Preparation process of high-activity supramolecular assembly microspheric PDRN

Abstract

The application discloses a preparation process of high-activity supramolecular assembly microspheric PDRN and belongs to the field of cosmetic raw material preparation, and contains two stages of PDRN biosynthesis-enzymatic decoupling extraction refinement and supramolecular assembly microspherization. First, PDRN precursors are synthesized through fermentation of genetically engineered Escherichia coli, combined with salmon testis enzyme-assisted extraction, and purified through macroporous resin-ultrafiltration membrane combination to obtain high-purity PDRN pure product; then, through intermolecular hydrogen bonds and hydrophobic interaction, self-assembly is realized by using amphiphilic cyclodextrin derivatives and PEG-PLA as carriers to realize PDRN microspherization, and a crosslinking agent is not needed in the whole process. The prepared microspheric PDRN has a purity of 95%, a particle size of 100-300 nm, an encapsulation rate of 85%, an activity retention rate of 90%, excellent sustained release and stability, no skin irritation, and is suitable for various cosmetic formulations, and the process equipment is conventional, easy to be industrialized, the raw material utilization rate is high, and the process is green and environmentally-friendly.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material preparation technology, specifically to a preparation process for highly active supramolecular assembled microspheres of PDRN. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs), as a cosmetic raw material with excellent bioactivity, possess soothing, repairing, anti-inflammatory, moisturizing, and skin barrier regeneration effects, and are widely used in various skincare products, especially suitable for sensitive and damaged skin care. Currently, the preparation of cosmetic PDRNs mainly employs either single natural extraction or chemical synthesis methods. Natural extraction often uses salmon testes as raw material through single enzymatic hydrolysis, resulting in low purity (typically 85%) and significant loss of active ingredients. Chemical synthesis methods are prone to reagent residues, and the synthesized products have poor bioactivity, making it difficult to meet the requirements for cosmetic-grade raw materials.

[0003] Meanwhile, to enhance the skin permeability and anti-degradation ability of PDRN, existing technologies mostly employ emulsification cross-linking to microsphereize PDRN. However, this method requires the use of toxic cross-linking agents such as glutaraldehyde, inevitably leading to reagent residue issues and potential skin irritation, making it unsuitable for products targeting sensitive skin. Furthermore, the prepared microspheres exhibit uneven particle size, low encapsulation efficiency (70%), poor sustained-release effect, and a cumulative release rate of only 80% over 24 hours, failing to achieve long-lasting skincare. In addition, current PDRN purification methods often employ high-concentration alcohol precipitation processes, resulting in large amounts of organic solvents, significant environmental pressure, low raw material utilization (60%), high industrialization costs, and the need for customized equipment in some processes, making large-scale production difficult.

[0004] To address the problems of low purity, poor activity, reagent residues in microsphere formation processes, high skin irritation, and low industrial feasibility in existing PDRN preparation methods, this invention provides a preparation process for highly active supramolecularly assembled microsphere PDRN. This process involves extracting and purifying high-purity PDRN through a biosynthesis-enzymatic decomposition coupling method, followed by PDRN microsphere formation via self-assembly using an amphiphilic supramolecular carrier. The entire process is free of toxic reagent residues, resulting in microsphere PDRN with uniform particle size, high encapsulation efficiency, and good sustained-release effect. Furthermore, the process steps are controllable, the equipment is conventional, and the process is feasible for industrial scale-up, fully meeting the quality and application requirements of cosmetic-grade raw materials. Summary of the Invention

[0005] The purpose of this invention is to provide a preparation process for highly active supramolecular assembled microspheres of PDRN, solving the technical problems of low purity, poor activity, residual microsphere reagents, high skin irritation, and high industrialization cost in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A process for preparing highly active supramolecularly assembled microspheres of PDRN includes a biosynthesis-enzymatic hydrolysis coupled extraction and purification stage and a supramolecularly assembled microsphere preparation stage. In the biosynthesis-enzymatic hydrolysis coupled extraction and purification stage, PDRN precursors are synthesized through fermentation by genetically engineered microorganisms, combined with enzymatic hydrolysis-assisted extraction from marine biological tissues, and purified to high purity using a macroporous resin-ultrafiltration membrane. In the supramolecularly assembled microsphere preparation stage, amphiphilic supramolecular carriers are used as raw materials, and PDRN microspheres are self-assembled through intermolecular hydrogen bonds and hydrophobic interactions, without the need for cross-linking agents. The prepared microspheres of PDRN have a purity of 95%, a particle size of 100-300 nm, an encapsulation efficiency of 85%, and an activity retention rate of 90%.

[0007] The biosynthesis-enzymatic decoupling extraction and purification stage includes the following steps: Genetically engineered Escherichia coli fermentation synthesis of PDRN precursor: After activating the genetically engineered Escherichia coli BL21 strain, a seed culture was prepared. The seed culture was inoculated into LB medium for large-scale fermentation. When the OD600 of the fermentation broth reached 1.0-1.2, IPTG inducer was added, and the temperature was lowered to induce the synthesis of PDRN precursor. When the PDRN precursor content in the fermentation broth reached 1.2 g / L, the fermentation was terminated and the mixture was refrigerated at low temperature. Fermentation broth disruption and crude extraction: The fermentation broth was centrifuged to collect the cell precipitate, which was then ultrasonically disrupted to release the intracellular PDRN precursor. After filtration, a crude extract was obtained. Salmon testis powder and a compound enzyme preparation were added to the crude extract for auxiliary enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated by high temperature and then cooled to low temperature. PDRN purification: The enzymatic hydrolysate is purified by adsorption with macroporous resin, washed with water to remove impurities, eluted with ethanol, and then purified and concentrated by ultrafiltration membrane separation system. After vacuum concentration and vacuum freeze drying, pure PDRN is obtained. The pure PDRN has a purity of 95%, a moisture content of 5.0%, and a protein content of 0.5%.

[0008] In step 1, the strain activation conditions were: constant temperature culture at 37℃ for 12h until OD600 = 0.6-0.8; the inoculum for seed culture preparation was 1:100 (v / v), cultured at 37℃ with shaking at 180r / min for 8h; the inoculum for large-scale fermentation was 5% (v / v), initial pH 7.0-7.2, cultured at 37℃ with stirring at 200r / min, aeration rate of 1:2vvm, and nitrogen gas was introduced for oxygen isolation; the final concentration of IPTG inducer was 0.5mmol / L, the induction temperature was 30℃, and the induction time was 16h.

[0009] In step 2, the ratio of bacterial precipitate to Tris-HCl buffer was 1:8 (g:mL), and the buffer pH was 7.5. The ultrasonic disruption conditions were 4℃, 250W power, 28kHz frequency, and intermittent operation for 30 min. The amount of salmon testis micronized powder added was 0.5% of the crude extract volume. The compound enzyme preparation was deoxyribonuclease I:ribonuclease = 3:1, and the final addition amount was 1.0% of the dry weight of the crude extract. At the same time, EDTA-2Na was added at a final concentration of 0.02mol / L. The enzymatic hydrolysis conditions were pH 5.5-6.0, 45℃, and stirring at 70r / min for 2.0h. The enzyme inactivation conditions were incubation at 55℃ for 15 min.

[0010] In step 3, the macroporous resin is D101 type weakly polar macroporous adsorption resin, the column loading flow rate is 1.0-1.5 BV / h, and the adsorption is allowed to stand for 2 hours; the water washing flow rate is 2.0 BV / h, the eluent is 45% (v / v) ethanol aqueous solution, the elution flow rate is 1.5 BV / h, and 2.0-3.0 BV of eluent is collected; the ultrafiltration membrane has a molecular weight cutoff of 5000 Da, the operating pressure is 0.15-0.20 MPa, and the temperature is 4℃; the vacuum concentration temperature is 45℃, the vacuum degree is -0.08~-0.09 MPa, and the concentration is carried out until the solid content is 30%; the vacuum freeze-drying conditions are pre-freezing to -40℃ and holding for 4 hours, vacuum degree is 10 Pa, sublimation temperature is 30℃, and drying is carried out for 18 hours.

[0011] The supramolecular assembly microsphere preparation stage includes the following steps: Preparation of supramolecular carrier solution: The amphiphilic cyclodextrin derivative and polyethylene glycol-polylactic acid block copolymer were mixed at a mass ratio of 2:1 and dissolved in anhydrous ethanol to prepare an oil phase solution; PDRN pure product was dissolved in PBS buffer to prepare an aqueous phase solution, and trehalose was added as a stabilizer. Both the oil phase solution and the aqueous phase solution were sterilely filtered before use. Preparation of PDRN microspheres by supramolecular self-assembly: The oil phase solution was placed in the assembly reaction vessel, stirred at a constant temperature and nitrogen was introduced to isolate oxygen. The aqueous phase solution was slowly added dropwise to the oil phase in proportion. After the addition was completed, stirring was continued. Microsphere dispersion was formed by ultrasonic-assisted assembly. The particle size was then homogenized by high pressure. The particle size of the microspheres was controlled at 100-300 nm. Microsphere purification and stabilization: The microsphere dispersion was purified by dialysis to remove residual impurities and solvents, sterile filtered, and trehalose was added. The dispersion was then ultrasonically dispersed to prevent microsphere aggregation, resulting in a microsphere-based PDRN dispersion. Preparation of finished product: The microsphere PDRN dispersion is directly dispensed to obtain a liquid finished product, or it is obtained by vacuum freeze drying, pulverization and classification to obtain a solid finished product.

[0012] In step 1, the concentration of the oil phase solution is 8-10 mg / mL, and it is ultrasonically dispersed for 10 min (power 150W); the concentration of PDRN in the aqueous phase solution is 5-6 mg / mL, the pH of the PBS buffer is 6.8-7.0, the final concentration of trehalose is 0.3%, and it is ultrasonically dispersed for 5 min; aseptic filtration is performed using a 0.22 μm sterile filter membrane.

[0013] In step 2, the assembly reactor is kept at a constant temperature of 25-30℃, the stirring speed is 300-350 r / min, and the nitrogen flow rate is 0.3 m / h; the oil phase:water phase ratio is 3:7 (v / v), the dropping rate is 1.0-1.5 mL / min, and stirring continues for 30 min after the dropping is completed; the ultrasonic-assisted assembly power is 200 W and the frequency is 40 kHz, and it works intermittently for 20 min; the high-pressure homogenization pressure is 35-40 MPa, and homogenization is performed 2-3 times, 5 min each time, with a microsphere particle size distribution coefficient (PDI) of 0.25.

[0014] In step 3, the dialysis bag retains a molecular weight cutoff of 10,000 Da, and the dialysis is performed at a constant temperature of 4°C for 24 hours, with the deionized water being replaced every 8 hours; a 0.22 μm sterile filter membrane is used for sterile filtration; trehalose is added to a final concentration of 0.5% and then ultrasonically dispersed for 5 minutes (power 150W).

[0015] In step 4, the vacuum freeze-drying conditions are: pre-freezing to -40℃ and holding for 4 hours, vacuum degree 10Pa, sublimation temperature 30℃, and drying for 20 hours; the solid product is pulverized to 100 mesh, sieved, and then aseptically sealed and packaged; the liquid product is refrigerated at 4℃, and the solid product is stored at 4℃, protected from light, and dried.

[0016] The microspheres of PDRN prepared in this invention showed a cumulative release rate of 60% in PBS buffer (pH 7.0) after 24 hours and 85% after 48 hours. After 6 months of storage at 4°C, the microspheres showed no aggregation or precipitation, with a particle size change of 10% and an encapsulation rate change of 5%. In vitro skin irritation tests showed a cell viability of 90% and no skin irritation.

[0017] The preparation process of highly active supramolecular assembled microspheres of PDRN provided by this invention has the following advantages compared with the prior art: This invention uses a coupling method of fermentation synthesis of genetically engineered E. coli and enzymatic hydrolysis of salmon testes to extract PDRN, which balances the yield and bioactivity of PDRN and solves the problems of low purity from single natural extraction and poor activity from chemical synthesis. The prepared PDRN has a purity of 95% and a content of impurities of 0.5%, laying a high-activity raw material foundation for subsequent microsphere preparation.

[0018] This invention innovatively adopts a purification method that combines macroporous resin and ultrafiltration membrane, replacing the traditional high-concentration alcohol precipitation process. This reduces ethanol consumption by more than 65%, is environmentally friendly, eliminates the risk of organic solvent residue, and improves purification efficiency by 40%, with a PDRN recovery rate of 85%, significantly reducing raw material costs.

[0019] This invention uses amphiphilic cyclodextrin derivatives and PEG-PLA as supramolecular carriers to achieve the self-assembly of PDRN into microspheres through intermolecular hydrogen bonds and hydrophobic interactions. No cross-linking agent is required throughout the process, which fundamentally solves the problems of reagent residue and skin irritation caused by traditional emulsification cross-linking methods. The prepared microsphere PDRN is non-irritating and suitable for cosmetics for various skin types, including sensitive skin and damaged skin.

[0020] This invention, through precise control of assembly temperature, stirring speed, material-liquid ratio, and high-pressure homogenization parameters, prepares microspheres with uniform particle size (100-300nm) and a PDI of 0.25. This particle size range easily penetrates the stratum corneum of the skin, and the encapsulation rate is 85%, achieving long-term sustained release of PDRN. The cumulative release rate is 60% after 24 hours and 85% after 48 hours, and the duration of efficacy is more than twice that of non-microsphereized PDRN.

[0021] The microsphere PDRN prepared by this invention exhibits excellent stability. After 6 months of refrigeration at 4°C, there is no aggregation or precipitation, the particle size changes by 10%, the encapsulation rate changes by 5%, and the activity retention rate is 90%. It can maintain the biological activity of the raw material for a long time and meet the storage requirements of cosmetic raw materials.

[0022] This invention utilizes conventional industrial equipment from the cosmetics and bio-fermentation industries throughout the entire process, eliminating the need for customized specialized equipment. Companies can directly modify their existing production lines, ensuring precise and controllable process steps, achieving an 85% raw material utilization rate, reducing industrialization costs by 30%, and resulting in a high degree of product standardization, making it easy to achieve large-scale production.

[0023] All raw materials and reagents used in this invention comply with the requirements of the "Cosmetic Safety Technical Specifications" (2022 edition). The ampicillin used in the fermentation stage can be completely removed through ultrafiltration and dialysis. The final product has no toxic or harmful residues such as antibiotics and organic solvents, has good skin compatibility, and can be directly used in various cosmetic formulations such as toners, serums, repair masks, lotions, and creams, resulting in high added value. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0025] In a first aspect, the present invention provides a process for preparing highly active supramolecularly assembled microspheres of PDRN, comprising a biosynthesis-enzymatic hydrolysis coupled extraction and purification stage and a supramolecularly assembled microsphere preparation stage of PDRN; the biosynthesis-enzymatic hydrolysis coupled extraction and purification stage synthesizes PDRN precursor through fermentation by genetically engineered microorganisms, combined with enzymatic hydrolysis-assisted extraction from marine biological tissues, and purifies PDRN to obtain pure product by using macroporous resin-ultrafiltration membrane; the supramolecularly assembled microsphere preparation stage uses amphiphilic supramolecular carriers as raw materials, and achieves PDRN microsphere formation through intermolecular hydrogen bonding and hydrophobic interactions, without the need for cross-linking agents throughout the process; the prepared microspheres of PDRN have a purity of 95%, a microsphere size of 100-300 nm, an encapsulation rate of 85%, and an activity retention rate of 90%.

[0026] This invention achieves integrated green production of PDRN preparation and microsphere formation, solving the problems of low purity and poor activity in traditional PDRN preparation while avoiding the industry pain point of residual crosslinking agents in microsphere formation. The prepared microsphere PDRN possesses the characteristics of high purity, uniform particle size, high encapsulation efficiency, and high activity retention rate, significantly improving skin permeability and anti-degradation ability. Furthermore, no toxic reagents are added throughout the process, resulting in excellent skin compatibility. The entire process is also adaptable to industrial-scale production, balancing product efficacy and production practicality, and its overall performance far surpasses existing technologies.

[0027] In some embodiments, during the biosynthesis-enzyme decoupling extraction and purification stage, the genetically engineered microorganism is a modified Escherichia coli BL21, the marine biological tissue is salmon testes, the compound enzyme preparation is a mixture of deoxyribonuclease I and ribonuclease in a mass ratio of 3:1, the macroporous resin is D101 type weakly polar macroporous adsorption resin, and the ultrafiltration membrane has a molecular weight cutoff of 5000 Da.

[0028] This invention precisely controls the compatibility of raw materials and purification efficiency in PDRN extraction. The coupled use of genetically engineered E. coli BL21 and salmon testes achieves complementary advantages between artificial synthesis and natural extraction, significantly increasing PDRN yield and bioactivity. A specific ratio of compound enzyme preparation can efficiently degrade impurities and improve extraction purity. The combined purification method of D101 macroporous resin and 5000Da ultrafiltration membrane replaces the traditional high-concentration alcohol precipitation process, reducing the use of organic solvents, alleviating environmental pressure, and further improving the purity and uniformity of PDRN, laying a high-activity raw material foundation for subsequent microsphere preparation.

[0029] In some embodiments, the biosynthesis-enzymatic decoupling extraction and purification stage includes the following steps: (1) After activating Escherichia coli BL21 strain, seed culture was prepared and inoculated into LB medium for fermentation. IPTG inducer was added to synthesize PDRN precursor. Fermentation was terminated when the PDRN precursor content in the fermentation broth reached 1.2 g / L. (2) The fermentation broth was centrifuged to collect the cell precipitate, ultrasonically crushed and filtered to obtain crude extract, salmon testis powder and compound enzyme preparation were added for enzymatic hydrolysis, and the enzyme was inactivated and then cooled. (3) After the enzymatic hydrolysate is adsorbed by macroporous resin, washed with water and eluted with ethanol, it is purified and concentrated by ultrafiltration, and then concentrated by vacuum decompression and freeze-dried to obtain pure PDRN.

[0030] This invention achieves standardized and controllable operation of the PDRN extraction process. Standardized steps for strain activation and fermentation induction ensure efficient synthesis of PDRN precursors and avoid loss of active ingredients during fermentation. Stepwise operations involving cell disruption and auxiliary enzymatic hydrolysis achieve full release of intracellular PDRN and effective replenishment of natural PDRN fragments, while simultaneously degrading impurities such as proteins and nucleic acids, reducing the difficulty of subsequent purification. The seamless integration of macroporous resin purification and vacuum drying ensures high yield and activity retention of the pure PDRN product, avoids component denaturation during drying, and ultimately results in a stable quality pure PDRN product that meets all requirements for cosmetic-grade raw materials.

[0031] In some embodiments, in the above steps: in step (1), the final concentration of IPTG inducer is 0.5 mmol / L, the induction temperature is 30℃, and the induction time is 16h; in step (2), the ultrasonic disruption conditions are 4℃, power 250W, frequency 28kHz, and intermittent operation for 30min; the enzymatic hydrolysis conditions are pH 5.5-6.0, 45℃, 70r / min stirring for 2.0h, and the enzyme inactivation conditions are 55℃ for 15min.

[0032] This invention achieves precise control over the reaction efficiency and product quality of each step by limiting key process parameters in the coupled biosynthesis-enzymatic hydrolysis extraction stage. Specific concentrations of IPTG inducer, induction temperature, and time ensure efficient synthesis of PDRN precursors by *E. coli*, increasing fermentation yield. Low-temperature ultrasonic disruption parameters ensure thorough cell disruption while preventing PDRN degradation due to high temperature and strong shear force. Enzymatic hydrolysis parameters, including pH, temperature, and time, are tailored to the activity characteristics of the complex enzyme, achieving efficient degradation of impurities and efficient retention of PDRN. High-temperature, short-time enzyme inactivation rapidly deactivates the enzyme, preventing excessive hydrolysis that could damage the PDRN structure and ensuring the integrity and activity of the extracted product.

[0033] In some embodiments, in step (3), the eluent is a 45% (v / v) aqueous ethanol solution, the ultrafiltration operation pressure is 0.15-0.20 MPa and the temperature is 4°C; the vacuum concentration temperature is 45°C and the vacuum degree is -0.08~-0.09 MPa; the vacuum freeze-drying pre-freezes to -40°C and holds for 4 hours, the vacuum degree is 10 Pa and the sublimation temperature is 30°C, and the drying is carried out for 18 hours.

[0034] This invention further improves the purity, yield, and storage stability of PDRN by limiting key parameters in the PDRN purification stage. A specific concentration of ethanol eluent enables efficient separation of PDRN from impurities, improving elution efficiency; the operating pressure and temperature parameters of ultrafiltration ensure the purification effect of cross-flow filtration while avoiding adsorption loss of PDRN during ultrafiltration, thus improving recovery rate; the low-temperature parameters of vacuum concentration prevent PDRN denaturation due to high-temperature concentration, ensuring activity; and the specific parameters of vacuum freeze-drying achieve rapid drying of the PDRN product, resulting in a white, loose powder with good solubility, low moisture content, and long-term storage without loss of activity.

[0035] In some embodiments, during the supramolecular assembly microsphere preparation stage, the amphiphilic supramolecular carrier is a mixture of an amphiphilic cyclodextrin derivative and a polyethylene glycol-polylactic acid block copolymer in a mass ratio of 2:1; the amphiphilic cyclodextrin derivative is 2-hydroxypropyl-cyclodextrin, and the polyethylene glycol-polylactic acid block copolymer contains PEG with a molecular weight of 2000 Da and PLA with a molecular weight of 8000 Da.

[0036] This invention constructs a highly efficient encapsulation carrier system adapted to PDRN by limiting the types and ratios of supramolecular carriers in the supramolecular assembly microsphere stage. The combination of 2-hydroxypropyl-cyclodextrin and PEG-PLA of a specific molecular weight utilizes their amphiphilic properties and complementary molecular structures to achieve efficient encapsulation of PDRN through intermolecular hydrogen bonds and hydrophobic interactions. Compared to a single carrier, the encapsulation efficiency of the combined carrier is significantly improved, and the resulting microsphere structure is stable and less prone to aggregation. The specific mass ratio is adapted to the molecular characteristics of PDRN, allowing for precise control of microsphere particle size. Furthermore, all carriers are made from cosmetic-grade raw materials, exhibiting good biocompatibility, biodegradability, and no skin irritation, making them suitable for various cosmetic formulations.

[0037] In some embodiments, the supramolecular assembly microsphere preparation stage includes the following steps: (1) The amphiphilic supramolecular carrier was dissolved in anhydrous ethanol to prepare an oil phase solution, and the pure PDRN was dissolved in PBS buffer to prepare an aqueous phase solution. Trehalose was added and then filtered aseptically. (2) The aqueous solution is added dropwise to the oil solution under constant temperature stirring, and the microspheres are assembled with ultrasonic assistance. High pressure homogenization is used to achieve uniform particle size. (3) After the microsphere dispersion was purified by dialysis and sterile filtered, trehalose was added for stabilization to obtain microsphere PDRN dispersion; (4) The microsphere PDRN dispersion is directly packaged as a liquid product, or freeze-dried, pulverized and graded into a solid product.

[0038] This invention achieves standardized, cross-linking agent-free production of PDRN microspheres by defining specific steps in supramolecular assembly microsphere formation. Aseptic preparation and filtration of the carrier solution prevent microbial contamination during microsphere formation, ensuring product hygiene and safety. Stepwise operations—aqueous addition of the aqueous phase, ultrasonic-assisted assembly, and high-pressure homogenization—achieve gradual microsphere shaping and uniform particle size, solving the problem of uneven particle size in traditional microsphere formation. The seamless integration of dialysis purification and stabilization removes residual solvents and free impurities, improving microsphere purity and storage stability. Stepwise preparation of liquid and solid products meets the raw material format requirements of different cosmetic formulations, enhancing product adaptability and application range.

[0039] In some embodiments, in the above steps: in step (1), the concentration of the oil phase solution is 8-10 mg / mL, the concentration of PDRN in the aqueous phase solution is 5-6 mg / mL, the pH of the PBS buffer is 6.8-7.0, and the initial and final concentration of trehalose is 0.3%; in step (2), the oil phase: aqueous phase = 3:7 (v / v), the assembly temperature is 25-30℃, the stirring speed is 300-350 r / min, the high pressure homogenization pressure is 35-40 MPa, and the homogenization is performed 2-3 times.

[0040] This invention achieves precise control over microsphere particle size, encapsulation efficiency, and structural stability by limiting key process parameters in the supramolecular assembly microsphere formation stage. The concentration parameters of the carrier solution and the PDRN aqueous phase ensure sufficient bonding between the carrier and PDRN, improving encapsulation efficiency. Specific oil-to-water ratios, assembly temperatures, and stirring speeds provide an optimal reaction environment for supramolecular self-assembly, promoting the full utilization of hydrogen bonding and hydrophobic interactions to form structurally stable microspheres. The pressure and number of homogenization cycles during high-pressure homogenization precisely control the microsphere particle size within the skin-penetrating range of 100-300 nm, ensuring uniform particle size distribution and enhancing the skin permeability and efficacy of the microspheres.

[0041] In some embodiments, in step (3), the dialysis bag retains a molecular weight cutoff of 10000 Da, and is dialyzed at a constant temperature of 4°C for 24 hours, with deionized water replaced every 8 hours; trehalose is added to a final concentration of 0.5%, and ultrasonically dispersed for 5 minutes at a power of 150W; 0.22m sterile filter membranes are used for sterile filtration.

[0042] This invention further enhances the purity, sterility, and storage stability of microsphere-based PDRN by limiting key parameters in the purification and stabilization processes. Specific molecular weight cutoff dialysis bags and parameters thoroughly remove residual anhydrous ethanol, unassembled carriers, and small molecule impurities, significantly improving microsphere purity and preventing skin irritation. Double filtration using a 0.22µm sterile membrane achieves sterilization of the microsphere dispersion, meeting the microbial limits for cosmetic raw materials. The added trehalose concentration and ultrasonic dispersion parameters form a stable protective barrier on the microsphere surface, preventing aggregation and sedimentation during storage and application, thus improving storage stability and formulation compatibility.

[0043] In some embodiments, the prepared microspheres of PDRN showed a cumulative release rate of 60% in PBS buffer (pH 7.0) over 24 hours and 85% over 48 hours. After 6 months of storage at 4°C, the microspheres showed no aggregation or precipitation, with a 10% change in particle size and a 5% change in encapsulation efficiency. In vitro skin irritation tests showed a cell viability of 90% and no skin irritation.

[0044] This invention ensures the efficacy, stability, and skin safety of the product by defining the core performance indicators of microsphere-derived PDRN. Specific sustained-release performance indicators achieve long-lasting release of PDRN, extending the duration of skin efficacy and resulting in a more sustained effect compared to non-microsphere-derived PDRN. Excellent storage stability indicators ensure that the microsphere structure, particle size, and encapsulation efficiency do not change significantly during long-term storage, extending the product's shelf life. Meeting the required in vitro cell viability indicators proves that the product is non-irritating to the skin and has excellent biocompatibility, making it suitable not only for healthy skin but also safe for use in skincare products for sensitive and damaged skin, significantly enhancing the product's application value and market competitiveness.

[0045] The present invention will be further described below with reference to several embodiments, but the present invention is not limited to these embodiments.

[0046] Example 1 A process for preparing highly active supramolecularly assembled microspheres of PDRN includes two stages: biosynthesis-enzymatic extraction and purification of PDRN, and supramolecular assembly and microsphere preparation of PDRN. The specific steps are as follows: Phase 1: Biosynthesis of PDRN - Enzymatic Decoupling, Extraction and Purification Genetically engineered E. coli fermentation synthesis of PDRN precursor Genetically engineered *Escherichia coli* strain BL21 was inoculated onto LB slant medium and cultured at 37°C for 12 h until OD600 = 0.7 to complete strain activation. The activated strain was then inoculated into LB seed medium at a 1:100 (v / v) ratio and placed in a constant-temperature shaking fermenter. The culture was carried out at 37°C and 180 rpm for 8 h with an aeration rate of 1:1.5 vvm to obtain seed culture. The seed culture was then inoculated into a 500L fermenter at a 5% (v / v) inoculation rate. The fermentation broth was cultured in LB medium (300 L) with an initial pH of 7.1 at 37 °C and stirred at 200 rpm. The aeration rate was 1:2 vvm, and nitrogen gas was introduced for oxygen isolation (flow rate 0.5 m / h). When the OD600 of the fermentation broth reached 1.1, IPTG inducer was added to a final concentration of 0.5 mmol / L. The temperature was lowered to 30 °C and fermentation continued for 16 h. The PDRN precursor content in the fermentation broth was measured to be 1.3 g / L. Fermentation was then stopped and the fermentation broth was cooled to 4 °C and refrigerated.

[0047] Fermentation broth crushing and crude extraction The fermentation broth refrigerated at 4°C was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 20 minutes at 4°C. The bacterial cell precipitate was collected. Tris-HCl buffer (pH 7.5) was added at a ratio of bacterial cell precipitate to Tris-HCl buffer of 1:8 (g:mL), and the mixture was stirred to obtain the bacterial cell solution. The bacterial cell solution was then transferred to an ultrasonic extraction vessel and subjected to intermittent ultrasonic treatment (4 minutes on, 2 minutes off) at 4°C, 250W, and 28kHz for 30 minutes. After ultrasonic disruption, the mixture was filtered through an 80-mesh plate and frame filter. A crude extract of PDRN precursor was obtained. Salmon testis micronized powder (0.5% by volume of crude extract) was added to the crude extract and stirred until well mixed. Then, a compound enzyme preparation (deoxyribonuclease I: ribonuclease = 3:1) and EDTA-2Na (0.02 mol / L) were added at a final addition amount of 1.0% of the dry weight of the crude extract. The pH was adjusted to 5.8, and the mixture was transferred to an enzymatic hydrolysis reactor. The mixture was stirred at 45°C and 70 r / min for 2.0 h for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the hydrolysate was rapidly heated to 55°C and kept at 55°C for 15 min to inactivate the enzyme, and then cooled to 4°C.

[0048] PDRN purification The enzymatic hydrolysate was adjusted to pH 7.5 with Tris-HCl buffer and filtered through a 0.45 μm microfiltration membrane to obtain the loading solution. The loading solution was loaded onto a pretreated D101 macroporous resin chromatography column at a flow rate of 1.2 BV / h and allowed to stand for 2 h for adsorption. The resin column was washed with deionized water at a flow rate of 2.0 BV / h until the refractive index of the effluent reached 1.005. Elution was performed with 45% (v / v) ethanol aqueous solution at a flow rate of 1.5 BV / h, collecting 2.0–3.0 BV of eluent. The eluent was then transferred to an ultrafiltration membrane separation system. The PDRN (molecular weight 5000 Da) was filtered under cross-flow conditions at an operating pressure of 0.18 MPa and 4°C, and then dialyzed with a small amount of deionized water. The purified ultrafiltration solution was transferred to a vacuum concentration tank and concentrated at 45°C and a vacuum of -0.085 MPa to a solid content of 32%. It was then transferred to a vacuum freeze dryer, pre-frozen to -40°C and kept at that temperature for 4 hours, and dried at a vacuum of 10 Pa and a sublimation temperature of 28°C for 18 hours to obtain pure PDRN. The purity was tested to be 96.2%, the moisture content was 4.5%, and the impurity protein content was 0.4%.

[0049] Phase 2: Supramolecular assembly and microsphere preparation of PDRN Preparation of supramolecular carrier solutions In a sterile operating room, 2-hydroxypropyl-cyclodextrin (HP-CD) and polyethylene glycol-polylactic acid block copolymer (PEG2000-PLA8000) were mixed at a mass ratio of 2:1, and anhydrous ethanol was added and stirred to dissolve, preparing an oil phase solution with a concentration of 9 mg / mL. The solution was then ultrasonically dispersed at 150 W for 10 min. Pure PDRN was taken and dissolved in PBS buffer at pH 6.9 with stirring, preparing an aqueous phase solution with a concentration of 5.5 mg / mL. Trehalose was added to a final concentration of 0.3%, and the solution was stirred and dispersed at 150 W for 5 min. The oil phase and aqueous phase solutions were then filtered through a 0.22 μm sterile filter membrane for later use.

[0050] Preparation of PDRN microspheres by supramolecular self-assembly The oil phase solution was transferred to a supramolecular assembly reactor. The temperature was controlled at 28°C using a thermostat, and high-speed stirring was started at 320 r / min. Nitrogen gas was introduced for oxygen isolation (flow rate 0.3 m / h). The aqueous phase solution was slowly added dropwise to the oil phase at a ratio of oil phase:water phase = 3:7 (v / v) using a constant flow pump at a rate of 1.2 mL / min. After the addition was completed, stirring was continued for 30 min. The ultrasonic module of the reactor was turned on, and the ultrasonic treatment was intermittently operated at 200W and 40kHz (operating for 3 min, pausing for 1 min) for 20 min. The assembled microsphere dispersion was transferred to a high-pressure homogenizer and homogenized twice at 38 MPa pressure for 5 min each time. The microsphere particle size was measured by a laser particle size analyzer, and the PDI was 0.21.

[0051] Microsphere purification and stabilization treatment The homogenized microsphere dispersion was transferred to a dialysis bag with a molecular weight cutoff of 10,000 Da, placed in a large amount of deionized water, and dialyzed at a constant temperature of 4°C for 24 hours, with the deionized water being replaced every 8 hours. After dialysis, the dispersion was filtered through a 0.22 μm sterile filter membrane. Trehalose was added to the filtered microsphere dispersion to a final concentration of 0.5%, and the mixture was stirred and dispersed by ultrasonication at 150 W for 5 minutes to obtain a microsphere-modified PDRN dispersion.

[0052] Finished product preparation Liquid finished product: Dispense the microsphere-sized PDRN dispersion into sterile sealed containers in a sterile operating table and store at 4°C; Solid product: The microsphered PDRN dispersion was transferred to a vacuum freeze dryer, pre-frozen to -40℃ and kept at that temperature for 4 hours, and then dried at a vacuum of 10 Pa and a sublimation temperature of 29℃ for 20 hours to obtain a white loose powder. The powder was then pulverized to 100 mesh using a pulverizer and grading machine, sieved, and then aseptically sealed and packaged. It was stored at 4℃, protected from light and in a dry place.

[0053] The microspheres of PDRN prepared in this embodiment were tested and found to have a purity of 96.2%, an encapsulation efficiency of 88.5%, and an activity retention rate of 92.3%. In PBS buffer (pH 7.0), the cumulative release rate was 56.2% after 24 hours and 87.8% after 48 hours. After refrigeration at 4°C for 6 months, the microspheres showed no aggregation or precipitation, a particle size change of 7.5%, and a change in encapsulation efficiency of 3.2%. In an in vitro skin irritation test, the cell viability was 94.6%, and there was no irritation.

[0054] Example 2 A preparation process for highly active supramolecular assembled microspheres of PDRN differs from that in Example 1 in that: The PDRN precursor content in the fermentation broth after IPTG induction during the fermentation stage was 1.2 g / L. The concentration of supramolecular carrier in the oil phase solution was 8 mg / mL, and the concentration of PDRN in the aqueous phase solution was 5 mg / mL. The supramolecular self-assembly temperature was 25℃, the stirring speed was 300 r / min, and the aqueous phase droplet acceleration rate was 1.0 mL / min. High-pressure homogenization was performed at a pressure of 35 MPa, and the homogenization was repeated 3 times.

[0055] Testing showed that the microspheres of PDRN prepared in this embodiment had a purity of 95.1%, a particle size of 180 nm, a PDI of 0.23, an encapsulation efficiency of 85.2%, and an activity retention rate of 90.5%. The cumulative release rate after 24 hours was 58.7%, and the cumulative release rate after 48 hours was 85.1%. After 6 months of refrigeration at 4°C, the particle size changed by 9.2%, and the encapsulation efficiency changed by 4.5%. In an in vitro skin irritation test, the cell viability rate was 91.3%, and there was no irritation.

[0056] Example 3 A preparation process for highly active supramolecular assembled microspheres of PDRN differs from that in Example 1 in that: The PDRN precursor content in the fermentation broth after IPTG induction during the fermentation stage was 1.4 g / L. The concentration of supramolecular carrier oil phase solution was 10 mg / mL, and the concentration of PDRN in aqueous phase solution was 6 mg / mL. The supramolecular self-assembly temperature was 30℃, the stirring speed was 350 r / min, and the aqueous phase droplet acceleration rate was 1.5 mL / min. High-pressure homogenization was performed at a pressure of 40 MPa, and the homogenization was repeated twice.

[0057] Testing showed that the microspheres of PDRN prepared in this embodiment had a purity of 97.0%, a particle size of 260 nm, a PDI of 0.20, an encapsulation efficiency of 90.1%, and an activity retention rate of 93.5%. The cumulative release rate after 24 hours was 54.3%, and the cumulative release rate after 48 hours was 89.2%. After 6 months of refrigeration at 4°C, the particle size changed by 6.8%, and the encapsulation efficiency changed by 2.8%. In an in vitro skin irritation test, the cell viability was 95.2%, and there was no irritation.

[0058] Proportional Design To verify the superiority of the process of this invention, seven comparative examples were set up. Each comparative example only changed the core process parameters / methods, while the remaining operations remained the same as in Example 1. The specific design is as follows: Comparative Example 1: PDRN was extracted using a single water extraction method from salmon testes without the coupling of genetically engineered E. coli fermentation synthesis and enzymatic hydrolysis. Specifically, salmon teste powder was added to 15 times its mass of deionized water and extracted in a 90°C water bath for 2 hours. After filtration, PDRN was purified by alcohol precipitation.

[0059] Comparative Example 2: PDRN purification was performed using a traditional 75% high-concentration ethanol precipitation process, replacing the macroporous resin-ultrafiltration membrane purification method of the present invention.

[0060] Comparative Example 3: Microspheres were formed using a traditional emulsification crosslinking method with glutaraldehyde as the crosslinking agent, replacing the supramolecular carrier self-assembly process of this invention.

[0061] Comparative Example 4: The supramolecular carrier used only 2-hydroxypropyl-cyclodextrin and was not compounded with PEG-PLA block copolymer.

[0062] Comparative Example 5: Supramolecular self-assembly without ultrasonic assistance steps, directly stirring and emulsifying to form microspheres.

[0063] Comparative Example 6: The microspheres were prepared without high-pressure homogenization and the microsphere particle size was not uniformly controlled.

[0064] Comparative Example 7: PDRN was not microsphere-treated; the pure PDRN prepared according to this invention was used directly.

[0065] Performance testing and results analysis The core performance indicators of the products of Examples 1-3 and Comparative Examples 1-7 were tested. The test items included PDRN purity, microsphere encapsulation rate, microsphere particle size and PDI, 24h / 48h cumulative release rate, particle size change rate after 6 months of refrigeration at 4℃, and in vitro cell viability. The test methods adopted were the general standard methods of the cosmetic raw material industry. The test results are shown in Tables 1 and 2 below.

[0066] Table 1 Results of basic performance indicators of the product Sample PDRN purity (%) Microsphere encapsulation efficiency (%) Microsphere size (nm) PDI Example 1 96.2 88.5 210 0.21 Example 2 95.1 85.2 180 0.23 Example 3 97.0 90.1 260 0.20 Comparative Example 1 78.5 72.3 280 0.35 Comparative Example 2 89.7 80.5 225 0.24 Comparative Example 3 96.0 68.2 350 0.42 Comparative Example 4 96.1 75.8 240 0.28 Comparative Example 5 96.2 82.1 310 0.38 Comparative Example 6 96.2 87.9 420 0.51 Comparative Example 7 96.2 - - - Table 2 Results of product sustained-release, stability and biocompatibility tests Sample 24-hour cumulative release rate (%) 48-hour cumulative release rate (%) Particle size change rate (%) after 6 months of refrigeration at 4℃ In vitro cell survival rate (%) Example 1 56.2 87.8 7.5 94.6 Example 2 58.7 85.1 9.2 91.3 Example 3 54.3 89.2 6.8 95.2 Comparative Example 1 75.3 92.5 15.8 88.2 Comparative Example 2 62.5 86.9 10.5 90.1 Comparative Example 3 82.6 95.3 22.7 75.4 Comparative Example 4 65.8 88.2 12.3 93.8 Comparative Example 5 70.2 89.5 18.6 94.2 Comparative Example 6 78.9 93.1 20.1 94.5 Comparative Example 7 98.5 99.8 - 94.8 The above test results show that: The microspheres of PDRN prepared in Examples 1-3 of this invention all had a purity of 95%, an encapsulation efficiency of 85%, and a microsphere particle size controlled between 100-300 nm with a PDI of 0.25, which are far superior to the comparative examples. This demonstrates that biosynthesis-enzymatic extraction can significantly improve the purity of PDRN. The combined process of amphiphilic supramolecular carrier compounding + ultrasound assistance + high-pressure homogenization can achieve uniform preparation of microspheres and improve the encapsulation effect.

[0067] Regarding sustained-release performance, the cumulative release rate of the product in the examples was 60% after 24 hours and 85% after 48 hours, achieving long-term sustained release. In contrast, the cumulative release rate of Comparative Example 1 (single water extraction), Comparative Example 3 (emulsified crosslinking), and Comparative Example 7 (unmicrosphereized) was only 75% after 24 hours, with extremely poor sustained-release effect. This demonstrates that the supramolecular self-assembly microsphereization process of the present invention can effectively regulate the release rate of PDRN and prolong the duration of efficacy.

[0068] In terms of stability, the particle size change rate of the product in the example after 6 months of refrigeration at 4°C was 10%, while the particle size change rate of comparative examples 1, 3, 5, and 6 was 15%, and some even exceeded 20%. This proves that supramolecular carrier compounding, ultrasound-assisted assembly, and high-pressure homogenization can significantly improve the storage stability of microspheres and prevent microsphere aggregation and particle size increase.

[0069] Regarding biocompatibility, the cell survival rate of the products in the examples and comparative examples 4-7 was 90%, while the cell survival rate of comparative example 1 (single water extract containing impurities) and comparative example 3 (emulsion cross-linking containing glutaraldehyde residue) was significantly reduced, especially comparative example 3, which had a survival rate of only 75.4%. This proves that the present invention uses cosmetic-grade raw materials throughout the process, with no toxic reagent residues, and the supramolecular self-assembly process is non-irritating to the skin, which is far superior to the traditional emulsion cross-linking method.

[0070] Although the unmicrosphered PDRN pure product in Comparative Example 7 showed good cell compatibility, its release rate was extremely fast, with a cumulative release rate of 98.5% over 24 hours, making it unable to achieve long-lasting skincare. This demonstrates that microsphere treatment is the key to enhancing the application value of PDRN cosmetics.

[0071] The preparation process of highly active supramolecular assembled microspheres of PDRN provided by this invention has the following advantages compared with the prior art: This invention uses a coupling method of fermentation synthesis of genetically engineered E. coli and enzymatic hydrolysis of salmon testes to extract PDRN, which balances the yield and bioactivity of PDRN and solves the problems of low purity from single natural extraction and poor activity from chemical synthesis. The prepared PDRN has a purity of 95% and a content of impurities of 0.5%, laying a high-activity raw material foundation for subsequent microsphere preparation.

[0072] This invention innovatively adopts a purification method that combines macroporous resin and ultrafiltration membrane, replacing the traditional high-concentration alcohol precipitation process. This reduces ethanol consumption by more than 65%, is environmentally friendly, eliminates the risk of organic solvent residue, and improves purification efficiency by 40%, with a PDRN recovery rate of 85%, significantly reducing raw material costs.

[0073] This invention uses amphiphilic cyclodextrin derivatives and PEG-PLA as supramolecular carriers to achieve the self-assembly of PDRN into microspheres through intermolecular hydrogen bonds and hydrophobic interactions. No cross-linking agent is required throughout the process, which fundamentally solves the problems of reagent residue and skin irritation caused by traditional emulsification cross-linking methods. The prepared microsphere PDRN is non-irritating and suitable for cosmetics for various skin types, including sensitive skin and damaged skin.

[0074] This invention, through precise control of assembly temperature, stirring speed, material-liquid ratio, and high-pressure homogenization parameters, prepares microspheres with uniform particle size (100-300nm) and a PDI of 0.25. This particle size range easily penetrates the stratum corneum of the skin, and the encapsulation rate is 85%, achieving long-term sustained release of PDRN. The cumulative release rate is 60% after 24 hours and 85% after 48 hours, and the duration of efficacy is more than twice that of non-microsphereized PDRN.

[0075] The microsphere PDRN prepared by this invention exhibits excellent stability. After 6 months of refrigeration at 4°C, there is no aggregation or precipitation, the particle size changes by 10%, the encapsulation rate changes by 5%, and the activity retention rate is 90%. It can maintain the biological activity of the raw material for a long time and meet the storage requirements of cosmetic raw materials.

[0076] This invention utilizes conventional industrial equipment from the cosmetics and bio-fermentation industries throughout the entire process, eliminating the need for customized specialized equipment. Companies can directly modify their existing production lines, ensuring precise and controllable process steps, achieving an 85% raw material utilization rate, reducing industrialization costs by 30%, and resulting in a high degree of product standardization, making it easy to achieve large-scale production.

[0077] All raw materials and reagents used in this invention comply with the requirements of the "Cosmetic Safety Technical Specifications" (2022 edition). The ampicillin used in the fermentation stage can be completely removed through ultrafiltration and dialysis. The final product has no toxic or harmful residues such as antibiotics and organic solvents, has good skin compatibility, and can be directly used in various cosmetic formulations such as toners, serums, repair masks, lotions, and creams, resulting in high added value.

[0078] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A preparation process for highly active supramolecular assembled microspheres of PDRN, characterized in that, The process includes a biosynthesis-enzymatic hydrolysis coupled extraction and purification stage for PDRN and a supramolecular assembly microsphere preparation stage for PDRN. The biosynthesis-enzymatic hydrolysis coupled extraction and purification stage involves synthesizing PDRN precursors through fermentation by genetically engineered microorganisms, followed by enzymatic hydrolysis-assisted extraction from marine biological tissues, and purification using a macroporous resin-ultrafiltration membrane to obtain pure PDRN. The supramolecular assembly microsphere preparation stage uses amphiphilic supramolecular carriers as raw materials, achieving PDRN microsphere formation through intermolecular hydrogen bonding and hydrophobic interactions, without the need for cross-linking agents. The prepared microspheres of PDRN have a purity of 95%, a particle size of 100-300 nm, an encapsulation efficiency of 85%, and an activity retention rate of 90%.

2. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 1, characterized in that, In the biosynthesis-enzyme decoupling extraction and purification stage, the genetically engineered microorganism is the modified Escherichia coli BL21, the marine biological tissue is salmon testes, the compound enzyme preparation is a mixture of deoxyribonuclease I and ribonuclease in a mass ratio of 3:1, the macroporous resin is D101 type weakly polar macroporous adsorption resin, and the ultrafiltration membrane has a molecular weight cutoff of 5000 Da.

3. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 2, characterized in that, The biosynthesis-enzymatic decoupling extraction and purification stage includes the following steps: (1) After activating Escherichia coli BL21 strain, seed culture was prepared and inoculated into LB medium for fermentation. IPTG inducer was added to synthesize PDRN precursor. Fermentation was terminated when the PDRN precursor content in the fermentation broth reached 1.2 g / L. (2) The fermentation broth was centrifuged to collect the cell precipitate, ultrasonically crushed and filtered to obtain crude extract, salmon testis powder and compound enzyme preparation were added for enzymatic hydrolysis, and the enzyme was inactivated and then cooled. (3) After the enzymatic hydrolysate is adsorbed by macroporous resin, washed with water and eluted with ethanol, it is purified and concentrated by ultrafiltration, and then concentrated by vacuum decompression and freeze-dried to obtain pure PDRN.

4. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 3, characterized in that, In step (1), the final concentration of IPTG inducer was 0.5 mmol / L, the induction temperature was 30℃, and the induction time was 16h. In step (2), the ultrasonic disruption conditions were 4℃, 250W power, 28kHz frequency, and intermittent operation for 30min. The enzymatic hydrolysis conditions were pH 5.5-6.0, 45℃, 70r / min stirring for 2.0h, and the enzyme inactivation conditions were 55℃ for 15min.

5. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 3, characterized in that, In step (3), the eluent is a 45% (v / v) aqueous ethanol solution, the ultrafiltration operation pressure is 0.15-0.20 MPa and the temperature is 4℃; the vacuum concentration temperature is 45℃ and the vacuum degree is -0.08~-0.09 MPa; the vacuum freeze-drying pre-freezes to -40℃ and holds for 4h, the vacuum degree is 10Pa and the sublimation temperature is 30℃, and the drying is carried out for 18h.

6. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 1, characterized in that, In the supramolecular assembly microsphere preparation stage, the amphiphilic supramolecular carrier is a mixture of amphiphilic cyclodextrin derivative and polyethylene glycol-polylactic acid block copolymer in a mass ratio of 2:1; the amphiphilic cyclodextrin derivative is 2-hydroxypropyl-cyclodextrin, and the polyethylene glycol-polylactic acid block copolymer has a molecular weight of PEG of 2000 Da and a molecular weight of PLA of 8000 Da.

7. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 6, characterized in that, The supramolecular assembly microsphere preparation stage includes the following steps: (1) The amphiphilic supramolecular carrier was dissolved in anhydrous ethanol to prepare an oil phase solution, and the pure PDRN was dissolved in PBS buffer to prepare an aqueous phase solution. Trehalose was added and then filtered aseptically. (2) The aqueous solution is added dropwise to the oil solution under constant temperature stirring, and the microspheres are assembled with ultrasonic assistance. High pressure homogenization is used to achieve uniform particle size. (3) After the microsphere dispersion was purified by dialysis and sterile filtered, trehalose was added for stabilization to obtain microsphere PDRN dispersion; (4) The microsphere PDRN dispersion is directly packaged as a liquid product, or freeze-dried, pulverized and graded into a solid product.

8. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 7, characterized in that, In step (1), the concentration of the oil phase solution is 8-10 mg / mL, the concentration of PDRN in the aqueous phase solution is 5-6 mg / mL, the pH of the PBS buffer is 6.8-7.0, and the initial and final concentration of trehalose is 0.3%; in step (2), the ratio of oil phase to aqueous phase is 3:7 (v / v), the assembly temperature is 25-30℃, the stirring speed is 300-350 r / min, the high-pressure homogenization pressure is 35-40 MPa, and the homogenization is performed 2-3 times.

9. The preparation process of highly active supramolecular assembled microspheres of PDRN according to claim 7, characterized in that, In step (3), the molecular weight cutoff of the dialysis bag is 10000 Da, and the dialysis is carried out at a constant temperature of 4℃ for 24 hours. The deionized water is replaced every 8 hours. Trehalose is added to a final concentration of 0.5%, and ultrasonic dispersion is performed for 5 minutes at a power of 150W. A 0.22m sterile filter membrane is used for sterile filtration.

10. The preparation process of highly active supramolecular assembled microspheres of PDRN according to any one of claims 1-9, characterized in that, The prepared microspheres of PDRN showed a cumulative release rate of 60% in PBS buffer after 24 hours and 85% after 48 hours. After 6 months of storage at 4°C, the microspheres showed no aggregation or precipitation, with a particle size change of 10% and an encapsulation efficiency change of 5%. In vitro skin irritation tests showed a cell viability of 90% and no skin irritation.