PDRN-containing anti-aging repair composition and lipid nanoparticles and application thereof

Abstract

The invention provides an anti-aging repair composition containing PDRN, and lipid nanoparticles and application thereof. The composition provided by the invention is prepared from polydeoxyribonucleotide, micro-current tetrapeptide-1 and palmitoyl tripeptide-5. According to the application, the polydeoxyribonucleotide, the micro-current tetrapeptide-1 and the palmitoyl tripeptide-5 are compounded for use, so that the composition has a synergistic effect in the aspects of promoting cell proliferation, inhibiting an intracellular ROS (reactive oxygen species) level and the like, and has remarkable antioxidant, anti-inflammatory, anti-aging, soothing and repairing effects. The PDRN-containing composition is prepared into the lipid nanoparticles, so that the stability of the PDRN-containing composition is improved, the permeability of the PDRN-containing composition on the skin surface is improved, the bioavailability of the PDRN-containing composition is improved, the cell survival rate and cell uptake are promoted, the expression of adenosine A2A receptors, the synthesis of collagen, angiogenesis and the inhibition of anti-inflammatory factors are promoted, and a remarkable anti-aging effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of daily chemical products, and in particular to an anti-aging and repairing composition containing PDRN, and lipid nanoparticles and applications thereof. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a polymer of DNA fragments extracted and purified from sperm cells of specific fish (usually salmon). Its molecular weight typically ranges from 50 to 1500 kDa and it is highly biocompatible. PDRN's mechanisms of action primarily involve activating A2A receptors, promoting cell proliferation and differentiation, modulating inflammatory responses, and promoting angiogenesis. When used as an active ingredient in cosmetics, it can effectively alleviate wrinkles caused by aging and help improve pigmentation.

[0003] Further improving the role of PDRN in promoting cell proliferation and differentiation, regulating inflammatory response, and promoting angiogenesis is one of the current research hotspots. Summary of the Invention

[0004] In view of this, the technical problem to be solved by this application is to provide an anti-aging and repair composition containing PDRN, its lipid nanoparticles and applications. The components in the composition provided by this application have a synergistic effect in promoting cell proliferation, inhibiting intracellular ROS levels, etc., and have significant antioxidant, anti-inflammatory, anti-aging, soothing and repair effects.

[0005] The present application provides a composition containing PDRN, including polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5.

[0006] This application uses a combination of polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5, which has a synergistic effect in promoting cell proliferation and inhibiting intracellular ROS levels, and has significant antioxidant, anti-inflammatory, anti-aging, soothing and repair effects.

[0007] Polydeoxyribonucleotide (PDRN) is a polymer of DNA fragments extracted and purified from sperm cells of specific fish (usually salmon). It has the potential to activate A2A receptors, promote cell proliferation and differentiation, regulate inflammatory responses, and promote angiogenesis. This application does not specifically limit the source of the PDRN; it can be purchased commercially.

[0008] The peptide sequence of microcurrent tetrapeptide-1 is LEAP, which can improve mitochondrial function and increase ATP production. This application has no special restrictions on the source of the microcurrent tetrapeptide-1, and it can be purchased from the market, such as the product called UplevityTM Products of e-Liftpeptide solution.

[0009] Palmitoyl tripeptide-5 can activate skin tissue growth factor (TGF-β), significantly increasing the production of type I, II, and IV collagen, enhancing skin elasticity, and reducing wrinkles and sagging. This application does not specifically limit the source of the palmitoyl tripeptide-5; it can be purchased commercially, such as the product under the trade name SYN@-COLL.

[0010] In some specific implementations, the mass ratio of the polydeoxyribonucleotide, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 is 0.1-5:0.000005-0.0005:0.0001-1. In some specific implementations, the mass ratio of the polydeoxyribonucleotide, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 is 0.5-4.5:0.00001-0.0003:0.0005-0.5. In some specific implementations, the mass ratio of the polydeoxyribonucleotide, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 is 1-4:0.00005-0.0001:0.001-0.1. In some specific implementations, the mass ratio of the polydeoxyribonucleotide, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 is 4:0.0001:0.04.

[0011] The present application also provides a lipid nanoparticle containing PDRN, comprising the composition described in the above technical solution and a lipid nanoparticle raw material, wherein the lipid nanoparticle raw material comprises at least two of phospholipids, an emulsifier, a polyol and water.

[0012] PDRN has the disadvantages of large molecular weight and strong polarity, making it difficult to penetrate the skin barrier and accumulate in the anti-aging target areas of the skin, and is easily degraded by nucleases on the skin surface. Microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 also have the problems of instability and difficulty in penetration. The present application prepares the composition containing PDRN into the form of lipid nanoparticles, improves the stability of the PDRN-containing composition and increases its permeability on the skin surface, improves its bioavailability, promotes cell survival rate and cell uptake, expression of adenosine A2A receptors, collagen synthesis, angiogenesis, and inhibits anti-inflammatory factors, thereby achieving significant anti-aging effects.

[0013] In some specific implementations, the lipid nanoparticles include:

[0014] 0.1wt%~5wt% polydeoxyribonucleotides;

[0015] 0.000005wt%~0.0005wt% of microcurrent tetrapeptide-1;

[0016] 0.0001wt%~1wt% palmitoyl tripeptide-5;

[0017] 0.1wt%~3wt% phospholipids;

[0018] 0.1wt%~3wt% non-phospholipid functional lipids;

[0019] 10wt%~40wt% emulsifier;

[0020] 10wt%~50wt% of alcohol compound;

[0021] The remaining amount of water.

[0022] In some specific implementations, the lipid nanoparticles include:

[0023] 0.5wt%~4.5wt% polydeoxyribonucleotides;

[0024] 0.00001wt%~0.0003wt% of microcurrent tetrapeptide-1;

[0025] 0.0005wt%~0.5wt% palmitoyl tripeptide-5;

[0026] 0.1wt%~3wt% phospholipids;

[0027] 0.1wt%~3wt% non-phospholipid functional lipids;

[0028] 10wt%~40wt% emulsifier;

[0029] 10wt%~50wt% of alcohol compound;

[0030] The remaining amount of water.

[0031] In some specific implementations, the phospholipids include but are not limited to lecithin (such as soybean lecithin, hydrogenated lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, etc.), distearoylphosphatidylcholine (DSPC), natural sphingomyelin (SM), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidyltryptophan, which can be one or more of them.

[0032] In some specific implementations, the non-phospholipid functional lipids include but are not limited to stearamide, oleyl fatty amine derivatives, stearic acid polyethylene glycol N-hydroxysuccinimide ester, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, etc., and can be one or more of them.

[0033] In some specific implementations, the emulsifier includes but is not limited to polyoxyethylene castor oil emulsifiers, polyoxyethylene hydrogenated castor oil emulsifiers, polyglycerol emulsifiers, poloxamer, cocoyl glucoside, polyglyceryl-10 laurate, glyceryl stearate citrate, sodium di(laurylamide glutamine) lysine, triglyceride emulsifiers (such as caprylic / capric triglyceride, palmitic triglyceride, 1,3-dioleyl-2-palmitic triglyceride, caprylic / capric / succinic triglyceride, behenyl glyceride, etc. The present invention may include but is not limited to: PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-20 stearate ...

[0034] In some specific implementations, the alcohol compound can be a monohydric alcohol, a dihydric alcohol or a polyhydric alcohol, including but not limited to glycerol, 1,2-propylene glycol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,3-propylene glycol, 1,2-pentanediol, ethoxydiglycol, 1,2-hexanediol, dipropylene glycol, isopropyl alcohol, methylpropylene glycol, polyethylene glycol-200, PPG-10 sorbitol and octyldodecanol, etc., and can be one or more of them.

[0035] The present application also provides a method for preparing the lipid nanoparticles described in the above technical solution, comprising the following steps:

[0036] (1) mixing polydeoxyribonucleotide, an emulsifier, water and a first mass portion of an alcohol compound to obtain a mixed solution 1;

[0037] Mixing the phospholipid, the non-phospholipid functional lipid and the second mass part of the alcohol compound to obtain a mixed solution 2;

[0038] Mixing microcurrent tetrapeptide-1, palmitoyl tripeptide-5, an emulsifier and a third part by mass of an alcohol compound to obtain a mixed solution 3;

[0039] (2) Mixed solution 3 is mixed with mixed solution 1, and then mixed with mixed solution 2 to obtain mixed solution 4;

[0040] (3) The mixed solution 4 is subjected to nano-processing to obtain lipid nanoparticles.

[0041] The present application first mixes polydeoxyribonucleotides, an emulsifier, and water with a first mass part of an alcohol compound under the protection of an inert gas, such as nitrogen, to obtain a mixed solution 1. Phospholipids, non-phospholipid functional lipids, and a second mass part of an alcohol compound are mixed to obtain a mixed solution 2. Microcurrent tetrapeptide-1, palmitoyl tripeptide-5, an emulsifier, and a third mass part of an alcohol compound are mixed to obtain a mixed solution 3. After obtaining mixed solutions 1, 2, and 3, respectively, mixed solution 3 is first mixed with mixed solution 1 and then mixed with mixed solution 2 to obtain mixed solution 4. Finally, mixed solution 4 is nanosized to obtain lipid nanoparticles.

[0042] In some specific implementations, the mixing in the above steps is performed at 10°C to 30°C.

[0043] In some specific implementations, the nano-processing is specifically:

[0044] The mixed liquid 4 is homogenized under the conditions of nitrogen protection, a pressure of 500-1200 bar, and homogenization 1-5 times.

[0045] In some specific implementations, after the nano-sizing treatment, the material is discharged at 20° C. under nitrogen protection conditions.

[0046] The method provided in this application enables phospholipids to encapsulate active ingredients, and the obtained lipid nanoparticles have a particle size of 100nm~300nm, a PDI of 0.1~0.6, and are relatively stable.

[0047] The present application also provides a composition containing lipid nanoparticles, comprising the lipid nanoparticles described in the above technical solution and a filtrate of a fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract.

[0048] Bifidobacterium / Lactobacillus / Soybean Seed Extract Ferment Filtrate (Huanfuyuan), national cosmetics original registration number 20230032, registered Chinese name is Bifidobacterium / Lactobacillus / Soybean Seed Extract Ferment Filtrate, INCI name is Bifidobacterium / Lactobacillus / Soybean Seed Extract Ferment Filtrate, which can be purchased from Yatsen e-commerce, or prepared by a method comprising the following steps: soaking black beans and water in a certain proportion, heating and water extraction to obtain soybean seed extract, sterilizing to obtain a fermentation base, then transferring the mixture into a fermentation tank, adding a strain composition (containing bifidobacteria and lactobacilli) for fermentation, and sterilizing and filtering to obtain a fermentation filtrate.

[0049] This application uses a combination of lipid nanoparticles and bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate, which can enable the active ingredients to efficiently penetrate the skin barrier and quickly reach the dermis deep in the skin, effectively stimulating fibroblast renewal, thereby promoting the synthesis of collagen and elastin, enhancing skin elasticity, and promoting skin renewal; as well as inhibiting the production of cellular inflammatory factors, enhancing the expression of adenosine A2A receptors, and delaying skin aging.

[0050] In some specific implementations, the mass ratio of the lipid nanoparticles to the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract is 1 to 10: 1. In some specific implementations, the mass ratio of the lipid nanoparticles to the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract is 2 to 15: 1.

[0051] The present application also provides a method for preparing a composition containing lipid nanoparticles, comprising the following steps:

[0052] The lipid nanoparticles were mixed with the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract, and then discharged at 20° C. under nitrogen protection.

[0053] In some specific implementations, the mixing is performed at 10-30° C. In some specific implementations, the mixing can be performed uniformly by stirring, shearing, or the like.

[0054] The PDRN-containing composition described in the above technical solution provided in this application, the lipid nanoparticles described in the above technical solution and / or the lipid nanoparticle-containing composition described in the above technical solution have anti-aging, anti-inflammatory, soothing and repairing effects, and can be used in products with corresponding effects.

[0055] The present application also provides a product, including the composition containing PDRN described in the above technical solution, the lipid nanoparticles described in the above technical solution and / or the composition containing lipid nanoparticles described in the above technical solution.

[0056] In some embodiments of the present invention, the above-mentioned products include, but are not limited to, personal care products (personal care products), medicines, etc. It will be understood by those skilled in the art that, in addition to the composition described in the above technical solution, other excipients may also be included, such as pharmaceutical excipients or excipients for personal care products.

[0057] In some embodiments of the present invention, the personal care product includes: a basic care product and / or a makeup product.

[0058] In some embodiments of the present invention, the cosmetic products include but are not limited to:

[0059] (1) Foundation makeup:

[0060] Liquid foundation / cream: used to even out skin tone and cover blemishes;

[0061] BB Cream / CC Cream: A lightweight makeup base that combines skincare and finishing touches.

[0062] Concealer / pen: for covering local areas such as acne, dark circles, etc.

[0063] Loose powder / face powder: sets makeup and reduces facial shine.

[0064] (2) Eye makeup:

[0065] Eyeshadow: adds color and depth to the eyes;

[0066] Eyeliner pencil / liquid / gel: outline the eyeliner to make the eyes look more vivid;

[0067] Mascara: lengthens and thickens eyelashes, adds depth to eyes;

[0068] Eyebrow pencil / eyebrow powder / eyebrow gel: fill in the gaps in the eyebrows and shape the ideal eyebrows.

[0069] (3) Cheek makeup:

[0070] Blush: Add natural redness to cheeks and enhance complexion;

[0071] Contouring cake / stick: Use shading techniques to make the facial contours more three-dimensional;

[0072] (4) Lip makeup:

[0073] Lipstick / Lip Glaze / Lip Gloss: Change or emphasize the color of the lips;

[0074] Lip liner: Draw a clear lip boundary to prevent lipstick from overflowing.

[0075] (5) Multifunctional cosmetics:

[0076] Highlighter stick / liquid / powder: Brighten the high points of the face (such as the bridge of the nose and cheekbones) to create a radiant effect.

[0077] In addition, there are products designed specifically for special occasions, such as waterproof and sweat-proof eyeliner and long-lasting lipstick.

[0078] In some embodiments of the present invention, the basic care products include but are not limited to:

[0079] Facial Cleanser / Face Wash: Gently removes dirt, oil and makeup residue from the face;

[0080] Makeup remover oil / makeup remover water / makeup remover cream: specially designed to completely remove makeup, especially waterproof makeup;

[0081] Toner / Lotion: Use after cleansing to further cleanse residue on the skin surface, replenish moisture to the skin, restore the skin's pH balance, and lay a good foundation for the absorption of subsequent skin care products;

[0082] Essence: Contains high concentrations of active ingredients, providing deep nourishment and repair for specific skin problems (such as anti-aging, moisturizing, whitening, etc.);

[0083] Eye cream: specially designed for the sensitive area around the eyes, it can reduce fine lines, dark circles and tighten the skin around the eyes. The texture is usually light and easy to absorb.

[0084] Day / night lotion or cream: has the functions of protecting, repairing and nourishing, promoting cell regeneration, etc., which can provide the skin with necessary moisture and lock in the replenished moisture;

[0085] Sunscreen: used for UV protection, prevention of photoaging, etc.;

[0086] Facial masks: Provide extra nourishment and care to the skin, such as hydration, pore cleansing or brightening.

[0087] In some specific implementations, the personal care product includes 0.1wt%~10wt% of the PDRN-containing composition described in the above technical solution, the lipid nanoparticles described in the above technical solution and / or the lipid nanoparticle-containing composition described in the above technical solution, preferably includes 0.5wt%~9.5wt%, and more preferably includes 1wt%~9wt%.

[0088] In some specific implementations, the personal care product can be an essence water, a typical formula of which includes: component A, component B, active ingredients and water. Based on the total mass of 100%, component A contains: 5% dipropylene glycol, 10% glycerol, 15% propylene glycol, and 0.15% carbomer 941; component B contains: 0.15% triethanolamine, 0.5% 1,2-hexanediol / caprylylhydroxamic acid / ethylhexylglycerol / propylene glycol; the active ingredient includes 0.1wt%~10wt% of the PDRN-containing composition described in the above technical solution, the lipid nanoparticles described in the above technical solution, and / or the lipid nanoparticle-containing composition described in the above technical solution; the balance is water.

[0089] This application uses a combination of polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5, which has a synergistic effect in promoting cell proliferation and inhibiting intracellular ROS levels, and has significant antioxidant, anti-inflammatory, anti-aging, soothing and repair effects.

[0090] Furthermore, the present application prepares lipid nanoparticles by combining polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 with phospholipids, non-phospholipid functional lipids, emulsifiers, alcohol compounds and water. The lipid nanoparticles are easily soluble in water, easy to use, have good stability and water dispersibility, increase the solubility of the active ingredients, and also improve the irritation of the active ingredients, so that the active ingredients can reach a sufficient concentration in the product to exert the corresponding functional effects. The lipid nanoparticles provided by the present application effectively improve the solubility of each active ingredient while providing a stable storage space for it, avoiding unnecessary degradation or inactivation of the active ingredients during storage and before use, which is conducive to increasing the concentration of the active substance during use.

[0091] The present application combines the lipid nanoparticles described in the above technical solution with the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract, which enables the active ingredients to efficiently penetrate the skin barrier and quickly reach the dermis deep in the skin, effectively stimulating fibroblast renewal, thereby promoting the synthesis of collagen and elastin, enhancing skin elasticity, and promoting skin renewal; as well as inhibiting the production of cellular inflammatory factors, enhancing the expression of adenosine A2A receptors, and delaying skin aging.

[0092] Experimental results show that the lipid nanoparticles and their compositions provided in this application have good skin permeability and retention properties after use, can be enriched in high concentrations in target tissues, retained for a long time, and have sustained and controlled release, and are effectively taken up by target cells, thereby improving the bioavailability of effective ingredients, enhancing anti-aging effects, and prolonging the duration of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The electrophoresis results provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0094] The present invention provides a composition containing PDRN, and its lipid nanoparticles and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. The methods and applications of the present invention have been described by preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0095] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0096] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0097] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0098] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0099] The present application provides an anti-aging repair composition containing PDRN, comprising polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5.

[0100] This application uses a combination of polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5, which has a synergistic effect in promoting cell proliferation and inhibiting intracellular ROS levels, and has significant antioxidant, anti-inflammatory, anti-aging, soothing and repair effects.

[0101] The present application also provides a lipid nanoparticle containing PDRN, comprising the composition described in the above technical solution and a lipid nanoparticle raw material, wherein the lipid nanoparticle raw material comprises at least two of phospholipids, non-phospholipid functional lipids, an emulsifier, an alcohol compound and water.

[0102] The present application prepares lipid nanoparticles by combining polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 with phospholipids, non-phospholipid functional lipids, emulsifiers, alcohol compounds and water. The lipid nanoparticles are easily soluble in water, easy to use, have good stability and water dispersibility, increase the solubility of the active ingredients, and also improve the irritation of the active ingredients, so that the active ingredients can reach a sufficient concentration in the product to exert the corresponding functional effects. The lipid nanoparticles provided by the present application effectively improve the solubility of each active ingredient while providing a stable storage space for it, avoiding unnecessary degradation or inactivation of the active ingredients before storage and use, which is conducive to increasing the concentration of the active substance during use.

[0103] The present application also provides a method for preparing the lipid nanoparticles described in the above technical solution, comprising the following steps:

[0104] (1) mixing polydeoxyribonucleotide, an emulsifier, water and a first mass portion of an alcohol compound to obtain a mixed solution 1;

[0105] Mixing the phospholipid, the non-phospholipid functional lipid and the second mass part of the alcohol compound to obtain a mixed solution 2;

[0106] Mixing microcurrent tetrapeptide-1, palmitoyl tripeptide-5, an emulsifier and a third part by mass of an alcohol compound to obtain a mixed solution 3;

[0107] (2) Mixed solution 3 is mixed with mixed solution 1, and then mixed with mixed solution 2 to obtain mixed solution 4;

[0108] (3) The mixed solution 4 is subjected to nano-processing to obtain lipid nanoparticles.

[0109] The method provided in this application enables phospholipids to encapsulate active ingredients, and the obtained lipid nanoparticles have a particle size of 100nm~300nm, a PDI of 0.1~0.6, and are relatively stable.

[0110] The present application also provides a composition containing lipid nanoparticles, comprising the lipid nanoparticles described in the above technical solution and a filtrate of a fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract.

[0111] This application uses a combination of lipid nanoparticles and bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate, which can enable the active ingredients to efficiently penetrate the skin barrier and quickly reach the dermis deep in the skin, effectively stimulating fibroblast renewal, thereby promoting the synthesis of collagen and elastin, enhancing skin elasticity, and promoting skin renewal; as well as inhibiting the production of cellular inflammatory factors, enhancing the expression of adenosine A2A receptors, and delaying skin aging.

[0112] The present application is further described below with reference to the following embodiments.

[0113] Example 1, Comparative Examples 1-6

[0114] According to the formulation shown in Table 1, the components were mixed to obtain a composition.

[0115] Table 1 Example 1 and comparative example formula

[0116]

[0117] Example 2

[0118] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0119] (2) 2% soybean lecithin, 0.5% phosphatidylglycerol, 10% 1,2-pentanediol, and 3% isopropanol are stirred at 30°C to form a homogeneous clear liquid 2.

[0120] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0121] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0122] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0123] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0124] Example 3

[0125] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0126] (2) 2% hydrogenated lecithin, 0.5% polyethylene glycol N-hydroxysuccinimide stearate, 10% 1,2-pentanediol, and 3% isopropyl alcohol are stirred at 30°C to form a homogeneous clear liquid 2.

[0127] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0128] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0129] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0130] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0131] Example 4

[0132] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0133] (2) Phosphatidylinositol 1%, hydrogenated lecithin 1%, polyethylene glycol N-hydroxysuccinimide stearate 0.5%, 1,2-pentanediol 10%, and isopropyl alcohol 3% are stirred at 30°C to form a homogeneous clear liquid 2.

[0134] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0135] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0136] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0137] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0138] Example 5

[0139] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0140] (2) 1% oleoyl fatty amine derivative, 1% stearylamide, 0.5% natural sphingomyelin, 10% 1,2-pentanediol, and 3% isopropanol are stirred at 30°C to form a homogeneous clear liquid 2.

[0141] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0142] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0143] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0144] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0145] Example 6

[0146] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0147] (2) Egg yolk phosphatidylcholine 1%, natural sphingomyelin 1%, phosphatidylglycerol 0.5%, 1,2-pentanediol 10%, isopropanol 3%, stirred at 30°C to form a homogeneous clear liquid 2.

[0148] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0149] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0150] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0151] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0152] Example 7

[0153] (1) PDRN 4%, methyl propylene glycol 20%, 1,3-butylene glycol 12%, laureth-23 5%, cocoyl glucoside 20%, Tween-80 10%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0154] (2) 1% phosphatidyltryptophan, 0.5% natural sphingomyelin, 0.5% phosphatidylglycerol, 0.5% N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, 10% 1,2-pentanediol, and 3% isopropanol are stirred at 30°C to form a homogeneous clear liquid 2.

[0155] (3) Add 0.0001% of microcurrent tetrapeptide-1, 0.04% of palmitoyl tripeptide-5, 5% of polyglyceryl-10 laurate, and 5% of 1,3-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0156] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0157] (5) Liquid 4 was homogenized three times with a high-pressure homogenizer under nitrogen protection and 800 bar to obtain PDRN lipid nanoparticles.

[0158] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0159] Example 8

[0160] (1) PDRN 2.5%, octyldodecanol 10%, ethoxydiglycol 8%, caprylic / capric / succinic triglyceride 10%, sodium di(laurylamide glutamine) lysine 15%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0161] (2) 1.5% soybean lecithin, 0.5% phosphatidylglycerol, and 10% isopropyl alcohol are stirred at 30°C to form a homogeneous clear liquid 2.

[0162] (3) Add 0.00008% of microcurrent tetrapeptide-1, 0.02% of palmitoyl tripeptide-5, 3% of glyceryl stearate citrate, 5% of PPG-26-butanol polyether-26, and 5% of glycerol and stir at 20°C to form a uniform clear liquid 3.

[0163] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0164] (5) Liquid 4 was homogenized once with a high-pressure homogenizer under nitrogen protection and 1000 bar to obtain PDRN lipid nanoparticles.

[0165] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0166] Example 9

[0167] (1) PDRN 1%, 1,2-pentanediol 10%, PPG-10 sorbitol 5%, trideceth-12 5%, PEG-20 hydrogenated castor oil 10%, laureth-23 5%, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0168] (2) 0.5% soybean lecithin, 0.5% phosphatidylglycerol, 3% 1,2-hexanediol, and 2% dipropylene glycol were stirred at 30°C to form a homogeneous clear liquid 2.

[0169] (3) Add 0.00005% of microcurrent tetrapeptide-1, 0.01% of palmitoyl tripeptide-5, 1% of sodium di(lauryl glutamine) lysine, and 6% of 1,2-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0170] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0171] (5) Liquid 4 was homogenized four times using a high-pressure homogenizer under nitrogen protection and 600 bar to obtain PDRN lipid nanoparticles.

[0172] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0173] Example 10

[0174] (1) 0.1% PDRN, 5% glycerol, 2,3-butylene glycol, 5% laureth-23, 3% poloxamer, and the balance water were stirred at 20°C under nitrogen protection to form a homogeneous clear liquid 1.

[0175] (2) 0.3% soybean lecithin, 0.2% phosphatidylglycerol, 1% 1,2-pentanediol, and 2% isopropanol are stirred at 30°C to form a homogeneous clear liquid 2.

[0176] (3) Add 0.00001% of microcurrent tetrapeptide-1, 0.001% of palmitoyl tripeptide-5, 3% of polyglyceryl-10 laurate, and 3% of 1,2-propylene glycol at 20°C and stir to form a uniform clear liquid 3.

[0177] (4) Pour liquid 3 into liquid 1 and mix evenly, then pour into liquid 2 and stir to form a uniform liquid 4.

[0178] (5) Liquid 4 was homogenized five times using a high-pressure homogenizer under nitrogen protection and 500 bar to obtain PDRN lipid nanoparticles.

[0179] In this embodiment, the percentages of each component are all weight percentages, and the total is 100%.

[0180] Example 11

[0181] Mix 20 parts of the PDRN lipid nanoparticles prepared in Example 2 and 1 part of the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0182] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 20:1.

[0183] Example 12

[0184] Mix 15 parts of the PDRN lipid nanoparticles prepared in Example 2 and 1 part of the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0185] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 15:1.

[0186] Example 13

[0187] Mix 10 parts of the PDRN lipid nanoparticles prepared in Example 2 and 1 part of the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0188] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 10:1.

[0189] Example 14

[0190] Mix 5 parts of the PDRN lipid nanoparticles prepared in Example 2 with 1 part of the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0191] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 5:1.

[0192] Example 15

[0193] Mix 2 parts of the PDRN lipid nanoparticles prepared in Example 2 and 1 part of the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0194] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 2:1.

[0195] Example 16

[0196] Mix 1 part of the PDRN lipid nanoparticles prepared in Example 2 and 1 part of the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) to obtain a PDRN lipid nanoparticle composition.

[0197] The mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) in the PDRN lipid nanoparticle composition is 1:1.

[0198] Example 17

[0199] This embodiment provides an essence for testing efficacy samples, the raw materials of which include component A, component B and water. Based on the total mass of 100%, component A includes: 5% dipropylene glycol, 10% glycerol, 15% propylene glycol, and 0.15% carbomer 941; component B includes: 0.15% triethanolamine, 0.5% 1,2-hexanediol / caprylhydroxamic acid / ethylhexylglycerin / propylene glycol; and the balance is water.

[0200] The preparation method of the essence comprises the following steps:

[0201] (1) Add the raw materials of component A to water and stir and dissolve at 35-45°C and 400-500 rpm until a clear, uniform liquid without agglomeration is obtained to obtain phase A.

[0202] (2) Add triethanolamine and 1,2-hexanediol / octanoylhydroxamic acid / ethylhexylglycerin / propylene glycol in component B to phase A while stirring at 400-5000 rpm, and then slowly stir at 200-300 rpm for 15 minutes until the liquid is uniform, thereby obtaining a blank essence solution.

[0203] The blank essence has the appearance of a transparent liquid with a pH between 5 and 7.

[0204] Test Example 1 Cell Proliferation Experiment

[0205] Test samples: the compositions prepared in Example 1 and Comparative Examples 1 to 6, the PDRN lipid nanoparticles prepared in Examples 2 to 4 and Examples 8 to 10, and the PDRN lipid nanoparticle compositions prepared in Examples 11 to 16.

[0206] HDF cells in logarithmic growth phase were cultured at a rate of 8×10 3The cells were seeded at a density of 100 μL per well in a 96-well plate and cultured at 37°C with 5% CO2 for 24 h. 100 μL of the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10 (the concentration of the PDRN lipid nanoparticles was 500 μg / mL), the PDRN lipid nanoparticle compositions prepared in Examples 11 to 16 (the concentration of the PDRN lipid nanoparticles was 500 μg / mL, and the concentrations of Huanfuyuan were 25 μg / mL, 33.3 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL, respectively), the composition prepared in Example 1 (the concentration of the active ingredient was consistent with that in the 500 μg / mL PDRN lipid particles, i.e., the concentrations of PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 were 20 μg / mL, 0.0005 μg / mL, and 0.2 μg / mL, respectively), and the compositions prepared in Comparative Examples 1 to 6 (the concentration of the active ingredient in Comparative Example 1, the concentration of PDRN was 20 μg / mL); Comparative Example 2 active substance concentration, microcurrent tetrapeptide-1 concentration is 0.0005 μg / mL; Comparative Example 3 active substance concentration, palmitoyl tripeptide-5 concentration is 0.2 μg / mL; Comparative Example 4 active substance concentration, PDRN, microcurrent tetrapeptide-1 concentration is 20 μg / mL and 0.0005 μg / mL respectively; Comparative Example 5 active substance concentration, PDRN, palmitoyl tripeptide-5 concentration is 20 μg / mL and 0.2 μg / mL respectively; Comparative Example 6 active substance concentration, microcurrent tetrapeptide-1, palmitoyl tripeptide-5 concentration is 0.0005 μg / mL and 0.2 μg / mL respectively) DMEM complete medium, blank control group is added with 100 μL DMEM complete medium, each group has 3 replicates. Continue to culture for 48 hours, CCK-8 method is used to measure cell proliferation rate, the results are shown in Table 2, Table 2 is the cell proliferation experiment results provided in the examples and comparative examples of the present application.

[0207] Table 2 Cell proliferation test results provided in the examples and comparative examples of this application

[0208]

[0209] Note: Compared with the control group, a p < 0.05, aa p < 0.01; compared with Example 1, b p < 0.05, bb p < 0.01; compared with Example 2, c p < 0.05, cc p < 0.01.

[0210] As shown in Table 2, compared with the blank control group, the cell survival rates of the compositions prepared in Examples 1 and Comparative Examples 1 to 6, the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10, and the PDRN lipid nanoparticle compositions prepared in Examples 11 to 16 were all improved; compared with Example 1, the cell survival rates of Comparative Examples 1 to 6 were significantly reduced, indicating that PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 have a triple composite synergistic effect in a free state, and the effect is better than a single or two-component combination. The synergistic effect of PDRN depends on a complete three-component system, and its effect is limited in a two-component free combination. Relative to the blank control group, the cell survival rates of Comparative Examples 1 to 6 increased by 5.32%, 3.81%, 2.54%, 10.98%, 9.36%, and 5.62%, respectively, and the cell survival rate of Example 1 increased by 20.36%. Taking Comparative Example 1 as Agent A and Comparative Example 6 as Agent B, the q value of Example 1 was calculated using the Jin Zhengjun method:

[0211] q=E A+B / (E A +E B -E A *E B )

[0212] The q value is 1.9, which further illustrates that the combination of PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 has a synergistic effect.

[0213] Compared with Example 1, the cell survival rate in Example 2 was significantly improved, indicating that after the active ingredient was encapsulated by the nanocarrier, the cell survival effect was significantly enhanced compared with the free active ingredient.

[0214] Compared with Example 2, the cell survival rate of the PDRN lipid nanoparticles prepared in Examples 3 to 4 was significantly reduced, and Example 2 had a better effect in promoting cell proliferation, indicating that the specific phospholipid combination may optimize the biocompatibility or cell affinity of the nanocarrier, thereby more effectively promoting cell survival rate and cell proliferation.

[0215] Compared with Example 2, the composition of PDRN lipid nanoparticles and Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) prepared in Examples 11 to 16 can promote cell proliferation, especially when the mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) is (2 to 15):1, the cell survival rate is significantly improved, indicating that PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) may synergistically promote the repair of the cell microenvironment, and the fermentation product may optimize the local environment of the skin, improve the bioavailability of PDRN and peptides, thereby enhancing the effect of PDRN lipid nanoparticles.

[0216] Test Example 2 ROS fluorescence intensity detection

[0217] Test samples: the compositions prepared in Example 1 and Comparative Examples 1 to 6, the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10, and the PDRN lipid nanoparticle compositions prepared in Examples 11 to 16.

[0218] HDF cells were cultured at 4 × 10 4 The cells were inoculated into 24-well plates at a density of 500 μL per well. After 24 h of incubation, the supernatant was discarded. The model group was added with 1 mL of DMEM medium containing 0.6 mmol / L H2O2, and the other sample groups were added with 1 mL of 0.6 mmol / L H2O2 and the PDRN lipid nanoparticles of Examples 2-4, 8-10 (the concentration of PDRN lipid nanoparticles was 500 μg / mL), the PDRN lipid nanoparticle compositions of Examples 11-16 (the concentration of PDRN lipid nanoparticles was 500 μg / mL, and the concentrations of Huanfuyuan were 25 μg / mL, 33.3 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL, respectively), the composition prepared in Example 1 (the concentration of the active ingredient in the PDRN lipid particles in 500 μg / mL was consistent, the concentrations of PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 were 20 μg / mL, 0.0005 μg / mL, and 0.2 μg / mL, respectively), and the compositions prepared in Comparative Examples 1-6 (the concentration of the active ingredient in Comparative Example 1 was 2.5 μg / mL, 0.0005 μg / mL, and 0.2 μg / mL, respectively). The PDRN concentration was 20 μg / mL; the active substance concentration of comparative example 2, the microcurrent tetrapeptide-1 concentration was 0.0005 μg / mL; the active substance concentration of comparative example 3, the palmitoyl tripeptide-5 concentration was 0.2 μg / mL; the active substance concentration of comparative example 4, the PDRN and microcurrent tetrapeptide-1 concentrations were 20 μg / mL and 0.0005 μg / mL respectively; the active substance concentration of comparative example 5, the PDRN and palmitoyl tripeptide-5 concentrations were 20 μg / mL and 0.2 μg / mL respectively; the active substance concentration of comparative example 6, the microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 concentrations were 0.0005 μg / mL and 0.2 μg / mL respectively) DMEM culture medium, and a blank control group without H2O2 was set up. After continuing to culture for 24 h, 1 mL of serum-free DMEM containing 20 μM DCFH-DA was added and incubated for 20 The cells were washed with PBS for 3 times, and the fluorescence intensity was observed under a fluorescence microscope. After the cells were collected, the fluorescence intensity was detected by flow cytometry. The results are shown in Table 3, which shows the ROS fluorescence intensity detection results of the examples and comparative examples of the present application.

[0219] Table 3 ROS fluorescence intensity detection results of the examples and comparative examples of the present application

[0220]

[0221] Note: Compared with the model group, a p < 0.05, aa p < 0.01; compared with Example 1, bb p < 0.01; compared with Example 2, cc p < 0.01.

[0222] As shown in Table 3, compared with the model group, the compositions prepared in Example 1 and Comparative Examples 1 to 6, the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10, and the PDRN lipid nanoparticle compositions prepared in Examples 11 to 16 can significantly inhibit the level of intracellular ROS;

[0223] Compared with Example 1, the intracellular ROS levels of Comparative Examples 1 to 6 were significantly increased, indicating that PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 have a triple compound synergistic effect in the free state, and the effect is better than that of a single or two-component combination. The synergistic effect of PDRN depends on a complete three-component system, and its effect is limited in a two-component free combination.

[0224] Relative to the model group, the ROS inhibition rates of the samples prepared in Comparative Examples 1 to 6 were 17.05%, 10.59%, 5.72%, 26.04%, 23.36%, and 18.13%, respectively. The ROS inhibition rate of the sample prepared in Example 1 was 44.18%. Comparative Example 1 was used as Agent A and Comparative Example 6 was used as Agent B. The q value of Example 1 was calculated using the Jin Zhengjun method:

[0225] q=E A+B / (E A +E B -E A *E B )

[0226] The q value is 1.4, which further indicates that the combination of PDRN, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5 has a synergistic effect;

[0227] Compared with Example 1, the intracellular ROS level of the PDRN lipid nanoparticles prepared in Examples 2 to 4 was significantly reduced, indicating that after the active ingredients were encapsulated by nanocarriers, the ROS inhibition and cellular antioxidant effects were significantly enhanced compared with the free active ingredients.

[0228] Compared with Example 2, the intracellular ROS level of the PDRN lipid nanoparticles prepared in Examples 3 to 4 was significantly increased, and Example 2 had a better cellular antioxidant effect, indicating that the specific phospholipid combination may optimize the biocompatibility or cell affinity of the nanocarrier, thereby more effectively inhibiting ROS and promoting cellular antioxidant effects.

[0229] Compared with Example 2, the composition of PDRN lipid particles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) prepared in Examples 11 to 16 can further inhibit ROS, especially when the mass ratio of PDRN lipid particles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) is (2 to 15): 1, the ROS inhibition effect is significant, further illustrating that PDRN lipid particles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) can further support its synergistic effect through the dual pathways of "reducing oxidative damage + promoting repair".

[0230] Test Example 3

[0231] 1. Stability test 1

[0232] The PDRN lipid nanoparticles in Examples 2 to 10 were placed at room temperature for 3 months, and then the particle size, PDI, and appearance were observed to see if they changed. The results are shown in Table 4, which shows the stability test results 1 of the lipid nanoparticles prepared in the examples of the present application.

[0233] Table 4 Stability test results of lipid nanoparticles prepared in the examples of this application 1

[0234]

[0235] As can be seen from Table 4, the average particle size of PDRN lipid nanoparticles is 100~300nm, and the PDI is 0.1~0.6. By comparing the effects of different phospholipid combinations on the stability of PDRN lipid nanoparticles in each embodiment, the results show that at room temperature, compared with Examples 2~5 and Examples 8~10, the particle size and PDI of Examples 6 and 7 are larger, and the PDRN lipid nanoparticles show stratification or chromatography, which shows that egg yolk lecithin, natural sphingomyelin, phosphatidylglycerol and phosphatidyltryptophan, natural sphingomyelin, phosphatidylglycerol, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, these two groups of phospholipid combinations have an effect on the stability of the carrier.

[0236] 2. Stability test 2

[0237] After the above experiments, the PDRN lipid nanoparticles prepared in Examples 2 to 5 and 8 to 10 with good room temperature stability were placed in a sealed container and placed at -20°C, 4°C, and 45°C for 3 months respectively to observe whether the appearance of the PDRN lipid nanoparticles showed stratification or precipitation. The results are shown in Table 5, which shows the stability test results 2 of the lipid nanoparticles prepared in the examples of the present application.

[0238] Table 5 Stability test results of lipid nanoparticles prepared in the examples of this application 2

[0239]

[0240]

[0241] As can be seen from Table 5, the PDRN lipid nanoparticles prepared in Example 5 showed stratification under high temperature conditions in the second month. The PDRN lipid nanoparticles prepared in Examples 2 to 4 and Examples 8 to 10 were placed at 4 ° C and 45 ° C for 3 months respectively, and no stratification or precipitation occurred, indicating that the PDRN lipid nanoparticles prepared in Examples 2 to 4 and Examples 8 to 10 had good stability. The phospholipid combination for preparing the PDRN lipid nanoparticles in Example 5 affected the stability of the carrier at high temperature.

[0242] Test Example 4: Evaluation of irritation to chicken embryo chorioallantoic membrane

[0243] Test samples: PDRN lipid nanoparticles prepared in Examples 2 to 4, 8 to 10, PDRN lipid nanoparticle compositions prepared in Examples 11 to 14, and the composition of Example 1.

[0244] Test method: After diluting the test sample 10-fold, draw 0.2 mL and drip it onto the surface of the chorioallantoic membrane. Observe the changes in the CAM blood vessels within 5 minutes and record the initial time of congestion, bleeding and coagulation in the CAM blood vessels. Calculate the stimulation score IS.

[0245] The irritationscore (IS) was calculated as follows:

[0246] IS=[(301-secH)×5+(301-secL)×7+(301-secC)×9] / 300

[0247] In the above formula, secH represents the initial time of congestion (s); secL represents the initial time of bleeding (s); and secC represents the initial time of coagulation (s).

[0248] The mean of repeated tests was calculated, and the irritation of the test substances was graded according to the size of the mean, where 0-0.9, 1.0-4.9, 5.0-8.9 and 9-21.0 were classified as no irritation, slight irritation, moderate irritation and severe irritation, respectively.

[0249] The test results showed that after the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10, the PDRN lipid nanoparticle composition prepared in Examples 11 to 14, and the free composition (Example 1) were diluted 10 times and contacted with the chicken embryo chorioallantoic membrane for 300 s, there was no capillary bleeding, vascular melting, or coagulation phenomenon, and the reaction integrals were 0.07, 0.06, 0.08, 0.07, 0.06, 0.08, 0.09, 0.08, 0.11, 0.09, and 0.08.

[0250] Test Example 5 Patch Test

[0251] Fifty subjects were selected and 10% of the PDRN lipid nanoparticles prepared in Examples 2-4 and Examples 8-10, the PDRN lipid nanoparticle composition prepared in Examples 11-14, and a blank control were applied to the flexed forearm of the subjects for 24 hours. After the patch tester was removed, the skin reaction was observed 30 minutes after the indentation disappeared. The skin reaction was observed again 24 and 48 hours after the patch tester was removed.

[0252] The results showed that none of the 50 subjects developed light red spots, erythema, edematous erythema, significant redness and swelling, infiltration or papules, and papules or blisters, etc., indicating that the PDRN lipid nanoparticles prepared in Examples 2 to 4 and 8 to 10 and the PDRN lipid nanoparticle compositions prepared in Examples 11 to 14 were non-irritating to human skin.

[0253] Test Example 6: Observation of skin penetration behavior using a laser confocal microscope

[0254] Transdermal permeation experiments on ex vivo porcine skin were conducted using a vertical Franz diffusion cell method. The skin was fixed between a receiving chamber and a donor chamber, and RhoB (added at 0.1%) was encapsulated in the aqueous phase as a water-soluble dye. Corresponding RhoB compositions were prepared according to the methods described in Examples 2, 8-16, and 16. RhoB (added at 0.1%) was added to Example 1 to prepare the corresponding RhoB free compositions. 0.5 g of each sample was placed in the donor chamber, and PBS was used as the receiving solution, with stirring and diffusion at 32°C. After 2 and 4 hours, the remaining sample on the skin was gently wiped off. The skin in the target area was removed, rinsed again, thoroughly cleaned, and dried to remove any residual moisture. The samples were cryosectioned and observed using a laser confocal microscope. The results are shown in Table 6, which shows the experimental results of skin permeation and cellular uptake behavior in the examples of this application.

[0255] Table 6 Experimental results of skin penetration behavior and cell uptake behavior of the examples of the present application

[0256]

[0257] Note: Comparison with Example 1, aa p < 0.01; compared with Example 2, b p < 0.05, bb p < 0.01.

[0258] As shown in Table 6, compared with Example 1, Examples 2 and Examples 8 to 10 have significant skin penetration effects, indicating that after being encapsulated by nanocarriers, the skin penetration of the active ingredients is significantly enhanced compared with the free active ingredients.

[0259] Compared to Example 2, at 4 hours, the skin penetration effect of the PDRN lipid nanoparticles in Examples 12-15 combined with the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) was significant, indicating that the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) has microecological regulation to assist penetration, improving the delivery efficiency of the active ingredient through physical penetration enhancement. Compared to Example 2, the skin penetration effect of the PDRN lipid nanoparticles in Example 16 combined with the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) was reduced. This suggests that a mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (2-15):1 is more effective. At relatively low concentrations, this ratio may primarily enhance penetration. However, an excessive ratio (e.g., 1:1) may lead to decreased nanoparticle stability, and organic acids or enzymes in high-concentration fermentation products may interfere with the lipid nanoparticle structure, thereby reducing the delivery efficiency of the PDRN nanoparticles.

[0260] Test Example 7: Flow cytometry detection of cell uptake behavior

[0261] Test samples: RhoB (0.1%) was incorporated into the aqueous phase as a water-soluble dye, and corresponding RhoB compositions were prepared according to the methods described in Examples 2 and 8-16. RhoB (0.1%) was added to Example 1 to prepare corresponding free RhoB compositions.

[0262] HDF cells were plated at 3.0 × 10 5 Cells were seeded at a density of 1000 cells / mL in a 6-well plate and incubated for 24 h to allow the cells to adhere. The old culture medium was discarded, and then a 1000-fold dilution of the RhoB compositions prepared in Examples 2, 8-16, and the free RhoB composition prepared in Example 1 (RhoB concentration of 1 μg / mL) in DMEM complete medium was added to each well. Untreated cells were used as negative controls. After further culture for 2 h and 4 h, the cells were washed with cold PBS, trypsinized, centrifuged, and the cell pellet was collected and resuspended in 0.5 mL of cold PBS solution. The intracellular fluorescence intensity was detected by flow cytometry. The results are shown in Table 7, which shows the results of the flow cytometric detection of cellular uptake behavior in the examples of the present application.

[0263] Table 7 Experimental results of cell uptake behavior detected by flow cytometry in the examples of the present application

[0264]

[0265] Note: Comparison with Example 1, aa p < 0.01; compared with Example 2, b p < 0.05, bb p < 0.01.

[0266] As shown in Table 7, compared with Example 1, Examples 2 and Examples 8 to 10 have significant cellular uptake effects, indicating that after being encapsulated by nanocarriers, the cellular uptake of the active ingredients is significantly enhanced compared with the free active ingredients.

[0267] Compared with Example 2, the PDRN lipid nanoparticles of Examples 12-14 combined with the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) showed significant cellular uptake effects, indicating that the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) has microecological regulation to assist penetration and synergistically enhance cellular uptake, synergistically improving the delivery efficiency of the active ingredient through physical penetration enhancement and biological activity. Compared with Example 2, there was no significant difference in the cellular uptake effects of the PDRN lipid nanoparticles of Examples 11 and 16 combined with the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan). This suggests that a mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (2-15):1 is more effective. This may be because the biosurfactant components in the fermentation filtrate (such as peptides or polysaccharides produced by fermentation) slightly modify the lipid nanoparticle surface, forming a more stable "invisible" hydrophilic layer (similar to the PEGylation effect), reducing macrophage clearance and enhancing specific uptake by target cells (such as fibroblasts). However, when the ratio of Huanfuyuan is too high (1:1), excessive fermentation components may over-wrap the lipid nanoparticles, resulting in surface charge neutralization or reversal (such as negatively charged fermentation products covering positively charged lipids), reducing electrostatic adsorption with the cell membrane; and when the ratio of Huanfuyuan is too low (20:1), the absorption-promoting effect is reduced; secondly, due to steric hindrance effects, the binding of lipid nanoparticles to cell membrane receptors may be hindered (such as CD36 or SR-B1-mediated endocytosis).

[0268] Test Example 8 Detection of Anti-Aging Factors

[0269] Test samples: PDRN lipid nanoparticles prepared in Examples 2 and 8 to 10, PDRN lipid nanoparticle compositions prepared in Examples 11 to 16, and the free composition of Example 1.

[0270] HDF cells were cultured at 4 × 10 4Cells were seeded into 24-well cell culture plates at a density of 500 μL per well and cultured at 5% CO2 and 37°C for 24 h. 1 mL of DMEM complete medium containing the PDRN lipid nanoparticles prepared in Example 2 (the concentration of the PDRN lipid nanoparticles was 500 μg / mL), the PDRN lipid nanoparticles prepared in Examples 8-10 (the concentration of the PDRN lipid nanoparticles was 500 μg / mL), the PDRN lipid nanoparticle compositions prepared in Examples 11-16 (the concentration of the PDRN lipid nanoparticles was 500 μg / mL, and the concentration of Huanfuyuan was the same as in Test Examples 1 and 2), and the free composition of Example 1 (the active substance test concentration was the same as in Test Examples 1 and 2) was added to each well. The blank control group was added with only 100 μL of DMEM complete medium. Three replicates were added to each group and incubated in a CO2 incubator for 24 h. The supernatant was taken and the Col I and Col III contents were tested using an ELISA kit. The results are shown in Table 8, which shows the results of the Col I and Col III contents in the examples of this application.

[0271] Table 8 Results of Col Ⅰ and Col Ⅲ content in the examples of this application

[0272]

[0273] Note: Compared with the model group, a p < 0.05, aa p < 0.01; compared with Example 1, b p < 0.05, bb p < 0.01; compared with Example 2, cc p < 0.01

[0274] As shown in Table 8, compared with the model group, Example 2, Examples 8 to 16, and Example 1 can all promote the secretion of Col Ⅰ and Col Ⅲ by cells; compared with Example 1, Example 2 and Example 8 can significantly increase the content of Col Ⅰ and Col Ⅲ, indicating that the active ingredients have more significant effects on promoting collagen synthesis and anti-aging after being encapsulated, which is significantly better than the free ingredients at the same dose. Compared with Example 2, the composition of PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) prepared in Examples 12 to 15 can significantly increase the content of COLⅠ and COLⅢ; the PDRN lipid nanoparticle compositions prepared in Examples 11 and 16 have no significant difference in the synthesis of COLⅠ and COLⅢ. The reason may be that the mass ratio of PDRN lipid nanoparticles to Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) (2-15):1 is more conducive to maintaining the integrity of lipid nanoparticles and ensuring the continuous release of PDRN to target cells; the surfactant components in the fermentation filtrate (such as the polysaccharides or peptides produced) may destroy the lipid bilayer structure at a high ratio (such as 1:1), resulting in premature release or degradation of PDRN; and the ratio of the fermentation filtrate is too low (such as 20:1), resulting in a decrease in the absorption-promoting effect and the inability of PDRN to be continuously released to the target cells. It shows that PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) may promote collagen synthesis through synergistic effects at a specific ratio, achieving significant anti-aging effects.

[0275] Test Example 9: Effects on Inflammatory Factors

[0276] Test samples: PDRN lipid nanoparticles prepared in Examples 2 and 8 to 10, PDRN lipid nanoparticle compositions prepared in Examples 11 to 16, and the free composition of Example 1.

[0277] HDF cells were cultured at a rate of 1.0 × 10 5 Cells were seeded at a density of 1 mL / well into a 24-well cell culture plate. After overnight culture, TNF-α / INF-γ (final concentration of 10 ng / mL) and 1 mL of sample were co-incubated. DMEM complete medium containing the test samples: PDRN lipid nanoparticles prepared in Example 2 and Examples 8-10 (PDRN lipid nanoparticle concentration of 500 μg / mL), PDRN lipid nanoparticle compositions prepared in Examples 11-16 (PDRN lipid nanoparticle concentration of 500 μg / mL, Huanfuyuan test concentrations were the same as in Test Examples 1 and 2), and the free composition of Example 1 (test concentration was the same as in Test Example 8) was added, with five replicates per group. After 24 hours, the cell supernatant was collected and the levels of IL-6 and TNF-α secretion by HDF cells were tested using an ELISA kit. The results are shown in Table 9, which shows the IL-6 and TNF-α content results of the examples of this application.

[0278] Table 9 IL-6 and TNF-α content results of the examples of this application

[0279]

[0280] Note: Compared with the model group, a p < 0.05, aa p < 0.01; compared with Example 1, b p < 0.05, bb p < 0.01; compared with Example 2, cc p < 0.01

[0281] As shown in Table 9, compared with the model group, Examples 2, Examples 8 to 10, Examples 11 to 16, and Example 1 all have the effect of inhibiting the secretion of IL-6 and TNF-α by cells; compared with Example 1, Examples 2 and Example 8 can significantly inhibit the content of IL-6 and TNF-α, indicating that the anti-inflammatory effect of the active ingredient after encapsulation is more significant, which is significantly better than the free ingredient at the same dose. Compared with Example 2, the PDRN lipid particles prepared in Examples 12 to 15 and the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) can significantly inhibit the secretion of IL-6 and TNF-α; the PDRN lipid particles prepared in Examples 11 and 16 and the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) have no significant effect on the inhibition of IL-6 and TNF-α secretion. The reason may be that the bifidobacterium / Lactobacillus / soybean seed fermentation product may contain probiotic metabolites (such as short-chain fatty acids and polypeptides), which have dual immunomodulatory effects; at an appropriate low concentration ratio, it may slightly activate TLR2 / 4 or PPAR-γ, promote the secretion of anti-inflammatory cytokines (such as IL-10), indirectly inhibit IL-6 / TNF-α, and synergize with PDRN to enhance the anti-inflammatory signal of the A2A receptor. However, high concentrations may overstimulate TLRs or NLRP3 inflammasomes, and organic acids in fermentation products (such as lactic acid) may lower pH, affecting PDRN stability or cellular uptake. Excessively low concentrations lead to decreased absorption and inability to sustainably release active ingredients into target cells, all of which reduce anti-inflammatory effects. This suggests that PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) at specific ratios may synergistically inhibit the secretion of inflammatory factors, achieving significant anti-inflammatory effects.

[0282] Test Example 10 Adenosine A2A Receptor Expression (qRT-PCR)

[0283] Test samples: PDRN lipid nanoparticles prepared in Examples 2 and 8 to 10, PDRN lipid nanoparticle compositions prepared in Examples 11 to 16, and the free composition of Example 1.

[0284] HDF cells were cultured at 3 × 10 5 Cells were seeded at a density of 500 μL per well into a 6-well cell culture plate and incubated at 5% CO₂ and 37°C for 24 h. 2 mL of DMEM complete medium containing the PDRN lipid nanoparticles prepared in Examples 2 and 8-10 (PDRN lipid nanoparticle concentration of 500 μg / mL), the PDRN lipid nanoparticle composition prepared in Examples 11-16 (PDRN lipid nanoparticle concentration of 500 μg / mL, with the same concentration of Huanfuyuan as in Test Examples 1 and 2), and the free composition of Example 1 (active ingredient concentration as in Test Examples 1 and 2) was added to each well. A blank control group was treated with only 100 μL of DMEM complete medium. The cells were incubated in a CO₂ incubator for 24 h. The cells were harvested, and total RNA was extracted using an RNA extraction kit. RNA concentration and purity were analyzed using a microplate reader. Complementary DNA synthesis was performed using an M-MLV reverse transcriptase kit. Fluorescence quantitative PCR analysis was performed using a real-time fluorescence quantitative PCR instrument according to the protocol of the One-Step Fluorescence Quantitative RT-PCR MasterMix Kit. Primer sequences and product sizes are shown in Table 10. Using ACTIN as an internal reference, the relative expression of A2A mRNA was quantified using the 2-ΔΔCt method based on the Δ cycle threshold (Ct). The control result was set as 1, and the ratio of the control result to the Ct value in the remaining groups was used as the relative expression level of the corresponding protein mRNA. Three replicates were performed for each sample, and the results were averaged. The results are shown in Table 11, which also shows the expression of adenosine A2A receptor (qRT-PCR) results.

[0285] Table 10 Primer sequences and product sizes

[0286]

[0287] Table 11 Adenosine A2A receptor expression (qRT-PCR) results.

[0288]

[0289] Note: Compared with the blank control group, a p < 0.05, aa p < 0.01; compared with Example 1, bb p < 0.01; compared with Example 2, cc p < 0.01.

[0290] As shown in Table 11, compared with the blank control group, Examples 2, Examples 8 to 10, Examples 11 to 16, and Example 1 all have the effect of expressing adenosine A2A receptors;

[0291] Compared with Example 1, the expression effect of adenosine A2A receptor in Examples 2 and 8 is significant, indicating that the expression effect of adenosine A2A receptor in the encapsulated PDRN lipid nanoparticles is more significant, significantly better than the free component at the same dose. Compared with Example 2, the expression effect of adenosine A2A receptor in the PDRN lipid nanoparticle compositions prepared in Examples 12 to 15 is significantly increased, which may be due to the synergistic enhancement of receptor expression at low concentrations by the fermentation filtrate, rather than inhibition; maintaining lipid nanoparticle stability to ensure effective delivery of PDRN. Compared with Example 2, there is no significant difference in the expression effect of adenosine A2A receptor in the PDRN lipid nanoparticle composition prepared in Example 16. It may be that a high proportion of fermentation filtrate leads to reduced adenosine production or downregulation of receptors due to metabolic interference, pH changes or carrier damage; too low a proportion of fermentation filtrate leads to a decreased absorption-promoting effect, and the active ingredient cannot be continuously released to the target cells, resulting in reduced adenosine production or downregulation of receptors. It shows that the combined use of PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) is better than that of single PDRN lipid nanoparticles, and PDRN lipid nanoparticles and Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan) maintain the PDRN-dominated adenosine-A2A receptor signaling pathway at a specific ratio.

[0292] Test Example 11 Nuclease Degradation Experiment

[0293] Test samples: PDRN lipid nanoparticles prepared in Example 2, PDRN lipid nanoparticle composition prepared in Example 12, and free composition in Example 1.

[0294] 20 μL of each of the PDRN lipid nanoparticles prepared in Example 2, the composition prepared in Example 12, and the free composition of Example 1 were placed in enzyme-free EP tubes. An equal volume of DNase I solution (enzyme concentration: 1 U) was added to each reaction system. The reaction system was placed in a thermostatic water bath, set to 37°C according to the kit instructions, and the reaction time was maintained to ensure experimental consistency. After completion, the reaction was terminated by adding an appropriate amount of EDTA to chelate free DNase I. The system was then heated at 65°C for 10 minutes to inactivate the DNase I enzyme. Then, 2.5 μL of 1× loading buffer and 7.5 μL of each sample were added to the spotting plate. After mixing, the samples were added to the sample wells of the gel plate using a 10 μL micropipette. After sample addition, the gel plate was immediately powered on for electrophoresis at 190V. Electrophoresis was stopped when the bromophenol blue moved to approximately 1 cm from the bottom edge of the gel plate. After electrophoresis, the gel was taken out and observed under UV light. If PDRN was present, a fluorescent band would appear. The gel was then photographed using a gel imaging system. Figure 1 , Figure 1 The electrophoresis results provided by the embodiment of the present invention.

[0295] like Figure 1 As shown, the PDRN fragment sizes in the PDRN lipid nanoparticles prepared in Example 2, the composition prepared in Example 12, and the free composition of Example 1 are in the range of 50 to 2000 bp, which is consistent with literature reports. After nuclease degradation, the bands of the free composition of Example 1 are concentrated below 100 bp, indicating that it has been degraded into small molecular fragments. The PDRN lipid nanoparticles prepared in Example 2 are degraded to a certain extent, but some macromolecular fragments are retained, which is better than the free composition of Example 1, indicating that the carrier can inhibit the complete degradation of the PDRN raw material by nucleases. The composition prepared in Example 12 is degraded to some extent, but more macromolecular fragments are retained, which is better than the PDRN lipid nanoparticles prepared in Example 2, indicating that the PDRN lipid nanoparticles prepared in Example 2, combined with the Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate (Huanfuyuan), have a synergistic effect in protecting PDRN.

[0296] Test Example 12 Human Efficacy Evaluation

[0297] Testing Method: 180 healthy women (aged 35-55) were divided into 18 groups, each with 10 participants. The products from Examples 2, 12, and 1 were used, respectively. Participants applied the samples twice daily, morning and evening, for 56 days. Participants were not permitted to use other products during the study. Results were assessed after 56 days of sample application. Before the start of the study, participants' skin wrinkles and skin elasticity were measured. After using a serum containing 5% of each sample (5% sample + 95% blank serum prepared in Example 17) for 7, 14, 28, and 56 days, wrinkles and skin elasticity were measured again. Facial wrinkles were measured using the Shanghai Fuhuan Vplus® Intelligent Skin Analysis System, and skin elasticity was measured using the Cutometer® dual MPA580 skin elasticity tester. The average values ​​of the 30 participants in each group were calculated.

[0298] Wrinkle change rate (%) = (average wrinkle count of each group after using the example / comparative example product - average wrinkle count of each group before use) / average wrinkle count of each group before use * 100%. The wrinkle improvement rate is the positive value of the change rate calculated using the formula. The results are shown in Table 12.

[0299] Table 12 Wrinkle improvement rate after using the product of Example for different time periods

[0300]

[0301] As shown in Table 12, the products of Examples 2 and 12 both exhibited significant wrinkle-removing effects after use, with longer usage resulting in improved wrinkle-removing effects. The wrinkle-removing and anti-aging properties of Examples 2 and 12 were significantly superior to those of Example 1, demonstrating that the synergistic effect of the three active ingredients, PDRN encapsulated in the nanocarriers of the present invention, microcurrent tetrapeptide-1, and palmitoyl tripeptide-5, maximizes the wrinkle-removing and anti-aging effects of the active ingredients. Compared to the free ingredients (Example 1), the wrinkle-removing and anti-aging properties of the encapsulated PDRN lipid nanoparticles were significantly superior to those of the free ingredients at the same dose. Comparing Example 2 with Example 12, the wrinkle-removing properties of the PDRN lipid nanoparticle combination (PDRN lipid nanoparticles combined with the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan)) were significantly superior to those of the PDRN lipid nanoparticles alone, demonstrating that the combination of PDRN lipid nanoparticles and the filtrate of the fermentation product of Bifidobacterium / Lactobacillus / soybean seed extract (Huanfuyuan) exhibits a synergistic effect, resulting in enhanced anti-aging efficacy.

[0302] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition containing PDRN, characterized in that, Includes polydeoxyribonucleotides, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5; The mass ratio of the polydeoxyribonucleotide, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 is 0.1-5:0.000005-0.0005:0.0001-1.

2. The composition according to claim 1, characterized in that The mass ratio of polydeoxyribonucleotide, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 is 0.5~4.5:0.00001~0.0003:0.0005~0.

5.

3. The composition according to claim 2, characterized in that The mass ratio of polydeoxyribonucleotide, microcurrent tetrapeptide-1 and palmitoyl tripeptide-5 is 1~4:0.00005~0.0001:0.001~0.

1.

4. A lipid nanoparticle containing PDRN, characterized in that, The method comprises the composition according to any one of claims 1 to 3 and a lipid nanoparticle raw material, wherein the lipid nanoparticle raw material comprises at least two of phospholipids, non-phospholipid functional lipids, an emulsifier, an alcohol compound and water.

5. The lipid nanoparticle according to claim 4, wherein include: 0.1wt%~5wt% polydeoxyribonucleotides; 0.000005wt%~0.0005wt% of microcurrent tetrapeptide-1; 0.0001wt%~1wt% palmitoyl tripeptide-5; 0.1wt%~3wt% phospholipids; 0.1wt%~3wt% non-phospholipid functional lipids; 10wt%~40wt% emulsifier; 10wt%~50wt% of alcohol compound; The remaining amount of water.

6. The lipid nanoparticle according to claim 5, characterized in that The phospholipid is one or more of phosphatidylcholine, distearoylphosphatidylcholine, natural sphingomyelin, phosphatidylglycerol, phosphatidylinositol and phosphatidyltryptophan; The non-phospholipid functional lipid is one or more of stearamide, oleyl fatty amine derivatives, stearic acid polyethylene glycol N-hydroxysuccinimide ester and N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt; The emulsifier is one or more of polyoxyethylene castor oil emulsifier, polyoxyethylene hydrogenated castor oil emulsifier, polyglycerol emulsifier, poloxamer, cocoyl glucoside, polyglyceryl-10 laurate, glyceryl stearate citrate, sodium di(lauroyl glutamine) lysine, triglyceride emulsifier, laureth-23, PPG-26-butaneth-26, trideceth-12, polyglyceryl ester, polysorbate-80, polysorbate-60, polysorbate-20, PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil and PEG-60 hydrogenated castor oil; The alcohol compound is one or more of glycerol, 1,2-propylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,3-propylene glycol, 1,2-pentanediol, ethoxydiglycol, 1,2-hexanediol, dipropylene glycol, isopropyl alcohol, methylpropylene glycol, polyethylene glycol-200, PPG-10 sorbitol and octyldodecanol.

7. The method for preparing lipid nanoparticles according to any one of claims 4 to 6, characterized in that: The following steps are involved: (1) mixing polydeoxyribonucleotide, an emulsifier, water and a first mass portion of an alcohol compound to obtain a mixed solution 1; Mixing the phospholipid, the non-phospholipid functional lipid and the second mass part of the alcohol compound to obtain a mixed solution 2; Mixing microcurrent tetrapeptide-1, palmitoyl tripeptide-5, an emulsifier and a third part by mass of an alcohol compound to obtain a mixed solution 3; (2) Mixed solution 3 is mixed with mixed solution 1, and then mixed with mixed solution 2 to obtain mixed solution 4; (3) The mixed solution 4 is subjected to nano-processing to obtain lipid nanoparticles.

8. A composition containing lipid nanoparticles, characterized in that comprising the lipid nanoparticles according to any one of claims 4 to 6 and a filtrate of a fermentation product of a bifidobacterium / lactobacillus / soybean seed extract; The mass ratio of the lipid nanoparticles to the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate is 1-20:

1.

9. A personal care product, characterized in that include: The composition according to any one of claims 1 to 3, the lipid nanoparticles according to any one of claims 4 to 6, and / or the lipid nanoparticle-containing composition according to claim 8.

10. Use of the composition according to any one of claims 1 to 3, the lipid nanoparticles according to any one of claims 4 to 6, and / or the composition containing lipid nanoparticles according to claim 8 in the preparation of anti-aging, anti-inflammatory, soothing and repair products.

Images