, and Zeta potential were determined using a laser particle size analyzer. The results are shown in [Figure number missing]. Figure 2 and 3Example 1 had an average particle size of 88.5 nm, a PDI of 0.158, and a Zeta of -3.47 mV. Example 7 had an average particle size of 131.4 nm, a PDI of 0.336, and a Zeta of 2.84.

[0119] 3. Encapsulation efficiency determination

[0120] (1) HPLC conditions

[0121] Chromatographic column: C18 column (Agilent, PN588905-902, 4.6×250 mm, 5 μm)

[0122] Mobile phase: 0.1% TFA solution: acetonitrile = 85:15

[0123] Detection wavelength: 220 nm

[0124] Column temperature: 25 ℃

[0125] Flow rate: 1 mL / min

[0126] Injection volume: 20 μL

[0127] (2) Standard curve

[0128] Accurately weigh 1 mg each of conopodyl peptide, hexapeptide, and snake venom peptide into 10 mL volumetric flasks, dissolve them in water, and dilute to the mark to obtain a composite peptide standard solution with a concentration of 100 μg / mL for each of the three peptides. Pipette 0.5, 1, 2, 5, and 10 mL of each solution into 10 mL volumetric flasks and dilute to the mark with water to obtain composite peptide standard solutions with concentrations of 5, 10, 20, 50, and 100 μg / mL, respectively. Under the above HPLC chromatographic conditions, the peak areas of the three peptides in the composite peptide standard solutions were determined according to the method. A standard curve was plotted with peak area (A) as the ordinate and concentration (C, μg / mL) as the abscissa. The standard curve equation for conopodyl peptide was A = 4.574C - 1.281, R... 2 =0.9999; the standard curve equation for the hexapeptide is A = 3.826C - 2.676, R 2 =0.9999; The standard curve equation for snake venom peptide is: A = 10.502C + 1.350, R 2 =0.9999. The results indicate that within the concentration range of 5-100 μg / mL, the peak area has a good linear relationship with the concentration. Typical HPLC chromatograms of the three peptides and the sample are shown in Figure (). Figure 4-7 ).

[0129] (3) Encapsulation efficiency determination:

[0130] Add 500 μL of the solution from Example 1 to an ultrafiltration tube and centrifuge at 10000 rpm for 10 min. Take 50 μL each of the outer tube solution and the solution before ultrafiltration into an EP tube and dilute with 450 μL of methanol. Centrifuge at 12000 rpm for 10 min. HPLC is used to determine the total concentration (C0) of each peptide and the concentration of peptides encapsulated in liposomes (Ce), and the encapsulation efficiency (EE) is calculated using the following formula:

[0131]

[0132] The encapsulation rates of conotoxin were 42.5%, hexapeptide was 69.8%, and snake venom peptide was 57.4%.

[0133] (4) Deformation test

[0134] The deformability of flexible liposomes was determined by measuring the ratio of the rate at which the liposomes deformed through a 0.22 μm microporous membrane to the rate at which water passed through the membrane under a certain external pressure, with pure water as a control. First, the time it took for 5g of pure water to pass through the membrane was recorded. Then, the time it took for 5g of the flexible liposomes from Example 1, the ordinary liposomes from Example 7, and the ordinary flexible liposomes from Example 8 to pass through the membrane was recorded. The relative permeation rate was calculated using the following formula, and the results are shown in Table 6.

[0135] Relative permeation rate = (Permeation rate of liposomes / Permeation rate of pure water) × 100%

[0136] Table 6 Results of Deformability Study of Flexible Liposomes

[0137]

[0138] The results showed that the ordinary liposomes in Example 7 could not completely permeate the microporous membrane, possibly due to their rigidity, which caused liposome breakage and blockage during membrane extrusion. Example 8, a typical flexible liposome, exhibited significantly increased deformability and the highest relative permeation rate. Examples 1 and 9, due to the addition of palmitic acid-modified peptides and lactic acid bacteria fermentation product lysate filtrate to the external aqueous phase, respectively, resulted in a slight increase in viscosity and a slight decrease in deformability during membrane extrusion. However, all three were flexible liposomes, possessing high deformability due to the addition of surfactants during the preparation of ordinary liposomes. The principle may be that the surfactants insert into the phospholipid bilayer of the liposome, disrupting the arrangement of the phosphatidyl chains, increasing the fluidity of the phospholipid bilayer, and thus providing greater flexibility and deformability.

[0139] II. In vitro release test

[0140] Take 45 mL of phosphate buffer (pH 6.8) and place it in a 50 mL beaker. Take 800 μL of aqueous solutions of three active peptides of the same concentration, as well as samples from Examples 1 and 7, in triplicate. Place each sample in a dialysis bag, tie both ends with a string, and place it in the beaker. When the temperature reaches 37°C, start magnetic stirring at 50 rpm. Then, take 1 mL samples at different time points and replenish with 1 mL of phosphate buffer as needed. Use HPLC to determine the content of each peptide at different time points. Simultaneously, take an appropriate amount of liposome solution, demulsify with Triton 100, vortex to mix evenly, centrifuge, and take the supernatant to determine the total amount of peptides. Calculate the release percentage of each peptide and plot its release curve. The results show that the peptides in the aqueous solution are released rapidly, with more than 85% released in 1 hour. In contrast, Examples 1 and 7 have a certain sustained-release effect, with 80% released in 4 hours. After 8 hours, the release curve tends to flatten out, and the release is complete in 12 hours. Flexible liposomes are slightly faster than ordinary liposomes, possibly because a certain amount of surfactant is inserted into the phospholipid bilayer of the liposomes, which not only increases the deformation of the membrane but also increases the release of active peptides.

[0141] III. Transdermal Permeability Detection

[0142] Using aqueous solutions of the same concentration of complex peptides, ordinary liposomes (Example 7), and flexible liposomes (Example 1) as experimental samples, each sample was tested in triplicate. The transdermal permeation of the three groups of samples was investigated using the Franz diffusion cell method and piglet skin (3-7 days). 0.5 mL of the experimental sample was added to the donor pool, and 10 mmol / L phosphate buffer solution (pH=7.4) was used as the receiving solution. Transdermal release was performed at 32°C and 180 rpm. 0.5 mL samples were taken at 3, 6, 12, and 24 h, and the same volume of receiving solution was added to the recipient pool. The samples at each time point were analyzed by HPLC. The cumulative transdermal permeation rate Q of the three peptides at each time point was calculated. n (Calculated using the following formula), with Q n A percutaneous osmosis histogram was plotted with time on the x-axis. The results are shown in [Figure number missing]. Figure 9-10 .

[0143]

[0144] In the formula, Q n Cumulative permeability per unit area (μg / cm²) 2 ), C n C represents the drug concentration (μg / mL) measured at the nth time point. i V represents the drug concentration (μg / mL) measured at the i-th time point, and V is the total volume of the receiving cell (mL). s S represents the volume of each sample taken (0.5 mL), and S represents the osmotic diffusion area (2.2 cm²). 2).

[0145] The results showed that none of the three peptides were detected in the samples at any time point in the peptide aqueous solution, indicating that the aqueous solutions of the three peptides had poor permeability on the skin. In Example 1, the transdermal permeation of flexible liposomes increased with time, with hexapeptide showing the best transdermal permeability, reaching a cumulative transdermal amount of 91.8 μg / cm³ at 24 h. 2 The improvement was significantly greater than that of ordinary liposomes in Example 7.

[0146] IV. Stability Test

[0147] The polypeptide aqueous solution of the same concentration and the flexible liposome sample of Example 1 were packaged in vials and placed under five conditions: -18℃, 4℃, room temperature, 40℃ and light exposure for 30 days. The appearance, content and particle size of the liposomes were examined. The sample placed at -18℃ was taken out and thawed at room temperature before being examined. The results are shown in Table 7.

[0148] Table 7 Results of the observation after 30 days under five conditions

[0149]

[0150] The results showed that after one month of storage under five conditions, the appearance of Example 1 did not change significantly. The particle size did not change significantly except for a slight increase at -18℃ and 40℃. The content of the three polypeptides decreased slightly, and the stability was significantly improved compared with the polypeptide aqueous solution of the same concentration.

[0151] V. Safety Evaluation

[0152] ① Subjects: 13 females, aged 20-55 years, test site: inner forearm, test time period: 0.5h, 24h, and 48h after removal of the spot treatment device.

[0153] ②Test Procedure: First, clean the arm and rest for 20 minutes in a constant temperature (20℃-25℃) and constant humidity (40%-60%) environment. Measure 0.020-0.025 g of sample and add it to the small chamber of the plaque applicator. Apply the plaque applicator to the flexor side of the subject's forearm, gently pressing it with the palm to ensure even adhesion to the skin, and leave it on for 24 hours. Observe the skin reaction at 0.5h, 24h, and 48h after removing the plaque applicator.

[0154] Table 8. Grading Criteria for Adverse Skin Reactions in Occlusive Patch Testing

[0155]

[0156] The results showed that all 13 subjects had negative results in the closed patch test, and no adverse skin reactions were observed (see [link to results]). Figure 11 This indicates that the prepared flexible liposomes have high safety.

[0157] VI. Anti-wrinkle test

[0158] ① Subjects: 6 males and 28 females, totaling 34 subjects, aged 20-55 years, tested on the face, tested for 0 days, 7 days, 14 days and 28 days.

[0159] ② Test sample: Take the flexible liposome from Example 1, dilute it 10 times with pure water, and use it as an essence water.

[0160] ③ Testing instruments: VisioFace 1000D (facial image analyzer), ElastiMeter (skin elasticity tester)

[0161] ④ Test Procedure: First, cleanse the face and rest for 20 minutes in a constant temperature (20℃-25℃) and constant humidity (40%-60%) environment. Collect facial images on day 0 and test skin elasticity. Apply evenly to the face after cleansing, twice daily (morning and evening), gently pressing until absorbed. Collect facial images and test skin elasticity on days 7, 14, and 28. Results are shown below. Figure 12 and Figure 13 .

[0162] ⑤ Test Results: The results showed that after 7 days of use, the volume of wrinkles in typical locations decreased by 11.15%, the area decreased by 6.35%, the area ratio decreased by 7.70%, and the immediate elasticity value increased by 7.48%; after 14 days of use, the volume of wrinkles in typical locations decreased by 20.03%, the area decreased by 11.30%, the area ratio decreased by 14.16%, and the immediate elasticity value increased by 13.82%.

[0163] After 28 days of use, the volume of wrinkles in typical locations decreased by 29.16%, the area decreased by 20.41%, the area ratio decreased by 22.45%, and the immediate elasticity value increased by 20.16%. This indicates that flexible liposomes have a significant effect on improving both wrinkles and skin elasticity.

[0164] Unlike traditional cosmetics, the functional cosmetics of this invention containing flexible liposomes can maximize the penetration of functional ingredients into the deep cells of the skin, providing nutrition, protection and treatment to tissues and cells for a longer period of time. They have multiple benefits, longer-lasting nutrition and stronger absorption, among other skin care advantages.

Claims

1. A flexible liposome containing a complex peptide for reducing wrinkles and fading fine lines, characterized in that, It consists of the following components by mass percentage: Acetylcholine-blocking peptides: 0.01–0.2% Liposome lipid materials 1~5% Liposome membrane softener 0.1~0.5% Solvent 5-10% Antioxidant 0.05~0.1% Stabilizer 1~10% Preservative 0.5~1.0% and water 80-95% The sum of the contents of all components is 100%; The solvent is a polyol; The stabilizer is a mixture of palmitic acid-modified peptides and lactic acid bacteria fermentation product lysate filtrate; the palmitic acid-modified peptides include one or more of palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4, and palmitoyl hexapeptide-12, with an addition amount of 0.05~0.1% of the total weight, and the addition amount of lactic acid bacteria fermentation product lysate filtrate is 5~10% of the total weight; The acetylcholine blocking polypeptide is composed of arginine / lysine polypeptide, acetyl hexapeptide-8, and dipeptide diaminobutyryl benzylamide diacetate. Phospholipids, cholesterol, membrane softener, antioxidant, and polyol were mixed and dissolved to obtain a lipid oil phase solution; acetylcholine-blocking peptides were added to purified water and dissolved by stirring, and then added to the lipid oil phase solution to obtain a W / O colostrum; Purified water was added to W / O colostrum, and the resulting crude liposome suspension was homogenized. Then, lactic acid bacteria fermentation product lysate filtrate and palmitic acid-modified active peptides were added, and the mixture was filtered to obtain flexible liposomes.

2. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 1, characterized in that: The liposome lipid material is composed of phospholipids and cholesterol, and the mass ratio of phospholipids to cholesterol is 4:1 to 10:

1.

3. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 2, characterized in that: The phospholipids include one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and distearate phosphatidylcholine; the cholesterol includes animal-derived cholesterol and plant-derived cholesterol.

4. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 3, characterized in that: The plant-derived cholesterol is one or more of sitosterol, campesterol, and stigmasterol.

5. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 2, characterized in that: The phospholipid is hydrogenated lecithin, and the cholesterol is sitosterol.

6. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 2, characterized in that: The polyols include propylene glycol, glycerol, butylene glycol, or pentanediol.

7. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 1, characterized in that: The liposome membrane softener is sodium deoxycholate or polysorbate 80, wherein the mass ratio of the liposome membrane softener to the liposome lipid material is 1:5-10.

8. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 1, characterized in that: The antioxidant is one or more of vitamin E, BHT and BHA, and the amount added accounts for 0.05 to 0.1% of the total mass of the composition.

9. The wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to claim 5, characterized in that: The preservative is one or more of sodium benzoate and phenoxyethanol.

10. A method for preparing a wrinkle-reducing and anti-wrinkle complex peptide flexible liposome according to any one of claims 1-9, comprising the following steps: Step S1, Preparation of lactic acid bacteria fermentation product lysate filtrate: Take frozen lactic acid bacteria glycerol and inoculate it onto MRS medium to activate it. Incubate at 30-42℃ for 20-30 h to obtain seed culture medium. Take the activated seed culture medium and inoculate it into MRS culture medium at a ratio of 1%-5%, and incubate at 30-42℃ for 20-30 hours to obtain lactic acid bacteria fermentation products; Take the lactic acid bacteria fermentation product, homogenize it once with a microfluidic high-pressure homogenizer at 18,000-20,000 psi, then increase the homogenization pressure to 23,000-25,000 psi and homogenize it twice more, while controlling the temperature at 0-10℃, to obtain the lactic acid bacteria fermentation product lysate; finally, centrifuge it in a refrigerated centrifuge at 12,000 rpm for 15 min, and take the supernatant as the lactic acid bacteria fermentation product lysate filtrate. Step S2, Preparation of W / O Colostrum: Weigh out phospholipids, cholesterol, membrane softener, and antioxidant and place them in a reaction vessel. Add polyol and stir in a water bath at 80-90℃ until completely dissolved. Cool to room temperature to obtain a lipid oil phase solution. Take purified water at a water phase to oil phase mass ratio of 1:2-5, add acetylcholine blocking peptides and stir until completely dissolved. Add the solution to the lipid oil phase under stirring to obtain W / O colostrum for later use. Step S3, preparation of liposomes using the W / O / W double emulsion method: Take 80% purified water and add W / O colostrum while stirring continuously for 30 minutes to obtain a milky white crude liposome suspension. Homogenize 3-5 times using a high-pressure microfluidic homogenizer at a pressure of 15000-20000 Psi. Then add lactic acid bacteria fermentation product lysate filtrate and palmitic acid-modified active peptides, stir until completely dissolved, and continue stirring for 1-2 hours. Finally, add preservatives and pure water to the total volume. Filter using a 0.1-0.45 μm polycarbonate membrane to obtain transparent to semi-transparent flexible liposomes.

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