Pdrn nanoliposome and preparation method thereof

Abstract

The application discloses a preparation method of PDRN nanoliposomes, which comprises the following steps: S1, taking hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol, heating and uniformly mixing to obtain an oil phase liquid; S2, taking macromolecular PDRN, small molecular PDRN and sodium hyaluronate, adding into water, heating and uniformly mixing to obtain an aqueous phase liquid; S3, under the state of stirring, adding the aqueous phase liquid into the oil phase liquid, and continuously stirring to obtain a crude liposome; S4, performing microjet treatment on the crude liposome under a pressure of ≤1000 bar to obtain the PDRN nanoliposomes; wherein, the step S1 and the step S2 do not have a sequence requirement. The application also provides the prepared PDRN nanoliposomes. The PDRN nanoliposomes provided by the application have high stability, can rapidly penetrate, and synergistically enhance the skin cell repair and anti-aging effects, and the preparation process is simple.

Description

Technical Field

[0001] This application relates to the field of liposome preparation technology, and in particular to a PDRN nanoliposome and its preparation method. Background Technology

[0002] Polydeoxyribonucleotide (PDRN) is a natural molecule derived from living organisms. It possesses biological activities such as promoting cell regeneration, anti-inflammation, anti-oxidation, and DNA damage repair, and is widely used in medical aesthetic injections and high-end skincare products. However, the large molecular weight and high polarity of PDRN result in a transdermal absorption rate of less than 5%, making it difficult for traditional topical products to reach an effective concentration.

[0003] Liposomes are nanoscale carriers that encapsulate active ingredients within a phospholipid bilayer, and are widely used for drug delivery and sustained release of functional ingredients. However, traditional liposomes have low encapsulation efficiency for high molecular weight substances (such as high molecular weight PDRN) and struggle to efficiently encapsulate both hydrophilic and hydrophobic components simultaneously. Furthermore, traditional liposome preparation processes are complex and unstable, easily leading to leakage of active ingredients. Additionally, the reagents used in the preparation process typically include ethanol and sodium deoxycholate, which may irritate the skin or trigger allergic reactions when used in cosmetics. Summary of the Invention

[0004] To address the aforementioned technical problems, the first objective of this invention is to provide a method for preparing PDRN nanoliposomes; the second objective is to provide PDRN nanoliposomes prepared by the above method. The PDRN nanoliposomes provided in this application are characterized by small particle size and high stability, enabling rapid penetration and transdermal absorption during use, resulting in high bioavailability and synergistic enhancement of skin cell repair and anti-aging effects. The liposome preparation process used in this invention is simple, the components are safe, and energy consumption is low, enabling large-scale production and increased production capacity.

[0005] The technical solution provided by this invention is as follows:

[0006] A method for preparing PDRN nanoliposomes includes the following steps:

[0007] S1. Take hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol, heat and mix them to obtain an oil phase liquid;

[0008] S2. Take macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate, add them to water, heat and mix well to obtain an aqueous phase solution;

[0009] S3. Add the aqueous phase to the oil phase while stirring, and continue stirring to obtain crude liposomes;

[0010] S4. Crude liposomes were subjected to microfluidic treatment at a pressure of ≤1000 bar to obtain PDRN nanoliposomes;

[0011] There is no requirement for the order of steps S1 and S2.

[0012] Preferably, the molecular weight of the macromolecule PDRN is 1000kDa-1800kDa;

[0013] The molecular weight of small molecule PDRN is 100 Da-500 kDa;

[0014] The molecular weight of hydrolyzed sodium hyaluronate is 400 Da-1600 Da.

[0015] Preferably, in steps S1 and S2, the heating temperature is 60-70°C.

[0016] Preferably, in steps S1 and S2, the heating temperature is 65-68°C.

[0017] Preferably, in step S4, the pressure of the microjet treatment is 400-1000 bar; and / or,

[0018] The number of microfluidic treatments is 1-5 times.

[0019] Preferably, the pressure of the microjet treatment is 800-900 bar; and / or,

[0020] The microfluidic treatment is performed 2-3 times.

[0021] Preferably, in step S3, the aqueous phase is added to the oil phase while stirring; stirring is continued for 3-5 minutes to obtain crude liposomes.

[0022] Preferably, the mass ratio of hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol is (2-4):(0.3-2):(0.3-1):(30-50);

[0023] The mass ratio of macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate is (0.01-0.2):(0.01-0.2):(0.05-0.5).

[0024] Preferably, the mass ratio of hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate, and glycerol is 3:0.5:0.5:45;

[0025] The mass ratio of macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate is 4:1:10.

[0026] A PDRN nanoliposome is prepared by any of the preparation methods described above.

[0027] The PDRN nanoliposomes provided in this application have a "core-shell-membrane" sandwich structure. The core consists of a high-concentration glycerol forming a high-osmotic-pressure environment, which drives the polydeoxyribonucleotides (PDRN) contained in the nanoliposomes to rapidly penetrate into the dermis. The middle layer is hydrolyzed sodium hyaluronate, which binds to the PDRN through hydrogen bonds to form a reservoir that facilitates sustained release. The outer shell is a dense bilayer membrane formed by hydrogenated lecithin and stearoyl glutamate (e.g., sodium stearoyl glutamate). Stearoyl glutamate imparts a negatively charged surface to the liposomes, reducing the contact and binding of nucleases through "like-charge repulsion," thus helping to reduce the risk of active exposure and burst release and providing a more stable microenvironment for PDRN. At the same time, polyglycerol-10 myristate is embedded in the dense bilayer membrane to regulate fluidity. Furthermore, due to the self-emulsifying effect of polyglycerol-10 myristate, nano-sized liposomes can be obtained without traditional high pressure (high pressure refers to greater than 1000 bar).

[0028] The applicant conducted multiple experiments, including particle size analysis, long-term high-temperature stability testing, type I collagen content testing, cell scratch repair experiments, and penetration experiments, and provided several comparative examples. These comparisons demonstrated that the PDRN nanoliposomes provided in this application possess small particle size and high stability, enabling rapid penetration and transdermal absorption with high bioavailability. Furthermore, the specific combination of large-molecule PDRN, small-molecule PDRN, and hydrolyzed sodium hyaluronate synergistically enhances the effects of skin cell repair and anti-aging. The liposomes used in this invention have a simple preparation process, safe components, low energy consumption, and can be mass-produced, increasing production capacity.

[0029] The PDRN nanoliposomes provided in this application contain both macromolecular PDRN and small molecule PDRN. They utilize a dual-molecular-weight polydeoxyribonucleotide and hydrolyzed sodium hyaluronate synergistic delivery technology to achieve transdermal absorption and long-lasting synergistic anti-aging and repair effects through a specific ratio combination.

[0030] In this application, the preferred molecular weight of macromolecular PDRN is 1000kDa-1800kDa; the molecular weight of small PDRN is 100Da-500kDa; and the molecular weight of hydrolyzed sodium hyaluronate is 400Da-1600Da. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The results of particle size stability of Examples 1, 4, and 5 of the present invention at different temperatures over 30 days;

[0033] Figure 2 This is a statistical analysis of the permeation test results of the present invention. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0035] The types and amounts of reagents used in Examples 1-3 and Comparative Examples 1-10 are shown in Table 1.

[0036] Table 1

[0037]

[0038] Examples 1-5 and Comparative Examples 1-10 in Table 1 were prepared using the following method:

[0039] S1. Take hydrogenated lecithin, sodium stearoyl glutamate, glycerol, and one of polyglycerol-10 myristate, polyglycerol-10 oleate, and polyglycerol-10 stearate (according to Table 1), heat them in a water bath at 65°C and stir to mix them evenly to obtain an oil phase liquid.

[0040] S2. Take macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate, add them to water, heat at 65℃ and stir to mix well to obtain an aqueous phase liquid;

[0041] S3. Add the aqueous phase to the oil phase while stirring, and continue stirring for 5 minutes to obtain crude liposomes;

[0042] S4. Crude liposomes were microfluidically treated at pressures of 500 bar, 800 bar, or 1000 bar to obtain PDRN nanoliposomes. Examples 1-5 and Comparative Examples 2-4 were treated at both 500 bar and 800 bar pressures to obtain the corresponding particle sizes under the two pressure treatments; Comparative Examples 1 and 5 were treated at only 1000 bar pressure. The PDRN nanoliposomes prepared under the above different pressure conditions were all transparent solutions.

[0043] There is no requirement for the order of steps S1 and S2.

[0044] I. Stability Testing

[0045] The microfluidization process, particle size, and stability of Examples 1, 4-5, and 1-5 are compared, and the results are shown in Table 2:

[0046] Table 2

[0047]

[0048] The stability / particle size comparison experiments are mainly to demonstrate the necessity and superiority of the "carrier ternary system" selected in this application. Examples 2-3 only changed the active ratio within the same carrier system (the content of the active ingredients in Examples 2-3: macromolecule PDRN, small molecule PDRN, and hydrolyzed sodium hyaluronate is different from that in Example 1, but the ratio remains the same as in Example 1, 4:1:10). Comparative Examples 6-10 are used for efficacy / synergistic verification, not for carrier stability determination. To avoid redundancy, Examples 2-3 and Comparative Examples 6-10 are not included here.

[0049] The results in Table 2 are analyzed in detail below:

[0050] The particle size stability results of Examples 1, 4, and 5 prepared at 800 bar at different temperatures over 30 days are as follows: Figure 1 As shown.

[0051] The stability results show that Examples 1, 4, and 5 exhibit small particle size and good stability. In contrast, Comparative Example 1, using only hydrogenated lecithin, is highly unstable, even exhibiting gelation at high temperatures; Comparative Example 2, using only hydrogenated lecithin and sodium stearoyl glutamate, shows larger particle size under the same pressure and poor stability at low temperatures; Comparative Example 3, using only hydrogenated lecithin and polyglycerol-10 myristate, produces very small particle size but a very high PDI, potentially indicating a tendency to form micelles, and is unstable at both high and low temperatures. Comparative Examples 4 and 5, using polyglycerol-10 oleate and polyglycerol-10 stearate respectively, combined with hydrogenated lecithin, are all unstable. Polyglycerol-10 oleate tends to form micelles under different pressures, while polyglycerol-10 stearate requires even higher pressures to achieve small particle sizes. Comparative Examples 4-5 verified the effect of emulsifier type on system stability, confirming the difference between polyglycerol-10 myristate and other polyglycerol emulsifiers, indicating that polyglycerol-10 myristate is the only suitable emulsifier among the compared polyglycerol emulsifiers for this system. The comparative examples exhibited problems such as increased particle size, turbidity, and even gelation at high temperatures. In contrast, the combination of "hydrogenated lecithin + polyglycerol-10 myristate + sodium stearoyl glutamate" used in the examples formed a stable structure, with particle size change ≤10% under high and low temperature and cycling conditions, and a PDI ≤0.28. It can form small-particle-size, highly stable PDRN liposomes without the need for high-pressure homogenization or the addition of additional bile salt activators. The comparative examples, lacking any core component or replacing the emulsifier, showed a significant decrease in stability.

[0052] II. Efficacy Test

[0053] (1) Experiment on the detection of type I collagen content

[0054] HFF-1 cells were seeded at 10,000 cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. Working solutions of the test substance were prepared within the cytotoxic concentration range according to the experimental design. Specific data are shown in Tables 3 and 5. Two groups of test samples were set up: a blank group, a positive control group, Example 1, and comparative examples 6-10. According to the test protocol, the drug was administered after the cell growth rate reached 80%-90%. After drug administration, the 96-well plates were incubated in an incubator (37°C, 5% CO2) for 24 hours. Cell supernatant was collected, and the content of type I collagen was detected using a reagent kit. GraphPad Prism 8.0 software was used for statistical analysis and graph plotting. Quantitative data are expressed as x±s. One-way ANOVA was used to analyze differences between groups. P<0.05 was considered statistically significant, P<0.05 was considered statistically significant, and P<0.01 was considered highly significant.

[0055] Table 3. Information on Type I Collagen Content Test Samples 1

[0056]

[0057] The experiments were conducted according to Table 3, and the average growth rate of type I collagen content in each group is shown in Table 4.

[0058] Table 4

[0059]

[0060] The results show that, compared with Comparative Example 7 (where the addition amounts of macromolecular PDRN, small molecule PDRN, and hydrolyzed sodium hyaluronate were all 0), Comparative Example 8 (hydrolyzed sodium hyaluronate only) mainly played a moisturizing and repairing role in this system, thus showing no significant synergistic effect on type I collagen, and achieving the highest promoting effect at a concentration of 0.05%. Comparative Example 9 (small molecule PDRN only) showed no synergistic effect at low concentrations of 0.005% and below, but its promoting effect on type I collagen increased with increasing concentration, also reaching a peak at 0.05%. Further increases in concentration may have led to cytotoxicity due to excessively rapid penetration, resulting in a slight decrease at 0.1%. Comparative Example 10 (macromolecular PDRN) had low transdermal efficiency, and there may have been experimental abnormalities at low concentrations, but it showed a significant promoting effect at a high concentration of 0.1%. Therefore, based on these experimental results, the components with the highest promoting effect (macromolecular PDRN: small molecule PDRN: hydrolyzed sodium hyaluronate = 4:1:10) can be combined for synergistic effect verification.

[0061] The synergistic effect was calculated using the Bliss model. In Example 1, the three components were combined in a 4:1:10 ratio, with a total concentration of 0.3%. Therefore, the concentration of the combined component was calculated as: 0.3% × experimental concentration. The concentration of each individual component in the combined component was:

[0062] Hydrolyzed sodium hyaluronate: 0.3% × 10 / (4 + 1 + 10) × (Experimental concentration in Example 1)

[0063] Small molecule PDRN: 0.3% × 1 / 15 × (Experimental concentration in Example 1)

[0064] Macromolecular PDRN: 0.3% × 4 / 15 × (Experimental concentration in Example 1).

[0065] The above content selects the optimal ratio of active ingredient concentration based on the peak value of the highest effect. For Comparative Examples 8-10, with consistent carrier concentrations, after correction with Comparative Example 7 (blank), the "single-component concentration gradient verification" is equivalent to "single-component verification at different concentrations." If there are more than 3 single-component gradient concentrations, no additional experiments are needed. The 4:1:10 ratio is initially selected based on the absolute peak value of the single components. After blank correction, the absolute peak values ​​of Comparative Examples 8 and 10 are consistent with the true effect peak value. Although the absolute peak value of Comparative Example 9 (small molecule PDRN) differs slightly from the true effect peak value, this concentration is still within the experimentally preset effective concentration range (small molecule PDRN 0.05% ≤ 0.2%), and does not exceed the safe and effective concentration boundary of the subsequent synergistic verification system. Furthermore, the Bliss model verified a significant synergistic effect in subsequent experiments, thus maintaining the rationality of the optimal embodiment.

[0066] Design the experimental group (Table 5). Calculate and explain whether the combination has a synergistic effect based on the Bliss model. The calculation formula is: Theoretical combined effect = 1 - (1 - E) A (1-E) B (1-E) C When the actual effect is greater than the theoretical effect, it indicates that the combination has synergistic effects; when the actual effect is less than the theoretical effect, it indicates that the combination does not have synergistic effects.

[0067] Table 5. Information on Type I Collagen Content Test Samples 2

[0068]

[0069] The results are as follows: Table 6 shows the average growth rate of type I collagen content in each group.

[0070] Table 6

[0071]

[0072] The results show that when the total concentration is 0.3% × 0.05% = 0.00015%, according to the Bliss model, E 理论效应 =1-(1-EA)(1-EB)(1-EC)=1-(1-7.25%)*(1-23.17%)*(1-9.10%)= 0.3522<0.3605, indicating that the combination of "hydrolyzed sodium hyaluronate + small molecule PDRN + large molecule PDRN" in the ratio of "10:1:4" has the best synergistic effect on promoting the expression of type I collagen.

[0073] Comparative Example 6 (active group only, without carrier) was used to compare the gain of the carrier on active delivery and efficacy, and Comparative Example 7 (blank liposomes, no activity) was used to confirm that the carrier itself would not introduce false positives. Neither was involved in the synergistic effect (Bliss) calculation in this study; they were only used for methodological and baseline reference explanations, illustrating the carrier effect and baseline, and were not included in the synergistic calculation to avoid statistical confounding. Blank group and positive control group (PC, TGF-β): provided zero-effect and positive reference baselines, respectively.

[0074] Comparative Examples 8 (hydrolyzed sodium hyaluronate only), 9 (small molecule PDRN only), and 10 (large molecule PDRN only) served as single-factor references in the theoretical joint effect calculation of the Bliss model and were used for synergy discrimination with Example 1. The experimental results show that Example 1 exhibited superior effects compared to the various comparative examples of "active only / single component only / blank carrier only," demonstrating more efficient delivery and a more stable effect.

[0075] (2) Cell scratch repair experiment

[0076] Using sterilized tweezers, place the bottom of the culture-insert 2 well into a 6-well plate and install the insert; seed HaCat cells at 60,000 cells / well into both wells of the culture-insert 2 well and incubate at 37°C, 5% CO2 for 24 h; design the experimental groups as shown in Table 7 according to the above logic and prepare the working solution of the test substance; according to the test protocol, remove the scratch insert and take pictures, and administer the drug to the groups, with 2 replicates per group; after drug administration, place the 6-well plate in an incubator (37°C, 5% CO2) for 24 h and observe the cell morphology under a microscope and take pictures. Image J software was used for statistical area analysis of the images. The scratch healing percentage was calculated using the formula: [Area of ​​scratch-free area (0H) - Area of ​​scratch-free area (24H) / Area of ​​scratch-free area (0H) * 100]. The scratch healing growth ratio was calculated using the formula: (Percentage of scratch healing in samples - Percentage of healing in positive scratches) / Percentage of healing in positive scratches. GraphPad Prism 8 software was used for statistical analysis of the data and to generate charts.

[0077] Table 7 Information on Cell Scratch Repair Test Samples

[0078]

[0079] The results are as follows, and the scratch healing growth rate of each group is shown in Table 8:

[0080] Table 8

[0081]

[0082] The results show that when the total concentration is 0.3% × 0.05% = 0.00015%, according to the Bliss model, E 理论效应 =1-(1-0.8221)(1-0.5932)(1-2.2972)=1.09287<1.8124, indicating that the combination of "hydrolyzed sodium hyaluronate + small molecule PDRN + large molecule PDRN" in the ratio of "10:1:4" has a synergistic effect in promoting scratch repair.

[0083] (3) Osmosis test

[0084] Samples from Example 1 and Comparative Example 6 were prepared by adding 0.02% sulfonylrhodamine salt to deionized water to form 20% fluorescent solutions for testing. 0.8-1 cm excised Bama miniature pig skin was used as the in vitro transdermal test material. PBS was used as the receiving medium, and the permeation absorption was measured using a Franz diffusion cell. The parameters were set at 37.5℃ and 600 rpm. After permeation for 30 min, the pig skin surface was cleaned with PBS, and sections were prepared. The distribution of fluorescence signals in the pig skin was observed under a fluorescence microscope, and the fluorescence penetration depth was analyzed using ImageJ software to evaluate the transdermal absorption effect of the encapsulated PDRN liposomes and the unencapsulated composite PDRN solution. The test results are as follows: Figure 2 As shown.

[0085] Comparing the fluorescence penetration depth of Example 1 and Comparative Example 6, Example 1 penetrated significantly deeper than Comparative Example 6, indicating that the encapsulated liposomes had a better penetration effect.

[0086] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present invention, but these improvements all fall within the protection scope of the present invention.

Claims

1. A method for preparing PDRN nanoliposomes, characterized in that, Includes the following steps: S1. Take hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol, heat and mix them to obtain an oil phase liquid; S2. Take macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate, add them to water, heat and mix well to obtain an aqueous phase solution; S3. Add the aqueous phase to the oil phase while stirring, and continue stirring to obtain crude liposomes; S4. Crude liposomes were subjected to microfluidic treatment at a pressure of ≤1000 bar to obtain PDRN nanoliposomes; There is no requirement for the order of steps S1 and S2; The mass ratio of macromolecule PDRN, small molecule PDRN and hydrolyzed sodium hyaluronate is 4:1:

10.

2. The method for preparing PDRN nanoliposomes according to claim 1, characterized in that, The molecular weight of macromolecule PDRN is 1000kDa-1800kDa; The molecular weight of small molecule PDRN is 100 Da-500 kDa; The molecular weight of hydrolyzed sodium hyaluronate is 400 Da-1600 Da.

3. The method for preparing PDRN nanoliposomes according to claim 1, characterized in that, In steps S1 and S2, the heating temperature is 60-70℃.

4. The method for preparing PDRN nanoliposomes according to claim 3, characterized in that, In steps S1 and S2, the heating temperature is 65-68℃.

5. The method for preparing PDRN nanoliposomes according to claim 1, characterized in that, In step S4, the pressure of the microjets is 400-1000 bar; and / or, The number of microfluidic treatments is 1-5 times.

6. The method for preparing PDRN nanoliposomes according to claim 5, characterized in that, The pressure for microfluidic treatment is 800-900 bar; and / or, The microfluidic treatment is performed 2-3 times.

7. The method for preparing PDRN nanoliposomes according to claim 1, characterized in that, In step S3, the aqueous phase is added to the oil phase while stirring; stirring is continued for 3-5 minutes to obtain crude liposomes.

8. The method for preparing PDRN nanoliposomes according to any one of claims 1-7, characterized in that, The mass ratio of hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol is (2-4):(0.3-2):(0.3-1):(30-50).

9. The method for preparing PDRN nanoliposomes according to claim 8, characterized in that, The mass ratio of hydrogenated lecithin, polyglycerol-10 myristate, stearoyl glutamate and glycerol is 3:0.5:0.5:

45.

10. A PDRN nanoliposome, characterized in that, It is prepared by any one of claims 1-9.

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