A polar gradient three-dimensional oil phase nano oil-soluble PDRN against skin cell aging and a preparation method and application thereof

Abstract

The application relates to a polar gradient three-dimensional oil phase nano oil-soluble PDRN against skin cell aging and a preparation method and application thereof. The oil-soluble PDRN comprises a polar gradient three-dimensional oil phase, a surfactant and an aqueous phase; wherein the polar gradient three-dimensional oil phase comprises nonpolar section oil, weakly polar section oil and moderately polar section oil. The application constructs a continuous spontaneous polar gradient oil phase of'moderate polarity-weak polarity-nonpolarity', utilizes the three-dimensional synergistic polar gradient effect, realizes high dispersibility, good stability and high efficient transdermal absorption of the PDRN in transparent essential oil, and solves the problems of low solubility of the PDRN in oil and the disadvantage of skin penetration. Meanwhile, specific double surfactants are introduced to reduce interface leakage and avoid particle aggregation. In addition, a composite aqueous phase system containing PDRN is constructed to confine the PDRN in a micro-scale closed environment, form a protective layer with a physical isolation effect, and realize multi-dimensional long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to a polar gradient three-dimensional oil-phase nano-oil-soluble PDRN for anti-skin cell aging, its preparation method and application. Background Technology

[0002] PDRN (Polydeoxyribonucleotide) is a mixture of DNA fragments extracted from the testes of fish such as salmon and trout. Its molecular weight typically ranges from 50 to 1500 bp. It possesses multiple biological activities, including promoting tissue repair, anti-inflammation, and collagen synthesis, and is widely used in skin repair, cosmetic fillers, and tissue regeneration. In the cosmetic ingredient catalog, the standard Chinese name for PDRN is sodium DNA. As a water-soluble component, PDRN currently faces two main challenges in the cosmetic field: First, PDRN is highly hydrophilic and has low solubility in oils, making it difficult to directly add to oil-based formulations. While it can be used in emulsion systems such as lotions and creams, it is not compatible with transparent essential oils, limiting its application in oil-based cosmetics. Second, the large molecular weight of PDRN hinders its penetration into the skin to exert its activity. Therefore, stably dispersing the large water-soluble PDRN molecules in anhydrous or low-water oil-phase systems presents a significant challenge.

[0003] To address the aforementioned issues, existing technologies have proposed some application improvement solutions. For example, microencapsulation technology can be used to encapsulate PDRN to improve its oil-phase dispersibility and transdermal efficiency. This method typically uses biocompatible materials such as phospholipids and cholesterol to prepare microcapsule carriers, encapsulating water-soluble PDRN within them and then dispersing it in an oil-phase matrix. However, this process is relatively complex, increasing production costs; furthermore, microcapsules may experience stability issues during storage or application, such as PDRN leakage or reduced activity due to encapsulation material degradation; such microcapsules cannot be used in transparent oil-based cosmetics, as they are prone to causing opacity in the liquid. Another approach uses a composite emulsification system to disperse PDRN aqueous solution into the oil phase, but existing emulsification systems mostly use PDRN oil dispersion systems prepared with a single polar oil phase, resulting in poor storage stability. Aggregation and precipitation are still prone to occur during long-term storage, failing to meet the requirements of transparent essential oil products for system transparency and storage stability. While existing solutions have alleviated some of the problems of PDRN's oil solubility and transdermal absorption to a certain extent, none of them have been able to fundamentally achieve its high dispersibility, good stability and efficient transdermal absorption in the oil phase at the same time. Especially in products such as transparent essence oils, which have extremely high requirements for system transparency and stability, existing technologies still have obvious limitations.

[0004] Therefore, there is an urgent need for an oil-soluble PDRN that achieves high dispersion stability, high biocompatibility, and transdermal absorption in the oil phase. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a polar gradient three-dimensional oil-phase nano-oil-soluble PDRN for anti-skin cell aging, its preparation method, and its application.

[0006] Existing single-polarity oil-phase systems cannot simultaneously meet the requirements of dispersion and stability of polar PDRN and compatibility with cosmetic matrices. This invention solves the technical problem that existing single-polarity oil-phase systems cannot adapt to the polarity characteristics of PDRN by constructing a three-dimensional oil-phase system with polarity gradient and a specific combination of dual surfactants. This ensures the transparency of the system while achieving long-term storage stability, further guaranteeing the performance and product quality of oil-soluble PDRN.

[0007] Existing technologies for improving PDRN applications, such as microencapsulation, suffer from problems such as complex processes, increased costs, and potential stability issues. Therefore, this invention provides a relatively simple, cost-effective method for preparing oil-soluble PDRN that ensures its bioactivity, overcoming the shortcomings of existing technologies. This method ensures that the prepared oil-soluble PDRN is not only stably dispersed in the oil phase and maintains a clear and transparent system, but also exhibits high biocompatibility with other commonly used oils in cosmetics, thus better enabling its application in transparent oil-based cosmetics.

[0008] The objective of this invention can be achieved through the following methods: In a first aspect, the present invention provides a polar gradient three-dimensional oil phase nano-oil-soluble PDRN for anti-skin cell aging, comprising: a polar gradient three-dimensional oil phase, a surfactant, and an aqueous phase; wherein the polar gradient three-dimensional oil phase comprises non-polar segment oil, weakly polar segment oil, and moderately polar segment oil; and wherein the aqueous phase contains PDRN.

[0009] As one embodiment of the present invention, the oil-soluble PDRN comprises, by weight, 55-70 parts of a three-stage polar gradient oil phase, 20-35 parts of a surfactant, and 6-15 parts of an aqueous phase.

[0010] In one embodiment of the present invention, the mass ratio of the non-polar grease, the weakly polar grease, and the medium polar grease is 1:5~9:1~3. Preferably, it is 1:6~8:1.5~2.5.

[0011] In one embodiment of the present invention, the non-polar segment oil comprises squalane; the weakly polar segment oil comprises camellia seed oil and ethyl oleate in a mass ratio of 1:1 to 2; and the moderately polar segment oil comprises diglyceride and lauroyl sarcosine isopropyl ester in a mass ratio of 1:0.5 to 1.5. The non-polar segment oil (squalane) provides viscosity buffering and film-forming closure, the weakly polar segment oil (camellia seed oil and ethyl oleate) constitutes the reverse micelle nucleation region and hydrogen bond network, and the moderately polar segment oil (diglyceride and lauroyl sarcosine isopropyl ester) provides interfacial anchoring and transdermal penetration promotion. The surface tension of the polar gradient three-dimensional oil phase is 30 to 32 mN / m.

[0012] In one embodiment of the present invention, the surfactant comprises polyglycerol fatty acid ester and polyether-modified long-chain alcohol in a mass ratio of 1:1 to 3, preferably 1:1.5 to 2.5.

[0013] Furthermore, the polyether-modified long-chain alcohol accounts for no less than 15% of the total mass of oil-soluble PDRN.

[0014] Furthermore, the polyglycerol fatty acid ester is selected from polyglycerol-10 dioleate and polyglycerol-10 trioleate, and the polyether-modified long-chain alcohol includes PPG-13-decyltetradecyl alcohol polyether-24.

[0015] As one embodiment of the present invention, the aqueous phase includes PDRN, polyol, polylysine, and water; wherein the mass ratio of PDRN to water is 1:10~50, the mass ratio of polyol to water is 1:4~8, and the mass ratio of PDRN to polylysine is 13~20:1.

[0016] Furthermore, the water accounts for 4.5-10% of the total mass of oil-soluble PDRN; the content of PDRN in oil-soluble PDRN is 0.1-0.5% (w / w).

[0017] Furthermore, the average molecular weight of the PDRN is ≤800bP, and the sodium DNA content is ≥90.0%; the polyol includes glycerol.

[0018] The oil-soluble PDRN of this invention remains clear and transparent after centrifugation at 25°C and 4000 rpm for 30 minutes.

[0019] Secondly, the present invention provides a method for preparing three-dimensional oil-phase nano-oil-soluble PDRN with polar gradient to resist skin cell aging, comprising the following steps: S1. Heat the three-dimensional oil phase with polar gradient, add surfactant, stir, homogenize and cool to obtain phase A; S2. First, stir and dissolve polylysine in water until clear, then add PDRN and stir until clear, then add polyol and stir to form phase B. S3. Add phase B to phase A, cool and stir to obtain the final product.

[0020] In one embodiment of the present invention, in step S1, the heating temperature is 50~60℃; the stirring time is 20-40min; the homogenization speed is 6000~8000rpm and the time is 3~5min; and the temperature is cooled to 40~45℃.

[0021] In one embodiment of the present invention, in step S2, the stirring time after adding PDRN is 2-3 hours; the temperature of the entire step is room temperature.

[0022] In one embodiment of the present invention, in step S3, the addition rate of phase B is 2-5 mL / min; and the temperature is lowered to 15-25°C.

[0023] Thirdly, this invention provides the application of the aforementioned polar gradient three-dimensional oil-phase nano-oil-soluble PDRN, which resists skin cell aging, in the preparation of cosmetics with anti-wrinkle, repair, and whitening effects.

[0024] As one embodiment of the present invention, the cosmetic includes one of the following: lotion, emulsion, cream, and essential oil.

[0025] The principle of this invention is as follows: First, this invention innovatively selects a three-dimensional oil phase with a polar gradient in its oil phase architecture, breaking through the traditional single-phase or binary mixed-phase oil phase structure. It constructs a continuous spontaneous polar gradient of "medium polarity - weak polarity - non-polarity," which is beneficial for forming a thermodynamically stable solvated shell structure. It has the following advantages: 1. (1) The medium polar segment uses diglyceride and lauroyl sarcosine isopropyl ester as the inner layer of the solvation shell; among them, diglyceride has a hydroxyl polar group, which can be used as a hydrogen bond donor; lauroyl sarcosine isopropyl ester contains amide and ester bipolar groups, which can be used as hydrogen bond acceptors; the synergistic use of these two oils can form hydrogen bonds or dipole interactions with the phosphate group and hydroxyl group on the sugar ring of the PDRN molecule, binding the PDRN in the polar micro-region of the oil phase, reducing the PDRN molecule association tendency, reducing the risk of precipitation and aggregation, and improving the storage stability of the composition; at the same time, these two components have a certain surface activity tendency, among which lauroyl sarcosine isopropyl ester is particularly prominent; in skin care applications, medium polar oils are easy to be oriented at the stratum corneum interface, and rely on the dynamic balance of intermolecular interactions to promote the migration of PDRN to the skin interface, increase the probability of contact between the active ingredient and the skin, achieve interface anchoring and assist transdermal absorption. (2) The weakly polar segment uses camellia seed oil and ethyl oleate to form the middle layer of the solubilization shell, which serves as the active transition layer. PDRN is a large molecule composed of chain deoxyribonucleotides. Its backbone (sugar-phosphate backbone) is polar, but it is difficult to dissolve directly in pure non-polar alkanes. Camellia seed oil (mainly large molecule triglycerides) and ethyl oleate (small molecule monoesters) are both weakly polar esters. The ester bonds and unsaturated carbon chains in the molecules can generate dipole-dipole interactions and hydrophobic interactions with the polar sites of PDRN, promoting the initial dispersion and swelling of PDRN in the oil phase. At the same time, the system creates a weakly polar microenvironment similar to human sebum, which is conducive to the spread and affinity of the composition on the skin surface and helps penetration. (3) The non-polar segment uses plant-derived squalane to form the solubilization shell outer layer, providing viscosity buffering and film-forming functions. Squalane is a saturated alkane with no polar groups, which can serve as an inert oil phase framework for oil-soluble PDRN compositions. On the one hand, it smooths the polarity drop in the three-dimensional polar gradient oil phase; on the other hand, squalane has a high viscosity, which can delay reverse micelle Brownian motion and inhibit PDRN aggregation. In cosmetic applications, squalane has excellent compatibility with the skin's sebum membrane and can form a breathable sealing film. Moreover, due to the chemical inertness of squalane, it will not interact with PDRN or interfere with the function of polar oils, providing a smooth, non-sticky skin feel base.

[0026] 2. The three-dimensional synergistic polarity gradient effect allows for a continuous transition from an outer non-polar layer, a middle weakly polar layer, to an inner medium polar layer, enabling a smooth polarity transition of the reverse micelles from the polar core to the outer oil phase. The uniform distribution of motion resistance across the PDRN molecular chain segments avoids localized stress concentration, effectively stabilizing the reverse micelle structure and reducing the particle size distribution (PDI) from 0.6 to 0.4. Traditional two-stage oil phases (such as squalane + vegetable oil) exhibit abrupt polarity changes at the interface, which can easily lead to interface instability and particle aggregation in reverse micelles.

[0027] 3. The specific selection of lauroyl sarcosine isopropyl ester also has additional advantages: First, the molecule contains a sarcosine structure, which is similar to the skin's natural moisturizing factor (NMF), which can further enhance the skin care efficacy of the composition; second, the molecule has a lauroyl hydrophobic chain, which has natural antibacterial potential and can reduce the pressure on the preservative system of oil-soluble PDRN compositions.

[0028] Secondly, this invention employs a compound of non-polar, weakly polar, and moderately polar oils to construct a three-dimensional polar gradient oil phase. Compared to single oils, binary oils, or oil phases randomly blended without gradient, this system exhibits a significant synergistic effect. 1. This oil phase has a gradient system with continuous polarity distribution characteristics, covering a wide range of solubility parameters from non-polar to moderately polar, which can effectively broaden the compatibility window between oil-soluble PDRN formulations and other external oil phases (such as vegetable oils, mineral oils, and synthetic esters).

[0029] 2. Weakly polar oils act as a polar transition layer, which can effectively alleviate the sudden change in interfacial tension during the mixing process with the external oil phase, reduce the system disturbance caused by polarity mismatch, improve the stability of the compatibility phase, and reduce the risks of phase separation, flocculation or system turbidity caused by polarity mismatch.

[0030] 3. When this formulation is applied to the oil phase of an oil-in-water (O / W) or water-in-oil (W / O) emulsion system, its built-in polar gradient structure enables rapid interfacial fusion and molecular-level dispersion with other compound oils of different polarities in the formulation. This eliminates the graininess or roughness caused by local polarity differences, improving the fineness and microscopic uniformity of the final emulsion product. Therefore, the oil-soluble PDRN formulation provided by this invention possesses excellent biocompatibility, good formulation versatility, and storage stability.

[0031] Furthermore, traditional single nonionic surfactants have a narrow polarity adaptation range, which cannot meet the stable emulsification requirements of high polarity PDRN aqueous solutions in the oil phase, and easily leads to problems such as excessively high system viscosity, particle agglomeration, or stratification during storage. This invention specifically introduces a dual surfactant system of polyglycerol-10 dioleate or polyglycerol-10 trioleate and PPG-13-decyltetradecyl alcohol polyether-24, which forms a strong synergistic effect in the three-dimensional polar gradient oil phase, and constructs a stable reverse micelle system with a "rigid shell-flexible tail" topology, thereby increasing PDRN loading and reducing interfacial leakage. The specific mechanism is as follows: (1) Formation of "rigid shell": PPG-13-decyltetradecyl alcohol polyether-24 has a large volume hydrophobic side chain and a long segment of polyether hydrophilic structure. The hydrophilic head group has strong steric hindrance and hydrogen bonding force, which can be closely arranged at the reverse micelle core interface, wrapping the high polarity PDRN aqueous solution and forming a dense rigid shell layer. This structure can effectively resist formulation shear disturbances, lock the core polar aqueous phase, effectively suppress PDRN molecular interface escape and leakage, and solve the defects of micelles in single surfactant systems that are easy to break and active substances are easy to precipitate. (2) Anchoring of "flexible tail": Polyglycerol-10 dioleate / trioleate has both a hydrophilic polyglycerol skeleton and a hydrophobic tail of dioleic acid, with a wide polarity range, which can adapt to the full-domain polar environment of the three-dimensional gradient oil phase of polar-weakly polar-nonpolar in this invention. The hydrophobic segments can penetrate through the multi-layer oil phase region to form a flexible anchoring structure. This can reduce the interfacial tension of reverse micelles, increase the solubilization space of the core, effectively improve the PDRN loading capacity, and at the same time avoid particle aggregation. The two surfactants complement each other: polyether surfactant is responsible for locking the core and stabilizing the interface, and polyglycerol ester surfactant is responsible for gradient adaptation, solubilization and expansion, solving the technical pain point that single-component surfactants cannot be compatible with high polarity aqueous solutions and wide polarity oils.

[0032] Finally, the oil-soluble PDRN described in this invention does not consist of a simple PDRN aqueous solution dispersion system, but rather an innovative quaternary coupling structure: "active ingredient-charge stabilizer-solvent-glass stabilizer," specifically a "PDRN-polylysine-water-polyol (glycerol)" composite system. PDRN is the core active ingredient, requiring an average molecular weight ≤800bp and a mass concentration of 0.1-1%. Excessive PDRN molecular weight or concentration can lead to increased molecular chain migration, causing cross-interface leakage of the active ingredient. Water, as the polar solvent of the reverse micelle core, has a strictly limited content, ≤10% in total. Excessive water will disrupt the polar balance of the micelle microenvironment, leading to system turbidity or phase separation and other instability issues. The polyol (glycerol) serves as the core stabilizing medium (glass forming agent and lyophilization protectant). Polylysine, as a key charge stabilizer and antibacterial functional component, is another core innovative element of this invention. This invention confines PDRN in a microscale closed environment through a quaternary coupling architecture, forming an amorphous glass protective layer with physical isolation effect, achieving multi-dimensional long-term stability. This invention innovatively introduces polylysine and glycerol as reverse micelle stabilizing media for PDRN, which has the following advantages: (1) Electrostatic charge pairing and locking, inhibiting PDRN interface leakage: The phosphate backbone of PDRN molecules carries a negative charge, and polylysine is a cationic polypeptide with a positive charge. The two can form a stable ion pairing complex through electrostatic attraction, which partially "locks" the PDRN molecule within the reverse micelle core. The purpose is to enhance the retention ability of PDRN in the water-in-oil system and help solve the technical problem that large PDRN molecules are prone to migration and leakage into the oil phase due to excessive molecular weight or high concentration. (2) Molecular-level hydrogen bond network constraint, inhibiting molecular aggregation and degradation: Glycerol molecules are rich in hydroxyl groups (-OH), which can simultaneously form a dense hydrogen bond network with oxygen atoms on the PDRN phosphate backbone and core water molecules, reducing the mobility of PDRN molecular chain segments, locking the molecules in a specific conformational state, inhibiting PDRN molecular entanglement and aggregation and structural degradation at the molecular level, and improving the structural stability of the active product. (3) Extremely low water activity (aw) environment, reducing hydrolysis risk and preservation pressure: Glycerin can reduce the water activity in the reverse micelle core, controlling the system aw to a level of <0.65. This low aw environment effectively inhibits the hydrolysis, oxidation and microbial degradation of PDRN during storage; and reduces the preservation pressure on the aqueous phase of the oil-soluble PDRN composition. On this basis, combined with the broad-spectrum antibacterial activity of polylysine itself, the preservation requirements for long-term storage can be met without the addition of additional preservatives. (4) Amorphous glassy physical barrier, achieving long-term storage stability: Glycerin, PDRN and water molecules synergistically form a dense amorphous sugar glass shell, constructing a physical isolation barrier in the reverse micelle core, further improving the precipitation of active substances, and achieving long-term storage stability of the oil-soluble PDRN composition.

[0033] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention constructs a continuous spontaneous polarity gradient oil phase of "medium polarity-weak polarity-nonpolarity," utilizing its three-dimensional synergistic polarity gradient effect to achieve high dispersibility, good stability, and efficient transdermal absorption of PDRN in transparent essential oils. This solves the problems of low solubility of PDRN in oils and its poor skin penetration. Simultaneously, it enhances the natural antibacterial potential of oil-soluble PDRN compositions, reducing the pressure on their preservative systems.

[0034] 2. This invention introduces specific dual surfactants to form a strong synergistic effect in a three-dimensional polar gradient oil phase, constructing a stable reverse micelle system with a "rigid shell-flexible tail" topology, which increases PDRN loading, reduces interfacial leakage, and avoids particle aggregation, thus solving the technical pain point that single-component surfactants cannot be compatible with highly polar aqueous solutions and widely polar oils.

[0035] 3. This invention constructs a quaternary coupling architecture of "active substance-charge stabilizer-solvent-glass stabilizer" in the internal aqueous phase, confining PDRN in a microscale closed environment to form an amorphous glass protective layer with physical isolation effect, thereby achieving multi-dimensional long-term stability.

[0036] 4. This invention employs nonionic reverse micelle lipid phase solubilization technology, utilizing the nonionic reverse micelle structure to achieve stable encapsulation, deep penetration, and high bioactivity of water-soluble components in the oil phase; furthermore, in the oil-soluble PDRN combination, the raw materials and commonly used cosmetic oils have high biocompatibility, and the composition itself is clear and transparent, making it applicable to oil-based cosmetics with high appearance requirements, such as transparent essential oils, thereby realizing the good application of PDRN in transparent oil-based cosmetics.

[0037] 5. The oil-soluble PDRN preparation method of this invention employs a three-step phase transition method of "high-temperature homogenization - medium-temperature dropping - low-temperature shearing" to replace the traditional one-step emulsification. It eliminates the need for high-pressure homogenization or microfluidic equipment, allowing for the production of a transparent oil solution using conventional shearing equipment, resulting in low energy consumption. The three-step phase transition method involves: first, high-temperature homogenization for dispersion, completely melting and miscibleizing the oil phase with the surfactant, allowing the surfactant to pre-form disordered aggregates; second, medium-temperature dropping for nucleation, injecting the aqueous phase to trigger spontaneous reverse micelle nucleation; and finally, low-temperature shearing for stabilization, solidifying the reverse micelles under low-temperature conditions, with the glassy stabilizer locking the structure. Attached Figure Description

[0038] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The results are the stability test results for the sample in Example 1; Figure 2 The diagram shows the liquid state of Example 1 and Comparative Examples 1-8; Figure 3 This is a graph showing the oil compatibility status of the samples from Example 1, Comparative Example 18, and Comparative Example 20. Figure 4 The images show the cell migration effects of Example 1 and Comparative Example 29. Figure 5 This is the particle size distribution curve of the sample from Example 1. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0040] In this embodiment, PDRN was purchased from Ruijiming (Shandong) Biotechnology Co., Ltd., under the trade name RJMPDRN-C02. It was derived from male salmon, with an average molecular weight of 780bP and a sodium DNA content of 97.7%.

[0041] Examples 1-5 Examples 1-5 relate to a method for preparing three-dimensional oil-phase nano-oil-soluble PDRN with polar gradient to resist skin cell aging. The content of each component in the oil-soluble PDRN is shown in Table 1. The specific preparation method includes the following steps: (1) Phase A includes oil phase base material (squalane, red camellia seed oil, ethyl oleate, dioleyl glycerol and lauroyl sarcosine isopropyl ester) and surfactant (polyglycerol-10 dioleate or polyglycerol-10 trioleate and PPG-13-decyltetradecyl alcohol polyether-24); Phase B is PDRN, polylysine, water and glycerol; (2) Mix the oil phase base material in Phase A, heat to 55°C and keep warm, add the surfactant to the oil phase base material respectively, stir for 30 min, homogenize (7000 rpm) for 5 min, and cool down to 45°C; (3) Stir the polylysine and water in Phase B at room temperature until completely dissolved, add PDRN and continue stirring for 2 h until the powder is completely dissolved, add glycerol and stir for 10 min; (4) Add the liquid in step (5) dropwise to Phase A at a rate of 3 mL / min; (6) After the dropwise addition is completed, cool down to 20°C and continue stirring for 1.5 h to obtain a transparent and clear oil solution.

[0042] Table 1. Components and dosages in the compositions of Examples 1-5

[0043] Comparative Examples 1-7 Comparative Examples 1-7: The difference from Example 1 is that the components and their amounts are different, as shown in Table 2.

[0044] Table 2. Components and dosages in the compositions of Examples 1 and Comparative Examples 1-7

[0045] The results were as follows: The liquid obtained in Example 1 was clear and transparent, with a slight viscosity. After an 8-week stability test (stability conditions: -18℃, 4℃, room temperature, 40℃, 48℃, -18℃ / 48℃ cycling), the liquid remained stable, as shown in the following results. Figure 1 As shown in the figure. Comparative Examples 1-2 and 4 were all opaque liquids; Comparative Examples 3 and 5-7 were all slightly permeable liquids. After standing at room temperature for 2 days, sediment was found at the bottom of all samples. The state of the liquids is shown in the figure. Figure 2 This indicates that employing a polar gradient three-dimensional oil phase is beneficial for the formation and storage stability of the oil-soluble PDRN reverse micelle transparent nanosystem.

[0046] Comparative Example 8 The difference from Example 1 is that the entire polar gradient three-dimensional oil phase was replaced with GTCC (caprylic / capric triglyceride). The result was that the prepared oil-soluble PDRN was an opaque solution, and the solution state is shown in [see Example 1]. Figure 2 .

[0047] Comparative Example 9 The difference from Example 1 is that the polar gradient three-dimensional oil phase was completely replaced with isohexadecane, resulting in a turbid liquid in phase A, which could not be used for further preparation.

[0048] Comparative Examples 10-14 Comparative Examples 10-14: The difference from Example 1 is that the surfactants used are different, as shown in Table 3.

[0049] Table 3. Components and dosages in the compositions of Examples 1 and Comparative Examples 10-14

[0050] The results were as follows: The liquid obtained in Example 1 was clear and transparent, with a slight viscosity. Comparative Example 10 yielded a slightly transparent liquid; Comparative Example 11 yielded a completely opaque liquid, which was an emulsion; Comparative Example 12 yielded a slightly transparent liquid, which, when left overnight at room temperature, produced sediment at the bottom; Comparative Example 13 yielded an opaque liquid; Comparative Example 14 yielded a slightly transparent liquid, which, when left overnight at room temperature, produced sediment at the bottom.

[0051] Comparative Example 15 The difference from Example 1 is that polyglycerol-10 dioleate was replaced with polyglycerol-10 oleate, resulting in an opaque liquid. After being left at room temperature overnight, a precipitate was formed at the bottom of the liquid.

[0052] Comparative Example 16 The difference from Example 1 is that polyglycerol-10 dioleate was replaced with water-in-oil polyglycerol nonionic surfactants commonly used in cosmetic raw materials (polyglycerol-2 distearate, polyglycerol-2 isostearate). In both cases, an opaque liquid was obtained, and when left overnight at room temperature, a precipitate was formed at the bottom of the liquid.

[0053] Comparative Example 17 The difference from Example 1 is that the two surfactants are replaced with one or more of polysorbate-60, polysorbate-80, sorbitan oleate, and sorbitan isostearate. Adding them in any proportion will result in an emulsified liquid.

[0054] Comparative Examples 18-21 The difference from Example 1, Comparative Example 16, and Comparative Example 17 is that a high-pressure homogenizer is added. The specific content of each component is shown in 4. The preparation process is as follows: (1) The oil phase raw material and surfactant are premixed at 55°C, stirred for 30 min, and homogenized (7000 rpm) for 3-5 min, and then cooled to 45°C; (2) The aqueous phase polylysine and water are stirred at room temperature until completely dissolved, PDRN is added and stirred for 2 h until the powder is completely dissolved, glycerol is added and stirred for 10 min; (3) The liquid in step (2) is added dropwise to the liquid in step (1) at a rate of 2-5 mL / min, and stirring is continued for 1.5 h after the addition is completed; (4) The liquid in step (3) is subjected to high-pressure homogenization (homogenization pressure 600 bar, homogenization times 6 times) to obtain a transparent oil solution.

[0055] Table 4. Components and dosages in the compositions of Examples 1 and Comparative Examples 18-21

[0056] The stability of the samples from Example 1 and Comparative Examples 18-21 was investigated for 8 weeks under cyclic conditions of -18℃, 4℃, room temperature, 40℃, 48℃, and -18℃ / 48℃. The results showed that: Comparative Examples 19 and 21 showed precipitation after 2 weeks at 48℃ under cyclic conditions and turbid precipitation after 3 weeks at 40℃; Comparative Examples 18 and 20 were normal under all conditions for the first 6 weeks, but precipitation and turbidity appeared in the 7th week at 48℃ under cyclic conditions.

[0057] The compatibility of the samples from Example 1, Comparative Examples 18 and 20 with commonly used cosmetic oils was investigated. The results are shown in Table 5. The experimental procedure was as follows: the oil-soluble PDRN sample was dissolved in oil at room temperature (mass fraction 1%), and the transparency of the final solution was observed. Experimental results: The results are shown in Table 5 and... Figure 3 The results show that the oil-soluble PDRN described in Example 1 of this invention, at a concentration of 1%, is soluble in most commonly used oils in cosmetics, demonstrating high oil compatibility.

[0058] Table 5. Oil compatibility test results of Samples 1 and 18 and 20 (Example 1)

[0059] Notes: (1) All the above observations were obtained by the naked eye under natural light; in order to make the photos show obvious differences and facilitate analysis and comparison, some examples were observed with the help of a flashlight. (2) Lin Qingxuan Camellia Anti-Wrinkle Repair Essence Oil is a compound oil. According to the ingredient list on the product box, the oils used include camellia seed oil, C15-19 alkyl, dioctyl ether, squalane, caprylic / capric triglyceride, coconut oil-caprylate / capric acid ester, sunflower seed oil, flaxseed oil, oat kernel oil, jojoba seed oil, and flaxseed oil.

[0060] Comparative Examples 22-25 The difference from Examples 1 and 5 is that the amount of polylysine added is different. The specific content of each component is shown in Table 6.

[0061] Experimental results: Compared with Examples 1 and 5, Comparative Examples 22-25 produced a milky white liquid instead of a clear and transparent liquid during aqueous phase preparation, making it impossible to proceed to the next step. After the milky white liquid was left overnight, precipitation occurred.

[0062] Table 6. Components and dosages in the compositions of Examples 1 and Comparative Examples 22-25

[0063] Comparative Examples 26-27 The difference from Example 1 is the amount of glycerol added; the specific content of each component is shown in Table 7. The water activity of the solutions in Example 1 and Comparative Examples 26-27 was compared, and the results are shown in Effect Example 1.

[0064] Comparative Example 28 The difference from Example 1 is that polylysine was not added; the specific content of each component is shown in Table 7. A corrosion challenge test was conducted on the solutions from Example 1 and Comparative Example 28, and the results are shown in Effect Example 2.

[0065] Table 7. Components and dosages in the compositions of Examples 1 and Comparative Examples 26-28

[0066] Comparative Example 29 This comparative example is a PDRN aqueous solution. The PDRN used in Example 1 was dissolved in pure water, with a mass fraction of 0.2% (the same as in Example 1).

[0067] Example 1 This embodiment provides the water activity test results of the solutions in Example 1 and Comparative Examples 26-27. The results are shown in Table 8.

[0068] Table 8. Water activity test results of the solutions in Example 1 and Comparative Examples 26-27

[0069] Water activity is an indicator in the evaluation system that reflects the free water content in a sample. Free water is the water that microorganisms can utilize for growth and reproduction. The lower the water activity, the more difficult it is for microorganisms to grow and reproduce in the sample, the less likely the sample is to spoil, and the lower the pressure on preservation. When Aw is below 0.75, the vast majority of microorganisms cannot grow and reproduce. For oil-based cosmetic raw materials, safety regulations generally require a water activity below 0.65, which eliminates the need for additional preservatives.

[0070] As shown in Table 8, with the increase of glycerol addition and the water content remaining constant, the water activity of the sample gradually decreased. The addition of glycerol in this invention effectively reduces the water activity of the system. The water activity of Example 1 was 0.642, which itself can inhibit microbial growth, eliminating the need for additional preservatives.

[0071] Example 2 This embodiment provides preservative challenge tests for Example 1, Comparative Example 26, and Comparative Example 28. The test methods and judgment criteria refer to the Cosmetic Preservative Challenge Test Evaluation Guidelines of the National Institutes for Food and Drug Control (NIFDC). A single inoculation method was used, and tests were conducted on day 0, day 7, day 14, and day 28, respectively. The results are shown in Table 9.

[0072] The results in Table 9 show that Comparative Example 28, with the addition of 1% glycerol to the aqueous phase and without any additional preservatives, achieved a Grade B preservative standard; while Comparative Example 26, with the addition of polylysine, achieved an even better Grade A preservative standard. This indicates that introducing glycerol and polylysine into the system can ensure the overall preservative effect.

[0073] Table 9. Corrosion resistance challenge test results of Example 1, Comparative Example 26, and Comparative Example 28

[0074] Example 3 This example demonstrates the transdermal absorption effects of Example 1 and Comparative Examples 4, 5, and 30. Specifically, PDRN permeability was analyzed using a Franz diffusion cell, and the amount of PDRN retained in the pig skin was tested. The results are shown in Table 10.

[0075] Table 10 In vitro permeation parameters

[0076] Table 10 shows that the polar gradient three-dimensional oil-phase nano-oil-soluble PDRN system significantly improved the transdermal effect of PDRN. In Comparative Example 29, the cumulative PDRN penetration was not detected, indicating that ordinary PDRN aqueous solution could not penetrate into the skin in a short time, and most of the PDRN active ingredients remained only on the skin surface. Combining the results of Example 1 with Comparative Examples 4 and 5, it can be seen that the weakly polar and moderately polar oils in the polar gradient three-dimensional oil-phase system can synergistically enhance PDRN transdermal absorption.

[0077] Example 4 This efficacy example provides the anti-wrinkle effects of Example 1 and Comparative Example 29. The specific test indicators are the expression level of type I collagen gene (COL1A1) and type III type I collagen.

[0078] (1) Experimental Principle: Type I collagen is the main component of skin collagen fibers and the most abundant collagen in human skin, playing a crucial role in skin elasticity and structural stability. Studies have shown that promoting the expression of COL1A1 can reduce skin lipid peroxidation, improve skin aging, and combat wrinkles, thereby improving skin quality. Type III collagen is also one of the main components of skin collagen fibers, usually coexisting with Type I collagen to form copolymer fibers. Its core function in the skin is to build and maintain skin elasticity, suppleness, and high-quality early repair capabilities. It is an indispensable key protein for maintaining youthful and healthy skin. Studies have shown that promoting the expression of Type III collagen can improve skin aging and combat wrinkles, thereby improving skin quality.

[0079] (2) Experimental basis: [1] Hu Xiaohui, Zhu Chengrui, Gao Fenghou, et al. Study on the mechanism of UVB-induced downregulation of type I and III collagen in human skin fibroblasts[J]. Chinese Journal of Aesthetic and Plastic Surgery, 2010(7):4. [2] Chiang HM, Chen HC, Chiu HH, et al. Neonauclea reticulata (Havil.) Merr Stimulates Skin Regeneration after UVB Exposure via ROS Scavenging and Modulation of the MAPK / MMPs / Collagen Pathway[J]. Evidence-Based Complementray and Alternative Medicine, 2013(7):324864. [3] Je YJ, Choi DK, Sohn KC, et al. Inhibitory role of Id1 on TGF-β-induced collagen expression in human dermal fibroblastsScienceDirect[J]. Biochemical & Biophysical Research Communications, 2014, 444(1):81-85. (3) Gene detection: 1) Cell seeding: Human skin fibroblasts were used. The cells were passaged to the logarithmic growth phase, and the appropriate cell density was adjusted and seeded into 12-well plates. The cells were incubated in an incubator for 24 hours until the confluence reached 70%-80%. 2) Sample processing: The culture medium (components: DMEM medium + 10% FBS + 1% triple antibody) was discarded. An equal volume of PBS was added and the cells were irradiated with UV. Then, culture medium containing the sample to be tested was added. The control group was added with an equal volume of complete culture medium and cultured for 24 hours. 3) RNA extraction and reverse transcription: The culture medium was discarded. The cells were washed twice with PBS. RNA was extracted from each group of cells according to the instructions of the kit (MolPure® 5 min Flash Cell RNA Kit 5 min Rapid Cell RNA Extraction Kit (Yisheng Biotechnology, catalog number: 19231ES50)). After the purity was tested, cDNA was synthesized by reverse transcription. 4) RT-qPCR (Real-time Quantitative PCR): Prepare the reaction system (fluorescent probe + target gene primer + cDNA), set the amplification program (denaturation-rejection-extension cycles 40 times), and detect the expression levels of COL1A1 and COL3A1 genes in each group of cells. 5) Data Analysis: Use 2... - Ct The relative expression level of the target gene is calculated using the following formula (with GAPDH as an internal reference): Ct=Ct 目的基因 -Ct 内参 Ct= Ct 处理组 - Ct 对照组 Relative gene expression level = 2 - Ct (4) Experimental results: The experimental results of anti-wrinkle related gene expression are shown in Table 11.

[0080] Table 11 Results of anti-wrinkle related gene expression

[0081] Note: Compared with the UV model group, *** indicates p < 0.001.

[0082] As shown in Table 11 of the experimental results, compared with the UV model group, the relative expression levels of COL1A1 and COL3A1 genes in Example 1 and Comparative Example 29 were significantly upregulated, indicating that both oil-soluble PDRN and water-soluble PDRN can promote the expression of anti-wrinkle-related genes in fibroblasts. The upregulation effect of Example 1 on the expression of the two genes was significantly better than that of Comparative Example 29 at the same PDRN concentration. The upregulation rate of COL1A1 was 32 percentage points higher than that of Comparative Example 29, and the upregulation rate of COL3A1 was 33 percentage points higher. This shows that the polar gradient three-dimensional oil phase nano-oil-soluble PDRN prepared in this invention can more effectively promote the expression of anti-wrinkle-related collagen genes in skin fibroblasts compared with conventional water-soluble PDRN, and has a better anti-wrinkle efficacy potential.

[0083] Example 5 This effect example provides the repair effect of Example 1 and Comparative Example 29.

[0084] (1) Experimental principle: Epidermal keratinocytes are important cells that make up the epidermal layer. When the skin surface is damaged, keratinocytes are stimulated to migrate and repair the damaged parts. When cells grow to a monolayer in vitro, a blank area is artificially created on the monolayer. Cells at the edge of the scratch will gradually enter the blank area to heal the scratch, which simulates the cell migration process in vivo to a certain extent. The repair efficacy of the test substance is evaluated by measuring the cell migration rate after sample treatment.

[0085] (2) Experimental basis: Research Techniques Made Simple: Analysis of CollectiveCell Migration Using the WoundHealing Assay. (3) Cell scratch assay: Scratches were made using a 10 μL pipette tip. After washing twice with PBS, 2 mL of DMEM basal medium containing different concentrations of the sample was added to each well. After 24 h of culture, the same field of view of the sample group was photographed and recorded. DMEM basal medium was used as a blank control. The scratch distance (d) at three locations at each time point was measured, and the cell migration rate was calculated. Cell migration rate (%) = (d... 0h -d 24h ) / d 0h ×100% (4) Experimental results: The effects of the samples on the cell migration rate of keratinocytes are shown in Table 12 and... Figure 4 As shown.

[0086] Table 12 Results of Cell Scratch Test

[0087] Note: Compared with the blank control group, * indicates p < 0.05, and *** indicates p < 0.001.

[0088] As shown in Table 12, both Sample 1 and Comparative Sample 29 promoted keratinocyte migration, and the cell migration rate of the oil-soluble PDRN of this invention was higher than that of PDRN in aqueous solution. The oil-soluble PDRN specifically incorporates skin-care oil components, which theoretically make it easier for skin keratinocytes to absorb. Experimental results indicate that the polar gradient three-dimensional oil-phase nano-oil-soluble PDRN is more effective than water-soluble PDRN in promoting keratinocyte migration and has superior repair effects on damaged skin areas.

[0089] Example 6 This example demonstrates the verification of nanoparticle size in Example 1. The instrument used for particle size testing was a nanoparticle size and zeta potential analyzer (model: Malvern Zetasizer Nano ZS90, UK). The test results are shown in Table 13 and... Figure 5 .

[0090] Table 13 Particle size test results of the sample from Example 1

[0091] As shown in Table 13, the Z-average particle size of the oil-soluble PDRN prepared by this invention is 65.29 nm, which is at the nanoscale. This indicates that the preparation method of this invention can obtain nanoscale oil-soluble PDRN with uniform particle size distribution, which is beneficial for its transdermal absorption and exerts corresponding skin care activity.

[0092] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A polar gradient three-dimensional oil-phase nano-oil-soluble PDRN for anti-skin cell aging, characterized in that, include: A three-phase polar gradient consisting of an oil phase, a surfactant phase, and an aqueous phase. The polar gradient three-dimensional oil phase includes non-polar oil, weakly polar oil, and moderately polar oil; wherein, the non-polar oil includes squalane, the weakly polar oil includes camellia seed oil and ethyl oleate, and the moderately polar oil includes dioleyl ester and lauroyl sarcosine isopropyl ester; the aqueous phase contains PDRN.

2. The oil-soluble PDRN according to claim 1, characterized in that, By weight, the oil-soluble PDRN comprises: 55-70 parts of a three-stage polar gradient oil phase, 20-35 parts of a surfactant, and 6-15 parts of an aqueous phase.

3. The oil-soluble PDRN according to claim 1, characterized in that, The mass ratio of the non-polar segment oil, the weakly polar segment oil, and the medium polar segment oil is 1:5~9:1~3; the mass ratio of the red camellia seed oil to ethyl oleate is 1:1~2, and the mass ratio of dioleate to lauroyl sarcosine isopropyl ester is 1:0.5~1.

5.

4. The oil-soluble PDRN according to claim 1, characterized in that, The surfactant comprises polyglycerol fatty acid ester and polyether-modified long-chain alcohol in a mass ratio of 1:1 to 3; wherein the polyglycerol fatty acid ester is selected from polyglycerol-10 dioleate and polyglycerol-10 trioleate; the polyether-modified long-chain alcohol comprises PPG-13-decyltetradecyl alcohol polyether-24, and the polyether-modified long-chain alcohol accounts for not less than 15% of the total mass of oil-soluble PDRN.

5. The oil-soluble PDRN according to claim 1, characterized in that, The aqueous phase comprises PDRN, polyol, polylysine, and water; wherein the mass ratio of PDRN to water is 1:10~50, the mass ratio of polyol to water is 1:4~8, the mass ratio of PDRN to polylysine is 13~20:1, water accounts for 4.5~10% of the total mass of oil-soluble PDRN, and the content of PDRN in oil-soluble PDRN is 0.1-0.5% (w / w).

6. The oil-soluble PDRN according to claim 5, characterized in that, The PDRN has an average molecular weight ≤800bP and a sodium DNA content ≥90.0%; the polyol includes glycerol.

7. A method for preparing oil-soluble PDRN as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Heat the three-dimensional oil phase with polar gradient, add surfactant, stir, homogenize and cool to obtain phase A; S2. First, stir and dissolve polylysine in water until clear, then add PDRN and stir until clear, then add polyol and stir to form phase B. S3. Add phase B to phase A, cool and stir to obtain the final product.

8. The preparation method according to claim 7, characterized in that, In step S1, the heating temperature is 50~60℃; the stirring time is 20-40min; the homogenization speed is 6000~8000rpm and the time is 3~5min; and the temperature is cooled to 40~45℃. In step S3, the addition rate of phase B is 2-5 mL / min; the temperature is then lowered to 15-25 °C.

9. The use of oil-soluble PDRN as described in any one of claims 1-6 in the preparation of cosmetics with anti-wrinkle, repairing, and whitening effects.

10. The application according to claim 9, characterized in that, The cosmetics include one of the following: toner, lotion, cream, and essential oil.

Images