A flexible liposome microcapsule with resistance to degradation polydeoxyribonucleic acid and its preparation method

Abstract

This invention provides a flexible liposome microcapsule with anti-degradation polydeoxyribonucleotides, composed of the following components in parts by weight: an aqueous core phase of 82-94 parts and a liposome membrane phase of 6-18 parts. The aqueous core phase comprises the following components in parts by weight: PDRN 0.1-1.5 parts, an anti-degradation modifier 0.02-0.8 parts, and water 81-92 parts. The liposome membrane phase comprises the following components in parts by weight: phospholipids 0.5-8 parts, a flexibility modifier 0.2-2.0 parts, a membrane stabilizer 0.1-0.5 parts, a humectant 3-15 parts, and an antioxidant 0.2-1.0 parts. The anti-degradation modifier is selected from one or more of EDTA-2Na, EDTA, sodium phytate, and sodium pyrophosphate. This invention belongs to the field of medical materials technology, fundamentally blocking the hydrolysis of PDRN by nucleases, significantly prolonging its activity maintenance time, and improving the deformability and transdermal efficiency of liposomes.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and particularly relates to a flexible liposome microcapsule with anti-degradation polydeoxyribonucleotides and its preparation method. Background Technology

[0002] Polydeoxyribonucleotide (PDRN) is an active ingredient extracted from salmon reproductive cells. Its base sequence is highly homologous to human DNA. It has clear effects such as promoting fibroblast proliferation, accelerating collagen and elastin synthesis, repairing the skin barrier, inhibiting inflammatory cascade reactions, and promoting angiogenesis. It is widely used in the medical aesthetics and skin care field.

[0003] However, PDRN is often limited by three inherent defects in clinical research and practical application: (1) easy degradation and inactivation, short action cycle: Deoxyribonuclease (DNase) is widely present in human skin tissue and body fluids. This enzyme can rapidly hydrolyze and break the phosphodiester bond of PDRN, resulting in a half-life of less than 4 hours in the skin, rapid loss of activity, and difficulty in achieving long-term repair effect; (2) low transdermal efficiency, difficult to reach the target: PDRN is a water-soluble macromolecular nucleic acid substance. The core target is located in the dermis of the skin, while the stratum corneum of the skin is a dense lipophilic barrier. Under normal conditions, free PDRN can hardly penetrate the stratum corneum on its own. The transdermal absorption rate is less than 5%, which makes it difficult to fully exert the drug effect; poor formulation stability and fast in vivo clearance: conventional PDRN aqueous solution or simple formulation is easy to aggregate and oxidize and inactivate. When administered in vivo, it is easily recognized and phagocytosed by the reticuloendothelial system. The systemic circulation time is short, making it difficult to achieve targeted delivery and long-term effect. Currently, research and development of PDRN formulations in the industry mainly focuses on simple encapsulation with conventional liposomes and nanoemulsions, such as Chinese patent applications CN 121102045 A and CN113368054 A. However, these applications still have shortcomings, such as difficulty in blocking degradation at the source, limited improvement in transdermal efficiency, difficulty in applying the preparation process to industrial production, and poor batch stability.

[0004] Therefore, it is of great significance to provide a flexible liposome microcapsule of polydeoxyribonucleotides suitable for industrial production and its preparation method. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a flexible liposome microcapsule with anti-degradation polydeoxyribonucleic acid and its preparation method. Through formulation optimization, it fundamentally blocks the hydrolysis of PDRN by nucleases, significantly prolongs its activity maintenance time, improves liposome deformability and transdermal efficiency, enhances bioavailability, and is suitable for industrial production and application.

[0006] The objectives of this invention will be further explained by the following detailed description.

[0007] This invention provides a flexible liposome microcapsule with anti-degradation polydeoxyribonucleotides, composed of the following components in parts by weight: an aqueous core phase of 82-94 parts and a liposome membrane phase of 6-18 parts; the aqueous core phase comprises the following components in parts by weight: polydeoxyribonucleotides of 0.1-1.5 parts, an anti-degradation modifier of 0.02-0.8 parts, and water of 81-92 parts; the liposome membrane phase comprises the following components in parts by weight: phospholipids of 0.5-8 parts, a flexibility modifier of 0.2-2.0 parts, a membrane stabilizer of 0.1-0.5 parts, a humectant of 3-15 parts, and an antioxidant of 0.2-1.0 parts; the anti-degradation modifier is selected from one or more of EDTA-2Na, EDTA, sodium phytate, and sodium pyrophosphate.

[0008] Using the above technical solution, the aqueous core is encapsulated within the liposome, and the liposome membrane forms a lipid bilayer shell. On one hand, this invention utilizes a dual anti-degradation mechanism of "nuclease inactivation + physical isolation for sustained release" to fundamentally block the hydrolysis of PDRN by nucleases, improving stability and significantly extending its activity maintenance time, overcoming the shortcomings of existing technologies where PDRN is easily degraded by nucleases and has a short active half-life. On the other hand, this invention constructs a highly flexible and deformable liposome membrane system, endowing the liposomes with excellent deformation capabilities, allowing them to be squeezed into the intercellular spaces of the stratum corneum under the action of skin osmotic pressure, significantly improving the cross-stratum corneum delivery efficiency and dermal targeting, potentially expanding its application scope in the pharmaceutical and cosmetic fields, overcoming the shortcomings of existing conventional liposomes such as poor deformability and low transdermal efficiency. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules provided by this invention can be further formulated into serums, gels, dressings, etc.

[0009] Preferably, the anti-degradation polydeoxyribonucleotide flexible liposome microcapsules are composed of the following components in parts by weight: 84-90 parts of an aqueous core phase and 10-16 parts of a liposome membrane phase; the aqueous core phase includes the following components in parts by weight: 0.2-1.2 parts of polydeoxyribonucleotide, 0.05-0.5 parts of an anti-degradation modifier, and 83-89 parts of water; the liposome membrane phase includes the following components in parts by weight: 2-6 parts of phospholipid, 1.0-1.8 parts of a flexible deforming agent, 0.1-0.4 parts of a membrane stabilizer, 4-12 parts of a humectant, and 0.3-0.7 parts of an antioxidant.

[0010] Preferably, the flexible deformable agent is selected from one or more of ceramides, Tween 80, oleic acid, dioleoglycerate, and sodium cholate. The flexible deformable agent can reduce membrane order, improve membrane fluidity and deformability, allowing liposomes to squeeze through the intercellular spaces of the stratum corneum. More preferably, ceramides are natural homologous lipids of the human skin stratum corneum, highly compatible with stratum corneum lipids, and can promote lipid adsorption, fusion, and exchange between the liposome membrane and the skin barrier, reducing the cross-barrier transport energy barrier, improving transdermal delivery efficiency, and simultaneously synergistically enhancing membrane structural uniformity and formulation stability with phospholipids and polyols.

[0011] Preferably, the molecular weight of the polydeoxyribonucleotide is 5000-20000 Da, and PDRN in this molecular weight range is beneficial for improving transdermal efficiency.

[0012] Preferably, the membrane stabilizer is selected from one or more of cholesterol, vitamin E, and phytosterols. The membrane stabilizer can improve the structural stability of the liposome membrane, reduce leakage, and simultaneously protect phospholipids and PDRN from oxidative degradation.

[0013] Preferably, the antioxidant is selected from one or more of p-hydroxyacetophenone, butylated hydroxytoluene, coenzyme Q10, and reduced glutathione. Antioxidants can inhibit oxidation, prevent nucleic acid chain breaks, and are safe and non-irritating.

[0014] Preferably, the phospholipid is selected from hydrogenated lecithin and / or soy lecithin. Hydrogenated lecithin and soy lecithin have good biocompatibility, high safety, and good film-forming stability.

[0015] Preferably, the moisturizer is selected from one or more of glycerin, butylene glycol, 1,2-hexanediol, and 1,3-propanediol. The moisturizer can improve dry skin and enhance the skin barrier repair effect; simultaneously, in the formulation system of this invention, it also acts as a dispersant, stabilizer, and antifreeze protectant, reducing system viscosity, preventing liposome microcapsule aggregation and sedimentation, maintaining uniform particle size and low PDI, reducing ice crystal formation under 4°C refrigeration conditions, and preventing damage to the liposome membrane structure.

[0016] Accordingly, the present invention also provides a method for preparing the aforementioned anti-degradation polydeoxyribonucleotide flexible liposome microcapsules, comprising the following steps: 1) Prepare the aqueous phase and oil phase separately: Add polydeoxyribonucleotides and anti-degradation modifiers to water, heat to 36-40℃, stir and mix well, cool to 24-32℃ and keep warm to obtain the aqueous phase; mix phospholipids, flexible deformation agents, membrane stabilizers, humectants and antioxidants, stir and mix well at 55-62℃, cool to 24-32℃ and keep warm to obtain the oil phase; 2) Preparation of the primary emulsion: The aqueous phase is slowly added to the oil phase, and the mixture is subjected to high-speed shearing for 8-15 minutes to form the primary emulsion; 3) Microfluidic homogenization and sterilization: The colostrum is homogenized using a microfluidic homogenizer, then cooled to room temperature and filtered through a filter membrane to obtain anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules.

[0017] By combining the above-mentioned preparation method with the innovative formulation of PDRN flexible liposome microcapsules and the microfluidic preparation process, the large-scale production of PDRN flexible liposome microcapsules has been achieved. This avoids high-temperature treatment, ensures product quality and stability, and has the advantages of high encapsulation efficiency, relatively uniform particle size distribution, and good batch stability. It solves the defects of existing processes such as uneven particle size and poor batch repeatability.

[0018] Preferably, the homogenization conditions include: 24-38°C, 5000-20000 psi, and 2-8 cycles. High-pressure homogenization using a microfluidic homogenizer avoids problems such as uneven shear force and localized overheating.

[0019] Preferably, the filter membrane has a particle size of 0.22 μm.

[0020] Preferably, the water is purified water.

[0021] Compared with the prior art, the beneficial effects of the present invention include: (1) This invention uses a dual anti-degradation mechanism of “nuclease inactivation + physical isolation and slow release” to block the hydrolysis of PDRN by nucleases from the source, improve stability and greatly prolong its activity maintenance time, and overcome the defects of PDRN being easily degraded by nucleases and having a short activity half-life in the prior art.

[0022] (2) By constructing a highly flexible and deformable liposome membrane system, this invention endows liposomes with excellent deformation ability, enabling them to squeeze into the intercellular spaces of the stratum corneum under the action of skin osmotic pressure, greatly improving the cross-stratum corneum delivery efficiency and dermal targeting, and is expected to expand its application scope in the fields of medicine and cosmetics, overcoming the defects of poor deformability and low transdermal efficiency of existing conventional liposomes.

[0023] (3) To match the PDRN flexible liposome microcapsule system, this invention develops a suitable microfluidic preparation method, realizing the large-scale production of PDRN flexible liposome microcapsules. This avoids high-temperature treatment, ensuring product quality and stability. It has advantages such as high encapsulation efficiency (≥90%), relatively uniform particle size distribution (average particle size 80-200nm, polydispersity index PDI≤0.3), and good batch stability (batch-to-batch variation rate≤3%), solving the defects of uneven particle size and poor batch repeatability in existing processes. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules provided by this invention were stored at 4℃ in the dark for 6 months. The test results showed that the particle size, polydispersity index and encapsulation efficiency all changed to a certain extent, but the fluctuation range was within an acceptable range. The appearance of the formulation was relatively uniform, and no defects such as particle aggregation, system stratification and leakage of contents were observed. Attached Figure Description

[0024] Figure 1 The particle size distribution of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 1 was measured.

[0025] Figure 2 The results of the detection of total drug content and free drug content in the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 1; wherein Figure 2 -A represents the total drug content detection result. Figure 2 -B represents the result of the free drug content detection.

[0026] Figure 3 The particle size distribution of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 1 after 6 months of storage was measured.

[0027] Figure 4 The results of the detection of total drug content and free drug content of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 1 after 6 months of storage; wherein Figure 4 -A represents the total drug content detection result. Figure 4 -B represents the result of the free drug content detection.

[0028] Figure 5 Transmission electron microscopy observation results of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 2.

[0029] Figure 6 The particle size distribution of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 2 was measured.

[0030] Figure 7 The results of the detection of total drug content and free drug content in the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 2; wherein Figure 7-A represents the total drug content detection result. Figure 7 -B represents the result of the free drug content detection.

[0031] Figure 8 The particle size distribution of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 2 after 6 months of storage was measured.

[0032] Figure 9 Example 2: Results of total drug content and free drug content detection after 6 months of storage of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules; Figure 9 -A represents the total drug content detection result. Figure 9 -B represents the result of the free drug content detection.

[0033] Figure 10 The particle size distribution of the liposome microcapsules prepared in Comparative Example 1 was measured.

[0034] Figure 11 Results of the detection of total drug content and free drug content in the liposome microcapsules prepared in Comparative Example 1; among which Figure 11 -A represents the total drug content detection result. Figure 11 -B represents the result of the free drug content detection.

[0035] Figure 12 The particle size distribution of the liposome microcapsules prepared in Comparative Example 1 after 6 months of storage.

[0036] Figure 13 The particle size distribution of the liposome microcapsules prepared in Comparative Example 2 was measured.

[0037] Figure 14 The results of the detection of total drug content and free drug content of the liposome microcapsules prepared in Comparative Example 2; among which Figure 14 -A represents the total drug content detection result. Figure 14 -B represents the result of the free drug content detection.

[0038] Figure 15 The particle size distribution of the liposome microcapsules prepared in Comparative Example 3 was measured.

[0039] Figure 16 Results of the detection of total drug content and free drug content in the liposome microcapsules prepared in Comparative Example 3; among which Figure 16 -A represents the total drug content detection result. Figure 16 -B represents the result of the free drug content detection.

[0040] Figure 17 A schematic diagram of the fluorescence color development of FITC and FITC-LipophoTyRan® NanoPDRN in pig skin.

[0041] Figure 18 Fluorescence intensity results of FITC and FITC-LipophoTyRan® NanoPDRN in pig skin. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0043] In this invention, the components, reagents, and equipment involved are all commercially available products or can be obtained through conventional technical means in the field. For example, PDRN is provided by Peptide Source (Guangzhou) Biotechnology Co., Ltd., product code: Fermentract®PDRN.

[0044] Encapsulation efficiency detection: The ultrafiltration tubes were pretreated to prevent PDRN adsorption due to membrane drying, thus reducing PDRN adsorption and ensuring separation efficiency. Then, the encapsulation efficiency of the anti-degradable polydeoxyribonucleic acid flexible liposome microcapsules was determined using ultrafiltration centrifugation, including the following steps: 1) 2 mL of the sample was diluted to 10 mL with chromatographic methanol, vortexed thoroughly, and then placed in an ultrasonic bath for complete demulsification, releasing all PDRN from the anti-degradable polydeoxyribonucleic acid flexible liposome microcapsules. After demulsification, the sample was filtered through a 0.22 μm organic filter. The filtered sample was then analyzed using Unano-1000, and the PDRN content was determined as the total drug content, denoted as R. total 2) Add 2 mL of the sample to be tested into an ultrafiltration centrifuge tube and centrifuge at 10,000 rpm at 4°C. After ultrafiltration centrifugation, dilute the filtrate with ultrapure water to 10 mL and filter it through a 0.22 μm organic filter. After filtration, the sample is analyzed by Unano-1000. The PDRN content obtained is the free drug content, denoted as R. free 3) Take blank liposome solution, process and measure it simultaneously according to the same experimental procedure, and subtract matrix background interference; 4) The encapsulation efficiency is calculated as follows: . Example 1: Anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules

[0045] The formulation of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules (based on a total mass of 100g) is as follows: 1. Core aqueous phase (89.0g): PDRN 1.0g, EDTA-2Na 0.2g, purified water 87.8g.

[0046] 2. Lipid membrane phase (11.0g): hydrogenated lecithin 3.0g, ceramide 0.6g, Tween 80 0.8g, cholesterol 0.15g, tocopherol 0.15g, butylene glycol 3.0g, glycerol 2.6g, 1,3-propanediol 0.2g, p-hydroxyacetophenone 0.5g.

[0047] A method for preparing anti-degradation polydeoxyribonucleotide flexible liposome microcapsules includes the following steps: 1) Preparation of aqueous and oil phases: PDRN and EDTA-2Na were added to purified water, heated to 38°C, stirred and mixed, cooled to 30°C and kept warm to obtain the aqueous phase; hydrogenated lecithin, ceramide, Tween 80, cholesterol, tocopherol, butylene glycol, glycerol, 1,3-propanediol and p-hydroxyacetophenone were mixed, stirred and mixed at 60°C, cooled to 30°C and kept warm to obtain the oil phase; 2) Preparation of the primary emulsion: The aqueous phase is slowly added to the oil phase, and the mixture is sheared at high speed for 10 min to form the primary emulsion; 3) Microfluidic homogenization and sterilization: The colostrum was homogenized using a microfluidic homogenizer. The homogenization conditions included: 35°C, 12000 psi, 5 cycles, followed by cooling to room temperature and filtration through a 0.22 μm filter membrane to obtain anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules.

[0048] The relevant quality parameters of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 1 were detected, and the results are as follows: Particle size / PDI: Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS detection, average particle size 87.3 nm, PDI = 0.205. Figure 1 As shown.

[0049] Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 2 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total =7469 ng / μL; following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 2 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =444.3ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate encapsulation efficiency according to step 4) of the aforementioned detection method =94.05%. Figure 2 In this context, absorbance (10 mm) refers to the absorbance measured at a standard optical path of 10 mm.

[0050] 6-month stability (4℃, protected from light): Particle size, PDI, and encapsulation efficiency were retested. Average particle size was 167.3 nm, and PDI was 0.227. Figure 3 As shown; Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 4 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total =6996 ng / μL; following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 4 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =650.4ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate the encapsulation rate according to step 4) of the aforementioned detection method =90.70%, with no leakage or delamination. Example 2: Anti-degradable polydeoxyribonucleic acid flexible liposome microcapsules

[0051] The formulation of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules (based on a total mass of 100g) is as follows: 1. Core aqueous phase (85.5g): PDRN 0.5g, EDTA-2Na 0.1g, purified water 84.0g.

[0052] 2. Lipid membrane phase (14.5g): soybean lecithin 3.0g, ceramide 0.3g, Tween 80 1.0g, tocopherol 0.2g, butylene glycol 5.0g, glycerol 4.0g, 1,2-hexanediol 0.5g, p-hydroxyacetophenone 0.5g.

[0053] A method for preparing anti-degradation polydeoxyribonucleotide flexible liposome microcapsules includes the following steps: 1) Preparation of aqueous and oil phases: PDRN and EDTA-2Na were added to purified water, heated to 40°C, stirred and mixed, cooled to 30°C and kept warm to obtain the aqueous phase; soybean lecithin, ceramide, Tween 80, tocopherol, butylene glycol, glycerol, 1,2-hexanediol and p-hydroxyacetophenone were mixed, stirred and mixed at 60°C, cooled to 30°C and kept warm to obtain the oil phase; 2) Preparation of the primary emulsion: The aqueous phase is slowly added to the oil phase, and the mixture is sheared at high speed for 12 min to form the primary emulsion; 3) Microfluidic homogenization and sterilization: The colostrum was homogenized using a microfluidic homogenizer. The homogenization conditions included: 33°C, 10000 psi, 3 cycles, followed by cooling to room temperature and filtration through a 0.22 μm filter membrane to obtain anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules.

[0054] The relevant quality parameters of the anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules prepared in Example 2 were detected, and the results are as follows: The liposome microcapsule sample prepared in Example 2 was appropriately diluted with ultrapure water to a low-concentration dispersion system (to avoid particle overlap). A small amount of sample was dropped onto the surface of a carbon membrane copper grid, allowed to stand, allowing the liposomes to adsorb onto the grid, and then excess liquid was gently absorbed with filter paper. 2% phosphotungstic acid staining solution was added for negative staining, and after staining, the sample was blotted dry and allowed to air dry at room temperature. Then, the sample was observed and images were acquired under a transmission electron microscope (80–200 kV). Figure 5 As shown. From Figure 5 It can be seen that the sample exhibits a regular spherical vesicle structure, good dispersibility, clear boundaries, and a relatively complete and uniform encapsulation structure.

[0055] Particle size / PDI: Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS detection, average particle size 84.55 nm, PDI = 0.207. Figure 6 As shown.

[0056] Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 7 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total =3585 ng / μL; following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 7 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =222.1ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate encapsulation efficiency according to step 4) of the aforementioned detection method =93.80%.

[0057] 6-month stability (4℃, protected from light): Particle size, PDI, and encapsulation efficiency were retested. Average particle size was 155.0 nm, and PDI was 0.277. Figure 8 As shown; Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 9 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total =2063 ng / μL; following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 9 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free=254.5 ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate the encapsulation rate according to step 4) of the aforementioned detection method =87.66%, with no leakage or delamination. Comparative Example 1 (without anti-degradation modifier)

[0058] The formulation of liposome microcapsules: Compared with Example 1, the only difference is that it does not contain EDTA-2Na and the amount of purified water is increased by 0.2g.

[0059] Preparation method: Same as in Example 1. Relevant quality parameters were tested, and the results are as follows: Particle size / PDI: Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS detection, average particle size 86.22 nm, PDI = 0.211. Figure 10 As shown.

[0060] Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 11 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total = 5492 ng / μL; Following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 11 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =534.2 ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate encapsulation efficiency = 90.27% according to step 4) of the aforementioned detection method.

[0061] Enzymatic hydrolysis for 30 min: DNase I incubated at 37℃ for 30 min, PDRN residue was determined by HPLC, PDRN residue was 32%.

[0062] 6-month stability (4℃, protected from light): Particle size, PDI, and encapsulation efficiency were retested. Average particle size was 179.8 nm, and PDI was 0.210. Figure 12 As shown, the encapsulation rate was 82.08%, indicating leakage. Comparative Example 2 (without film stabilizer)

[0063] The formulation of liposome microcapsules is the only difference from Example 1: the absence of cholesterol and tocopherol.

[0064] Preparation method: Same as in Example 1. Relevant quality parameters were tested, and the results are as follows: Particle size / PDI: Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS detection, average particle size 109.8 nm, PDI = 0.298. Figure 13 As shown.

[0065] Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 14 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total = 6709 ng / μL; Following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 14 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =997.3 ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate encapsulation efficiency according to step 4) of the aforementioned detection method =85.13%.

[0066] Clear aggregation and stratification will be observed after one month of storage at 4℃ away from light.

[0067] 6-month stability (4℃, protected from light): average particle size 439.6nm, PDI=0.388; encapsulation efficiency 70.06%, significant leakage. Comparative Example 3 (Non-microfluidic homogeneous)

[0068] The formulation of liposome microcapsules is the same as in Example 1.

[0069] Preparation method: Compared with Example 1, the microfluidic homogenization (35°C, 12000 psi, 5 cycles) was replaced with 55°C, high-pressure homogenization at 800 bar, 5 cycles. Relevant quality parameters were measured, and the results are as follows: Particle size / PDI: Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS detection, average particle size 214.8 nm, PDI = 0.270. Figure 15 As shown.

[0070] Encapsulation efficiency: Following step 1) of the aforementioned detection method, the filtered sample was tested using Unano-1000, and the results are as follows. Figure 16 As shown in -A, the measured PDRN content is the total drug content, denoted as R. total =7273 ng / μL; following step 2 of the aforementioned detection method, the filtered sample was analyzed using Unano-1000, and the results are as follows. Figure 16 As shown in -B, the measured PDRN content is the free drug content, denoted as R. free =1396 ng / μL; Subtract matrix background interference according to step 3) of the aforementioned detection method; Calculate encapsulation efficiency according to step 4) of the aforementioned detection method =80.81%.

[0071] Batch variation: 12.7%, poor stability.

[0072] 6-month stability (4℃, protected from light): average particle size 1011nm, PDI=0.349; encapsulation rate 65.07%, significant leakage. Test Example: Permeability Test

[0073] The permeability of the liposome microcapsule sample prepared in Example 2 (ex vivo porcine skin) was detected using the Franz diffusion cell in vitro transdermal absorption method. The experimental method included the following steps: 1) Sample addition to the donor cell: The quantitative sample to be tested was evenly coated on the surface of the diffusion barrier (the donor cell was in an open state to simulate the exposure scenario of cosmetic external use) with a pipette. The surface was gently smoothed to remove air bubbles. The start time of the experiment was recorded the moment the sample addition was completed; 2) After diffusion for 1 hour, stirring and constant temperature water bath were stopped. The clamp was slowly disassembled, the diffused porcine skin was taken out, rinsed with pure water, and the fluorescence intensity of the target area of ​​the porcine skin was detected by a fluorescence imaging system. The epidermal fluorescence distribution and signal intensity were observed by laser confocal fluorescence microscopy.

[0074] Experimental results are as follows Figure 17 and Figure 18 As shown. By Figure 17 It was observed that both the FITC group and the FITC-LipophoTyRan® NanoPDRN group (liposome microcapsule samples prepared in Example 2) showed clear yellow-green specific fluorescence signals in porcine skin, indicating that both samples could be infiltrated through ex vivo porcine skin. Figure 18 It can be seen that the green fluorescence intensity of the FITC-LipophoTyRan® NanoPDRN group is significantly improved compared with that of the FITC group, with a fluorescence intensity increase of 3.8 times, indicating that the liposome microcapsule sample prepared by the present invention has good transdermal permeability and absorption characteristics.

[0075] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A flexible liposome microcapsule with resistance to degradation polydeoxyribonucleotides, characterized in that: It is composed of the following components in parts by weight: 82-94 parts of the aqueous core phase and 6-18 parts of the liposome membrane phase; the aqueous core phase includes the following components in parts by weight: 0.1-1.5 parts of polydeoxyribonucleotides, 0.02-0.8 parts of anti-degradation modifier, and 81-92 parts of water; the liposome membrane phase includes the following components in parts by weight: 0.5-8 parts of phospholipids, 0.2-2.0 parts of flexible deforming agent, 0.1-0.5 parts of membrane stabilizer, 3-15 parts of humectant, and 0.2-1.0 parts of antioxidant; the anti-degradation modifier is selected from one or more of EDTA-2Na, EDTA, sodium phytate, and sodium pyrophosphate.

2. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1, characterized in that: It is composed of the following components in parts by weight: 84-90 parts of the aqueous core phase and 10-16 parts of the liposome membrane phase; the aqueous core phase includes the following components in parts by weight: 0.2-1.2 parts of polydeoxyribonucleotides, 0.05-0.5 parts of anti-degradation modifier, and 83-89 parts of water; the liposome membrane phase includes the following components in parts by weight: 2-6 parts of phospholipids, 1.0-1.8 parts of flexible deforming agent, 0.1-0.4 parts of membrane stabilizer, 4-12 parts of humectant, and 0.3-0.7 parts of antioxidant.

3. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The flexible deforming agent is selected from one or more of ceramide, Tween 80, oleic acid, dioleoyl glycerol and sodium cholate.

4. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The molecular weight of the polydeoxyribonucleotide is 5000-20000 Da.

5. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The membrane stabilizer is selected from one or more of cholesterol, vitamin E, and phytosterols.

6. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The antioxidant is selected from one or more of p-hydroxyacetophenone, butylated hydroxytoluene, coenzyme Q10, and reduced glutathione.

7. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The phospholipids are selected from hydrogenated lecithin and / or soybean lecithin.

8. The anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 1 or 2, characterized in that: The moisturizer is selected from one or more of glycerin, butylene glycol, 1,2-hexanediol and 1,3-propanediol.

9. The method for preparing the anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to any one of claims 1 to 8, characterized in that: Includes the following steps: 1) Prepare the aqueous phase and oil phase separately: Add polydeoxyribonucleotides and anti-degradation modifiers to water, heat to 36-40℃, stir and mix well, cool to 24-32℃ and keep warm to obtain the aqueous phase; mix phospholipids, flexible deformation agents, membrane stabilizers, humectants and antioxidants, stir and mix well at 55-62℃, cool to 24-32℃ and keep warm to obtain the oil phase; 2) Preparation of the primary emulsion: The aqueous phase is slowly added to the oil phase, and the mixture is subjected to high-speed shearing for 8-15 minutes to form the primary emulsion; 3) Microfluidic homogenization and sterilization: The colostrum is homogenized using a microfluidic homogenizer, then cooled to room temperature and filtered through a filter membrane to obtain anti-degradation polydeoxyribonucleic acid flexible liposome microcapsules.

10. The method for preparing the anti-degradation polydeoxyribonucleotide flexible liposome microcapsules according to claim 9, characterized in that: The homogenization conditions include: 24-38°C, 5000-20000 psi, and 2-8 cycles.

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