Preparation and application of sulfhydrylated radix bistortae polysaccharide hydrogel

Abstract

The application discloses a kind of preparation and application of sulfhydrylated radix bletillae polysaccharide hydrogel, first using the way of amidation to prepare sulfhydrylated radix bletillae polysaccharide (tBSP), tBSP and four-arm polyethylene glycol norbornene (PEGNB) are mixed and added to photo initiator I2959, sulfhydrylated radix bletillae polysaccharide-four-arm polyethylene glycol norbornene hydrogel (tBP hydrogel) is obtained under ultraviolet light irradiation, the concentration of 0-2wt% tannic acid (TA) solution is configured and added to tBP hydrogel and reacts, i.e., sulfhydrylated radix bletillae polysaccharide hydrogel (tBP@TA hydrogel) is obtained.The tBP@TA hydrogel prepared in the application has swelling and strong adhesion performance first, can be firmly adhered to wound and absorb wound exudate pus;Secondly, tBP@TA hydrogel has antibacterial and antioxidant properties, so that the wound can avoid the healing delay caused by microbial infection and oxidative stress, and finally promote the healing of infected wounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to preparation and application of sulfhydrylated radix bistortae polysaccharide hydrogel. BACKGROUND

[0002] Wound healing is a complex process, including four stages of hemostasis, inflammation, proliferation and remodeling. It involves complex mechanisms such as removal of foreign matter, inflammation, granulation tissue formation, and mature scar production. Most skin wounds will heal within one to two weeks, but infected wounds, especially infected full-thickness skin wounds, can cause serious complications such as pain, sepsis, amputation, and even death. Bacterial infection is one of the biggest obstacles to wound healing, and microorganisms can induce severe tissue damage, long-term inflammatory response and delayed wound healing. In clinical practice, antibiotics are often used to prevent wound infection. However, improper or overuse of antibiotics has led to the emergence of multiple drug-resistant strains, reduced antibiotic efficacy, and made the skin always susceptible to infection, even leading to the formation of chronic wounds. In addition, the excessive reactive oxygen species (ROS) produced by bacterial infection can cause persistent inflammation at the infection site, causing serious damage to blood vessels and cells, making it difficult for the wound to heal. Therefore, reducing bacterial infection and reducing oxidative stress are key to promoting the healing of infected wounds.

[0003] In order to eliminate bacterial infection, various antibacterial agents have emerged, and existing antibacterial materials can be roughly divided into the following four types: antibiotics, antibacterial nanoparticles, cationic organic agents and other antibacterial materials. The main antibacterial mechanism of antibiotics includes inhibiting bacterial cell wall synthesis, blocking key metabolic pathways of bacteria, interfering with bacterial protein synthesis, and inhibiting bacterial nucleic acid synthesis, thereby causing bacterial death. The antibacterial mechanism of nanoparticles is to cause bacterial death by directly contacting the bacterial cell wall, releasing toxic metal ions or generating ROS. The antibacterial mechanism of cationic antibacterial agents is first to interact with the negatively charged lipids on the outer surface of the bacterial cell membrane through the large number of positive charge groups on their own, and then the hydrophobic segment of the cationic antibacterial agent increases the permeability of the bacterial cell membrane, leading to the outflow of bacterial cell contents and bacterial death. The types and antibacterial mechanisms of other antibacterial materials are complex. Many natural product extracts are included in other antibacterial materials, and their main antibacterial mechanism is to inhibit bacterial cell membrane synthesis and damage bacterial cell membranes, leading to bacterial death. However, the overuse of antibiotics can lead to the emergence of drug-resistant bacteria, which seriously endangers human health. The metal ions in nanoparticle antibacterial agent hydrogels have high cost; they are highly toxic, release toxic metal ions or generate ROS to cause bacterial death, and also have an impact on normal tissues. Cationic antibacterial agent hydrogels have insufficient sterilization effect, relatively poor mechanical properties after forming hydrogels, and are prone to rupture when subjected to external forces or have certain toxicity. Other antibacterial material hydrogels cannot have both antibacterial ability and cytotoxicity or have high antibacterial cost.

[0004] Oxidative stress is a biochemical process in which excess oxidants in cells disrupt the redox balance. Specifically, during oxidative stress, free radicals and other reactive oxidants in cells damage biomolecules such as proteins, lipids, and DNA, leading to cell damage, excessive inflammation, tissue aging, and delayed wound healing. Therefore, removing excess ROS or reactive nitrogen is an important strategy to alleviate oxidative stress. Antioxidants are divided into synthetic antioxidants and natural antioxidants. Synthetic antioxidants can delay the peroxidation process of lipids in food by removing free radicals, thereby extending the shelf life. However, synthetic antioxidants have certain biological toxicity, so they are not used in the treatment of diseases caused by oxidative stress. In view of the biological toxicity and adverse effects of synthetic antioxidants, scientists have explored plant extracts as antioxidants to resist the negative effects of ROS or reactive nitrogen caused by oxidative stress. The main sources of natural antioxidants are phenolic and saccharide substances in plants. The antioxidant mechanism of phenolic substances is that there are hydroxyl groups connected to the aromatic ring in phenolic substances, which have strong electron-donating effects and are easily oxidized by losing electrons, thereby having antioxidant activity. The antioxidant mechanism of saccharide substances is more complex, including enhancing antioxidant enzyme activity, inhibiting the production of lipid peroxides, and affecting the expression of related proteins. These natural antioxidants extracted from plants have good antioxidant capacity and biocompatibility, and can safely and effectively remove free radicals in the body to improve oxidative stress, and have good medical application potential.

[0005] Hydrogel is a hydrophilic polymer with a three-dimensional porous structure formed by physical or chemical cross-linking of polymer chains. Hydrogel has soft texture, strong water retention capacity, and good biocompatibility, and is widely used in cell culture, drug delivery, tissue engineering and other biomedical fields. On the one hand, hydrogel can be used as a drug delivery carrier to effectively release therapeutic drugs, antibacterial agents, antioxidants and other substances, greatly improving the utilization rate of antibacterial agents and antioxidants. On the other hand, the substance composed of hydrogel itself has antibacterial and antioxidant properties, reducing the toxic effects of antibiotics or non-natural antioxidants on cells, and having significant advantages in wound treatment. Therefore, hydrogel as a substitute material for treating infected wounds is increasingly attracting attention. SUMMARY

[0006] The present application aims to prepare a hydrogel dressing capable of treating infected wounds. First, for the phenomenon of pus exudation of infected wounds, the hydrogel has the property of swelling to absorb the pus exudated from the wound. In addition, the exudation of pus is accompanied by the forced wet environment of the wound, and the hydrogel with poor adhesion is difficult to adhere to the wound and easy to fall off from the wound, so the hydrogel should have high adhesion ability and can firmly adhere to the surface of the wound. Thirdly, for the phenomenon of bacterial infection of infected wounds, in order to prevent multiple drug-resistant strain infection, it is best to avoid the use of antibiotics, therefore, the hydrogel needs to have good antibacterial ability to reduce the inflammation and complications caused by bacterial infection. Finally, for the characteristics of ROS accumulation in infected wounds, the hydrogel also needs to have good antioxidant activity to avoid cell damage and excessive inflammation caused by oxidative stress at the wound site. The present application solves the status quo of difficult healing of infected wounds through the above four factors. Therefore, the present application aims to prepare a hydrogel dressing with good swelling performance, adhesion performance, and antibacterial and antioxidant ability to promote the rapid healing of infected wounds.

[0007] Traditional Chinese herbs have a long history in treating various types of wounds. Bletilla striata is a traditional Chinese medicinal material listed as one of the herbal species in the Chinese Pharmacopoeia, and is known for its hemostatic, heat-clearing, anti-inflammatory, and wound healing properties. Bletilla striata polysaccharide (BSP) is an effective active ingredient extracted from the traditional Chinese herb Bletilla striata, which is a natural glucomannan containing multiple hydroxyl groups composed of alpha-mannose, beta-mannose and beta-glucose. Modern pharmacological studies have confirmed that BSP is mainly used as a hemostatic, anticancer and antitumor agent, as well as a wound healing promoter, an antibacterial agent, a mucosal protective agent, an anti-ulcer agent, an antioxidant agent, an anti-aging agent and an immunomodulator. Due to the multifunctionality of BSP, the present application selects BSP as the raw material. However, this glucomannan has three disadvantages: first, BSP contains a large number of hydroxyl groups and a small number of aldehyde groups, which cannot adhere to the skin through the bonding of multiple groups; second, previous studies have verified that BSP has weak antioxidant activity, making it difficult to treat damage caused by oxidative stress in infected wounds; third, the hydroxyl groups and a small number of aldehyde groups in BSP limit the method of preparing hydrogels, and the network structure of the hydrogel formed by hydrogen bonds is easy to break and prone to damage when subjected to stress. Therefore, the present application proposes a preparation scheme for tBSP, which has the following advantages over BSP: first, the thiol groups in tBSP are easy to react with free radicals, enhancing its antioxidant activity; second, the thiol groups in tBSP can cross-link with double-bonded substances through photo-click chemistry to form hydrogels, which are often faster in reaction speed, more uniform in gel pore size, and more stable in gel stability than hydrogels formed by hydrogen bonds. Third, tBSP has four functional groups: hydroxyl, aldehyde, carboxyl, and thiol, which can form hydrogen bonds, carbon-nitrogen double bonds, ionic bonds, and disulfide bonds with various substances on the skin, thereby adhering firmly to the skin. The preparation of tBSP first grafts the formic acid group of chloroacetic acid to the hydroxyl group of BSP through etherification to prepare carboxymethylated Bletilla striata polysaccharide (CBSP), and then grafts L-cysteine to the formic acid group of CBSP through amidation to prepare tBSP. At this time, tBSP has four types of functional functional groups: hydroxyl, aldehyde, carboxyl, and thiol, which provide active sites for cross-linking reactions, antioxidant properties, and skin adhesion of hydrogels.

[0008] Four-arm PEGNB is a kind of functional polymer, which has four arms in structure, based on polyethylene glycol as the basic structural unit, and four arms are grafted with norbornene groups through amidation, with good water solubility, biodegradability and biocompatibility. Under the action of photoinitiator and ultraviolet light, PEGNB will crosslink with tBSP through thiol-ene photo click chemistry reaction to form thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel (tBP hydrogel). Compared with the traditional chain growth type photopolymerization reaction, the polymerization mode of thiol-ene photo click chemistry reaction is free radical mediated stepwise growth, the reaction is faster, the free oxygen growth is less and the formed grid is more uniform. This uniform grid hydrogel structure has four advantages, first, the uniform hydrogel network can more effectively transmit and disperse stress when subjected to external force, and is not easy to appear local stress concentration and cause rupture or damage, thereby showing better stability. Second, the uniform grid is conducive to the uniform diffusion and absorption of water molecules in the hydrogel interior, making the hydrogel more stable during the swelling process. Third, in the uniform grid, the diffusion path of small molecule substances such as drug molecules is more regular, which can be quickly transported in the hydrogel interior and accompanied by regular gel degradation. Fourth, the uniform structure may reduce the potential immunogenicity sites in the hydrogel, reduce the risk of triggering immune response, and improve the safety and biocompatibility of the hydrogel.

[0009] TA is a natural polyphenol, which is a hydrolysable, amphiphilic tannin derivative of gallic acid, and has antibacterial, antioxidant and anti-inflammatory activities. The tBP hydrogel is prepared by immersing TA into the tBP hydrogel. The TA has three advantages by immersing into the tBP hydrogel. First, the metal chelating ability and phenolic hydroxyl group of TA make TA have antioxidant and antibacterial ability, and adding TA into the tBP hydrogel enhances the antibacterial and antioxidant ability of the hydrogel. Second, TA contains rich phenolic hydroxyl groups, which can interact with the hydroxyl, carboxyl and sulfhydryl groups in the tBP hydrogel to form hydrogen bonds, thereby enhancing the cross-linking structure inside the tBP@TA hydrogel. The tBP@TA hydrogel has two network structures: one is the network formed by the cross-linking of tBSP and PEGNB through thiol-ene photo-click chemistry reaction, and the other is the network formed by the hydrogen bonds between the phenolic hydroxyl groups on TA and the hydroxyl groups of tBP. The double network structure enhances the cohesion of the hydrogel, and the cohesion enhances the adhesion of the tBP@TA hydrogel. Third, the surface of the tBP@TA hydrogel formed by immersing TA into the tBP hydrogel has a layer of mucous membrane, and the main component of the mucous membrane is TA. The rich phenolic hydroxyl groups of TA can form hydrogen bonds or hydrophobic interactions with the surface of the skin tissue, thereby improving the tissue adhesion of the hydrogel. The second point mentioned above is relatively important for the improvement of the adhesion of the hydrogel.

[0010] In summary, the present application first prepares tBSP as a raw material for hydrogel, and then tBSP and PEGNB are cross-linked under ultraviolet light to form tBP hydrogel through thiol-ene photo-click chemistry reaction. The tBP hydrogel is immersed in a TA solution of different concentrations to prepare tBP@TA hydrogel. The tBP@TA hydrogel has swelling and strong adhesion performance, can adhere to the wound tightly and absorb the exudate of the wound; secondly, the tBP@TA hydrogel has antibacterial and antioxidant properties, which can avoid the delay of wound healing caused by microbial infection and oxidative stress, and finally promote the healing of infected wounds.

[0011] The present application is realized by the following technical means:

[0012] The present application first discloses a preparation method of thiolated bletilla striata polysaccharide hydrogel, comprising:

[0013] (1) preparing thiolated bletilla striata polysaccharide:

[0014] To the carboxymethylated Bletilla striata polysaccharide solution, 0.5-5 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added and activated for 40-70 min, 0.5-3 g L-cysteine hydrochloride was added for reaction, 20 wt% NaOH was used to adjust the pH value to 4.0, and stirring was carried out at 25°C for 1-5 h, then the pH value of the reaction mixture was adjusted to 6.0, and stirring was carried out at 25°C for 0.5-5 h, the pH value of the reaction mixture was adjusted to neutral, and dialysis was carried out in RO water using a dialysis bag with a molecular weight of 3500 Da for 72 h, and then freeze-drying was carried out, to obtain thiolated Bletilla striata polysaccharide;

[0015] (2) Preparation of thiolated Bletilla striata polysaccharide hydrogel:

[0016] 1-3 wt% four-arm polyethylene glycol norbornene, 5-25 wt% thiolated Bletilla striata polysaccharide, and 0.11 wt% I2959 were added into an EP tube to be dissolved in RO water to obtain a hydrogel precursor, the hydrogel precursor solution was irradiated under a UV lamp to initiate crosslinking, to form a thiolated Bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel, then 0-2 wt% tannic acid solution was added into the thiolated Bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel, and the obtained mixture was placed in the dark, to obtain the thiolated Bletilla striata polysaccharide hydrogel.

[0017] Further, the carboxymethylated Bletilla striata polysaccharide solution in step (1) is prepared by the following method:

[0018] 0.5-5 g carboxymethylated Bletilla striata polysaccharide was dissolved in 100 mL RO water, to obtain the carboxymethylated Bletilla striata polysaccharide solution.

[0019] Further, the carboxymethylated Bletilla striata polysaccharide is prepared by the following method:

[0020] 0.5-5 g Bletilla striata polysaccharide was dissolved in 20 mL, 20 wt% NaOH under ice bath conditions, and stirring was carried out until complete dissolution, to obtain a first mixture;

[0021] 30 mL isopropyl alcohol was added into the first mixture and stirred for 10-60 min, then 0.1-2 mL of chloroacetic acid solution was added in 10 portions, and stirring was carried out at 55°C for 30-120 min, to obtain a second mixture;

[0022] 40 wt% glacial acetic acid was used to adjust the pH value to neutral, dialysis was carried out in RO water using a dialysis bag, and then freeze-drying was carried out, to obtain the carboxymethylated Bletilla striata polysaccharide.

[0023] Further, the chloroacetic acid solution is prepared by the following method: 1-7 g chloroacetic acid was dissolved in 1-20 mL RO water, to obtain the chloroacetic acid solution;

[0024] The dialysis bag has a molecular weight of 3500 Da, a dialysis time of 72 h, and a freeze-drying time of 72 h.

[0025] Further, the hydrogel precursor solution in step (2) is irradiated under a UV lamp with a wavelength of 376 nm and an intensity of 5 mWcm -2 The concentration of the tannin solution is 2%, and the volume ratio of the thiolated Bletilla striata polysaccharide-tetra-arm polyethylene glycol norbornene hydrogel to the tannin solution is 1:2.

[0026] Further, the concentration of the tannin solution is 2%, and the volume ratio of the thiolated Bletilla striata polysaccharide-tetra-arm polyethylene glycol norbornene hydrogel to the tannin solution is 1:2.

[0027] Further, the volume ratio of the tetra-arm polyethylene glycol norbornene, the thiolated Bletilla striata polysaccharide, and I2959 in step (2) is 10:39:1.

[0028] Further, the light-shielding placement condition in step (2) is 24-48 h of placement at 37 DEG C in the dark.

[0029] The application further discloses a thiolated Bletilla striata polysaccharide hydrogel prepared according to any one of the preparation methods.

[0030] The application further discloses application of the thiolated Bletilla striata polysaccharide hydrogel in preparation of medical dressings.

[0031] The application has the following beneficial effects:

[0032] The application first prepares tBSP as a raw material of the hydrogel, then tBSP and PEGNB are subjected to thiol-ene photo-click chemistry reaction under UV irradiation to form cross-linking, i.e., tBP hydrogel, and then the tBP hydrogel is soaked in a TA solution with different concentrations to prepare tBP@TA hydrogel.

[0033] The adhesion performance is improved: the tBP@TA hydrogel is combined with tBP and TA, the adhesion performance of the tBP@TA hydrogel is improved, the tBP hydrogel has hydroxyl, aldehyde, carboxyl, thiol, four groups, the TA solution contains rich phenolic hydroxyl, the TA is doped into the tBP hydrogel by the immersion method, the phenolic hydroxyl in the TA can form a hydrogen bond with the hydroxyl in the tBP hydrogel, a second network structure is formed in the original tBP hydrogel network, the tBP@TA hydrogel has a double network structure, the double network structure provides stronger cohesion than the single network structure, and the cohesion gives the tBP@TA hydrogel stronger adhesion performance.

[0034] The stability is enhanced: the tBP@TA hydrogel is combined with tBP and TA, the stability of the tBP@TA hydrogel is improved, the TA is doped into the tBP hydrogel by the immersion method, and the hydrogel has a double network structure, and the double network structure gives the tBP@TA hydrogel stronger stability.

[0035] (1) The advantages of material performance: first, after the BSP is grafted with a thiol group, the tBSP has stronger antioxidant activity than the BSP because the thiol group is easy to react with a free radical; second, the tBSP and the TA in the components of the tBP@TA hydrogel contain hydroxyl, aldehyde, carboxyl, thiol and phenolic hydroxyl groups, and these groups can form hydrogen bonds and various covalent bonds with the surface of skin tissue, thereby enhancing the adhesion force of the skin combination; third, the tBSP and the TA in the components of the tBP@TA hydrogel have antibacterial and antioxidant activities, and can better treat bacterial infection and oxidative stress in infected wounds.

[0036] (2) Advantages of the hydrogel network: tBP@TA hydrogel is prepared by two steps, first, tBSP and PEGNB are used to form tBP hydrogel by thiol-ene photo-click chemistry, then tBP hydrogel is soaked in TA solution to form tBP@TA hydrogel. tBP@TA hydrogel has three advantages, first, thiol-ene photo-click chemistry is a free radical mediated step-growth, the reaction is faster, less free oxygen growth and the grid formed is more uniform; second, tBP@TA hydrogel has a double network structure (the first network is the network generated by the crosslinking of tBSP and PEGNB by thiol-ene photo-click chemistry, and the second network is the network generated by the hydrogen bond between the phenolic hydroxyl group on TA and the hydroxyl group of tBP), this double network structure provides stronger cohesion than single network structure, and the abundant functional groups and the cohesion provided by the double network endow tBP@TA hydrogel with good adhesion capacity. Third, the uniform network structure of tBP@TA hydrogel provides a good channel for absorbing the exudate of infected wounds, and the cohesion provided by the double network also provides good swelling capacity. The combination of strong adhesion and high swelling ensures that while absorbing the exudate of infected wounds, it can also adhere to the surface of the wound to play the functions of antibacterial and antioxidant, thereby promoting the wound to heal as soon as possible.

[0037] (3) Biocompatibility advantage: PEGNB, tBSP and TA in tBP@TA hydrogel have good biocompatibility; thiol-ene photo-click chemistry is a free radical mediated step-growth at room temperature, less free oxygen is generated, which greatly reduces the oxidative damage of free oxygen to biological macromolecules. Therefore, from the perspective of material or the preparation method of hydrogel, this hydrogel has good biocompatibility.

[0038] In summary, the hydrogel designed in the present application scheme can treat infected wounds. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The nuclear magnetic resonance hydrogen spectrum of BSP, CBSP and tBSP;

[0040] Figure 2 The DPPH free radical scavenging rate of BSP and tBSP;

[0041] Figure 3 The tBP and tBP@2%TA hydrogel gelation state comparison;

[0042] Figure 4 The DPPH free radical scavenging rate of Examples 26-29;

[0043] Figure 5The swelling ratios are for Examples 26–29;

[0044] Figure 6 Examples 26–29 demonstrate the antibacterial properties of E. coli and S. aureus;

[0045] Figure 7 Examples 26–29 show the inhibition rates against E. coli.

[0046] Figure 8 Examples 26–29 show the antibacterial rates against S. aureus.

[0047] Figure 9 For the hemolysis rates of Examples 26–29;

[0048] Figure 10 The cell compatibility of mouse mononuclear macrophages (Raw264.7), mouse fibroblasts (L929), and human umbilical vein endothelial cells (EC) in Examples 26–29 was studied.

[0049] Figure 11 Examples 26–29 demonstrate cytotoxicity in mouse mononuclear macrophages for 1–3 days;

[0050] Figure 12 Examples 26–29 show cytotoxicity in mouse fibroblasts for 1–3 days;

[0051] Figure 13 Examples 26–29 show cytotoxicity in human umbilical vein endothelial cells for 1–3 days;

[0052] Figure 14 Scanning electron microscope image of tBSP@2%TA hydrogel in Example 28;

[0053] Figure 15 The elastic modulus of the tBSP@2%TA hydrogel in Example 28;

[0054] Figure 16 This is a schematic diagram of the adhesion of tBSP@2%TA hydrogel on pigskin in Example 28;

[0055] Figure 17 For example 28, tBSP@2%TA was used to promote in vivo wound healing of infected wounds in SD rats. Detailed Implementation

[0056] Compared with the prior art, the present invention provides a method for preparing thiolized Bletilla striata polysaccharide hydrogel. The specific definitions of the English abbreviations involved in this invention are as follows:

[0057] BSP: Bletilla striata polysaccharide; the molecular weight of BSP used in this invention is 6 × 10⁻⁶. 4 ~1.5×10 5Da.

[0058] tBSP: thiolated bletilla striata polysaccharide;

[0059] CBSP: carboxymethylated bletilla striata polysaccharide;

[0060] PEGNB: four-arm polyethylene glycol norbornene; the molecular weight of PEGNB used in the present application is 5000 Da on average.

[0061] TA: tannic acid;

[0062] tBP hydrogel: thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel;

[0063] tBP@TA hydrogel: thiolated bletilla striata polysaccharide hydrogel;

[0064] The related solutions involved in the present application are prepared by the following methods:

[0065] Prepare a BSP (bletilla striata polysaccharide) solution with a mass fraction of 1-5 wt% for standby, and the solvent is RO water.

[0066] Prepare a tBSP (thiolated bletilla striata polysaccharide) solution with a mass fraction of 5-25 wt% for standby, and the solvent is RO water.

[0067] Prepare a PEGNB (four-arm polyethylene glycol norbornene) solution with a mass fraction of 1-5 wt% for standby, and the solvent is RO water.

[0068] Prepare an I2959 solution with a mass fraction of 0.11 wt% for standby, and the solvent is RO water.

[0069] Prepare a TA (tannic acid) solution with a mass fraction of 0-3 wt% for standby, and the solvent is RO water. The 0 wt% TA solution is RO water of the same volume.

[0070] A preparation method of a thiolated bletilla striata polysaccharide hydrogel, comprising:

[0071] Prepare a 1.5 mL centrifuge tube, and add tBSP solution (5-25 wt%), PEGNB solution (1-5 wt%), and I2959 (0.11 wt%) solution into the centrifuge tube. Mix the mixed solution by ultrasonic oscillation and vortex. The volume ratio of tBSP solution, PEGNB solution, and I2959 solution is 39:10:1. Place the mixed solution under a 376 nm ultraviolet lamp (UV) with an intensity of 5 mWcm -2 to form a tBP hydrogel.

[0072] The prepared tBP hydrogel was immersed in TA solution (0-3wt%) and reacted at 37°C in dark for 24h-48h to obtain thiolated tBSP hydrogel (tBP@0-3%TA). The volume ratio of tBP hydrogel to TA solution was 0.5-2:1-4.

[0073] Example 1

[0074] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 5wt%, and the mass percentage of PEGNB was 1wt%.

[0075] Example 2

[0076] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 5wt%, and the mass percentage of PEGNB was 2wt%.

[0077] Example 3

[0078] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 5wt%, and the mass percentage of PEGNB was 3wt%.

[0079] Example 4

[0080] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 5wt%, and the mass percentage of PEGNB was 4wt%.

[0081] Example 5

[0082] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 5wt%, and the mass percentage of PEGNB was 5wt%.

[0083] Example 6

[0084] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 10wt%, and the mass percentage of PEGNB was 1wt%.

[0085] Example 7

[0086] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 10wt%, and the mass percentage of PEGNB was 2wt%.

[0087] Example 8

[0088] The tBP hydrogel was prepared according to the above method, and the mass percentage of tBSP in this example was 10wt%, and the mass percentage of PEGNB was 3wt%.

[0089] Example 9

[0090] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 10wt%, and the mass percentage of PEGNB was 4wt%.

[0091] Example 10

[0092] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 10wt%, and the mass percentage of PEGNB was 5wt%.

[0093] Example 11

[0094] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 15wt%, and the mass percentage of PEGNB was 1wt%.

[0095] Example 12

[0096] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 15wt%, and the mass percentage of PEGNB was 2wt%.

[0097] Example 13

[0098] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 15wt%, and the mass percentage of PEGNB was 3wt%.

[0099] Example 14

[0100] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 15wt%, and the mass percentage of PEGNB was 4wt%.

[0101] Example 15

[0102] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 15wt%, and the mass percentage of PEGNB was 5wt%.

[0103] Example 16

[0104] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 20wt%, and the mass percentage of PEGNB was 1wt%.

[0105] Example 17

[0106] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 20wt%, and the mass percentage of PEGNB was 2wt%.

[0107] Example 18

[0108] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 20wt%, and the mass percentage of PEGNB was 3wt%.

[0109] Example 19

[0110] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 20wt%, and the mass percentage of PEGNB was 4wt%.

[0111] Example 20

[0112] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 20wt%, and the mass percentage of PEGNB was 5wt%.

[0113] Example 21

[0114] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 25wt%, and the mass percentage of PEGNB was 1wt%.

[0115] Example 22

[0116] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 25wt%, and the mass percentage of PEGNB was 2wt%.

[0117] Example 23

[0118] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 25wt%, and the mass percentage of PEGNB was 3wt%.

[0119] Example 24

[0120] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 25wt%, and the mass percentage of PEGNB was 4wt%.

[0121] Example 25

[0122] tBP hydrogel was prepared according to the above method, the mass percentage of tBSP in this example was 25wt%, and the mass percentage of PEGNB was 5wt%.

[0123] Example 26

[0124] tBP@0-3%TA hydrogel was prepared according to the above method. In this example, the mass percentage of tBSP was 15 wt%, the mass percentage of PEGNB was 2 wt%, and the mass percentage of TA solution was 0 wt%. The hydrogel prepared in this example is named tBP0@0%TA hydrogel.

[0125] Example 27

[0126] tBP@0-3%TA hydrogel was prepared according to the above method. In this example, the mass percentage of tBSP was 15 wt%, the mass percentage of PEGNB was 2 wt%, and the mass percentage of TA solution was 1 wt%. The hydrogel prepared in this example is named tBP0@1%TA hydrogel.

[0127] Example 28

[0128] The tBP0-3%@TA hydrogel was prepared according to the above method. In this example, the mass percentage of tBSP was 15 wt%, the mass percentage of PEGNB was 2 wt%, and the mass percentage of TA solution was 2 wt%. The hydrogel prepared in this example is named tBP0@2%TA hydrogel.

[0129] Example 29 Hydrogel

[0130] The tBP0-3%@TA hydrogel was prepared according to the above method. In this example, the mass percentage of tBSP was 15 wt%, the mass percentage of PEGNB was 2 wt%, and the mass percentage of TA solution was 3 wt%. The hydrogel prepared in this example is named tBP0@3%TA hydrogel.

[0131] Experimental Example 1

[0132] Preparation of tBSP

[0133] (1) NMR detection of tBSP

[0134] To verify whether L-cysteine ​​hydrochloride is grafted onto the sugar chain of BSP, nuclear magnetic resonance (NMR) spectroscopy was used to detect the proton NMR spectra of BSP, CBSP, and tBSP at a frequency of 400 MHz. The three samples were dissolved in deuterium oxide (D₂O), and the solutions were placed in NMR tubes and then analyzed by the instrument to observe the appearance of corresponding characteristic peaks.

[0135] like Figure 1 As shown, in the 1H NMR spectrum, the OH peak of deuterated water appears at δ 4.79 ppm, and the sugar backbone (CH2) of BSP appears at 3.1–4.0 ppm. The newly formed peak at 2.9 ppm corresponds to the characteristic peak of CH2 on L-cys, indicating that the grafting of thiol group was successful.

[0136] (2) Detection of antioxidant activity of tBSP

[0137] To verify whether the antioxidant property of tBSP is improved, the antioxidant activity of BSP and tBSP was determined using DPPH radical scavenging experiment. DPPH was dissolved in anhydrous ethanol to prepare a DPPH solution of 0.1 mM, tBSP and BSP were prepared into a mixture solution of 100 μL of different concentrations (1 mg / mL, 2.5 mg / mL, 5 mg / mL), the mixture solution of different concentrations was mixed with 2 mL of DPPH solution in the dark for 30 min, and then the absorbance was detected at 517 nm, and the DPPH radical scavenging rate was calculated according to formula (1-1):

[0138]

[0139] wherein Ao is the absorbance of the DPPH blank group (DPPH + ethanol); Ay is the absorbance of the DPPH sample group (DPPH + ethanol + sample).

[0140] The DPPH radical scavenging ability of tBSP and BSP at different concentrations is shown in Table 1. Figure 2 As shown in Table 1, the antioxidant activity of tBSP is obviously improved compared with BSP. The DPPH radical scavenging rate of BSP is 47% at low concentration (1 mg / mL), and the DPPH radical scavenging rate of tBSP reaches 68%, and the DPPH radical scavenging rate of BSP is 52% at high concentration (5 mg / mL), and the DPPH radical scavenging rate of tBSP reaches 96%. It is proved that compared with BSP, tBSP after grafting mercapto can endow the hydrogel wound dressing with stronger antioxidant activity.

[0141] Test Example 2

[0142] Screening of tBP hydrogel

[0143] 50 uL of tBP solution in Examples 1-25 was dropped on a cover glass, and the gel state was observed by ultraviolet light irradiation, the cover glass was turned over every 5 s until the hydrogel was completely formed and did not flow, the gel time was recorded, and the gel state was observed.

[0144] In the experiment of hydrogel formation, the time of hydrogel formation and the speed of gelation increased with the increase of tBSP and PEGNB concentration. It was found that when the concentration of PEGNB was 3wt% or more, the gel was brittle and difficult to adhere to the wound surface. When the concentration of tBSP was more than 15wt%, the formed gel was opaque and contained flocculent substances, indicating that tBSP was not completely dissolved. It was found that when the concentration of PEGNB was 2wt% and the concentration of tBSP was 15wt%, the hydrogel had the best form. Therefore, the concentrations of PEGNB and tBSP in tBP hydrogel were set to 2wt% and 15wt%, and examples 26-29 were carried out at these concentrations.

[0145] Test Example 3

[0146] Screening of tBP@TA hydrogel

[0147] (1) Swelling of tBP@TA hydrogel

[0148] The samples prepared in examples 26-29 were subjected to swelling performance test. 200 u mg of hydrogel was weighed and dried (W d ). Then the hydrogel was immersed in PBS, taken out at a certain time, wiped off the excess water, weighed the wet weight of the hydrogel (W t ), and the swelling rate was calculated according to formula (1-2):

[0149]

[0150] Swelling is a basic property of hydrogel, which reflects the absorption capacity of hydrogel to wound exudate. As Figure 5 It can be seen that the swelling rate of tBSP@0%TA hydrogel can reach 93.5% at 15min and 259.3% at 120min. The swelling capacity of tBSP@1%TA, tBP@2%TA and tBP@3%TA hydrogels decreased compared with tBP@0%TA hydrogel, but at 15min, the swelling rate of tBP@2%TA and tBP@3%TA hydrogels reached 90%, and at 30min, the swelling rate reached about 100%, indicating that tBSP@0%TA, tBP@2%TA and tBP@3%TA hydrogels can absorb wound exudate in a short time.

[0151] (2) Antioxidant performance test of tBP@TA hydrogel

[0152] The samples prepared in Examples 26-29 were subjected to antioxidant activity testing. Four groups of hydrogels were prepared according to different concentrations of TA, designated as tBP@0%TA, tBP@1%TA, tBP@2%TA, tBP@3%TA. DPPH was dissolved in anhydrous ethanol to prepare a 0.1 mM DPPH solution, then 50 μL of hydrogel was mixed with 2 mL of DPPH solution and incubated in the dark for 30 min, then the absorbance was detected at 517 nm, and the DPPH radical scavenging rate was calculated according to formula (1-3):

[0153]

[0154] where A o is the absorbance of the DPPH blank group (DPPH + ethanol); A h is the absorbance of the DPPH hydrogel group (DPPH + ethanol + sample)

[0155] As Figure 4 shown, the DPPH radical scavenging ability of tBP@TA hydrogel increases with increasing tannic acid concentration, the DPPH radical scavenging rate of tBSP@0%TA is 15.1%, the DPPH radical scavenging rate of tBSP@1%TA is 46.7%, the DPPH radical scavenging rate of tBSP@2%TA is 80%, and the DPPH radical scavenging rate of tBSP@3%TA reaches 96.7%. The experiment proves that tBSP@2%TA planted in Example 28 and tBSP@3%TA prepared in Example 29 have good antioxidant capacity and can remove reactive oxygen to accelerate wound healing, showing great potential for wound dressings.

[0156] (3) Antimicrobial performance test of tBP@TA hydrogel

[0157] The samples prepared in Examples 26-29 were subjected to antimicrobial performance testing. E. coli (ATCC 87393) and S. aureus (ATTC 29213) were used to evaluate the activity of the hydrogel. First, LB broth was used to culture E. coli and S. aureus to a good state of bacterial growth.

[0158] 10 μL of bacterial suspension (1 x 10 5CFU / mL) and 40 μL sterilized PBS were added to the surface of hydrogel. The well plate was wrapped with tin foil to keep the hydrogel in the dark, and then incubated at 37 °C for 6 h. After that, the non-killed bacteria were resuspended with 100 μL sterilized PBS, and then 20 μL of the bacterial suspension was spread on the agar plate. 10 μL of the bacterial suspension was diluted in 100 μL sterilized PBS, and 20 μL was spread on the agar plate as a control group. The above agar plates were incubated at 37 °C for 24 h, and then the colonies were counted. The experiment was repeated three times for each group, and the results were expressed as the mortality rate, and the antibacterial rate was calculated according to equation (1-4):

[0159]

[0160] The dressing with antibacterial properties can kill bacteria and prevent wound infection. If bacteria adhere to the surface of the dressing and continue to proliferate, a biofilm will be formed. Once a biofilm is formed, it is difficult for bacteria to be killed by antibacterial agents. Many persistent infections are believed to be related to biofilms. TA has the characteristics of inhibiting the formation of E. coli and S. aureus biofilms, and has great potential in preventing infections caused by biofilms on the surface of biological materials. The antibacterial activity of hydrogel was detected with E. coli and S. aureus. As shown in Figure 6 , for E. coli, tBSP@0%TA and tBSP@1%TA had no inhibitory effect, but instead promoted the proliferation of E. coli, indicating that tBSP could promote the proliferation of E. coli, and low concentration of TA had no good antibacterial effect on E. coli. As shown in Figure 7 , with the increase of TA concentration, the antibacterial rate of tBSP@2%TA on E. coli reached 81.6%, and the antibacterial rate of tBSP@2%TA on E. coli reached 88.1%, indicating that high concentration of TA had obvious antibacterial effect on E. coli. As shown in Figure 6 , for S. aureus, tBSP@0%TA, tBSP@1%TA, tBSP@2%TA, tBSP@3%TA all showed antibacterial effect, indicating that tBSP and TA had inhibitory effect on S. aureus. As shown in Figure 8 , with the increase of TA concentration, the antibacterial rates of the four groups of hydrogel on S. aureus reached 58.1%, 79.2%, 96.2%, and 99.4%, respectively. The experimental results showed that tBSP@2%TA and tBSP@3%TA had good antibacterial effect on E. coli and S. aureus.

[0161] (4) Hemolysis rate detection of tBP@TA hydrogel

[0162] The samples prepared in Examples 26-29 were subjected to hemolysis rate detection. Rat citric acid whole blood was used, the whole blood was diluted with PBS to a volume ratio of 5:4, the hydrogel was placed in a centrifuge tube, 10 mL of PBS was added and incubated at 37°C for 30 min, the same volume of PBS and ultrapure water were placed in the centrifuge tube as negative and positive controls, respectively, 200 μL of diluted whole blood was added to each centrifuge tube, and incubated at 37°C for 60 min. The incubated reaction solution was loaded into a new centrifuge tube and centrifuged at 3000 rpm for 5 min. 100 μL of supernatant was taken and added to a 96-well plate, and the absorbance value was measured at 540 nm. The hemolysis rate was calculated according to formula (1-5):

[0163]

[0164] Here A s is the absorbance value of the hydrogel; A p and A n are the absorbance values of the positive and negative controls.

[0165] Figure 9 As shown, as the concentration of TA solution increases, the hemolysis rate of tBP@TA hydrogel also increases. The hemolysis rate of tBSP@0%TA is 0.17%, the hemolysis rate of tBSP@1%TA is 0.47%, the hemolysis rate of tBSP@2%TA is 2%, and the hemolysis rate of tBSP@3%TA is close to 4%, which is lower than the national relevant standard (5%). It is proved that tBP@0%TA, tBP@1%TA, and tBP@2%TA hydrogels have good blood compatibility.

[0166] (5) Cell compatibility detection of tBP@TA hydrogel

[0167] The samples prepared in Examples 26-29 were subjected to cell compatibility detection. After the hydrogel was formed, it was washed with PBS for 3 times to remove unreacted solution. Then the hydrogel was soaked in high glucose DMEM containing 15% FBS and 1% penicillin / streptomycin, and incubated at 37°C for 24 h. The hydrogel extract was obtained by filtration. L929 cells were cultured in high glucose DMEM containing 15% FBS and 1% penicillin / streptomycin. When the cells grew to 90% on the bottom of the cell culture bottle, the cells were inoculated into a 48-well cell culture plate with a cell density of 2×10 4 / well. After cell attachment, the culture medium was replaced with one containing hydrogel extract, and the cells were cultured at 37°C in a cell incubator containing 5% CO2 for 1, 2, and 3 days. Cell viability was assessed using a CCK-8 assay kit, with absorbance measured at 450 nm using a microplate reader. RAW264.7 cells were viable using the same method. EC cells were cultured in DMEM / F-12 medium, and cell viability was measured using the same procedure for the remaining cells. Cell viability was calculated using formula (1-6):

[0168]

[0169] Where V s V is the absorbance of the hydrogel group. c V is the absorbance of the blank control group. b It is the absorbance of the culture medium containing 10% CCK-8 reagent.

[0170] After culturing the cells for 1-3 days according to the above steps, carefully remove the cell culture medium, wash 3 times with PBS, add 2.5% glutaraldehyde to fix the cells, remove the glutaraldehyde after 24 hours, wash 3 times with PBS, then add Rhodamine stain to the cell culture wells, remove it after 30 minutes, wash 3 times with PBS, and then place the well plate under a fluorescence microscope to observe the cell morphology.

[0171] Cell compatibility is crucial for the clinical application of wound dressings. For example... Figure 10 As shown, under a fluorescence microscope, cells from different groups exhibited similar morphologies. RAW264.7 cells were round, L929 cells were all spindle-shaped, and EC cells were irregular polygonal. Compared to the control group, the cell morphology of the hydrogel extract group showed no significant change. Figures 11-13 As shown, the activity of L929 and EC cells decreased with increasing TA concentration in tBSP@0%TA, tBSP@1%TA, tBSP@2%TA, and tBSP@3%TA groups, but remained higher than 80% of the control group. For RAW264.7 cells, these groups showed a certain degree of cell proliferation promotion. The results indicate that the four hydrogels (tBP@0%TA, tBP@1%TA, tBP@2%TA, and tBP@3%TA) prepared according to the present invention exhibit good cell compatibility.

[0172] After the tBP@0%TA, tBP@1%TA, tBP@2%TA, tBP@3%TA hydrogels prepared in Examples 26-29 were subjected to the above swelling test, antioxidant test, antibacterial test, hemolysis rate test and cell compatibility test, the results showed that tBP@0%TA exhibited higher swelling, low antioxidant activity, single antibacterial activity against S. aureus, good blood compatibility and cell compatibility; tBP@1%TA exhibited higher swelling, low antioxidant activity, single antibacterial activity against S. aureus, good blood compatibility and cell compatibility; tBP@2%TA hydrogel exhibited good swelling, high antioxidant activity, antibacterial activity against E. coli and S. aureus, good hemolysis rate and cell compatibility, tBP@3%TA hydrogel exhibited good swelling, high antioxidant activity, good antibacterial activity and hemolysis rate, but poor cell compatibility, therefore, after comprehensive consideration, tBP@2%TA hydrogel was selected for subsequent experiments.

[0173] Test Example 4

[0174] Performance characterization and application of tBP@2%TA hydrogel

[0175] (1) Micro-morphology of tBP@2%TA hydrogel

[0176] The surface morphology of the hydrogel was detected by scanning electron microscopy. The tBP@2%TA hydrogel prepared in Example 28 was placed in an EP tube, frozen at -40°C for 24h, and then broken. The sample was vacuum dried for 48h. The freeze-dried sample was attached to the conductive glue, and the gold was sprayed for 20min in a vacuum environment. The cross-sectional microstructure was observed by scanning electron microscopy at a voltage of 3.0KV.

[0177] As shown in Figure 14 , tBP@2%TA presents a connected porous structure, and the pore size is small and uniformly distributed. This uniform and interconnected porous microstructure can provide an entry for water vapor, facilitate nutrient and oxygen supply, and waste removal required for cell survival.

[0178] (2) Rheological test of tBP@2%TA hydrogel

[0179] Excellent mechanical properties are a prerequisite for hydrogels as wound dressings in clinical applications. The rheological properties of the tBP@2%TA hydrogel prepared in Example 28 were determined using a rotational rheometer. The tBP@2%TA hydrogel was prepared into a 5mm cylinder with a diameter of 12mm, placed between parallel plates, and tested at 37°C. The elastic modulus of the hydrogel was recorded during the process of angular velocity from 0 to 10 rad / s.

[0180] As shown in Figure 15As shown, during the continuous increase of angular velocity, the storage modulus (G') of tBP@2%TA hydrogel is always greater than the loss modulus (G”), indicating that tBP@2%TA hydrogel remains in a relatively stable state. Mechanical strength plays a crucial role in hydrogel wound dressings. Excessive mechanical properties cannot guarantee the softness of biomaterials, making them prone to brittle fracture, while poor mechanical properties may lead to tearing, peeling, and deformation of hydrogel dressings.

[0181] The storage modulus of tBP@2%TA hydrogel is similar to that of skin (300-2000 Pa), and it has the potential to be used as a wound dressing.

[0182] (3) Tissue adhesion properties test of tBP@2%TA hydrogel

[0183] The tissue adhesion properties of the hydrogel were evaluated by stretching and adhesion to pigskin.

[0184] like Figure 16 As shown, after the tBP@2%TA hydrogel prepared in Example 28 was adhered to pigskin, it still adhered well to the pigskin even when stretched or twisted. tBSP contains functional groups such as carboxyl, hydroxyl, and thiol groups, while TA contains abundant phenolic hydroxyl groups. These abundant functional groups can form physical and chemical bonds with various groups on the skin, contributing to improved interfacial adhesion of the tBSP@2%TA hydrogel. Adhesive hydrogel dressings that can completely adhere to and bind to tissue can prevent wound infection, ensuring a close fit between the hydrogel and the wound site, providing a favorable microenvironment for wound healing.

[0185] (4) Rat full-cortical defect infection wound healing experiment

[0186] Compared to ordinary wounds, bacterially infected skin wounds with a high infection rate significantly delay healing time. Therefore, the use of *S. aureus* (10...) 10 CFU / mL) and E. coli (10 10 To evaluate the healing ability of this hydrogel on bacterial wounds, an infected full-thickness skin defect model was established in animals infected with a mixed bacterial solution (CFU / mL).

[0187] First, male SD rats (200-250g) that had acclimatized to their environment for one week were anesthetized and their dorsal hair was removed. A circular, full-thickness skin defect with a diameter of 8mm was created on the rat's back using a biopsy punch. Next, 20μL of a mixed bacterial solution was dripped onto the wound, and the wound was covered with a sterile, sealed PU membrane for 24 hours. The PU membrane was then removed to obtain a bacterial infection wound model. At this point, a prepared hydrogel sample was used to treat the bacterial full-thickness skin wound. The wound treatment groups for each rat were designed as follows:

[0188] ①PBS group;

[0189] ②Control (mixed bacteria solution + PBS);

[0190] ③Commercial dressing group (mixed bacteria solution + 3M dressing);

[0191] ④tBP@2% TA hydrogel group (mixed bacteria solution + tBP@2% TA hydrogel);

[0192] ⑤tBP hydrogel group (mixed bacteria solution + tBP hydrogel).

[0193] Afterwards, the rats were regularly monitored for any signs of discomfort and inflammation in the wounds. At the same time, the healing of the wounds was observed, and photographs were taken and samples were obtained at time nodes of 3 days, 7 days, 14 days, 21 days, etc.

[0194] The experimental results are shown in Figure 17 The wounds of the Control group and the commercial 3M dressing group still showed inflammation and yellowish exudate on the third day, but the wounds treated with tBP@2% TA hydrogel and the wounds treated with tBP hydrogel showed no signs of inflammation. Figure 16 The wounds treated with the hydrogel method all showed obvious contraction at 3, 7, and 14 days. Among them, the wounds treated with tBP hydrogel showed more obvious wound contraction than the blank (Control) group. This shows that the tBSP itself has a relatively strong wound healing effect. Among all the groups, the wounds treated with tBP@2% TA hydrogel contracted the fastest, which is mainly due to the tBSP, TA, and as a material for forming a hydrogel, which has been proven to have good antibacterial and tissue repair-promoting ability. In summary, tBSP@2% TA hydrogel can eliminate bacterial infection, relieve oxidative stress, and promote the healing of infected wounds.

[0195] The above description of disclosed embodiments enables one skilled in the art to make or use the invention. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not to be limited to the embodiments shown herein but is to be accorded the scope consistent with the principles and novel features disclosed herein. For example, by varying the molecular weight of the white tuber polysaccharide, the molecular weight / structure of the four-arm polyethylene glycol norbornene, and the volume ratio of tBP hydrogel to tannic acid, etc.

Claims

1. A method for preparing a thiolized Bletilla striata polysaccharide hydrogel, comprising: (1) Preparation of thiolized Bletilla striata polysaccharide: 0.5–5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added to the carboxymethylated Bletilla striata polysaccharide solution for activation for 40–70 min. Then, 0.5–3 g of L-cysteine ​​hydrochloride was added for reaction. The pH was adjusted to 4.0 with 20 wt% NaOH and stirred in a 25°C water bath for 1–5 h. The pH of the reaction mixture was then adjusted to 6.0 and stirred at 25°C for 0.5–5 h. The pH of the reaction mixture was then adjusted to neutral and dialyzed with RO water using a dialysis bag with a molecular weight of 3500 Da for 72 h. Finally, the mixture was freeze-dried to obtain thiolated Bletilla striata polysaccharide. (2) Preparation of thiolized Bletilla striata polysaccharide hydrogel: A 1-3 wt% aqueous solution of tetra-arm polyethylene glycol norbornene RO, a 5-25 wt% aqueous solution of thiolated Bletilla striata polysaccharide RO, and a 0.11 wt% aqueous solution of I2959 RO were added to an EP tube to obtain a hydrogel precursor. The hydrogel precursor solution was placed under a UV lamp to induce cross-linking, forming a thiolated Bletilla striata polysaccharide-tetra-arm polyethylene glycol norbornene hydrogel. Subsequently, a 1-3 wt% tannic acid solution was added to the thiolated Bletilla striata polysaccharide-tetra-arm polyethylene glycol norbornene hydrogel. The resulting mixture was placed in the dark to obtain the thiolated Bletilla striata polysaccharide hydrogel.

2. The preparation method according to claim 1, wherein: The carboxymethylated Bletilla striata polysaccharide solution in step (1) is prepared by the following method: Dissolve 0.5-5 g of carboxymethylated Bletilla striata polysaccharide in 100 mL of RO water to obtain a carboxymethylated Bletilla striata polysaccharide solution.

3. The preparation method according to claim 2, wherein: The carboxymethylated Bletilla striata polysaccharide was prepared by the following method: Under ice bath conditions, 0.5–5 g of Bletilla striata polysaccharide was dissolved in 20 mL of 20 wt% NaOH and stirred until completely dissolved to obtain the first mixture; Add 30 mL of isopropanol to the first mixture and stir for 10-60 min. Then add 0.1-2 mL of chloroacetic acid solution in 10 portions and stir in a 55 ℃ water bath for 30-120 min to obtain the second mixture. The pH was adjusted to neutral using 40 wt% glacial acetic acid, and the mixture was dialyzed in RO water using a dialysis bag, followed by freeze drying to obtain carboxymethylated Bletilla striata polysaccharide.

4. The preparation method according to claim 3, wherein: The chloroacetic acid solution is prepared by dissolving 1-7 g of chloroacetic acid in 1-20 mL of RO water. The dialysis bag has a molecular weight of 3500 Da, a dialysis time of 72 h, and a freeze-drying time of 72 h.

5. The preparation method according to claim 1, wherein: The hydrogel precursor solution in step (2) is placed at a wavelength of 376 nm and an intensity of 5 mW / cm. 2 Irradiated under ultraviolet light; The concentration of the tannin solution is 1-2 wt%; The volume ratio of the thiolized Bletilla striata polysaccharide-tetraarm polyethylene glycol norbornene hydrogel to the tannin solution is 0.5~2:1~4; The concentration of the four-arm polyethylene glycol norbornene was 2 wt%, and the concentration of thiolated Bletilla striata polysaccharide was 15 wt%.

6. The preparation method according to claim 5, wherein: The concentration of the tannin solution is 2 wt%; The volume ratio of the thiolated Bletilla striata polysaccharide-tetraarm polyethylene glycol norbornene hydrogel to the tannin solution is 1:

2.

7. The preparation method according to claim 1, wherein: In step (2), the volume ratio of the four-arm polyethylene glycol norbornene RO aqueous solution, the thiolated Bletilla striata polysaccharide RO aqueous solution, and the I2959 RO aqueous solution is 10:39:

1.

8. The preparation method according to claim 1, wherein: The light-protected placement conditions described in step (2) are: placing in the dark at 37℃ for 24 to 48 hours.

9. A thiolized Bletilla striata polysaccharide hydrogel prepared according to any one of claims 1 to 8.

10. The application of the thiolated Bletilla striata polysaccharide hydrogel according to claim 9 in the preparation of medical dressings.

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