Chlorella PDRN, chlorella PDRN liposome, and preparation method and application thereof

Abstract

The present application relates to the field of biological extraction and medical technology, and particularly relates to a chlorella PDRN, chlorella PDRN liposome and a preparation method and application thereof. The chlorella PDRN is obtained by a process of wall breaking, purification and fragmentation, and has high purity and specific molecular weight. The liposome is a liposome encapsulation technology based on silk fibroin, and can significantly improve the stability, water solubility and transdermal performance of the PDRN by constructing a core-shell structure carrier, solve the problems of easy degradation and low transdermal efficiency, and significantly improve the bioavailability and efficacy of the PDRN, and has a wide application prospect in the field of skin care and repair.

Description

Technical Field

[0001] This invention relates to the fields of bio-extraction and pharmaceutical technology, and in particular to a Chlorella PDRN, Chlorella PDRN liposomes, their preparation methods and applications. Background Technology

[0002] PDRN (polydeoxyribonucleic acid) is a mixture of DNA fragments of varying lengths obtained by degrading DNA using specific techniques; in other words, it consists of small fragments of DNA. Algal PDRN and its liposomes are widely used in the biomedical field due to their plant origin and their low allergenicity and high biocompatibility.

[0003] Currently, the main methods for extracting PDRN from algae involve a combination of crushing, salting out, and alcohol precipitation. For example, Chinese Patent No. CN121343981A discloses a method for obtaining high-purity PDRN from microalgae through a combination of anionic surfactant treatment, ultra-high pressure crushing, high-concentration metal salt salting out, and alcohol precipitation. Meanwhile, Chinese Patent No. CN120699959A discloses a method for extracting and preparing small-molecule PDRN from algae and its application, which involves optimizing the solid-liquid ratio of the algal solution and using repeated freeze-thaw cell disruption combined with ammonium acetate salting out and alcohol precipitation to extract small-molecule PDRN from algae. While these methods can extract high-purity algal PDRN, the resulting PDRN fragments are relatively random and non-specific.

[0004] Meanwhile, due to the hydrophilicity of PDRN, if extracted PDRN is directly used in the biopharmaceutical field, not only is the transdermal absorption efficiency extremely low, but it is also easily degraded by nucleases in vivo. To overcome this defect, the commonly used method is to construct core-shell liposomes using phospholipids as the shell to improve the stability and delivery efficiency of PDRN nucleic acid drugs. For example, Chinese patent number CN119331936BPDRN, supramolecular liposomes containing this PDRN, and their preparation method and uses, discloses supramolecular composite nanoparticles of PDRN formed by electrostatic interaction using fatty acids extracted from marine microalgae as lipid carriers. However, due to the limitations of the core material and the core-shell connection method, the PDRN liposomes prepared by the existing methods have insufficient structural stability and bring about a contradiction between transdermal and sustained release. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose a Chlorella PDRN, Chlorella PDRN liposomes, their preparation method and application, in order to solve the problems that the algal PDRN fragments extracted in the prior art are relatively random and nonspecific, and that the prepared PDRN liposomes have poor structural stability and bring about the contradiction between transdermal and sustained release.

[0006] To achieve the above objectives, the present invention provides a Chlorella PDRN, which is extracted using a method comprising the following steps:

[0007] Raw material pretreatment: Select Chlorella strain Sorokinae, culture it in a sterile fermentation environment for 5-7 days, centrifuge to obtain algal mud, and then perform sterile washing and drying treatment on the algal mud to obtain algal powder.

[0008] The algae powder is added to a buffer solution to obtain an algae powder suspension. The algae powder suspension is subjected to high-pressure homogenization 2-3 times and ultrasonic disruption 5-30 minutes to obtain a disrupted suspension. The disrupted suspension is centrifuged and filtered to obtain a crude extract.

[0009] Extraction and purification: Add surfactant and protease to the crude extract, mix and extract for 1-2 hours, then remove impurities, purify and reconstitute to obtain pure extract;

[0010] Enzymatic digestion and fragmentation: Add restriction endonuclease solution to the purified extract, mix well, and carry out the enzyme digestion reaction at a constant temperature of 25~37℃ for 2~4h to obtain PDRN fragments of specific molecular weight.

[0011] The PDRN fragments of a specific molecular weight were purified by ultrafiltration, chromatography, dialysis and freeze-drying to obtain powdered Chlorella sologenophylla PDRN.

[0012] Secondly, the present invention also provides a Chlorella PDRN liposome, wherein the liposome has a core-shell structure, wherein the core is composed of a complex formed by Chlorella PDRN and silk fibroin through electrostatic interaction, and the shell is a phospholipid bilayer surrounding the core.

[0013] The mass ratio of Chlorella PDRN to silk fibroin is (1:1) to (1:3);

[0014] The phospholipid bilayer is composed of lecithin and cholesterol in a mass ratio of (2:1) to (5:1).

[0015] Furthermore, this invention also provides a method for preparing Chlorella PDRN liposomes, which are prepared by an aqueous self-assembly method, the aqueous self-assembly comprising the following steps:

[0016] The powdered Chlorella sologenophylla PDRN was dissolved in sterile, enzyme-free water, and then a silk fibroin solution was added to adjust the pH to 5.5-6.5. The mixture was stirred until homogeneous to obtain the aqueous phase.

[0017] Lecithin and cholesterol were dissolved in anhydrous ethanol to obtain an organic phase;

[0018] Under stirring conditions, the organic phase is added dropwise to the aqueous phase until the volume ratio of the organic phase to the aqueous phase is 1:9. The mixture is stirred until the organic phase is evenly dispersed, and then subjected to rotary evaporation and ultrasonic homogenization to obtain a crude liposome suspension.

[0019] The crude liposome suspension was centrifuged, filtered, and concentrated to a concentration of 1-5 mg / mL to obtain a liposome concentrate.

[0020] Add a freeze-drying protectant to the liposome concentrate, mix well, and freeze-dry to obtain Chlorella PDRN liposomes.

[0021] Finally, the present invention also provides an application of Chlorella PDRN liposomes for the preparation of cosmetics, skin care products or topical drug formulations with anti-aging, skin repair, anti-inflammatory or tissue regeneration effects.

[0022] The beneficial effects of this invention are:

[0023] (1) The extraction process of Chlorella PDRN is based on "efficient cell disruption, precise purification, and controllable fragmentation". By combining cell disruption, enzymatic fragmentation and column chromatography purification, high-purity PDRN products with specific molecular weights can be obtained. The final Chlorella PDRN has the following properties: ① HPLC or agarose gel electrophoresis shows that the purity of a single band / single main peak is ≥90%; ② Polyacrylamide gel electrophoresis (PAGE) shows that the molecular weight is concentrated in the range of 50~300bp; ③ UV spectrophotometry shows that A260 / A280 = 1.8~2.0 and the nucleic acid content is ≥90%; ④ Sterile and free of endotoxins (<0.1EU / mg); ⑤ After 14 days of accelerated extraction at 45℃, there is no significant change in purity and molecular weight.

[0024] (2) The positively charged amino acids such as lysine and arginine contained in the silk fibroin molecules of Chlorella PDRN liposomes can interact electrostatically with the negatively charged phosphate groups in the PDRN molecules to form a stable PDRN-silk fibroin complex. Subsequently, phospholipid molecules self-assemble in the aqueous phase to form a bilayer that encapsulates the complex and forms liposomes. Among them, silk fibroin can not only enhance the binding force between PDRN and liposomes, but also improve the biocompatibility and transdermal performance of liposomes. The phospholipid bilayer can physically isolate the external environment, protect the activity of PDRN, and reduce degradation. By constructing a core-shell structure carrier, the stability, water solubility and transdermal performance of PDRN are significantly improved, solving the problems of easy degradation and low transdermal efficiency, and laying the foundation for subsequent applications. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this invention 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 for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of Chlorella sorokinense culture according to an embodiment of the present invention;

[0027] Figure 2 This is an electrophoretic comparison of total PDRN extracted in lane 2 and PDRN after enzyme digestion in an embodiment of the present invention (lane 1);

[0028] Figure 3 This is a particle size distribution diagram of the liposomes prepared in accordance with the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0030] Explanation of technical terms:

[0031] Chlorella sorokinina;

[0032] PDRN: Polydeoxyribonucleotide;

[0033] BG11: Blue-Green11;

[0034] EDTA: Ethylenediaminetetraacetic acid;

[0035] Tris-HCl: Tris(hydroxymethyl)aminomethane hydrochloride

[0036] DEAE: Diethylaminoethyl;

[0037] PDI: Polymer Dispersion Index, also known as Polydispersion Index or Polydispersion Coefficient.

[0038] In order to address the problems of random and non-specific algal PDRN fragments extracted by existing technologies and the poor structural stability of the prepared PDRN liposomes, which leads to the contradiction between transdermal and sustained release, this invention provides Chlorella PDRN, Chlorella PDRN liposomes, their preparation method and applications.

[0039] As one possible implementation method, Embodiment 1 of the present invention provides Chlorella PDRN, which is extracted using a method including the following steps. The entire extraction process of Chlorella PDRN requires a sterile environment to avoid nucleic acid degradation.

[0040] For raw material pretreatment, Sorokin Chlorella strain was selected and cultured under aseptic conditions at a temperature of 25-28℃ and a light intensity of 3000-5000 lux for 5-7 days with aeration. After centrifugation, algal mud was obtained. The algal mud was then aseptically washed and dried to obtain algal powder.

[0041] Specifically, algal strain selection: Select Chlorella strains with high nucleic acid content to ensure that the basic nucleic acid content in the Chlorella strains is ≥8%, providing a guarantee for subsequent extraction.

[0042] Culture medium and culture conditions: such as Figure 1 As shown, BG11 liquid medium was used for culture in a sterile fermentation environment, with the temperature controlled at 25℃, light intensity at 4000 lux, and aeration maintained for 6 days until the cell density reached the target level. At this point, the nucleic acid content of the algal cells reaches its peak.

[0043] Harvesting and pretreatment: The algal mud was collected by centrifugation at 8000 rpm for 10 min. The algal mud was washed twice with sterile deionized water to remove culture medium residue and impurities. The algal powder was then obtained by freeze-drying or spray drying. The algal powder was sealed and stored to prevent moisture absorption and degradation.

[0044] The algae powder is added to a buffer solution to obtain an algae powder suspension. The algae powder suspension is subjected to high-pressure homogenization 2-3 times at a pressure of 800-1000 bar and ultrasonic disruption for 5-30 minutes at a power of 100-300 W to obtain a disrupted suspension. The disrupted suspension is centrifuged and filtered to obtain a crude extract.

[0045] Specifically, the cell walls of Chlorella sorokinensis are tough, and a single cell disruption method is inefficient. A combination of high-pressure homogenization and ultrasonic disruption is used to ensure a cell disruption rate of ≥95%, thus fully releasing intracellular nucleic acids.

[0046] Suspension preparation: Algae powder is suspended in Tris-HCl buffer (pH 8.0) containing 0.1% EDTA at a material-to-liquid ratio of 1:40. EDTA can complex metal ions, inhibit nuclease activity, and protect nucleic acids from degradation, thus obtaining an algae powder suspension.

[0047] Combined cell wall disruption: The algal powder suspension is first subjected to high-pressure homogenization at 1000 bar, and the high-pressure homogenization is repeated 3 times to break the cell wall skeleton; then ultrasonic disruption is performed at 300 W for 10 min to further break the cell debris and release nucleic acids; the entire process is carried out in an ice bath to avoid nucleic acid denaturation caused by high temperature.

[0048] Sludge removal and filtration: Centrifuge the broken suspension at 12000 rpm for 15 min to remove cell debris and insoluble impurities, and take the supernatant; then filter it through a 0.45 μm filter membrane to obtain the crude extract for later use.

[0049] Extraction and purification: Add surfactant and protease to the crude extract, mix and extract for 1-2 hours, then remove impurities, purify and reconstitute to obtain pure extract.

[0050] Specifically, the crude extract contains impurities such as proteins, polysaccharides, and lipids, and requires a series of purification steps to obtain high-purity total DNA in preparation for subsequent fragmentation.

[0051] Nucleoprotein lysis: Add 1% SDS (sodium dodecyl sulfate) and 0.5 mg / mL proteinase K to the crude extract and incubate at 55°C for 1.5 h. SDS can disrupt the cell membrane and nuclear membrane, and proteinase K can degrade proteins bound to nucleic acids, releasing free DNA.

[0052] Protein removal: Add an equal volume of a solution of phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 to the nucleoprotein lysis buffer, shake vigorously for 10 min, allow to stand for layering, centrifuge at 12000 rpm for 15 min, and take the upper aqueous phase containing DNA; repeat the extraction twice to completely remove protein impurities.

[0053] DNA precipitation: Add 0.6 times the volume of isopropanol to the aqueous phase, let stand at -20℃ for 30 min to allow DNA to precipitate fully; then centrifuge at 12000 rpm for 15 min, discard the supernatant to obtain the precipitate, wash the precipitate twice with 75% ethanol to remove residual salts and organic solvents; dry at room temperature to obtain total DNA precipitate.

[0054] Reconstitution: Dissolve the total DNA precipitate in sterile, enzyme-free water, adjust the concentration to 10-20 mg / mL, and set aside. Avoid nuclease contamination throughout the process to obtain a pure extract.

[0055] Enzymatic digestion and fragmentation: Add restriction endonuclease solution to the purified extract, mix well, and carry out the enzyme digestion reaction at 37℃ for 2-4 hours to obtain PDRN fragments of specific molecular weight.

[0056] Specifically, total DNA is a long-chain nucleic acid that needs to be cleaved by restriction endonucleases to obtain PDRN fragments with a molecular weight of 50-300 bp. This range of fragments has the best transdermal permeability and biological activity.

[0057] Enzyme digestion system preparation (500μL): Take 10mg of total DNA purified extract, 50U of restriction endonuclease (EcoRI or BamHI, HindIII, and SmaI are preferred as they have high digestion efficiency and uniform fragment distribution), 50μL of 10× enzyme digestion buffer, and add sterile enzyme-free water to 500μL. Mix gently.

[0058] Enzyme digestion reaction: Incubate the enzyme digestion reaction at 37℃ for 3 hours to ensure that the long DNA chain is fully digested; after the reaction is completed, incubate in a water bath at 65℃ for 10 minutes to inactivate the endonuclease and terminate the reaction to avoid over-digestion and obtain PDRN fragments of a specific molecular weight.

[0059] The PDRN fragments of a specific molecular weight were purified by ultrafiltration, chromatography, dialysis and freeze-drying to obtain powdered Chlorella sologenophylla PDRN.

[0060] Specifically, ultrafiltration desalting involves centrifuging PDRN fragments of a specific molecular weight using a 10kDa ultrafiltration tube to remove small molecule impurities, salts, and inactivated enzymes from the enzyme digestion system, thereby concentrating the PDRN solution and increasing its concentration.

[0061] Column chromatography enrichment: PDRN main peak components were collected by DEAE-cellulose ion exchange chromatography with gradient NaCl elution to further remove impurities such as nucleic acids and small molecules.

[0062] Desalting and sterilization: The enriched PDRN solution was dialyzed for 24 hours using a 3kDa dialysis bag to remove residual salts; then it was sterilized by filtration through a 0.22μm filter membrane to obtain a sterile PDRN solution.

[0063] Freeze-drying preservation: Freeze-dry the sterile PDRN solution to obtain a white powdered PDRN product, seal and store in a refrigerator at 4°C, avoiding light and moisture absorption.

[0064] The extracted powdered Chlorella sologenophylla PDRN was subjected to the following performance tests:

[0065] ① Purity: Detected by HPLC or agarose gel electrophoresis, single band / single main peak, purity ≥90%; ② If Figure 2 As shown, molecular weight: detected by polyacrylamide gel electrophoresis (PAGE), the fragments are concentrated in the range of 50~300bp;

[0066] ③ Nucleic acid content is shown in Table 1: Detected by ultraviolet spectrophotometry, A260 / A280 = 1.8~2.0, nucleic acid content ≥90%;

[0067] Table 1. Nucleic acid content detection results

[0068]

[0069] ④ Microbiological: Sterile, endotoxin-free (<0.1 EU / mg);

[0070] ⑤ Stability: After 14 days of accelerated testing at 45℃, there was no significant change in purity or molecular weight.

[0071] As one possible implementation, Embodiment 1 of the present invention provides a Chlorella PDRN liposome, wherein the liposome has a core-shell structure, the core is composed of a complex formed by Chlorella PDRN and silk fibroin through electrostatic interaction, and the shell is a phospholipid bilayer surrounding the core.

[0072] The mass ratio of Chlorella PDRN to silk fibroin is (1:1) to (1:3);

[0073] The phospholipid bilayer is composed of lecithin and cholesterol in a mass ratio of (2:1) to (5:1).

[0074] Specifically, the positively charged amino acids such as lysine and arginine in silk fibroin molecules can electrostatically interact with the negatively charged phosphate groups in PDRN molecules to form a stable PDRN-silk fibroin complex. Subsequently, phospholipid molecules self-assemble in the aqueous phase to form a bilayer that encapsulates the complex, forming liposomes. Silk fibroin not only enhances the binding force between PDRN and liposomes but also improves the biocompatibility and transdermal properties of liposomes. The phospholipid bilayer physically isolates the external environment, protecting PDRN activity and reducing degradation.

[0075] As one possible implementation method, Example 1 of the present invention provides a method for preparing Chlorella PDRN liposomes, which is prepared by an aqueous self-assembly method.

[0076] Principle: Silk fibroin has good biocompatibility, biodegradability and positive charge properties, and can form a stable complex with negatively charged PDRN. By combining with phospholipid bilayer to construct liposomes, a core-shell structure of "PDRN-silk fibroin complex as core and phospholipid bilayer as shell" is formed, which can effectively protect PDRN from degradation, while improving its water solubility and transdermal efficiency. The encapsulation process adopts aqueous self-assembly method, which is simple to operate and has a high encapsulation rate, making it suitable for laboratory and pilot production.

[0077] (1) Experimental reagents

[0078] Core ingredients: Chlorella PDRN freeze-dried powder (purity ≥90%), silk fibroin (purity ≥95%), lecithin (cosmetic grade), cholesterol (reagent grade);

[0079] Solvents and excipients: anhydrous ethanol (analytical grade), sterile enzyme-free water, trehalose (protectant), Tris-HCl buffer (pH 5.5–6.5);

[0080] Other: EDTA-2Na (chelating agent), used to inhibit liposome degradation caused by metal ions.

[0081] (2) Experimental equipment

[0082] Magnetic stirrer, rotary evaporator, ultrasonic homogenizer, high-speed centrifuge, laser particle size analyzer, HPLC, sterile clean bench, freeze dryer.

[0083] The aqueous phase self-assembly includes the following steps:

[0084] The powdered Chlorella pulmonale PDRN was dissolved in sterile, enzyme-free water, and then a silk fibroin solution was added to adjust the pH to 5.5-6.5. The mixture was stirred until homogeneous to obtain the aqueous phase.

[0085] Specifically, take 10 mg of Chlorella pulmonale PDRN lyophilized powder, dissolve it in 10 mL of sterile enzyme-free water, and stir for 5 min (300 rpm) until completely dissolved to obtain a PDRN solution; add silk fibroin solution to the PDRN solution, controlling the mass ratio of PDRN to silk fibroin to be 1:3, stir for 10 min until a homogeneous solution is formed; adjust the pH to 6 with Tris-HCl buffer, let stand for 5 min, and obtain the aqueous phase.

[0086] This step is to provide a suitable environment for the electrostatic binding of PDRN and silk fibroin, ensuring the formation of a stable complex. The pH must be strictly controlled; deviations from this range will reduce the binding force and affect subsequent encapsulation effects. A pH between 5.5 and 6.5 is suitable for the performance of the final Chlorella PDRN liposome product.

[0087] Lecithin and cholesterol were dissolved in anhydrous ethanol to obtain an organic phase.

[0088] Specifically, weigh the raw materials according to the ratio of lecithin to cholesterol = 2:1 (w / w); dissolve the weighed raw materials in anhydrous ethanol, stir until completely dissolved, and prepare an organic phase solution with a concentration of 10 mg / mL.

[0089] This step is to construct the phospholipid bilayer framework of the liposomes; the ratio of lecithin to cholesterol needs to be precise, as cholesterol can increase the stability of liposomes, but too high a ratio will affect transdermal permeability.

[0090] Under stirring conditions, the organic phase is added dropwise to the aqueous phase until the volume ratio of the organic phase to the aqueous phase is 1:9. The mixture is stirred until the organic phase is evenly dispersed, and then subjected to rotary evaporation and ultrasonic homogenization to obtain a crude liposome suspension.

[0091] Specifically, the organic phase was added dropwise to the aqueous phase, with the organic phase:aqueous phase ratio controlled at 1:9 (v / v) and the dropping rate at 1 drop / second. The mixture was magnetically stirred at 25°C for 30 minutes (400 rpm) to ensure uniform dispersion of phospholipid molecules. The dispersed mixture was then transferred to a rotary evaporator and evaporated at 35°C under reduced pressure for 15 minutes to remove anhydrous ethanol. After removing the ethanol, 5 mL of sterile, enzyme-free water was added, and the mixture was sonicated for 10 minutes using an ultrasonic homogenizer (300W) under ice bath conditions to obtain a crude liposome suspension.

[0092] This step is to enable phospholipid molecules to self-assemble into a bilayer that encapsulates the PDRN-silk fibroin complex; ice bath sonication is used to avoid high temperatures damaging the liposome structure; rotary evaporation requires temperature control to prevent PDRN degradation.

[0093] The crude liposome suspension was centrifuged, filtered, and concentrated to a concentration of 1-5 mg / mL to obtain a liposome concentrate.

[0094] Specifically, the crude liposome suspension is centrifuged at 10,000 rpm for 10 min to remove unencapsulated PDRN and silk fibroin; the supernatant is taken, filtered through a 0.22 μm filter membrane for sterilization, and then concentrated by centrifugation using a 3 kDa ultrafiltration tube to concentrate the crude liposome suspension to a concentration of 1-5 mg / mL, thus obtaining a concentrated liposome solution.

[0095] This step is to obtain high-purity liposomes, remove free impurities, and ensure encapsulation efficiency; the centrifugation speed and time must be precise to avoid liposome rupture.

[0096] Add a freeze-drying protectant to the liposome concentrate, mix well, and freeze-dry to obtain Chlorella PDRN liposomes.

[0097] Specifically, 5%~10% trehalose is added to the liposome concentrate as a freeze-drying protectant and stirred evenly; then pre-frozen at -80℃ for 2 hours, transferred to a vacuum freeze dryer, and freeze-dried for 12 hours to obtain white and loose silk fibroin liposome-PDRN freeze-dried powder, namely Chlorella PDRN liposome powder; sealed in packaging and stored at 4℃ away from light.

[0098] This step is to improve the storage stability of liposomes, prevent liposome rupture during freeze-drying, and ensure reconstitution. The amount of trehalose used must be appropriate; too much will affect the purity of the product, while too little will not provide protection.

[0099] The prepared Chlorella PDRN liposomes were subjected to the following performance tests:

[0100] (1) Particle size and uniformity, such as Figure 3As shown: Laser particle size analyzer detection, D50=100~300nm (optimal range for transdermal absorption), PDI<0.2, ensuring good uniformity of liposomes and avoiding aggregation.

[0101] (2) Encapsulation rate: The content of PDRN before and after encapsulation was determined by HPLC. The encapsulation rate was ≥85%. If the encapsulation rate was too low, PDRN would be easily degraded and its activity would be reduced.

[0102] (3) Stability: ① Accelerated stability: After being sealed at 45℃ for 14 days, the particle size and encapsulation rate changed by <10%, with no precipitation or stratification; ② Long-term stability: After being stored at 4℃ in the dark for 12 months, there were no significant changes in any of the indicators; ③ Resolubility: After the lyophilized powder was added to sterile enzyme-free water, it completely dissolved within 5 minutes without precipitation.

[0103] (4) Transdermal permeability: In vitro transdermal test (Franz diffusion cell) showed a transdermal rate of ≥60% after 24 hours, ensuring that liposomes can effectively promote the penetration of PDRN into the deep layers of the skin.

[0104] (5) Microbiology: Sterility test, no bacterial, mold, or yeast contamination, and no endotoxins.

[0105] As one possible implementation method, Example 2 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the ratio of organic phase to water is 1:5.

[0106] As one possible implementation method, Example 3 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the ratio of organic phase to water is 1:2.

[0107] As one possible implementation method, Example 4 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the mass ratio of Chlorella PDRN to silk fibroin is 1:1.

[0108] As one possible implementation method, Example 5 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the mass ratio of Chlorella PDRN to silk fibroin is 1:3.

[0109] As one possible implementation method, Example 6 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the phospholipid bilayer is composed of lecithin and cholesterol in a mass ratio of 3:1.

[0110] As one possible implementation method, Example 7 of the present invention provides a Chlorella PDRN liposome, which is the same as Example 1, except that the phospholipid bilayer is composed of lecithin and cholesterol in a mass ratio of 5:1.

[0111] To verify the significant effects of the technical solutions in the embodiments of the present invention, the present invention also provides the following comparative examples:

[0112] Comparative Example 1 provides a Chlorella PDRN liposome, which is the same as Example 1, except that it does not contain silk fibroin and has no core-shell structure.

[0113] Comparative Example 2 provides a Chlorella PDRN liposome, which is the same as in Example 1, except that chitosan is used instead of silk fibroin.

[0114] Comparative Example 3 provides a Chlorella PDRN liposome, which is the same as in Example 1, except that it does not have a phospholipid shell.

[0115] The liposomes prepared in the above examples and comparative examples were subjected to relevant performance tests, and the test results are shown in Table 2.

[0116] Table 2. Liposome performance test results

[0117]

[0118] The test results above show that the liposomes prepared in Example 1 have the best performance. Comparison of the data from each example and the comparative example shows that silk fibroin plays a decisive role in the structural integrity and encapsulation efficiency of the liposomes. This is because silk fibroin self-assembles into a β-sheet conformation under specific pH conditions, forming a dense and compact core with PDRN through electrostatic interactions. Simultaneously, due to the abundance of hydroxyl and amino groups in the silk fibroin side chains, it can form a dense hydrogen bond network with the polar bonds of phospholipids, firmly "anchoring" the phospholipid shell to the core surface. This provides a regular and stable surface for the orderly arrangement and tight adsorption of the phospholipid bilayer, thus achieving small particle size and high encapsulation efficiency. The performance results of using chitosan instead of silk fibroin further confirm that not any positively charged polymer can achieve the same effect.

[0119] The synergistic mechanical effect of the "hard core-soft shell" is the structural basis for achieving efficient transdermal absorption. Transdermal efficiency requires the carrier to possess both the deformable flexibility to penetrate the stratum corneum and the mechanical rigidity to maintain structural integrity. In this invention, the hard core, composed of silk fibroin, provides mechanical support and prevents carrier rupture during transdermal deformation, while the soft shell, composed of phospholipids / cholesterol, endows the carrier with sufficient deformability. In contrast, shell-less liposomes lack the deformation lubrication of the phospholipid layer and struggle to penetrate the stratum corneum; coreless liposomes, on the other hand, experience structural collapse during deformation, leading to leakage of their contents.

[0120] The Chlorella PDRN liposome core-shell structure prepared by this invention, upon entering the skin, is first degraded by skin esterases into the shell-phospholipid bilayer, exposing the silk fibroin-PDRN core. Subsequently, skin proteases slowly degrade the silk fibroin, releasing PDRN fragments, thereby forming a storage and sustained long-lasting release of product performance in the dermis. Shell-less liposomes are directly exposed to the enzymatic environment, resulting in rapid degradation and clearance of PDRN. While coreless liposomes possess some sustained-release capacity, their dermal retention effect is far inferior to that of the core-shell structure of this invention due to the lack of hard core protection and sustained-release regulation.

[0121] The above experimental results fully demonstrate that the silk fibroin core-shell structured liposomes of the present invention achieve a unified high encapsulation rate, small particle size, high transdermal rate and long-lasting dermal retention through the synergistic effect of multiple mechanisms such as silk fibroin self-assembly into a core, hydrogen bond anchoring, mechanical synergy and hierarchical time-sequential degradation.

[0122] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in the details for the sake of brevity.

[0123] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A Chlorella PDRN, characterized in that, The following steps were used to extract the following: Raw material pretreatment: Select Chlorella strain Sorokinae, culture it in a sterile fermentation environment for 5-7 days, centrifuge to obtain algal mud, and then perform sterile washing and drying treatment on the algal mud to obtain algal powder. The algae powder is added to a buffer solution to obtain an algae powder suspension. The algae powder suspension is subjected to high-pressure homogenization 2-3 times and ultrasonic disruption 5-30 minutes to obtain a disrupted suspension. The disrupted suspension is centrifuged and filtered to obtain a crude extract. Extraction and purification: Add surfactant and protease to the crude extract, mix and extract for 1-2 hours, then remove impurities, purify and reconstitute to obtain pure extract; Enzymatic digestion and fragmentation: Add restriction endonuclease solution to the purified extract, mix well, and carry out the enzyme digestion reaction at a constant temperature of 25~37℃ for 2~4h to obtain PDRN fragments of specific molecular weight. The PDRN fragments of a specific molecular weight were purified by ultrafiltration, chromatography, dialysis and freeze-drying to obtain powdered Chlorella sologenophylla PDRN.

2. The Chlorella PDRN according to claim 1, characterized in that, The raw material pretreatment was carried out by aerated culture using BG11 liquid medium at a temperature of 25-28°C and a light intensity of 3000-5000 lux.

3. The Chlorella PDRN according to claim 1, characterized in that, The buffer solution used in the disruption process is a Tris-HCl buffer solution containing 0.1% EDTA, and the ratio of algal powder to the buffer solution is 1:

40.

4. The Chlorella PDRN according to claim 1, characterized in that, The pressure for high-pressure homogenization during the crushing process is 800~1000 bar, and the ultrasonic power is 100~300W.

5. The Chlorella PDRN according to claim 1, characterized in that, The surfactant used in the extraction and purification process is sodium dodecyl sulfate at a concentration of 1% to 3%, and the protease is proteinase K at a concentration of 0.5 to 1 mg / mL.

6. The Chlorella PDRN according to claim 1, characterized in that, The restriction endonuclease solution used in the enzyme digestion fragmentation contains 50-100 U of restriction endonuclease and 50-100 μL of 10× digestion buffer, wherein the restriction endonuclease is at least one of EcoRI, BamHI, HindIII, and SmaI.

7. The Chlorella PDRN according to claim 1, characterized in that, The specific molecular weight is 50~300bp.

8. A liposome containing Chlorella PDRN, characterized in that, The liposome comprises Chlorella PDRN as described in any one of claims 1 to 7, wherein the liposome has a core-shell structure, wherein the core is composed of a complex formed by Chlorella PDRN and silk fibroin through electrostatic interaction, and the shell is a phospholipid bilayer surrounding the core. The mass ratio of Chlorella PDRN to silk fibroin is (1:1) to (1:3); The phospholipid bilayer is composed of lecithin and cholesterol in a mass ratio of (2:1) to (5:1).

9. A method for preparing Chlorella PDRN liposomes according to claim 8, characterized in that, The aqueous phase self-assembly method is used to prepare the product, which includes the following steps: The powdered Chlorella sologenophylla PDRN was dissolved in sterile, enzyme-free water, and then a silk fibroin solution was added to adjust the pH to 5.5-6.

5. The mixture was stirred until homogeneous to obtain the aqueous phase. Lecithin and cholesterol were dissolved in anhydrous ethanol to obtain an organic phase; Under stirring conditions, the organic phase is added dropwise to the aqueous phase until the volume ratio of the organic phase to the aqueous phase is (1:2) to (1:9). The mixture is stirred until the organic phase is evenly dispersed, and then subjected to rotary evaporation and ultrasonic homogenization to obtain a crude liposome suspension. The crude liposome suspension was centrifuged, filtered, and concentrated to a concentration of 1-5 mg / mL to obtain a liposome concentrate. Add a freeze-drying protectant to the liposome concentrate, mix well, and freeze-dry to obtain Chlorella PDRN liposomes.

10. An application of the Chlorella PDRN liposome according to claim 8, characterized in that, The PDRN liposomes described above are used to prepare cosmetics, skin care products, or topical drug formulations with anti-aging, skin repair, anti-inflammatory, or tissue regeneration effects.

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