PDRN-loaded dynamic cross-linked polysaccharide-based hydrogel and preparation method thereof

Abstract

The invention discloses PDRN-loaded dynamic cross-linked polysaccharide-based hydrogel and a preparation method thereof, and belongs to the technical field of hydrogel. The preparation method comprises the following steps: adding modified carboxymethyl cellulose, polyvinyl alcohol and PDRN into deionized water, and dissolving to obtain a solution A; performing oxidation modification on the sodium hyaluronate by using sodium periodate to obtain oxidized sodium hyaluronate; dissolving oxidized sodium hyaluronate and sodium metaborate in deionized water to obtain a solution B; uniformly mixing the solution A and the solution B, and standing at room temperature to obtain the hydrogel. According to the invention, polyvinyl alcohol and sodium metaborate are utilized to form a dynamic borate bond, and modified carboxymethyl cellulose and oxidized sodium hyaluronate are utilized to form a dynamic Schiff base bond, so that the problem of insufficient self-healing property of hydrogel is solved; the excellent biocompatibility and anti-inflammatory and bactericidal properties of PDRN are utilized, so that the tissue repair and regeneration capacity is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and specifically relates to a dynamically crosslinked polysaccharide-based hydrogel loaded with PDRN and a preparation method thereof. Background Art

[0002] The skin is one of the most important organs of the human body. It can directly contact the external environment, sense external stimuli, regulate body temperature, and protect the body from external injuries, etc. Although most common skin injuries can heal on their own, wound healing is a complex dynamic process, and various factors can cause interruptions in the healing process and form chronic wounds. However, a variety of factors may lead to various wound healing problems, making it difficult to repair wounds. Hydrogels are considered ideal substitutes in tissue repair engineering because they can resist infection, absorb wound exudate, maintain water balance and gas exchange, load and deliver bioactive factors, and have biocompatibility. An ideal hydrogel wound dressing has appropriate mechanical properties, self-healing properties, adhesion ability, antibacterial properties, etc.

[0003] Chinese Patent No. CN 118286150 A discloses a double-dynamically crosslinked polysaccharide-based hydrogel loaded with rhein and a preparation method thereof. It dissolves rhein in the hydrogel precursor solution to achieve direct loading of rhein, solving the problems of poor water solubility and low bioavailability of rhein; however, its self-healing ability is not yet ideal. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a dynamically crosslinked polysaccharide-based hydrogel loaded with PDRN (polydeoxyribonucleotide) and a preparation method thereof.

[0005] To achieve the above purpose, the present invention is realized through the following technical solutions: A preparation method of a dynamically crosslinked polysaccharide-based hydrogel loaded with PDRN, comprising the following steps: S1: Modify carboxymethyl cellulose with 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanylylidene))bis(2-amino-5-oxopentanoic acid) to obtain modified carboxymethyl cellulose; S2: Dissolve the modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN in deionized water to obtain solution A; S3: Oxidatively modify sodium hyaluronate with sodium periodate to obtain oxidized sodium hyaluronate; S4: Dissolve the oxidized sodium hyaluronate and sodium metaborate in water to obtain solution B; S5: Mix solution A and solution B evenly and let it stand at room temperature to obtain a hydrogel.

[0006] The 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) is prepared by the following method: Under nitrogen protection, glutathione and triethylamine are added to a mixed solution of acetonitrile and saturated borax buffer solution, and SO2F2 gas is introduced, followed by reaction at room temperature to obtain the product.

[0007] The modified carboxymethyl cellulose is prepared by the following method: Under nitrogen protection, carboxymethyl cellulose is dissolved in deionized water, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added in sequence, and after stirring, 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) is added, followed by stirring at room temperature for 20 - 30 h to obtain the product.

[0008] The feeding mass ratio of the carboxymethyl cellulose, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) is 10:(0.4 - 2):(0.2 - 1):(1 - 5).

[0009] In step S2, the concentration of the modified carboxymethyl cellulose in solution A is 3 wt%, the concentration of polyvinyl alcohol is 4 wt%, and the concentration of PDRN is 0.05 - 0.5 wt%.

[0010] The oxidized sodium hyaluronate is prepared by the following method: Sodium hyaluronate and sodium periodate are dissolved in deionized water, and the reaction is carried out in the dark at room temperature for 20 - 26 h. Then, ethylene glycol is added to terminate the reaction, and the product is dialyzed with ultrapure water and freeze-dried to obtain the oxidized sodium hyaluronate.

[0011] The feeding mass ratio of the sodium hyaluronate and sodium periodate is 2:(1 - 5).

[0012] In step S4, the feeding mass ratio of the oxidized sodium hyaluronate and sodium metaborate is 5:1.

[0013] In step S4, the mixing volume ratio of solution A and solution B is 1:1.

[0014] A dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN is prepared by the above-mentioned method.

[0015] Due to the adoption of the above technical solutions, the beneficial effects of the present invention include: (1)The present invention modifies carboxymethyl cellulose after forming a disulfide bond with glutathione, forms a dynamic Schiff base with oxidized sodium hyaluronate, and forms a dynamic borate ester bond with polyvinyl alcohol and sodium metaborate, solving the problems of poor mechanical properties and insufficient self-healing ability of the hydrogel.

[0016] (2)The present invention loads PDRN on a dynamically crosslinked polysaccharide-based hydrogel, and utilizes the excellent biocompatibility, anti-inflammatory and bactericidal properties of PDRN to enhance the tissue repair and regeneration functions of the hydrogel.

[0017] (3)As a bioactive molecule, glutathione endows the hydrogel with good compatibility. The presence of disulfide bonds improves its self-healing ability and antibacterial properties, can effectively relieve the oxidative stress at the wound site, and improve the speed of wound repair. Description of the Drawings

[0018] Figure 1 It is a diagram of the healing situation of the hydrogel under the microscope in Example 2 and Comparative Example 2; Figure 2 It is a test diagram of the antibacterial properties of the hydrogel in Example 2 and Comparative Example 1; Figure 3 It is a photographed record diagram of the wound healing situation of the infected animal model; Figure 4 It is a HE staining diagram of the wound healing situation of the infected animal model. Detailed Embodiments

[0019] The following is further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0020] Example 1: Preparation of 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(aza-2,1-diyl))bis(2-amino-5-oxopentanoic acid): Under nitrogen protection, 0.05 mol of glutathione (reduced form) and 0.05 g of triethylamine were added to 100 ml of acetonitrile / saturated borax buffer solution (volume ratio 1:1), and SO2F2 gas was introduced. The mixture was vigorously stirred at room temperature for 1 h. The excess SO2F2 gas was introduced into an acetonitrile solution containing thiol and triethylamine, diluted with 300 ml of ethyl acetate, washed three times with 50 ml of saturated brine, the organic layer was dried with 10 g of anhydrous Na2SO4, filtered, and distilled under reduced pressure at 60 °C for 2 h to obtain 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(aza-2,1-diyl))bis(2-amino-5-oxopentanoic acid), and its structural formula is as follows:

[0021] The nuclear magnetic resonance hydrogen spectrum data thereof are as follows: 11H NMR (300 MHz, DMSO- d 6) δ 12.66 (s, 2H), 9.64 (s, 2H), 8.59 - 8.47 (m, 4H), 6.78 (d, J = 6.5 Hz, 4H), 4.44 (dt, J = 8.1, 4.2 Hz, 2H), 3.80 (dd, J = 6.5, 2.4 Hz, 4H), 3.71 (p, J = 6.4 Hz, 2H), 3.01 - 2.91 (m, 2H), 2.70 (dd, J = 13.9, 4.1 Hz, 2H), 2.48 - 2.19 (m, 4H), 2.11 (dq, J = 15.6, 6.7 Hz, 2H), 1.85 (dq, J = 15.4, 6.5 Hz, 2H).

[0022] Example 2: Preparation of PDRN-loaded dynamically crosslinked polysaccharide-based hydrogel: S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water. Then, 0.04 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.02 g of N-hydroxysuccinimide were added successively, and the mixture was stirred for 30 min. Next, 0.1 g of 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added, and the mixture was stirred at room temperature for 20 h. Then, it was put into a dialysis bag (6000 Da) and dialyzed for 3 days (changing water every 12 h), and freeze-dried at -20 °C for 36 h to obtain modified carboxymethyl cellulose; S2: Preparation of solution A: Modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN were dissolved in 50 ml of deionized water to prepare solution A. The final concentrations were: 3 wt% of modified carboxymethyl cellulose, 4 wt% of polyvinyl alcohol, and 0.05 wt% of PDRN; S3: Preparation of oxidized sodium hyaluronate: 2 g of sodium hyaluronate and 1 g of sodium periodate were dissolved in 50 ml of deionized water, and the reaction was carried out in the dark at room temperature for 20 h. Then, 2 g of ethylene glycol was added to terminate the reaction, and it was dialyzed with ultrapure water (8 kDa) for 3 days (changing water every 12 days), and freeze-dried at -20 °C for 36 h to obtain oxidized sodium hyaluronate; S4: Preparation of solution B: 1.5 g of oxidized sodium hyaluronate and 0.3 g of sodium metaborate were dissolved in 50 ml of deionized water to obtain solution B; S5: Preparation of hydrogel: 5 ml of solution A and 5 ml of solution B were mixed evenly and allowed to stand at room temperature for 12 h to form a hydrogel.

[0023] Example 3: Preparation of PDRN-loaded dynamically crosslinked polysaccharide-based hydrogel: S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water. Then, 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide were added successively, and the mixture was stirred for 30 min. Next, 0.3 g of 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added, and the mixture was stirred at room temperature for 24 h. Then, it was placed in a dialysis bag (6000 Da) and dialyzed for 3 days (changing water every 12 h), and freeze-dried at -20 °C for 36 h to obtain modified carboxymethyl cellulose; S2: Preparation of solution A: Modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN were dissolved in 50 ml of deionized water to prepare solution A. The final concentrations were: 3 wt% for modified carboxymethyl cellulose, 4 wt% for polyvinyl alcohol, and 0.25 wt% for PDRN; S3: Preparation of oxidized sodium hyaluronate: 2 g of sodium hyaluronate and 3 g of sodium periodate were dissolved in 50 ml of deionized water, and the reaction was carried out in the dark at room temperature for 24 h. Then, 3 g of ethylene glycol was added to terminate the reaction, and it was dialyzed with ultrapure water (10 kDa) for 3 days (changing water every 12 days), and freeze-dried at -20 °C for 36 h to obtain oxidized sodium hyaluronate; S4: Preparation of solution B: 1.5 g of oxidized sodium hyaluronate and 0.3 g of sodium metaborate were dissolved in 50 ml of deionized water to obtain solution B; S5: Preparation of hydrogel: 2 ml of solution A and 2 ml of solution B were mixed evenly and left to stand at room temperature for 12 h to form a hydrogel.

[0024] Example 4: Preparation of PDRN-loaded dynamically crosslinked polysaccharide-based hydrogel: S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water. Then, 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 g of N-hydroxysuccinimide were added successively, and the mixture was stirred for 30 min. Next, 0.5 g of 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added, and the mixture was stirred at room temperature for 30 h. Then, it was placed in a dialysis bag (6000 Da) and dialyzed for 3 days (changing water every 12 h), and freeze-dried at -20 °C for 36 h to obtain modified carboxymethyl cellulose; S2: Preparation of Solution A: Dissolve modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN in 50 ml of deionized water to prepare Solution A. Final concentrations: modified carboxymethyl cellulose is 3 wt%, polyvinyl alcohol is 4 wt%, and the concentration of PDRN is 0.5 wt%. S3: Preparation of oxidized sodium hyaluronate: Dissolve 2 g of sodium hyaluronate and 5 g of sodium periodate in 50 ml of deionized water, react in the dark at room temperature for 24 h, add 4 g of ethylene glycol to terminate the reaction, dialyze with ultrapure water (12 kDa) for 3 days (change water every 12 days), and freeze-dry at -20 °C for 36 h to obtain oxidized sodium hyaluronate. S4: Preparation of Solution B: Dissolve 1.5 g of oxidized sodium hyaluronate and 0.3 g of sodium metaborate in 50 ml of deionized water to obtain Solution B. S5: Preparation of hydrogel: Mix 2 ml of Solution A and 2 ml of Solution B evenly, and let it stand at room temperature for 12 h to form a hydrogel.

[0025] Comparative Example 1 A dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN, the preparation method of which is basically the same as that of Example 3, except that the modified carboxymethyl cellulose in step S2 is replaced by the modified carboxymethyl cellulose prepared by the following method: Under nitrogen protection, dissolve 1 g of carboxymethyl cellulose in 50 g of deionized water, sequentially add 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide, stir for 30 min, add 0.3 g of dopamine hydrochloride, stir at room temperature for 24 h, put it into a dialysis bag (6000 Da) and dialyze for 3 days (change water every 12 h), and freeze-dry at -20 °C for 36 h to obtain dopamine-modified carboxymethyl cellulose.

[0026] Comparative Example 2 A dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN, the preparation method of which is basically the same as that of Example 3, except that the modified carboxymethyl cellulose in step S2 is replaced by carboxymethyl cellulose.

[0027] Comparative Example 3 A dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN, the preparation method of which is basically the same as that of Example 3, except that PDRN is not added in step S2.

[0028] Comparative Example 4 The hydrogel prepared by the method of Example 1 of the Chinese invention patent with the publication number CN118286150A.

[0029] Comparative Example 5 S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water. Then, 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide were added successively, and the mixture was stirred for 30 min. Next, 0.3 g of 5,5'-((disulfanediyldi(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added, and the mixture was stirred at room temperature for 24 h. Then, it was placed in a dialysis bag (6000 Da) and dialyzed for 3 days (changing water every 12 h), and freeze-dried at -20 °C for 36 h to obtain modified carboxymethyl cellulose; S2: Preparation of Solution A: Modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN were dissolved in 50 ml of deionized water to prepare Solution A. The final concentrations were: 3 wt% of modified carboxymethyl cellulose, 4 wt% of polyvinyl alcohol, and 0.25 wt% of PDRN; S3: Preparation of oxidized sodium alginate: 2.0 g of sodium alginate and 2.16 g of sodium periodate were added to 200 mL of deionized water, and the reaction was carried out in the dark at room temperature for 5 h. Then, 3 mL of ethylene glycol was added to terminate the reaction. After that, the reaction solution was placed in a dialysis bag (3500 kD) and dialyzed for 3 days (changing water every 12 days), and freeze-dried at -20 °C for 36 h to obtain oxidized sodium alginate; S4: Preparation of Solution B: 1.5 g of oxidized sodium alginate and 0.3 g of sodium metaborate were dissolved in 50 ml of deionized water to obtain Solution B; S5: Preparation of hydrogel: 2 ml of Solution A and 2 ml of Solution B were mixed evenly and left to stand at room temperature for 12 h to form a hydrogel.

[0030] In Examples 2, 3 and the comparative example of this application, the PDRN (polydeoxyribonucleotide) model used was RJMPDRN-M, purchased from Regimen (Shandong) Biotechnology Co., Ltd.; the PDRN model used in Example 4 was PDRN-MD, purchased from Bloomage Biotechnology Co., Ltd.; the polyvinyl alcohol model was PVA-1799; the carboxymethyl cellulose model was CM-32, purchased from Shanghai Kanglang Biotechnology Co., Ltd.; the sodium hyaluronate model was MEDIHYA ® S, purchased from Shandong Zhongshan Biotechnology Co., Ltd.; Preparation method of saturated borax buffer solution: 19.07 g of borax was dissolved in deionized water and transferred to a 1000 ml volumetric flask for volume fixation to obtain a saturated borax buffer solution.

[0031] (1) Self-healing property test: The hydrogels prepared in Examples 2-4 and Comparative Examples 1-5 were subjected to self-healing tests. The test method was as follows: The hydrogel was evenly cut in half, and then the two separated halves were reassembled. After a certain period of time, the healing situation was observed under a microscope. The healing time is shown in Table 1.

[0032] Table 1 Self-healing time table

[0033] Figure 1 Fig. is the healing situation diagram of Example 2 and Comparative Example 2 under the microscope. It can be seen that Figure 1 the hydrogel prepared in the example had healed at 3 minutes. The hydrogel prepared in Comparative Example 2 relied only on the formation of borate ester bonds between polyvinyl alcohol (PVA) and sodium metaborate. Although it had a certain self-healing ability, its self-healing efficiency was low. In the hydrogels prepared in Comparative Example 1 and Comparative Example 4, the borate ester bond and the Schiff base bond formed by the reaction of aldehyde group and amino group formed a dynamic crosslinking mechanism, which had good self-healing properties. The hydrogel prepared in Example 2 introduced disulfide bonds, and the synergistic effect of Schiff base bonds and disulfide bonds further enhanced the self-healing performance of the hydrogel.

[0034] (2) Antibacterial test: The hydrogels prepared in Example 2 and Comparative Example 1 were subjected to antibacterial tests. 100 μL of Staphylococcus aureus solution and 100 μL of Escherichia coli were respectively and evenly coated on the surface of a petri dish. The hydrogels prepared in Example 2 and Comparative Example 1 were cut into circles with a diameter of 1 cm and placed on the plate to co-culture with Staphylococcus aureus and Escherichia coli at 37 °C for 24 h, and the change in the diameter of the inhibition zone was observed.

[0035] The diameters of the inhibition zones of the hydrogels prepared in Example 2 and Comparative Example 1 are shown in Figure 2 . After observation, the diameter of the inhibition zone of the hydrogel prepared in Example 2 was larger and the antibacterial performance was stronger. The reason was that the antioxidant property of PDRN could inhibit the production of reactive oxygen species (ROS), thus affecting the growth environment of bacteria, and the introduced disulfide bond had antibacterial effects.

[0036] (3) Infection healing test: The wounds of mice were infected with Staphylococcus aureus to obtain an infected animal model. The infected animal model was divided into four groups, namely the control group, Comparative Example 1, Comparative Example 3, and Example 2 group. Among them, the control group did not perform any treatment on the infected animal model, and the remaining groups used the corresponding hydrogel to perform dressing treatment on the infected animal model. The wound healing situations of the infected animal models in each group were photographed and recorded on the 0th day, 3rd day, 6th day, and 10th day from the start of treatment.

[0037] The wound healing situations of the four groups are as shown in Figure 3As shown, the recovery of the Example 2 group was significantly better than that of other groups. The reason is that the hydrogel can keep the wound moist, provide a protective barrier for the wound, and at the same time load PDRN with anti-inflammatory and bactericidal properties, jointly promoting wound healing. The healing of Comparative Example 1 and 3 was relatively poor compared to Example 2, indicating that the hydrogel prepared from modified carboxymethyl cellulose with disulfide bonds has the ability to synergistically promote wound anti-inflammatory and healing.

[0038] HE staining was performed on the wound healing conditions of the control group, Comparative Example 1, Comparative Example 3, and Example 2 on the 10th day, as shown in Figure 4 , and the internal structure of the skin tissue at different magnifications was studied. The emergence of new epidermis was observed in the healing of the four groups of infected wounds, indicating that the uniformly dispersed PDRN in the hydrogel dressing prepared in Example 2 can play an anti-inflammatory role, and the hydrogel can better promote the healing of infected wound tissues.

[0039] (4) Blood compatibility test The hemolysis rate tests of the hydrogels prepared in Examples 2-4 and Comparative Example 4 were carried out. Fresh blood was taken from the mouse orbit and injected into a disposable vacuum blood collection tube, centrifuged at 3000 rpm for 3 min to obtain red blood cells. The red blood cells were washed 3 times with physiological saline and diluted to a concentration of 2% (v / v) to obtain a red blood cell suspension.

[0040] Preparation of the hydrogel extract: The hydrogel was freeze-dried and ground into powder, and a suspension with a concentration of 8.0 mg / mL was prepared with physiological saline, incubated at 37 °C for 6 h, and centrifuged at 3000 rpm for 5 min to obtain the extract.

[0041] For each of Examples 2-4 and Comparative Example 4, 3 test tubes were used. Each test tube was added with 1 mL of a 2% red blood cell suspension, and then 0.25 mL of the extract was added to each test tube. The mixture was cultured at 37 °C for 30 min, and then centrifuged at 3000 rpm for 3 min. The absorbance of the supernatant was measured at 540 nm. For the positive control group, 3 test tubes were taken, each test tube was added with 1 mL of a 2% red blood cell suspension, and then 0.25 mL of deionized water was added. The mixture was cultured at 37 °C for 30 min, and then centrifuged at 3000 rpm for 3 min. The absorbance of the supernatant was measured at 540 nm.

[0042] The hemolysis rate was calculated based on the measured absorbance. The specific calculation formula is as follows: Hemolysis rate (%) = A s / A p × 100%; where, A s is the absorbance value of the experimental group at 540 nm, and A p is the absorbance value of the positive control group at 540 nm. The average value was calculated for each group of three test tubes, and the test results are shown in Table 2.

[0043] Table 2 Blood Compatibility Test Table

[0044] As can be seen from Table 2, the hydrogel prepared in this application has excellent compatibility. The reason is that PDRN is a natural polynucleotide extracted from organisms, and its chemical structure is similar to that of DNA in organisms, having good biocompatibility; and glutathione, as a bioactive molecule, plays a positive role in biocompatibility.

[0045] As described above, the above are only the preferred embodiments of the present invention and are not used to limit the present invention; however, for those of ordinary skill in the art, without departing from the scope of the technical solution of the present invention, any slight changes, modifications, and equivalent changes made using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN, characterized in that: The following steps are involved: S1: Carboxymethyl cellulose is modified with 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) to obtain modified carboxymethyl cellulose; S2: dissolving modified carboxymethyl cellulose, polyvinyl alcohol and PDRN in deionized water to obtain solution A; S3: oxidizing sodium hyaluronate with sodium periodate to obtain oxidized sodium hyaluronate; S4: dissolving oxidized sodium hyaluronate and sodium metaborate in water to obtain solution B; S5: Mix solution A and solution B evenly, and let stand at room temperature to obtain a hydrogel.

2. The method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that: The 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) is prepared by the following method: Under nitrogen protection, glutathione and triethylamine are added to a mixed solution of acetonitrile and saturated borax buffer, and SO2F2 gas is introduced to react at room temperature to obtain the product.

3. The preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that, The modified carboxymethyl cellulose is prepared by the following method: Under nitrogen protection, carboxymethyl cellulose was dissolved in deionized water, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added in sequence, and the mixture was stirred. 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) was added, and the mixture was stirred at room temperature for 20-30 hours to obtain the product.

4. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 3, characterized in that, The feed mass ratio of the carboxymethyl cellulose, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) is 10:(0.4-2):(0.2-1):(1-5).

5. The method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that, In step S2, the concentration of modified carboxymethyl cellulose in solution A is 3wt%, the concentration of polyvinyl alcohol is 4wt%, and the concentration of PDRN is 0.05-0.5wt%.

6. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that, The oxidized sodium hyaluronate is prepared by the following method: Sodium hyaluronate and sodium periodate were dissolved in deionized water, and reacted in the dark at room temperature for 20-26 hours. Ethylene glycol was added to terminate the reaction, and the mixture was dialyzed with ultrapure water and freeze-dried to obtain oxidized sodium hyaluronate.

7. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 6, characterized in that, The mass ratio of sodium hyaluronate to sodium periodate is 2:(1-5).

8. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that, In step S4, the mass ratio of the oxidized sodium hyaluronate to the sodium metaborate is 5:

1.

9. A method for preparing a dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, characterized in that, In step S4, the solution A and solution B are mixed in a volume ratio of 1:

1.

10. A dynamically cross-linked polysaccharide-based hydrogel loaded with PDRN, characterized in that: Prepared by the method according to any one of claims 1 to 9.

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