Polydeoxyribonucleotide-containing lipophilic liposome as well as preparation method and application thereof

Abstract

The invention discloses a lipophilic liposome containing polydeoxyribonucleotide as well as a preparation method and application of the lipophilic liposome. The lipophilic liposome comprises the following components in parts by weight: 0.001-3 parts of polydeoxyribonucleotide; 0.5 to 3 parts of phospholipid; 0.1-2 parts of a cationic emulsifier; 0.1-3 parts of an anionic emulsifier; 40 to 70 parts of polyol; the total amount is 100 parts. According to the invention, an emulsifier system formed by combining lecithin with anionic and cationic compound surfactants is adopted, so that the transdermal absorption of PDRN is facilitated. The lipophilic liposome containing polydeoxyribonucleotide prepared by the invention can effectively resist nuclease degradation, prolong the acting time of PDRN in skin, and improve the skin anti-aging and repairing effects of the liposome. The anti-aging effect of the active ingredients is improved, and the bioavailability of the active ingredients is improved. The cosmetic composition can be widely applied to cosmetic formulas of various dosage forms.

Description

Technical Field

[0001] This invention belongs to the fields of colloid chemistry and surface chemistry and cosmetics, specifically relating to a lipophilic liposome containing polydeoxyribonucleotides, its preparation method, and its application. Background Technology

[0002] PDRN, short for polydeoxyribonucleotide, is a hot ingredient for cell and DNA damage repair and anti-aging, and is currently widely used in skin regeneration and wound healing.

[0003] PDRN provides purines and pyrimidines, repairs cellular DNA damage, and activates cellular energy. By precisely inhibiting the secretion of SASP factors by some senescent cells, it achieves bidirectional inhibition of skin inflammation, demonstrating excellent efficacy in combating inflammatory aging.

[0004] However, the application of PDRN in conventional cosmetics faces challenges due to its difficulty in penetrating the skin and its tendency to degrade. Therefore, PDRN is primarily available in injectable products, requiring professional administration, and its high cost significantly limits its application in traditional skincare products. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a lipophilic liposome containing polydeoxyribonucleotides, comprising, by weight parts,

[0007] Polydeoxyribonucleotides 0.001-3 parts;

[0008] Phospholipids 0.5-3 parts;

[0009] 0.1-2 parts of cationic emulsifier;

[0010] 0.1-3 parts of anionic emulsifier;

[0011] 40-70 parts of polyols;

[0012] Add water to make up to 100 servings.

[0013] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention: by mass fraction, comprising,

[0014] Polydeoxyribonucleotides 0.1-2.5 parts;

[0015] Phospholipids 0.6-2.5 parts;

[0016] 0.2-1 part cationic emulsifier;

[0017] 0.5-2 parts of anionic emulsifier;

[0018] 50-65 parts of polyols;

[0019] Add water to make up to 100 servings.

[0020] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention, the anionic emulsifier includes anionic peptide emulsifiers.

[0021] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention, the cationic emulsifier is lauroyl arginine ethyl ester hydrochloride and / or PCA cocoyl arginine ethyl ester salt.

[0022] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention, the anionic emulsifier is sodium di(lauramide-glutamine)lysine and / or sodium subtilisin.

[0023] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention, the phospholipid is lecithin and / or hydrogenated lecithin.

[0024] As a preferred embodiment of the lipophilic liposomes containing polydeoxyribonucleotides described in this invention, the polyol includes one or more of glycerol, 1,3-propanediol, 1,3-butanediol, 1,2-hexanediol, 1,2-pentanediol, isopentanediol, PEG-400, PEG-1500, and sorbitol.

[0025] This invention also provides a method for preparing the lipophilic liposomes containing polydeoxyribonucleotides, comprising the following steps:

[0026] Anionic emulsifier, lecithin and polyol were mixed, heated and stirred until homogeneous, and then cooled to room temperature to obtain phase A1.

[0027] The cationic emulsifier was dissolved in water and slowly added dropwise to phase A1 to obtain a transparent solution phase A2; polydeoxyribonucleotides were dissolved in water to obtain phase B;

[0028] Phase B and phase A2 were mixed evenly and then mixed using high-pressure microfluidic mixing to obtain lipophilic liposomes containing polydeoxyribonucleotides.

[0029] The present invention also provides a method for preparing lipophilic liposomes containing polydeoxyribonucleotides, wherein the heating and stirring to homogenize includes heating to 75-85°C and stirring to homogenize; and the high-pressure microjet is performed at a pressure of 900-1000 bar.

[0030] The present invention also provides the application of the lipophilic liposomes containing polydeoxyribonucleotides in the preparation of cosmetics.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention employs an emulsifier system combining lecithin with anionic and cationic compounds, which is beneficial for the transdermal absorption of PDRN. The anionic and cationic compound emulsifier of this invention has a synergistic effect in promoting transdermal absorption.

[0033] (2) The lipophilic liposomes containing polydeoxyribonucleotides prepared in this invention can effectively resist nuclease degradation, prolong the action time of PDRN in the skin, and improve the anti-aging and repair effects of liposomes on the skin.

[0034] (3) This invention improves the anti-aging efficacy of active ingredients and enhances their bioavailability.

[0035] (4) The present invention overcomes the technical problem that both anionic emulsifiers and cationic emulsifiers are prone to precipitation, and preferably uses a compound emulsifier.

[0036] (5) The lipophilic liposomes containing polydeoxyribonucleotides prepared by this invention are easy to use and add in cosmetic formulations, and can be widely used in cosmetic formulations of various dosage forms. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0038] Figure 1 This is a diagram showing the results of a transdermal assay.

[0039] Figure 2 This is a graph showing the test results for type I collagen.

[0040] Figure 3 This is a graph showing the results of the type IV collagen test.

[0041] Figure 4 The image shows the test results for type XVII collagen.

[0042] Figure 5 This is a graph showing the results of a DNA repair test. Detailed Implementation

[0043] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0044] The raw materials used in this invention are all commercially available products.

[0045]

[0046]

[0047] Example 1:

[0048] Samples were prepared according to the raw material ratios in Table 1:

[0049] Preparation method:

[0050] Anionic emulsifier, lecithin and polyol were mixed, dissolved and stirred evenly at 80°C, and then cooled to room temperature to obtain phase A1;

[0051] The cationic emulsifier was dissolved in water and slowly added dropwise to phase A1 to obtain a transparent solution phase A2; polydeoxyribonucleotide (PDRN) was dissolved in water to obtain phase B;

[0052] Phase B and phase A2 were mixed evenly and subjected to high-pressure microjet, 1000 bar, 3 times, 5 minutes each time (calculated based on a total feed amount of 100g each time) to obtain lipophilic liposomes containing polydeoxyribonucleotides.

[0053] Table 1 Raw Material Proportions (Unit: wt%)

[0054]

[0055] As shown in Table 1, both the use of other cationic emulsifiers in combination with the anionic emulsifier selected in this invention and the use of other anionic emulsifiers in combination with the cationic emulsifier selected in this invention result in the formation of precipitates due to the binding of anions and cations, making it impossible to form stable, transparent, lipophilic liposomes. The preferred combination of anionic and cationic emulsifiers in this invention is stable and does not exhibit precipitation.

[0056] Table 2 Raw Material Proportioning Table (Unit: wt%)

[0057]

[0058]

[0059] Table 3 Raw Material Proportioning Table (Unit: wt%)

[0060]

[0061] Example 2:

[0062] Liposome stability:

[0063] The samples were divided into room temperature samples, heat-resistant samples, and cold-resistant samples. Room temperature samples were stored away from light, heat-resistant samples were placed in a constant temperature oven at (50±1℃), and cold-resistant samples were placed in a -18℃ refrigerator. The appearance, color, and odor of the samples were observed periodically. The results are shown in Table 4.

[0064] Table 4 Stability Test

[0065] Sample High temperature stability Room temperature stability Low temperature stability 1 4 weeks stable 4 weeks stable 4 weeks stable 2 4 weeks stable 4 weeks stable 4 weeks stable 3 4 weeks stable 4 weeks stable 4 weeks stable 4 4 weeks stable 4 weeks stable 4 weeks stable 5 Precipitate out Precipitate out Precipitate out 6 Precipitate out Precipitate out Precipitate out 7 Precipitate out Precipitate out Precipitate out 8 Precipitate out Precipitate out Precipitate out 9 4 weeks stable 4 weeks stable 4 weeks stable 10 4 weeks stable 4 weeks stable 4 weeks stable 11 4 weeks stable 4 weeks stable 4 weeks stable 12 4 weeks stable 4 weeks stable 4 weeks stable 13 4 weeks stable 4 weeks stable 4 weeks stable 14 4 weeks stable 4 weeks stable 4 weeks stable 15 1 day out 1 day out 1 day out 16 1 day out 1 day out 1 day out 17 1 week out 1 week out 1 day out 18 1 week out 1 week out 1 day out

[0066] Example 3:

[0067] Prepare the emulsion according to the raw material ratios and preparation methods in Table 5:

[0068] Table 5

[0069]

[0070]

[0071] Experimental results show that stable liposomes can be obtained using the preparation method of this invention. The method using a mixture of all emulsifiers in sample 19 resulted in poor product stability and precipitation in the later stages.

[0072] Example 4:

[0073] Transdermal test: Throughout the experiment, intact and undamaged pigskin of similar thickness must be selected for the transdermal test. The pigskin is cut into pieces of about 2.5cm × 2.5cm and placed between the receiving pool and the supply pool of the Franz diffusion cell, with the stratum corneum facing the supply pool.

[0074] The receiving chamber contains PBS buffer (pH = 7.4) containing 5% Tween 80;

[0075] Add 2 mL of sample to the supply tank;

[0076] The assembled Franz diffusion cell was placed in a transdermal diffusion apparatus, and the experimental conditions were set as follows: temperature 37℃, stirring speed 400rpm, and transdermal time 12h.

[0077] After transdermal treatment, the pigskin was removed from the diffusion chamber, and the remaining sample on the surface of the pigskin was wiped clean with absorbent cotton. Then, the content of Dendrobium polysaccharides in the stratum corneum was determined using the tape stripping method. 21 layers of skin on the surface of the pigskin were removed with 3M tape. The first layer was discarded, and the remaining 20 layers were placed in a 15mL centrifuge tube. 2mL of isopropanol was added to the tube, and the mixture was sonicated for 30min. After centrifugation at 5000rpm for 30min, the supernatant was filtered through a 0.22μm organic filter membrane, and the content of PDRN was determined, which is the content of PDRN in the stratum corneum.

[0078] The remaining skin surface was heated at 60℃ for 15 seconds. The viable epidermal layer was carefully cut off with a scalpel, minced, and placed in a 15mL centrifuge tube. 2mL of isopropanol was added, and the mixture was sonicated for 30 minutes. After centrifugation at 5000rpm for 30 minutes, the supernatant was filtered through a 0.22μm organic filter membrane, and the PDRN content was measured. This is the PDRN content measured in the viable epidermal layer.

[0079] After the remaining skin was cut into small pieces, it was placed in a 15 mL centrifuge tube, 2 mL of isopropanol was added, and the mixture was sonicated for 30 min. Then it was centrifuged at 5000 rpm for 30 min. The supernatant was filtered through a 0.22 μm organic filter membrane and the ergothionein content was determined, which is the PDRN content in the dermis.

[0080] Method for determining PDRN concentration: Refer to GB / T 34796-2017 for the determination of nucleic acid concentration and purity in aqueous solution.

[0081] The test samples are shown in Table 6.

[0082] Table 6

[0083]

[0084]

[0085] Transdermal test results as follows Figure 1 As shown in the results, liposomes exhibit better transdermal absorption than free PDRN aqueous solution in both the active epidermis and dermis. The increase in cationic emulsifiers reduces the polarity of liposomes, enhances their lipophilicity, and further promotes transdermal absorption of PDRN.

[0086] The synergistic effect of the samples was calculated using the Bliss model:

[0087] Bliss co-value:

[0088] ΔE=E AB -(EA+E B -EA.E B )

[0089] Where E AB Transdermal value of sample 1 - transdermal value of sample 14;

[0090] EA is the transdermal value of sample 12 minus the transdermal value of sample 14;

[0091] E B Transdermal value of sample 13 - transdermal value of sample 14;

[0092] Calculations showed that ΔE = 1.82 and ΔE > 0, indicating that the cationic emulsifier and anionic emulsifier in sample 1 had a synergistic effect.

[0093] Example 5:

[0094] Anti-aging efficacy test:

[0095] Test samples: Samples 1, 12, 13 and 14 in Example 4.

[0096] Promotion of type I collagen expression in fibroblasts:

[0097] The specific method is as follows:

[0098] Human skin fibroblasts were grown in complete culture medium (high glucose DMEM medium containing 10% FBS and 1% penicillin and antibiotics). Cells were digested with trypsin at a concentration of 1 × 10⁶ cells / mL. 5 100 μL of culture medium was seeded per well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then aspirated and the remaining medium was washed away with PBS. 100 μL of PBS was added, and the UVA intensity was set to 10 J / cm². 2 After irradiation, 100 μL of sample and DMEM were added (the control group was formed by adding DMEM after UVA irradiation, and the blank group was formed by not being irradiated with UVA). The sample concentration was prepared to be 0.05%-0.5%, and the samples were incubated in an incubator. After 24 hours, the upper culture medium was collected, centrifuged at 13000 rpm for 5 minutes, and the supernatant was collected. The content of type I collagen and type III collagen was determined using an ELISA Kit.

[0099] The synergistic effect of the samples was calculated using the Bliss model:

[0100] Bliss co-value:

[0101] ΔE=E AB -(EA+E B -EA·E B )

[0102] Where E AB The expression level of type I collagen in sample 1 is compared with the expression level of type I collagen in sample 14.

[0103] EA represents the expression level of type I collagen in sample 12 - the expression level of type I collagen in sample 14;

[0104] E B The expression level of type I collagen in sample 13 is compared with the expression level of type I collagen in sample 14.

[0105] Calculations showed that ΔE > 0 for all sample concentrations, indicating that sample 1 had a synergistic effect compared to samples 12 and 13.

[0106] Depend on Figure 2 It was found that all three samples promoted the expression of type I collagen within a concentration range of 0.05%-0.5%, exhibiting firming and anti-aging effects. Among them, the PDRN encapsulated in the lipophilic liposomes prepared in this invention showed significantly better efficacy than the ordinary liposomes prepared in samples 12 and 13, and the combination of cationic and anionic emulsifiers in this invention had a significant synergistic effect.

[0107] Promotion effect on the expression of type IV and type XVII collagen:

[0108] The specific method is as follows:

[0109] Human skin fibroblasts were grown in complete culture medium (high glucose DMEM medium containing 10% FBS and 1% penicillin and antibiotics). Cells were digested with trypsin at a concentration of 1 × 10⁶ cells / mL. 5 100 μL of culture medium was seeded per well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then aspirated and the remaining medium was washed away with PBS. 100 μL of PBS was added, and the UVA intensity was set to 10 J / cm². 2 After irradiation, 100 μL of sample and DMEM were added to each cell (the control group was formed by adding DMEM after UVA irradiation, while the blank group was formed by not being irradiated with UVA). Cells were incubated in an incubator. After 24 hours, the supernatant was collected, cells were lysed with lysis buffer, and the lysis buffer was collected. The cells were centrifuged at 13000 rpm for 5 minutes, and the supernatant was collected. The content of type IV and type VII collagen was measured using an ELISA Kit.

[0110] Human immortalized keratinocytes were grown in complete culture medium (high glucose DMEM medium containing 10% FBS and 1% penicillin and antibiotics). Cells were digested with trypsin at a concentration of 1 × 10⁶ cells / mL. 5 100 μL of culture medium was seeded per well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then aspirated and the remaining medium was washed away with PBS. 100 μL of PBS was added, and the UVB intensity was 1 J / cm². 2 After irradiation, 100 μL of sample and DMEM were added to each cell (the control group was formed by adding DMEM after UVB irradiation, and the blank group was formed by not being irradiated with UVB). Cells were incubated in an incubator. After 24 hours, the supernatant was collected, cells were lysed with lysis buffer, the lysis buffer was collected, and the cells were centrifuged at 13000 rpm for 5 minutes. The supernatant was then collected, and the content of type XVII collagen was determined using an ELISA kit.

[0111] Depend on Figure 3 It can be seen that the test samples promoted the expression of type IV and type XVII collagen in the concentration range of 0.05%-0.5%, and had the effect of basement membrane repair.

[0112] Example 6:

[0113] DNA repair:

[0114] Test sample: Sample 1 in Example 1.

[0115] Human immortalized keratinocytes were grown in complete culture medium (high-glucose DMEM medium containing 10% FBS and 1% antibiotics). Cells were digested with trypsin and seeded at 2 × 10⁵ cells / well in 12-well plates (1 mL per well), and incubated at 37°C in a 5% CO₂ incubator for 24 h. The culture medium was aspirated, and residual medium was washed away with PBS. 1 mL of PBS was added, and the UVB intensity was 1 J / cm². 2 After irradiation, 1 mL of sample and DMEM were added (the control group was formed by adding DMEM after UVB irradiation, while the blank group was formed by not being irradiated with UVB). The samples were incubated in an incubator. After 24 hours, staining was performed using a DNA damage detection kit (γ-H2AX immunofluorescence assay), and images were taken under a fluorescence microscope.

[0116] When DNA is attacked, especially after a double-strand break (DSB), serine 139 of histone H2AX is rapidly phosphorylated to generate phosphorylated H2AX, i.e., γ-H2AX. γ-H2AX accumulates in large quantities at DSB sites, forming focal points that participate in DNA damage repair, and the number of γ-H2AX focal points is directly proportional to the number of DSBs. Current research indicates that γ-H2AX is a specific biomarker for characterizing DNA damage and can serve as a specific indicator of genotoxic effects.

[0117] Figure 4 The results showed that the PDRN liposomes prepared in this invention have significant effects on DNA repair.

[0118] Example 7:

[0119] The serum formula is shown in Table 8:

[0120] Table 8

[0121]

[0122] The essence was divided into three samples: room temperature samples, heat-resistant samples, and cold-resistant samples. Room temperature samples were stored away from light, heat-resistant samples were placed in a constant temperature oven at (45±1℃), and cold-resistant samples were placed in a -18℃ refrigerator. After 3 months, the appearance, color, and odor of the samples were observed. The results are shown in the table below.

[0123] The formulation stability test results are shown in Table 7:

[0124] Table 7

[0125]

[0126] Experimental results show that the sample provided by this invention, after being combined with the matrix, did not show significant changes after being placed at room temperature, high temperature, and low temperature for 3 months, which can meet the needs of practical applications.

[0127] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A lipophilic liposome containing polydeoxyribonucleotides, characterized in that: By weight, including, 2. The lipophilic liposome containing polydeoxyribonucleotides according to claim 1, characterized in that: By weight, including, 3. The lipophilic liposome containing polydeoxyribonucleotides according to claim 1 or 2, characterized in that: The anionic emulsifier includes anionic peptide emulsifiers.

4. The lipophilic liposome containing polydeoxyribonucleotides according to claim 1 or 2, characterized in that: The cationic emulsifier is lauroyl arginine ethyl ester hydrochloride and / or PCA coconut arginine ethyl ester salt.

5. The lipophilic liposome containing polydeoxyribonucleotides according to claim 3, characterized in that: The anionic emulsifier is sodium di(lauramide-glutamine)lysine and / or sodium subtilisin.

6. The lipophilic liposome containing polydeoxyribonucleotides according to claim 1 or 2, characterized in that: The phospholipid is lecithin and / or hydrogenated lecithin.

7. The lipophilic liposome containing polydeoxyribonucleotides according to claim 1 or 2, characterized in that: The polyols include one or more of glycerol, 1,3-propanediol, 1,3-butanediol, 1,2-hexanediol, 1,2-pentanediol, isopentanediol, PEG-400, PEG-1500, and sorbitol.

8. The method for preparing lipophilic liposomes containing polydeoxyribonucleotides according to claim 1, characterized in that: Includes the following steps, Anionic emulsifier, lecithin and polyol were mixed, heated and stirred until homogeneous, and then cooled to room temperature to obtain phase A1. The cationic emulsifier was dissolved in water and slowly added dropwise to phase A1 to obtain a transparent solution phase A2. Polydeoxyribonucleotides were dissolved in water to obtain phase B; Phase B and phase A2 were mixed evenly and then mixed using high-pressure microfluidic mixing to obtain lipophilic liposomes containing polydeoxyribonucleotides.

9. The method for preparing lipophilic liposomes containing polydeoxyribonucleotides according to claim 8, characterized in that: The heating and stirring process includes heating to 75-85°C and stirring until uniform; the high-pressure microjet has a pressure of 900-1000 bar.

10. The application of the lipophilic liposomes containing polydeoxyribonucleotides as described in claim 1 in the preparation of cosmetics.

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