Collagen microspheres loaded with PDRN, and preparation method and application thereof

Abstract

The present application relates to a kind of PDRN-loaded collagen microspheres and its preparation method and application, belong to the cosmetic technical field.The preparation method provided by the present application can realize the stable loading of PDRN while ensuring the stability of microsphere structure, thereby improving the thermal stability of microsphere, particle size uniformity, in vitro degradation behavior and PDRN retention rate.Because the stable crosslinking network has been formed before the microsphere is loaded with PDRN, the pore and surface characteristics of the microsphere can effectively fix PDRN, which can slow release PDRN to some extent, reduce the risk of rapid diffusion and degradation, and prolong the action time.Using the strategy of first preparing collagen microspheres and then loading PDRN through physical adsorption, the active molecules can be gently protected, and the loss of PDRN during preparation can be reduced, which can improve the loading capacity while enhancing the activity retention rate and loading stability of PDRN.

Description

Technical Field

[0001] This invention relates to a collagen microsphere loaded with PDRN, its preparation method and application, belonging to the field of cosmetic technology. Background Technology

[0002] Collagen, as a natural polymer material, possesses excellent biocompatibility and biodegradability, and can provide a suitable microenvironment for cells, playing a positive role in cell adhesion, proliferation, and tissue repair. It is now widely used in the fields of medical aesthetics and tissue engineering. To improve the mechanical properties, structural stability, and retention time of collagen raw materials in vivo, current technologies typically prepare collagen in the form of microspheres, microparticles, or porous structures for loading active ingredients and achieving sustained release.

[0003] Chinese patent document CN120860321A (application number 202511377887.0) reports a technical solution for preparing collagen microspheres via emulsification crosslinking. This method involves dispersing a collagen solution in an oil phase to form an emulsion, followed by crosslinking and curing to obtain collagen microspheres with controllable particle size. These microspheres improve the structural stability of collagen raw materials to a certain extent and can serve as carriers for drugs or bioactive ingredients. In practical applications, the performance of the microspheres directly affects the stability and release behavior of the active ingredients they carry.

[0004] Polydeoxyribonucleotides (PDRNs) are bioactive substances composed of deoxyribonucleotide fragments. They have been widely studied in recent years for their effects on promoting cell proliferation, inhibiting apoptosis, promoting tissue repair, and slowing down aging. However, as a water-soluble biomolecule, PDRNs are easily diffused and degraded in vivo. Their application is typically done directly in solution form or simply mixed with carriers such as hyaluronic acid and hydrogels for medical aesthetics and tissue repair. The lack of a stable loading structure makes it difficult to effectively protect and slowly release PDRNs, thus limiting their duration of action and bioavailability.

[0005] Collagen has a relatively slow onset of action but a long duration of action, while PDRN has a faster onset of action but a relatively limited duration of action. The two are complementary in terms of the duration of action. At the same time, collagen can also be prepared into microsphere structures to provide relatively stable loading and protection for PDRN. Therefore, the combined application of the two has certain practical application value.

[0006] Chinese patent document CN118681062A (application number 202410697320.0) discloses a PDRN-composite recombinant collagen microsphere and its preparation method. However, the preparation method involves simultaneously introducing collagen and PDRN into microspheres during emulsification and cross-linking. The state of PDRN in the cross-linking or spheroidization environment is highly susceptible to change, thus affecting the maintenance of its bioactivity. Therefore, how to maintain the structural stability of collagen microspheres while achieving effective PDRN loading and activity retention warrants further investigation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a PDRN-loaded collagen microsphere, its preparation method, and its application. The preparation method provided by this invention can achieve stable loading of PDRN while ensuring the structural stability of the microspheres, thereby improving the thermal stability, particle size uniformity, in vitro degradation behavior, and PDRN retention rate of the microspheres.

[0008] The technical solution of the present invention is as follows: A method for preparing PDRN-loaded collagen microspheres includes the following steps: (1) Add recombinant collagen to buffer solution at a mass ratio of 0.5%~1.5% and homogenize to obtain collagen solution; (2) The collagen solution and the oil phase are mixed at a volume ratio of 1: (1~3) and homogenized until a stable emulsion system is formed. A cross-linking agent is added to the emulsion system and the mixture is stirred at room temperature and 300~800 rpm for 1~4 hours. The supernatant is discarded by centrifugation, and the precipitate is washed with water for injection 3~5 times to obtain the washed collagen microspheres. (3) Add PDRN to the buffer solution at a mass ratio of 0.5% to 1.5%, and stir at 45 to 55°C until completely dissolved to obtain a PDRN solution; (4) Disperse the washed collagen microspheres in PDRN solution, control the mass ratio of recombinant collagen to PDRN to be 1:(0.5~1.5), and load PDRN by shaking at 50~120 rpm for 4~8 h at room temperature; (5) Centrifuge and discard the supernatant, wash the precipitate 3-5 times with water for injection, and dry it under low temperature to obtain collagen microspheres loaded with PDRN.

[0009] According to a preferred embodiment of the present invention, the recombinant collagen is type I recombinant collagen, type III recombinant collagen, or a mixture of the two.

[0010] More preferably, the recombinant collagen is a mixture of type I recombinant collagen and type III recombinant collagen, wherein the type I recombinant collagen and type III recombinant collagen are compounded in a mass ratio of (1-6):1.

[0011] More preferably, the type I recombinant collagen and type III recombinant collagen are compounded in a mass ratio of 4:1.

[0012] According to a preferred embodiment of the present invention, the components of the buffer solution in steps (1) and (3) are: 7~10 wt‰ NaCl, 0.16~0.30 wt‰ Na2HPO4, 0.03~0.10 wt‰ NaH2PO4, and the solvent is water for injection.

[0013] According to a preferred embodiment of the present invention, the preparation method of the oil phase in step (2) is as follows: Span 80 is added to liquid paraffin at a mass-volume ratio (g / mL) of 1% to 3%, and stirred until homogeneous at room temperature.

[0014] According to a preferred embodiment of the present invention, the crosslinking agent in step (2) is EDC and NHS.

[0015] More preferably, the amount of EDC added is 1% to 3% of the recombinant collagen protein, and the mass ratio of EDC to NHS is 1:1.

[0016] According to a preferred embodiment of the present invention, the homogenization conditions in step (1) are: rotation speed 10,000~12,000 rpm, homogenization for 5 min, shutdown for 5 min, repeated 4~6 times; the homogenization conditions in step (2) are: rotation speed 10,000~12,000 rpm, homogenization for 3 min, shutdown for 2 min, repeated 3~5 times.

[0017] According to a preferred embodiment of the present invention, the centrifugation conditions described in steps (2) and (5) are: centrifugation at 4,500 to 5,000 rpm for 10 to 15 min.

[0018] The collagen microspheres loaded with PDRN prepared by the above preparation method.

[0019] According to a preferred embodiment of the present invention, the microspheres have a particle size distribution D50 value of 45~58μm, a D90 value of 75~89μm, and a thermal denaturation temperature of 56~66℃.

[0020] The PDRN-loaded collagen microspheres have collagen microspheres as the core and PDRN as the coating layer. The PDRN is adsorbed on the surface or in the internal pores of the collagen microspheres.

[0021] The application of PDRN-loaded collagen microspheres prepared by the above preparation method or the above-mentioned PDRN-loaded collagen microspheres in the preparation of tissue repair or medical aesthetic products.

[0022] Beneficial effects: This invention prepares collagen microspheres loaded with PDRN. Because the microspheres have already formed a stable cross-linked network before PDRN loading, their porosity and surface properties effectively immobilize PDRN, providing a sustained-release effect, reducing the risk of rapid diffusion and degradation, and prolonging the reaction time. The strategy of first preparing collagen microspheres and then loading PDRN through physical adsorption gently protects the active molecules, reduces PDRN loss during preparation, and enhances both the loading capacity and the activity retention rate and loading stability of PDRN.

[0023] PDRN is a water-soluble biomolecule that readily diffuses and degrades in vivo. It possesses biological functions such as promoting cell proliferation, inhibiting apoptosis, promoting tissue repair, and slowing aging. Collagen microspheres, on the other hand, have a stable structure and slow degradation, promoting cell adhesion and proliferation, supporting tissue repair, and improving the local microenvironment. By using collagen microspheres as the core and PDRN as the coating layer, the two can complement each other in terms of their timing of action: PDRN exerts its effect initially, while collagen gradually exerts its effect later, thereby enhancing the overall effect.

[0024] This invention uses a mixture of type I and type III recombinant collagen to prepare microspheres. The resulting microspheres have a stable structure and can be used for loading and releasing PDRN. Attached Figure Description

[0025] Figure 1 The image shows an electron microscope image of the PDRN-collagen microspheres prepared in Example 1. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0028] Example 1 A method for preparing PDRN-loaded collagen microspheres, the specific steps of which are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0029] Solution 2: Weigh 1.0 g of type I recombinant collagen (purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., SPA), add 99.0 g of solution 1, and process using a homogenizer at a speed of 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0030] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0031] Solution 4: Weigh 1.0 g of PDRN (purchased from Ruijiming (Shandong) Biotechnology Co., Ltd., RJMPDRN-M) and add it to 99 g of Solution 1. Stir at 50℃ until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0032] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, homogenize for 3 min, stop for 2 min, and repeat 3 to 5 times until a stable emulsion system is formed.

[0033] (3) After emulsification, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to the emulsion system as crosslinking agents. The crosslinking reaction was carried out at 500 rpm and room temperature for 2 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0034] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0035] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 60 rpm for 6 h at room temperature to load PDRN.

[0036] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0037] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0038] The electron micrograph of the PDRN-collagen microspheres is shown below. Figure 1 As shown, the microsphere is spherical or near-spherical with an irregular, rough surface structure. It has a collagen microsphere as its core and PDRN as its coating layer. PDRN is adsorbed on the surface or in the internal pores of the collagen microsphere, and the surface of the microsphere has an irregular, rough structure.

[0039] Example 2 A method for preparing PDRN-loaded collagen microspheres, differing from Example 1 in that solution 2 is prepared with type III recombinant collagen, and the specific steps are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0040] Solution 2: Weigh 1.0 g of type III recombinant collagen (purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., SFA01), add 99.0 g of solution 1, and process using a homogenizer at a speed of 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0041] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0042] Solution 4: Weigh 1.0 g of PDRN and add it to 99 g of Solution 1. Stir at 50°C until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0043] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, and the homogenization is stopped for 2 minutes after 3 min. Repeat this process 3 to 5 times to form a stable emulsion system.

[0044] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 500 rpm and room temperature for 2 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0045] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0046] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 60 rpm for 6 h at room temperature to load PDRN.

[0047] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0048] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0049] Example 3 A method for preparing PDRN-loaded collagen microspheres differs from Example 1 in that solution 2 is prepared with both type I and type III recombinant collagen in a mass ratio of 1:1. The specific steps are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0050] Solution 2: Weigh 0.5 g of type I recombinant collagen and 0.5 g of type III recombinant collagen, add 99.0 g of solution 1, and process using a homogenizer at a speed of 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0051] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0052] Solution 4: Weigh 1.0 g of PDRN and add it to 99 g of Solution 1. Stir at 50°C until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0053] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, and the homogenization is stopped for 2 minutes after 3 min. Repeat this process 3 to 5 times to form a stable emulsion system.

[0054] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 500 rpm and room temperature for 2 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0055] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0056] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 60 rpm for 6 h at room temperature to load PDRN.

[0057] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0058] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0059] Example 4 A method for preparing PDRN-loaded collagen microspheres differs from Example 1 in that solution 2 is prepared with both type I and type III recombinant collagen in a mass ratio of 4:1. The specific steps are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0060] Solution 2: Weigh 0.8 g of type I recombinant collagen and 0.2 g of type III recombinant collagen, add 99.0 g of solution 1, and process using a homogenizer at 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0061] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0062] Solution 4: Weigh 1.0 g of PDRN and add it to 99 g of Solution 1. Stir at 50°C until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0063] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, and the homogenization is stopped for 2 minutes after 3 min. Repeat this process 3 to 5 times to form a stable emulsion system.

[0064] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 500 rpm and room temperature for 2 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0065] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0066] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 60 rpm for 6 h at room temperature to load PDRN.

[0067] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0068] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0069] Example 5 A method for preparing PDRN-loaded collagen microspheres differs from Example 1 in that some parameters used in the preparation method are different. The specific steps are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0070] Solution 2: Weigh 1.0 g of type I recombinant collagen, add 99.0 g of solution 1, and process using a homogenizer at 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0071] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0072] Solution 4: Weigh 1.5 g of PDRN and add it to 98.5 g of Solution 1. Stir at 50°C until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0073] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, homogenize for 3 min, stop for 2 min, and repeat 3 to 5 times until a stable emulsion system is formed.

[0074] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 500 rpm and room temperature for 3 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0075] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0076] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 60 rpm for 6 h at room temperature to load PDRN.

[0077] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 4,500 rpm for 15 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0078] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0079] Example 6 A method for preparing PDRN-loaded collagen microspheres differs from Example 1 in that some parameters used in the preparation method are different. The specific steps are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0080] Solution 2: Weigh 1.0 g of type I recombinant collagen, add 99.0 g of solution 1, and process using a homogenizer at 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain collagen solution, i.e., solution 2.

[0081] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0082] Solution 4: Weigh 0.5 g of PDRN and add it to 99.5 g of Solution 1. Stir at 50°C until completely dissolved to obtain PDRN solution, i.e., Solution 4.

[0083] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, homogenize for 3 min, stop for 2 min, and repeat 3 to 5 times until a stable emulsion system is formed.

[0084] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 800 rpm and room temperature for 1.5 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0085] (4) After the cross-linking reaction is completed, the collagen microspheres are separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove residual oil phase, emulsifier and unreacted cross-linking agent.

[0086] (5) Disperse the washed collagen microspheres obtained in step (4) in solution 4 and place them in a shaker. Shake at 80 rpm for 4 h at room temperature to load PDRN.

[0087] (6) After loading is completed, the collagen microspheres loaded with PDRN are recovered by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres are rinsed with water for injection and washed more than 3 times to remove unloaded PDRN.

[0088] Finally, the obtained washed PDRN-loaded collagen microspheres were placed in... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer and dry. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0089] Comparative Example 1 A method for preparing PDRN-loaded collagen microspheres, the specific steps of which are as follows: (1) Prepare the solution as follows: Solution 1: Weigh 9.0 g NaCl, 0.22 g Na2HPO4 and 0.05 g NaH2PO4, add 988.3 g water for injection, stir until completely dissolved to obtain buffer solution, i.e., solution 1.

[0090] Solution 2: Weigh 1.0 g PDRN and add it to 98.0 g Solution 1. Stir at 50°C until completely dissolved. Then add 1.0 g type III recombinant collagen and process it using a homogenizer at 12,000 rpm for 5 min, stop for 5 min, and repeat 6 times to obtain Solution 2.

[0091] Solution 3: Measure 200 mL of liquid paraffin as an oily medium, add 4 g of Span 80 to it, and stir until homogeneous at room temperature to obtain the oil phase, i.e., solution 3.

[0092] (2) Using solution 2 as the aqueous phase, add it to solution 3, and then use a homogenizer for emulsification. The homogenization speed is 12,000 rpm, homogenize for 3 min, stop for 2 min, and repeat 3 to 5 times until a stable emulsion system is formed.

[0093] (3) After emulsification, EDC and NHS were added to the emulsion system as crosslinking agents, and the crosslinking reaction was carried out at 500 rpm and room temperature for 2 h to solidify the collagen microsphere structure. The amount of EDC added was 2% of the collagen protein mass, and the mass ratio of EDC to NHS was 1:1.

[0094] (4) After the cross-linking reaction was completed, the collagen microspheres were separated from the oil phase by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the microspheres were rinsed with water for injection, and the washing was repeated at least 3 times to remove residual oil phase, emulsifier, and unreacted cross-linking agent. Finally, the obtained washed PDRN-loaded collagen microspheres were... Pre-freeze at 20℃ for 12 hours, then place in a freeze dryer. The collagen microspheres loaded with PDRN were freeze-dried at 60℃ for 24 h to obtain the product (PDRN-collagen microspheres).

[0095] The PDRN-collagen microspheres have a spherical or near-spherical structure, and the interior of the microspheres has a collagen cross-linking network, with PDRN uniformly dispersed and embedded in the collagen cross-linking network.

[0096] Experimental Example 1 Particle size detection of PDRN-collagen microspheres The particle size of the collagen microspheres was determined using a Malvern particle size analyzer. The PDRN-collagen microspheres prepared in each example were dispersed in deionized water, ultrasonically dispersed, and then tested and recorded for particle size distribution. This method was used to characterize the particle size of microspheres prepared in different examples, providing reference data for the analysis of the preparation process and microsphere performance. The results are shown in Table 1. In the examples, the microspheres prepared with type III collagen had smaller particle sizes than those prepared with type I collagen. The mixture of type I and type III collagen improved the particle size uniformity of microspheres prepared with type I collagen alone.

[0097] Table 1. Particle size distribution of each group of samples

[0098] Experiment Example 2 Structural thermal stability assay of PDRN-collagen microspheres The PDRN-collagen microspheres and collagen raw materials prepared in each example and comparative example were diluted with solution 1 prepared in the above examples to a concentration of 1 mg / mL. The differential scanning calorimeter was set to a temperature range of 15℃~90℃ and a heating rate of 1.5℃ / min. The structural thermal denaturation temperature of the collagen raw materials and the PDRN-collagen microspheres prepared in each example was measured, and the corresponding endothermic peak position was recorded as the reference temperature. The results are shown in Table 2. The structural thermal denaturation temperature of the PDRN-collagen microspheres was significantly higher than that of the collagen raw materials. Among them, the microspheres prepared from type I collagen had the highest structural thermal denaturation temperature, exhibiting higher stability. Meanwhile, the structural thermal denaturation temperature of the microspheres prepared by mixing type I and type III collagen was higher than that of the microspheres prepared from type III collagen alone. Although the thermal denaturation temperature of the microspheres prepared in Comparative Example 1 was higher than that of the collagen raw materials, it was lower than that of the examples.

[0099] Table 2. Structural thermal denaturation temperatures of each group of samples

[0100] Experimental Example 3 Detection of the anti-collagenase degradation performance of PDRN-collagen microspheres The above samples were dispersed in PBS buffer (pH 7.4) containing 0.36 mg / mL collagenase (Thermo Fisher Scientific, catalog number 17100017) and treated in a shaker at 37°C. Samples were taken at set time points. The microspheres were recovered by centrifugation, dried at low temperature, and weighed to calculate the degradation rate of the microspheres, thereby evaluating the controllable degradation performance of the microspheres in a simulated in vivo collagenase environment.

[0101] Weigh 1 g of the PDRN-collagen microspheres prepared in the above examples or comparative examples and place them in a stoppered test tube. Add 10 mL of Solution 1 (the Solution 1 prepared in the examples) to fully suspend them. Then add 1 mL of PBS buffer containing 0.36 mg / mL collagenase. Incubate at 37°C with shaking at 100 rpm. Take samples at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, 1 mL each time. After sampling, centrifuge at 12,000 rpm for 15 min, discard the supernatant, and dry the precipitate at 105°C to constant weight. Use the dry weight of the 0 h sample as a control to calculate the degradation rate of the microspheres. The calculation formula is as follows:

[0102] Where m0 represents the dry weight of the sample at 0h, m t This indicates the dry weight of the sample taken at each time point.

[0103] The results are shown in Table 3. The anti-degradation ability of the collagen microspheres prepared in each example was determined under a simulated in vivo collagenase environment. The results showed that there were some differences in the anti-degradation performance of the microspheres in each example, but all were higher than those of the collagen raw material. The microspheres prepared from type I collagen showed higher anti-degradation ability. The anti-degradation performance of the microspheres prepared by mixing type I collagen and type III collagen was significantly improved compared with that of the microspheres prepared from type III collagen alone. Although the anti-degradation ability of the microspheres prepared in Comparative Example 1 was higher than that of the type III collagen raw material, its anti-degradation ability was significantly lower than that of the examples.

[0104] Table 3. Degradation rate of each group of samples

[0105] Experiment Example 4 Detection of the anti-nuclease degradation performance of PDRN-collagen microspheres Weigh 0.05 g of the PDRN-collagen microspheres prepared in the above examples or comparative examples and place them in a stoppered test tube. Add 10 mL of solution 1 (the solution 1 prepared in the examples) to suspend them fully. Then add 0.5 mL of DNase I with an enzyme activity of 50 U (purchased from Thermo Fisher Scientific, catalog number 18047019). The reaction is carried out at 37°C with shaking at 120 rpm for 90 min. After the reaction is completed, add 2 mL of 0.2 M EDTA and 0.3 mL of 20% SDS (m / v, g / mL) to the reaction system to terminate the enzymatic hydrolysis reaction.

[0106] After terminating the reaction, the reaction system was centrifuged at 12,000 rpm for 10 min, and the microsphere precipitate was collected. 5 mL of collagenase solution (collagenase concentration 1 mg / mL) was added to the precipitate, and the mixture was incubated at 37°C for 4 h to allow the collagen microspheres to fully degrade and release any residual PDRN. After the reaction, the supernatant was centrifuged, and the PDRN content was determined using the diphenylamine method. Three replicates were prepared for each group.

[0107] Using untreated PDRN-collagen microspheres as a control group, the total PDRN content in the samples was determined using the same collagenase digestion method. The PDRN retention rate was calculated based on the change in PDRN content before and after DNase I treatment to evaluate the protective ability of PDRN-collagen microspheres against nuclease degradation. The calculation formula is as follows:

[0108] The results are shown in Table 4. The collagen microspheres prepared by the present invention have a higher PDRN retention rate under the action of DNase compared with the control group. The microspheres prepared by mixing type I collagen and type III collagen have a relatively high PDRN retention rate, indicating that the preparation method can effectively maintain a stable PDRN load.

[0109] This is primarily because the microspheres have already formed a stable cross-linked network before being loaded with PDRN. The porosity and surface properties of the microspheres effectively immobilize PDRN, slowing its release rate and preventing premature degradation. Furthermore, the cross-linked network formed by the mixture of type I and type III collagen microspheres is more stable. In contrast, the comparative preparation method, due to the pre-mixing of PDRN, may result in uneven collagen cross-linking or excessively large pores, thus affecting the structural stability of the microspheres and leading to poor PDRN loading stability. Therefore, the method of this invention, by stabilizing the microsphere structure before loading with PDRN, effectively improves the loading stability and controlled release effect of PDRN.

[0110] Table 4. PDRN retention rate of each group of samples

[0111] The results of experiments 1-4 show that the microspheres prepared by mixing type I and type III collagen in a 4:1 ratio exhibit superior overall performance in terms of thermal stability, particle size uniformity, in vitro degradation behavior, and PDRN retention rate. This further demonstrates that this method can improve the loading stability of PDRN while maintaining the structural stability of the microspheres.

[0112] Experimental Example 5 Anti-inflammatory activity assay of PDRN-collagen microspheres Human fibroblasts (purchased from Pronosai, CP-H103) were seeded in 96-well plates at a density of 3000-5000 cells per well. After incubation at 37°C and 5% CO2 until adherence and 50-70% confluence, the cells were divided into different treatment groups, with three replicates per group. 1) Negative control group: Cultured for 24 hours without LPS stimulation and without adding any sample; 2) Damage control group: LPS was added to a final concentration of 1 μg / mL and cultured for 24 h to induce an inflammatory cell model. No other samples were added. 3) Positive control group: LPS was added to a final concentration of 1 μg / mL and cultured for 24 h to induce an inflammatory cell model. Then, pure PDRN was added to a final concentration of 10 μg / mL to control the anti-inflammatory effect of PDRN. 4) Each sample group: LPS was added to a final concentration of 1 μg / mL and cultured for 24 h to induce the establishment of an inflammatory cell model. Then, PDRN-collagen microspheres prepared in the various embodiments or comparative examples of the present invention were added to a final concentration of 10 μg / mL.

[0113] After culturing for another 24 hours, cell supernatant was collected, and IL-6 concentration was measured by ELISA as an anti-inflammatory evaluation index. The negative control group represented the baseline expression level of IL-6 (0%), and the damage control group represented the IL-6 level after inflammation activation (100%).

[0114] Table 5. IL-6 concentration levels of each group of samples

[0115] The results are shown in Table 5. The PDRN-collagen microspheres prepared in each group of embodiments of the present invention significantly reduced IL-6 levels in LPS-stimulated inflammatory cell models, exhibiting significant anti-inflammatory effects close to those of pure PDRN, indicating minimal loss of PDRN activity during microsphere loading. In contrast, the anti-inflammatory effect of Comparative Example 1 was relatively weak, further demonstrating that the collagen microsphere loading method of the present invention is beneficial for maintaining the bioactivity of PDRN and exerting a good anti-inflammatory effect.

Claims

1. A method for preparing collagen microspheres loaded with PDRN, characterized in that, Includes the following steps: (1) Add recombinant collagen to buffer solution at a mass ratio of 0.5%~1.5% and homogenize to obtain collagen solution; (2) The collagen solution and the oil phase are mixed at a volume ratio of 1: (1~3) and homogenized until a stable emulsion system is formed. A cross-linking agent is added to the emulsion system and the mixture is stirred at room temperature and 300~800 rpm for 1~4 hours. The supernatant is discarded by centrifugation, and the precipitate is washed with water for injection 3~5 times to obtain the washed collagen microspheres. (3) Add PDRN to the buffer solution at a mass ratio of 0.5% to 1.5%, and stir at 45 to 55°C until completely dissolved to obtain a PDRN solution; (4) Disperse the washed collagen microspheres in PDRN solution, control the mass ratio of recombinant collagen to PDRN to be 1:(0.5~1.5), and load PDRN by shaking at 50~120 rpm for 4~8 h at room temperature; (5) Centrifuge and discard the supernatant, wash the precipitate 3-5 times with water for injection, and dry it under low temperature to obtain collagen microspheres loaded with PDRN.

2. The preparation method according to claim 1, characterized in that, The recombinant collagen is type I recombinant collagen, type III recombinant collagen, or a mixture of both.

3. The preparation method according to claim 2, characterized in that, The recombinant collagen is a mixture of type I recombinant collagen and type III recombinant collagen, with the type I recombinant collagen and type III recombinant collagen mixed in a mass ratio of (1-6):

1.

4. The preparation method according to claim 3, characterized in that, The type I recombinant collagen and type III recombinant collagen are compounded at a mass ratio of 4:

1.

5. The preparation method according to claim 1, characterized in that, One or more of the following conditions must be met: ① The components of the buffer solution mentioned in steps (1) and (3) are: 7~10 wt‰ NaCl, 0.16~0.30 wt‰ Na2HPO4, 0.03~0.10 wt‰ NaH2PO4, and the solvent is water for injection; ②The preparation method of the oil phase in step (2) is as follows: add Span 80 to liquid paraffin at a mass-volume ratio (g / mL) of 1%~3% and stir until uniform at room temperature; ③ The crosslinking agents mentioned in step (2) are EDC and NHS; ④ The homogenization conditions described in step (1) are: rotation speed 10,000~12,000 rpm, homogenization for 5 min, stop for 5 min, repeat 4~6 times; ⑤ The homogenization conditions described in step (2) are: rotation speed 10,000~12,000 rpm, homogenization for 3 min, stop for 2 min, repeat 3~5 times; ⑥ The centrifugation conditions described in steps (2) and (5) are: centrifugation at 4,500~5,000 rpm for 10~15 min.

6. The preparation method according to claim 5, characterized in that, The amount of EDC added is 1% to 3% of the recombinant collagen protein, and the mass ratio of EDC to NHS is 1:

1.

7. Collagen microspheres loaded with PDRN prepared by the preparation method according to any one of claims 1 to 6.

8. The PDRN-loaded collagen microspheres as described in claim 7, wherein the microspheres have a particle size distribution D50 value of 45~58μm, a D90 value of 75~89μm, and a thermal denaturation temperature of 56~66℃.

9. The application of PDRN-loaded collagen microspheres prepared by the preparation method according to any one of claims 1 to 6, or PDRN-loaded collagen microspheres according to claim 7 or 8, in the preparation of tissue repair or medical aesthetic products.

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