A PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel and its preparation method

Abstract

The present invention discloses a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN and a preparation method thereof, belonging to the field of hydrogel technology. The preparation method comprises the following steps: adding modified carboxymethyl cellulose, polyvinyl alcohol and PDRN to deionized water to dissolve to obtain solution A; oxidatively modifying sodium hyaluronate with sodium periodate to obtain oxidized sodium hyaluronate; dissolving oxidized sodium hyaluronate and sodium metaborate in deionized water to obtain solution B; and evenly mixing solution A and solution B and standing at room temperature to obtain a hydrogel. The present invention utilizes polyvinyl alcohol and sodium metaborate to form a dynamic borate ester bond, and utilizes modified carboxymethyl cellulose and oxidized sodium hyaluronate to form a dynamic Schiff base bond, thereby solving the problem of insufficient self-healing property of the hydrogel; utilizing the excellent biocompatibility and anti-inflammatory and bactericidal properties of PDRN to enhance tissue repair and regeneration capabilities.

Description

Technical Field

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

[0002] The skin is one of the most important organs in the human body, in direct contact with the external environment, sensing external stimuli, regulating body temperature, and protecting the body from external harm. Although most common skin injuries can heal themselves, wound healing is a complex and dynamic process. Various factors can cause the healing process to be interrupted and chronic wounds to form. However, these factors can lead to a variety of wound healing problems, making wound repair difficult. Hydrogels are considered to be ideal alternatives in tissue repair engineering due to their ability to resist infection, absorb wound exudate, maintain water balance and gas exchange, load and deliver bioactive factors, and have biocompatibility. Ideal hydrogel wound dressings have suitable mechanical properties, self-healing properties, adhesion ability, antibacterial properties, etc.

[0003] Chinese invention patent publication number CN 118286150 A discloses a dual-dynamic cross-linked polysaccharide-based hydrogel loaded with rhein and a preparation method thereof. Rhein is dissolved in a hydrogel precursor solution, enabling direct loading of rhein and addressing the issues of rhein's poor water solubility and low bioavailability. However, its self-healing ability is still unsatisfactory. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN (polydeoxyribonucleotide) and a preparation method thereof.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A method for preparing a PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel comprises the following steps:

[0007] 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;

[0008] S2: Dissolve modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN in deionized water to obtain solution A;

[0009] S3: oxidatively modifying sodium hyaluronate with sodium periodate to obtain oxidized sodium hyaluronate;

[0010] S4: dissolving sodium hyaluronate and sodium metaborate in water to obtain solution B;

[0011] S5: Mix solution A and solution B evenly, and let stand at room temperature to obtain a hydrogel.

[0012] The 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) was prepared by the following method:

[0013] Under nitrogen protection, glutathione and triethylamine are added to a mixed solution of acetonitrile and saturated borax buffer, and SO2F2 gas is introduced, and the reaction is carried out at room temperature to obtain the product.

[0014] The modified carboxymethyl cellulose is prepared by the following method:

[0015] Under nitrogen protection, carboxymethyl cellulose was dissolved in deionized water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added in sequence, and stirred. 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) was added, and stirred at room temperature for 20-30 hours to obtain the product.

[0016] The 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).

[0017] In step S2, the concentration of 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 %.

[0018] The oxidized sodium hyaluronate is prepared by the following method:

[0019] Sodium hyaluronate and sodium periodate were dissolved in deionized water, reacted in the dark at room temperature for 20-26 hours, ethylene glycol was added to terminate the reaction, dialyzed with ultrapure water, and freeze-dried to obtain oxidized sodium hyaluronate.

[0020] The mass ratio of sodium hyaluronate to sodium periodate is 2:(1-5).

[0021] In step S4, the mass ratio of the oxidized sodium hyaluronate to the sodium metaborate is 5:1.

[0022] In step S4, the volume ratio of the solution A and the solution B is 1:1.

[0023] A PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel is prepared by the above-described method.

[0024] Due to the adoption of the above technical solution, the beneficial effects of the present invention include:

[0025] (1) The present invention modifies carboxymethyl cellulose by forming a disulfide bond with glutathione, and forms a dynamic Schiff base with oxidized sodium hyaluronate. Dynamic borate ester bonds are formed using polyvinyl alcohol and sodium metaborate, thereby solving the problems of poor mechanical properties and insufficient self-healing properties of hydrogels.

[0026] (2) The present invention loads PDRN onto a dynamically cross-linked polysaccharide-based hydrogel, utilizing the excellent biocompatibility and anti-inflammatory and bactericidal properties of PDRN to enhance the hydrogel's tissue repair and regeneration functions.

[0027] (3) Glutathione, as a bioactive molecule, gives the hydrogel good compatibility. The presence of disulfide bonds improves its self-healing ability and antibacterial properties, which can effectively relieve oxidative stress at the wound site and increase the speed of wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figures are the healing status of the hydrogels under microscope of Example 2 and Comparative Example 2;

[0029] Figure 2 This is a test diagram of the antibacterial performance of the hydrogels of Example 2 and Comparative Example 1;

[0030] Figure 3 Take photos and record the wound healing of infected animal models;

[0031] Figure 4 HE staining of wound healing in infected animal models. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to these embodiments.

[0033] Example 1: Preparation of 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid):

[0034] 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 (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. 300 ml of ethyl acetate was added to dilute the mixture, and the mixture was washed three times with 50 ml of saturated brine. The organic layer was dried over 10 g of anhydrous Na2SO4, filtered, and distilled at 60°C under reduced pressure for 2 h to obtain 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid), the structural formula of which is as follows:

[0035]

[0036] Its H NMR spectrum data are as follows: 1 H 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, 4.2Hz, 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, 6.7Hz, 2H), 1.85 (dq, J=15.4, 6.5 Hz, 2H).

[0037] Example 2: Preparation of PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel:

[0038] S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water, and 0.04 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.02 g of N-hydroxysuccinimide were added in sequence. The mixture was stirred for 30 min, and 0.1 g of 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added. The mixture was stirred at room temperature for 20 h, and dialyzed in a dialysis bag (6000 Da) for 3 days (water was changed every 12 h), and freeze-dried at -20°C for 36 h to obtain modified carboxymethyl cellulose.

[0039] 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 of modified carboxymethyl cellulose were 3 wt %, polyvinyl alcohol was 4 wt %, and PDRN was 0.05 wt %.

[0040] S3: Preparation of oxidized sodium hyaluronate: Dissolve 2 g of sodium hyaluronate and 1 g of sodium periodate in 50 ml of deionized water. React in the dark at room temperature for 20 h. Add 2 g of ethylene glycol to terminate the reaction. Dialyze against ultrapure water (8 kDa) for 3 days (change the water every 12 days), and freeze-dry at -20°C for 36 h to obtain oxidized sodium hyaluronate.

[0041] S4: Preparation of Solution B: Dissolve 1.5 g of sodium hyaluronate and 0.3 g of sodium metaborate in 50 ml of deionized water to obtain Solution B;

[0042] 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.

[0043] Example 3: Preparation of PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel:

[0044] S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water, and 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide were added in sequence. The mixture was stirred for 30 min, and 0.3 g of 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added. The mixture was stirred at room temperature for 24 h, and dialyzed in a dialysis bag (6000 Da) for 3 days (water was changed every 12 h), and freeze-dried at -20°C for 36 h to obtain modified carboxymethyl cellulose.

[0045] 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 of modified carboxymethyl cellulose were 3 wt %, polyvinyl alcohol was 4 wt %, and PDRN was 0.25 wt %.

[0046] S3: Preparation of oxidized sodium hyaluronate: Dissolve 2 g of sodium hyaluronate and 3 g of sodium periodate in 50 ml of deionized water. React in the dark at room temperature for 24 h. Add 3 g of ethylene glycol to terminate the reaction. Dialyze against ultrapure water (10 kDa) for 3 days (change the water every 12 days), and freeze-dry at -20°C for 36 h to obtain oxidized sodium hyaluronate.

[0047] S4: Preparation of Solution B: Dissolve 1.5 g of sodium hyaluronate and 0.3 g of sodium metaborate in 50 ml of deionized water to obtain Solution B;

[0048] S5: Preparation of hydrogel: 2 ml of solution A and 2 ml of solution B were mixed evenly and allowed to stand at room temperature for 12 h to form a hydrogel.

[0049] Example 4: Preparation of PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel:

[0050] S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water, and 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 g of N-hydroxysuccinimide were added in sequence. The mixture was stirred for 30 min, and 0.5 g of 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added. The mixture was stirred at room temperature for 30 h, and dialyzed in a dialysis bag (6000 Da) for 3 days (water was changed every 12 h), and freeze-dried at -20°C for 36 h to obtain modified carboxymethyl cellulose.

[0051] 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 of modified carboxymethyl cellulose were 3 wt %, polyvinyl alcohol was 4 wt %, and PDRN was 0.5 wt %.

[0052] 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 against ultrapure water (12 kDa) for 3 days (change the water every 12 days), and freeze-dry at -20°C for 36 h to obtain oxidized sodium hyaluronate.

[0053] S4: Preparation of Solution B: Dissolve 1.5 g of sodium hyaluronate and 0.3 g of sodium metaborate in 50 ml of deionized water to obtain Solution B;

[0054] S5: Preparation of hydrogel: 2 ml of solution A and 2 ml of solution B were mixed evenly and allowed to stand at room temperature for 12 h to form a hydrogel.

[0055] Comparative Example 1

[0056] A PDRN-loaded dynamically cross-linked polysaccharide-based hydrogel, 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 modified carboxymethyl cellulose prepared by the following method:

[0057] Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water, and 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide were added in sequence. The mixture was stirred for 30 min, and 0.3 g of dopamine hydrochloride was added. The mixture was stirred at room temperature for 24 h, and dialyzed in a dialysis bag (6000 Da) for 3 days (water was changed every 12 h). The mixture was freeze-dried at -20 °C for 36 h to obtain dopamine-modified modified carboxymethyl cellulose.

[0058] Comparative Example 2

[0059] A dynamically 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.

[0060] Comparative Example 3

[0061] A dynamically 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.

[0062] Comparative Example 4

[0063] The hydrogel was prepared using the method of Example 1 of Chinese invention patent publication number CN118286150A.

[0064] Comparative Example 5

[0065] S1: Preparation of modified carboxymethyl cellulose: Under nitrogen protection, 1 g of carboxymethyl cellulose was dissolved in 50 g of deionized water, and 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 g of N-hydroxysuccinimide were added in sequence. The mixture was stirred for 30 min, and 0.3 g of 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) (prepared in Example 1) was added. The mixture was stirred at room temperature for 24 h, and dialyzed in a dialysis bag (6000 Da) for 3 days (water was changed every 12 h), and freeze-dried at -20°C for 36 h to obtain modified carboxymethyl cellulose.

[0066] 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 of modified carboxymethyl cellulose were 3 wt %, polyvinyl alcohol was 4 wt %, and PDRN was 0.25 wt %.

[0067] 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 reacted at room temperature in the dark for 5 h. 3 mL of ethylene glycol was then added to terminate the reaction. The reaction solution was then dialyzed in a dialysis bag (3500 kD) for 3 days (with water changed every 12 days), and freeze-dried at -20°C for 36 h to obtain oxidized sodium alginate.

[0068] S4: Preparation of Solution B: Dissolve 1.5 g of oxidized sodium alginate and 0.3 g of sodium metaborate in 50 ml of deionized water to obtain Solution B;

[0069] S5: Preparation of hydrogel: 2 ml of solution A and 2 ml of solution B were mixed evenly and allowed to stand at room temperature for 12 h to form a hydrogel.

[0070] The PDRN (polydeoxyribonucleotide) used in Examples 2, 3 and the comparative example of the present application is model RJMPDRN-M, purchased from Ruijiming (Shandong) Biotechnology Co., Ltd.; the PDRN used in Example 4 is model PDRN-MD, purchased from Huaxi Biotechnology Co., Ltd.; the polyvinyl alcohol is model PVA-1799; the carboxymethyl cellulose is model CM-32, purchased from Shanghai Kanglang Biotechnology Co., Ltd.; the hyaluronic acid is model MEDIHYA ® S was purchased from Shandong Zhongshan Biotechnology Co., Ltd.; preparation method of saturated borax buffer: dissolve 19.07 g of borax in deionized water and transfer to a 1000 ml volumetric flask to obtain saturated borax buffer.

[0071] (1) Self-healing test:

[0072] The hydrogels prepared in Examples 2-4 and Comparative Examples 1-5 were subjected to a self-healing test. The test method was as follows: the hydrogel was evenly cut into two halves, and then the two halves were reassembled. After a certain period of time, the healing state was observed under a microscope. The healing time is shown in Table 1.

[0073] Table 1 Self-healing timeline

[0074]

[0075] Figure 1 The healing condition diagrams under the microscope of Example 2 and Comparative Example 2 are shown. Figure 1As can be seen, the hydrogel prepared in Example 2 had healed within 3 minutes. The hydrogel prepared in Comparative Example 2 relied solely on borate bonds formed between polyvinyl alcohol (PVA) and sodium metaborate. While exhibiting some self-healing ability, its healing efficiency was relatively low. In the hydrogels prepared in Comparative Examples 1 and 4, borate bonds and Schiff base bonds formed between aldehyde and amino groups formed a dynamic crosslinking mechanism, resulting in superior self-healing properties. The hydrogel prepared in Example 2 incorporated disulfide bonds, and the synergistic effect of the Schiff base and disulfide bonds further enhanced the hydrogel's self-healing properties.

[0076] (2) Antibacterial test:

[0077] The hydrogels prepared in Example 2 and Comparative Example 1 were subjected to antibacterial testing. 100 μL of Staphylococcus aureus solution and 100 μL of Escherichia coli were respectively evenly coated on a surface dish. The hydrogels prepared in Example 2 and Comparative Example 1 were cut into circles with a diameter of 1 cm and placed on a plate. They were co-cultured with Staphylococcus aureus and Escherichia coli at 37°C for 24 hours, and the changes in the diameter of the inhibition zone were observed.

[0078] The diameters of the inhibition zones of the hydrogels prepared in Example 2 and Comparative Example 1 are shown in Table 2. Figure 2 . It was observed that the hydrogel prepared in Example 2 had a larger inhibition zone diameter and stronger antibacterial performance. The reason is that the antioxidant property of PDRN can inhibit the production of reactive oxygen species (ROS) and thus affect the growth environment of bacteria, and the introduced disulfide bond has an antibacterial effect.

[0079] (3) Infection healing test:

[0080] Mouse wounds were infected with Staphylococcus aureus to obtain infected animal models, which were then divided into four groups: a control group, comparative example 1, comparative example 3, and example 2. The control group received no treatment for the infected animal models, while the other groups received patch treatment with the corresponding hydrogels. The wound healing status of each group of infected animal models was recorded on day 0, day 3, day 6, and day 10 after the start of treatment.

[0081] The wound healing results of the four groups were as follows Figure 3 As shown, the recovery of 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 to the wound, and at the same time load PDRN with anti-inflammatory and bactericidal properties to jointly promote wound healing; the healing of Comparative Examples 1 and 3 was relatively poorer than that of Example 2, indicating that the hydrogel prepared by modified carboxymethyl cellulose with disulfide bonds has the ability to synergize wound anti-inflammatory healing.

[0082] HE staining was performed on the wound healing conditions of the control group, comparative example 1, comparative example 3 and embodiment 2 on the 10th day. Figure 4, the internal structure of skin tissue at different magnifications was studied. The emergence of new epidermis could be seen from the healing of four groups of infected wounds, indicating that the PDRN evenly dispersed in the hydrogel dressing prepared in Example 2 could play an anti-inflammatory role, and the hydrogel could better promote the healing of infected wound tissue.

[0083] (4) Blood compatibility test

[0084] The hydrogels prepared in Examples 2-4 and Comparative Example 4 were subjected to a hemolysis rate test. Fresh blood was collected from the eye sockets of mice and injected into a disposable vacuum blood collection tube. The blood was centrifuged at 3000 rpm for 3 minutes to obtain red blood cells. The red blood cells were washed three times with physiological saline and diluted to a concentration of 2% (v / v) to obtain a red blood cell suspension.

[0085] Preparation of hydrogel extract: The hydrogel was freeze-dried and ground into powder, and prepared into a suspension with a concentration of 8.0 mg / mL in physiological saline, incubated at 37° C. for 6 h, and centrifuged at 3000 rpm for 5 min to obtain an extract.

[0086] In Examples 2-4 and Comparative Example 4, 3 test tubes were added to each test tube with a 2% red blood cell suspension, and 0.25 mL of extract was added to each test tube. The cells were cultured at 37 ° C for 30 min, and then centrifuged at 3000 rpm for 3 min. The absorbance of the supernatant was tested at 540 nm. The positive control group took 3 test tubes, 1 mL of 2% red blood cell suspension was added to each test tube, and 0.25 mL of deionized water was added. The cells were cultured at 37 ° C for 30 min, and then centrifuged at 3000 rpm for 3 min. The absorbance of the supernatant was tested at 540 nm.

[0087] The hemolysis rate is calculated based on the absorbance obtained from the test. The calculation formula is as follows:

[0088] Hemolysis rate (%) = A s / A p ×100%;

[0089] Among them, A s is the absorbance value of the experimental group at 540 nm, A p The absorbance of the positive control group at 540 nm was calculated as the average value of three test tubes in each group. The test results are shown in Table 2.

[0090] Table 2 Blood compatibility test table

[0091]

[0092] 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, and it has good biocompatibility; and glutathione, as a bioactive molecule, plays a positive role in biocompatibility.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. However, any equivalent changes, modifications and evolutions made by ordinary technicians in this field without departing from the scope of the technical solution of the present invention by 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 essential technology of the present invention are still within the scope of protection 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: Dissolve modified carboxymethyl cellulose, polyvinyl alcohol, and PDRN in deionized water to obtain solution A; S3: oxidatively modifying sodium hyaluronate with sodium periodate to obtain oxidized sodium hyaluronate; S4: dissolving 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 preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, wherein The 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) was 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, and the reaction is carried out 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, wherein The modified carboxymethyl cellulose is prepared by the following method: Under nitrogen protection, carboxymethyl cellulose was dissolved in deionized water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added in sequence, and stirred. 5,5'-((disulfanediylbis(3-((carboxymethyl)amino)-3-oxopropane-1,2-diyl))bis(azadiyl))bis(2-amino-5-oxopentanoic acid) was added, and stirred at room temperature for 20-30 hours to obtain the product.

4. The preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 3, wherein The 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 preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, wherein In step S2, the concentration of 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 %.

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

7. The preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 6, wherein The mass ratio of sodium hyaluronate to sodium periodate is 2:(1-5).

8. The preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, wherein In step S4, the mass ratio of the oxidized sodium hyaluronate to the sodium metaborate is 5:

1.

9. The preparation method of a dynamic cross-linked polysaccharide-based hydrogel loaded with PDRN according to claim 1, wherein In step S4, the volume ratio of the solution A and the solution B is 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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