PDRN-containing double-network hydrogel dressing using marine biomass resources, preparation method and application thereof

Abstract

The application provides a PDRN-containing double-network hydrogel dressing using marine biomass resources, a preparation method and application. The preparation method of the dressing comprises the following steps: S1, preparing a chitosan solution; S2, preparing a PDRN solution; S3, slowly dropping the PDRN solution into the chitosan solution, and stirring to form a PDRN-chitosan mixed solution; S4, preparing an AgNPs-GSH solution; S5, slowly pouring the AgNPs-GSH solution into the PDRN-chitosan mixed solution, adding a gelatin aqueous solution, gently stirring, adding a KCl solution, rapidly stirring, and standing to form a hydrogel dressing. The PDRN-containing double-network hydrogel dressing has the dual effects of anti-inflammation and antibiosis + healing promotion; carrageenan and chitosan promote cell migration and regeneration; chitosan / PDRN complex protects nucleic acids from enzymatic hydrolysis; GSH buffers Ag + release, and is co-distributed with carrageenan in the matrix to provide a long-acting bactericidal barrier; the anti-inflammatory repair effect of PDRN is complementary to that of the above-mentioned components; and the overall system realizes sustained release and maintenance of biological activity.

Description

Technical Field

[0001] This invention relates to the field of wound dressings and hydrogel technology, and particularly to a PDRN-containing dual-network hydrogel dressing using marine biomass resources, its preparation method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are naturally derived low-molecular-weight DNA derivatives with molecular weights ranging from 50 kDa to 1500 kDa. The most representative molecular weights for PDRNs are 80 kDa to 200 kDa, with a Gaussian peak around 132 kDa. PDRNs have a base length of 50 bp to 2000 bp and are linear polymers of deoxyribonucleotides with phosphodiester bonds. PDRNs contain 50% double-stranded deoxyribonucleotides, with the two polydeoxyribonucleotides linked by hydrogen bonds between base pairs to form a double helix structure. PDRNs are primarily extracted and purified from the sperm cells of salmon or rainbow trout.

[0003] PDRN's base composition shares 98% similarity with human DNA. Based on this biological effect, its clinical applications are gradually expanding, showing significant effects in tissue repair, wound healing, anti-ischemia, and anti-inflammation. In vitro and in vivo experiments have shown that PDRN's most relevant mechanism of action is as an agonist of adenosine A2A receptors, activating A2A receptors and producing a variety of physiological effects.

[0004] Chinese patent CN114642606A discloses a composition with skin barrier repair function, its preparation method, and its application. The composition includes the following components: 0.01%-0.5% high molecular weight hyaluronic acid or its salt, 0.1%-1.0% hydrolyzed hyaluronic acid or its salt, 0.01%-10% silk fibroin, 0.05%-1% polydeoxyribonucleic acid (PDRN), and 0.1%-10% phenolphthalein (PHX). The high molecular weight hyaluronic acid or its salt can target the stratum corneum of the skin, forming a moisturizing and breathable protective film on the skin surface. The extremely low molecular weight hydrolyzed hyaluronic acid or its salt can be absorbed transdermally into the dermis, stimulating angiogenesis. PDRN, silk fibroin, and phenolphthalein promote collagen synthesis and cell migration. These components work synergistically to promote wound healing, repair damaged skin barriers, and achieve rapid repair of skin wounds.

[0005] Chinese patent CN112932988A discloses an anti-aging composition containing polydeoxyribonucleic acid (PDA), comprising honey and PDA; the invention also discloses a skincare product comprising the PDA-containing anti-aging composition and cosmetically acceptable adjuvants. The disclosed anti-aging composition and the skincare product including it enhance the effects of promoting skin cell regeneration and anti-aging through the synergistic effect of honey and PDA.

[0006] Currently, PDRN suffers from poor stability, which limits its application in repair products.

[0007] Marine biomass, such as carrageenan, is a natural polymer extracted from red algae. Due to its excellent biocompatibility, biodegradability, and functional activity, it exhibits broad application potential. The synergistic use of PDRN with carrageenan and other marine biomass is expected to further enhance its tissue repair capabilities and stability, demonstrating broad application prospects. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a PDRN-containing dual-network hydrogel dressing using marine biomass resources, its preparation method and application, and to significantly improve the stability of PDRN and demonstrate a synergistic effect in repair by realizing a chitosan / carrageenan dual-network composite hydrogel and selecting reasonable component combinations.

[0009] The technical solution of the present invention:

[0010] In a first aspect, the present invention provides a method for preparing a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources, comprising the following steps:

[0011] S1: Add chitosan to glacial acetic acid solution, heat and stir until completely dissolved, then cool to obtain chitosan solution;

[0012] S2: Slowly dissolve PDRN in PBS buffer at room temperature to obtain a PDRN solution;

[0013] S3: Slowly add the PDRN solution dropwise to the chitosan solution and stir to form a PDRN-chitosan mixture;

[0014] S4: Add carrageenan to deionized water, heat and stir until dissolved to obtain a carrageenan solution; slowly add nano-silver colloid to the above carrageenan solution and stir evenly to obtain a mixed solution; dissolve glutathione in warm water and slowly add it to the mixed solution while stirring to obtain an AgNPs-GSH solution;

[0015] S5: Slowly pour the AgNPs-GSH solution into the PDRN-chitosan mixture, add the gelatin aqueous solution, stir gently, then add the KCl solution, stir quickly, and let stand to form a hydrogel dressing.

[0016] PDRN-containing dual-network hydrogel dressings possess dual anti-inflammatory, antibacterial, and healing-promoting effects; carrageenan and chitosan promote cell migration and regeneration; the chitosan / PDRN complex protects nucleic acids from enzymatic degradation; and GSH buffers Ag. + Released and co-distributed with carrageenan in the matrix, it provides a long-lasting bactericidal barrier. The anti-inflammatory and repairing effects of PDRN complement it, and the overall system achieves continuous release and maintenance of bioactivity.

[0017] In some embodiments, the chitosan concentration in the chitosan solution in S1 is 0.5-1.5 g / 100 mL.

[0018] In some embodiments, the concentration of PDRN in the PDRN solution in S2 is 0.1-0.5 g / 100 mL.

[0019] In some embodiments, the volume ratio of the PDRN solution and the chitosan solution in S3 is 1:1-1.5; more preferably, the volume ratio of the PDRN solution and the chitosan solution is 1:1.5.

[0020] In some embodiments, the concentration of carrageenan in the carrageenan solution in S4 is 0.1-1.0 g / 100 mL.

[0021] In some embodiments, the concentration of silver nanoparticles in the AgNPs-GSH solution is 5-25 ppm, the particle size of the silver nanoparticles is 10-50 nm, and the amount of glutathione added is 0.05-0.2 g / 100 mL.

[0022] In some embodiments, the volume ratio of AgNPs-GSH solution to PDRN-chitosan mixture in S5 is 1:1-2; more preferably, the volume ratio of AgNPs-GSH solution to PDRN-chitosan mixture is 1:1-1.5.

[0023] In some embodiments, the concentration of the gelatin aqueous solution in S5 is 5% (w / v) gelatin, and the amount added is 5-10% of the total volume of the AgNPs-GSH solution and the PDRN-chitosan mixture.

[0024] In some embodiments, the concentration of the KCl solution in S5 is 0.05-0.1M KCl, and the amount added is 5-10% of the total volume of the AgNPs-GSH solution and the PDRN-chitosan mixture.

[0025] In a second aspect, the present invention provides a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources, prepared by the method described above.

[0026] Finally, this invention provides the application of the above-mentioned PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources in the preparation of wound repair products.

[0027] Preferably, the repair product includes cosmetics and pharmaceuticals.

[0028] More preferably, the repair promotes wound healing, has anti-inflammatory and antibacterial properties.

[0029] Beneficial effects:

[0030] 1. The PDRN-containing dual-network hydrogel dressing provided by this invention utilizes marine biomass resources. In this dressing, PDRN is stably encapsulated by chitosan, and AgNPs are embedded in the marine biomass resource-carrageenan matrix. The gelling system forms a cross-linked network, which has dual effects of anti-inflammatory, antibacterial and healing promotion. It activates A2A receptors, has anti-inflammatory and repair effects, and carrageenan and chitosan promote cell migration and regeneration. It has broad application prospects in wound dressing and hydrogel technology.

[0031] 2. Chitosan / PDRN complex protects nucleic acids, GSH buffers Ag + The system achieves continuous release and maintenance of bioactivity, with a good controlled-release mechanism, prolonging the action time of PDRN and improving the stability of the dressing gel.

[0032] 3. The gelation process does not require organic solvents, high temperatures or radiation, making it suitable for direct application to wounds. The gel formation is gentle. Carrageenan is extracted from marine red algae, has high biocompatibility, and enhances the mechanical properties of the gel, forming a protective barrier for the wound. Detailed Implementation

[0033] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0034] Unless otherwise specified, all chemical reagents used in this invention are commercially available analytical grade. Chitosan was purchased from Shandong Aokang Biotechnology Co., Ltd.; PDRN was purchased from Shaanxi Baichanghong Technology Co., Ltd.; PBS buffer was purchased from Thermo Fisher Scientific; carrageenan was purchased from Guangdong Mingcheng Biotechnology Co., Ltd.; and gelatin was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd.

[0035] Preparation of silver nanoparticles

[0036] Weigh 17 mg AgNO3, dissolve it in 100 mL of deionized water, and store it in the dark to obtain a 100 ppm AgNO3 solution; take 20 mL of the 100 ppm AgNO3 solution, add 0.5 mL of 1% (w / v) PVP-K30 solution, add 1 mL of 1% sodium citrate solution, stir well, and react in a 60℃ water bath for 30 minutes. Then, use deionized water to make up to 100 mL to obtain 20 ppm AgNPs colloid.

[0037] The particle size distribution was confirmed using dynamic light scattering (DLS) with a target average particle size of 15-30 nm.

[0038] Example 1

[0039] S1: Add 1g of chitosan to 100ml of 0.5% glacial acetic acid solution, heat and stir at 50℃ for 2 hours until completely dissolved.

[0040] Cool to room temperature to obtain a chitosan solution;

[0041] S2: Slowly dissolve PDRN in PBS buffer at room temperature, adjust the pH to obtain a PDRN solution with pH=6.0 and a concentration of 0.3 g / mL;

[0042] S3: Slowly add 50ml of PDRN solution to 50ml of chitosan solution and stir to form a PDRN-chitosan mixture;

[0043] S4: Add 0.15g carrageenan to 50mL of deionized water, heat and stir at 60℃ for 30min until completely dissolved to obtain a carrageenan solution; slowly add 50mL of 20ppm nano-silver colloid to the above carrageenan solution, stir evenly to obtain a mixture; dissolve 0.1g of glutathione in 5mL of 35℃ warm water, slowly add it to the above mixture and stir for 10min.

[0044] A solution of AgNPs-GSH was obtained;

[0045] S5: Slowly pour 100ml of AgNPs-GSH solution into 100ml of PDRN-chitosan mixture, add 20ml of 5% (w / v) gelatin aqueous solution, stir gently, then add 20ml of 0.1M KCl solution, stir quickly for 10s, and let stand at 37℃ for 30min to form a hydrogel dressing.

[0046] Example 2

[0047] The experimental steps were basically the same as in Example 1, except that the mixing volumes of the PDRN solution and chitosan solution in S3 were 50 ml and 75 ml, respectively.

[0048] Comparative Example 1

[0049] The experimental steps were basically the same as in Example 1, except that the mixing volumes of the PDRN solution and chitosan solution in S3 were 50 ml and 200 ml, respectively.

[0050] Comparative Example 2

[0051] S1: Add 1g of chitosan to 100ml of 0.5% glacial acetic acid solution, heat and stir at 50℃ for 2 hours until completely dissolved.

[0052] Cool to room temperature to obtain a chitosan solution;

[0053] S2: Slowly dissolve PDRN in PBS buffer at room temperature, adjust the pH to obtain a PDRN solution with pH=6.0 and a concentration of 0.3 g / mL;

[0054] S3: Slowly add 50 ml of PDRN solution to 50 ml of chitosan solution and stir to form a complex; slowly add 50 ml of 20 ppm silver nanoparticle colloid to the above complex and stir evenly to obtain a mixture; dissolve 0.1 g of glutathione in 5 ml of 35°C warm water and slowly add it to the above mixture and stir for 10 min to obtain a chitosan-AgNPs solution;

[0055] S4: Add 0.15g of carrageenan to 50mL of deionized water, heat and stir at 60℃ for 30min until completely dissolved to obtain a carrageenan solution;

[0056] S5: Slowly pour 100ml of chitosan-AgNPs solution into 100ml of carrageenan solution, add 20ml of 5% (w / v) gelatin aqueous solution, stir gently, then add 20ml of 0.1M KCl solution, stir quickly for 10s, and let stand at 37℃ for 30min to form a hydrogel dressing.

[0057] Comparative Example 3

[0058] Basically the same as in Example 1, steps S1-S3 are the same, the only difference is that steps S4-S5 are modified as follows:

[0059] S4: Add 0.15g of carrageenan to 50mL of deionized water, heat and stir at 60℃ for 30min until completely dissolved to obtain a carrageenan solution;

[0060] S5: Slowly pour 100ml of carrageenan solution into 100ml of PDRN-chitosan mixture, add 20ml of 5% (w / v) gelatin aqueous solution, stir gently, then add 20ml of 0.1M KCl solution, stir quickly for 10s, and let stand at 37℃ for 30min to form a hydrogel dressing.

[0061] Comparative Example 4

[0062] S1: Slowly dissolve PDRN in PBS buffer at room temperature, adjust the pH to obtain a PDRN solution with pH=6.0 and a concentration of 0.3 g / mL;

[0063] S2: Add 0.3g of carrageenan to 50mL of deionized water, heat and stir at 60℃ for 30min until completely dissolved to obtain a carrageenan solution; slowly add 50mL of 20ppm nano silver colloid to the above carrageenan solution and stir evenly to obtain a mixed solution; dissolve 0.1g of glutathione in 5mL of 35℃ warm water, slowly add it to the above mixed solution and stir for 10min to obtain an AgNPs-GSH solution;

[0064] S3: Slowly pour 100ml of AgNPs-GSH solution into 100ml of PDRN solution, add 20ml of 5% (w / v) gelatin aqueous solution, stir gently, then add 20ml of 0.1M KCl solution, stir quickly for 10s, and let stand at 37℃ for 30min to form a hydrogel dressing.

[0065] Comparative Example 5

[0066] S1: Add 1g of chitosan to 100ml of 0.5% glacial acetic acid solution, heat and stir at 50℃ for 2h until completely dissolved, cool to room temperature to obtain chitosan solution;

[0067] S2: Add 0.15g of carrageenan to 50mL of deionized water, heat and stir at 60℃ for 30min until completely dissolved to obtain a carrageenan solution; slowly add 50mL of 20ppm nano silver colloid to the above carrageenan solution and stir evenly to obtain a mixed solution; dissolve 0.1g of glutathione in 5mL of 35℃ warm water, slowly add it to the above mixed solution and stir for 10min to obtain an AgNPs-GSH solution;

[0068] S3: Slowly pour 100ml of AgNPs-GSH solution into 100ml of chitosan solution, add 20ml of 5% (w / v) gelatin aqueous solution, stir gently, then add 20ml of 0.1M KCl solution, stir quickly for 10s, and let stand at 37℃ for 30min to form a hydrogel dressing.

[0069] Result detection

[0070] 1. Antibacterial test:

[0071] The hydrogels prepared in the examples and comparative examples were subjected to antibacterial tests. 100 μL of Staphylococcus aureus solution was evenly spread on a petri dish. The hydrogels prepared in the examples and comparative examples were cut into circles with a diameter of 1 cm and placed on petri dishes. They were co-cultured at 37°C for 24 h, and the changes in the diameter of the inhibition zone were observed.

[0072] 2. Infection healing test:

[0073] Mouse wounds were infected with Staphylococcus aureus to obtain an infectious animal model. The infected animal models were divided into 9 groups: a control group, Example 1-2 groups, and Comparative Examples 1-5 groups. The control group received no treatment, while the other groups received treatment with corresponding hydrogels applied to their infected animal models. Wound healing status was recorded on days 0, 3, and 10 of treatment, and the percentage of the current wound area relative to the original wound area was calculated.

[0074] 3. Stability testing:

[0075] Take 0.5g of the hydrogel dressing prepared in the examples and comparative examples, add it to 50mL of water, then add DNase I (addition amount is 100u / mL according to enzyme activity), seal, and place in a constant temperature water bath shaker for enzymatic hydrolysis (enzymatic hydrolysis temperature 37℃, enzymatic hydrolysis time 2h, stirring speed 100rpm). After enzymatic hydrolysis, add 20mL of EDTA standard solution (concentration 0.01mol / L) and 1mL of sodium dodecyl sulfate aqueous solution (mass fraction 10%), stir at 1500rpm for 20min to obtain a mixture, and place the mixture in a centrifuge and centrifuge at 10000rpm for 20min. After centrifugation, take the supernatant and test the PDRN content by the diphenylamine method, and record it as M1. Each group has a blank treatment group that does not undergo enzymatic hydrolysis, and the PDRN content of the blank treatment group is tested by the diphenylamine method, and recorded as M0.

[0076] The retention rate is calculated using the following formula: Retention rate = M1 / M0 × 100%.

[0077] The test results are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] The data in Table 1 show that when the product of this invention is applied to the wound surface, it exhibits a significant healing-promoting and repairing effect, with Example 2 showing the best repair effect. Furthermore, the dual-network gel of this invention has a long-lasting sustained-release effect, demonstrating a continuous repairing effect over a 9-day experimental period after a single application.

[0082] The hydrogel prepared in Example 2 has a larger antibacterial zone diameter and stronger antibacterial performance. This is because the nano-silver particles and carrageenan are co-distributed in the matrix to provide a long-lasting bactericidal barrier. The anti-inflammatory and repairing effects of PDRN complement each other, synergistically enhancing the bactericidal effect and providing a repair barrier for the wound.

[0083] The results of the comparative examples and embodiments show that the nanocomposite formed by PDRN and chitosan is an important component, preventing enzymatic degradation on the wound surface, forming an interpenetrating network with carrageenan, and GSH protecting the stability of cells and nucleic acids. All of the above key materials are indispensable. The results of Comparative Example 1 show that the ratio of raw materials affects the sustained release of PDRN, and choosing an appropriate ratio is more conducive to wound repair. Comparative Example 2 did not form a PDRN / chitosan nanocomposite, exhibited lower stability in the stability experiment, and also affected the activity of PDRN, resulting in a decrease in the repair effect.

[0084] This invention can also be implemented in various other ways. Without departing from the spirit and essence of this invention, those skilled in the art can make various corresponding changes and modifications according to this invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.

Claims

1. A method for preparing a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources, characterized in that, Includes the following steps: S1: Add chitosan to glacial acetic acid solution, heat and stir until completely dissolved, then cool to obtain chitosan solution; S2: Slowly dissolve PDRN in PBS buffer to obtain a PDRN solution; S3: Slowly add the PDRN solution dropwise to the chitosan solution and stir to form a PDRN-chitosan mixture; S4: Add carrageenan to deionized water, heat and stir until dissolved to obtain a carrageenan solution; slowly add nano-silver colloid to the above carrageenan solution and stir evenly to obtain a mixed solution; dissolve glutathione in warm water and slowly add it to the mixed solution while stirring to obtain an AgNPs-GSH solution; S5: Slowly pour the AgNPs-GSH solution into the PDRN-chitosan mixture, add the gelatin aqueous solution, stir gently, then add the KCl solution, stir quickly, and let stand to form a hydrogel dressing; In S1, the chitosan concentration in the chitosan solution is 0.5-1.5 g / 100 mL; In step S2, the concentration of PDRN in the PDRN solution is 0.1-0.5 g / 100 mL; In step S3, the volume ratio of the PDRN solution to the chitosan solution is 1:1-1.

5.

2. The method for preparing a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources according to claim 1, characterized in that, The concentration of carrageenan in the carrageenan solution is 0.1-1.0 g / 100 mL.

3. The method for preparing a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources according to claim 1, characterized in that, In AgNPs-GSH solutions, the concentration of silver nanoparticles is 5-25 ppm, and the particle size of silver nanoparticles is 10-50 nm; the amount of glutathione added is 0.05-0.2 g / 100 mL.

4. The method for preparing a PDRN-containing dual-network hydrogel dressing utilizing marine biomass resources according to claim 1, characterized in that, The volume ratio of AgNPs-GSH solution to PDRN-chitosan mixture in S5 is 1:1-2.

5. The PDRN-containing dual-network hydrogel dressing using marine biomass resources prepared by the method according to any one of claims 1-4.

6. The application of the PDRN-containing dual-network hydrogel dressing based on marine biomass resources as described in claim 5 in the preparation of wound repair products.

7. The application of the PDRN-containing dual-network hydrogel dressing based on marine biomass resources according to claim 6 in the preparation of wound repair products, characterized in that, The repair process promotes wound healing, has anti-inflammatory and antibacterial properties; the products include cosmetics and pharmaceuticals.

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