Preparation and application of sulfhydrylated bletilla striata polysaccharide hydrogel

Abstract

The invention discloses preparation and application of sulfhydrylated bletilla striata polysaccharide hydrogel, firstly sulfhydrylated bletilla striata polysaccharide (tBSP) is prepared in an amidation mode, the tBSP and four-arm polyethylene glycol norbornene (PEGNB) are mixed, a photoinitiator I2959 is added, sulfhydrylated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel (tBP hydrogel) is obtained under ultraviolet irradiation, and the sulfhydrylated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel (tBP hydrogel) is added into the mixture to prepare the sulfhydrylated bletilla striata polysaccharide hydrogel. And preparing a tannic acid (TA) solution with the concentration of 0-2wt%, adding the tannic acid (TA) solution into the tBP hydrogel, and reacting to obtain the sulfhydrylated bletilla striata polysaccharide hydrogel (tBP-TA hydrogel). The tBP-TA hydrogel prepared by the invention firstly has swelling and strong adhesion properties, and can be firmly adhered to a wound and absorb exosmosis fester of the wound; secondly, the tBP (at) TA hydrogel has antibacterial and antioxidant properties, so that the wound can be prevented from healing delay caused by microbial infection and oxidative stress, and finally the healing of the infected wound is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the preparation and application of thiolated bletilla striata polysaccharide hydrogel. Background Art

[0002] Wound healing is a complex process that includes four stages: hemostasis, inflammation, proliferation, and remodeling. It involves complex mechanisms such as removal of foreign bodies, inflammation, granulation tissue formation, and mature scar formation. Most skin wounds heal within one to two weeks, but infected wounds, especially infected full-thickness skin wounds, can lead to serious complications such as pain, sepsis, amputation, and even death. Bacterial infection is one of the biggest obstacles to wound healing. Microorganisms can induce severe tissue damage, prolonged inflammatory responses, and delayed wound healing. Clinically, antibiotics are often used to prevent wound infections. However, improper or excessive use of antibiotics has led to the emergence of multidrug-resistant strains, reduced antibiotic efficacy, and caused the skin to remain in an infected state, and even led to the formation of chronic wounds. In addition, excessive reactive oxygen species (ROS) produced by bacterial infection can cause persistent inflammatory responses at the site of infection, causing serious damage to blood vessels and cells, making it difficult for wounds to heal. Therefore, alleviating bacterial infection and reducing oxidative stress are the key to promoting the healing of infected wounds.

[0003] In order to eliminate bacterial infections, various antibacterial agents have emerged. 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 mechanisms of antibiotics include inhibiting bacterial cell wall synthesis, blocking key bacterial metabolic pathways, interfering with bacterial protein synthesis, and inhibiting bacterial nucleic acid synthesis, thereby causing bacterial death. The antibacterial mechanism of nanoparticles is to directly contact the bacterial cell wall, release toxic metal ions or produce ROS, thereby causing bacterial death. 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 positively charged groups they carry, and then the hydrophobic segment of the cationic antibacterial agent will increase the permeability of the bacterial cell membrane, resulting in the outflow of bacterial cell contents and bacterial death. The types and antibacterial mechanisms of other antibacterial materials are complex. Other antibacterial materials contain many natural product extracts, and their main antibacterial mechanism is to inhibit bacterial cell membrane synthesis and damage the bacterial cell membrane, resulting in bacterial death. However, excessive use of antibiotics can lead to the emergence of drug-resistant bacteria, which seriously endangers human health. The metal ions in nanoparticle antimicrobial hydrogels are expensive and toxic, releasing toxic metal ions or producing ROS, which can lead to bacterial death and affect normal tissues. Cationic antimicrobial hydrogels have insufficient bactericidal effects, and the mechanical properties of the hydrogels are relatively poor after formation. They are prone to rupture when subjected to external forces or have certain toxicity. Other antimicrobial hydrogels cannot have both antimicrobial ability and cytotoxicity or have high antimicrobial costs.

[0004] Oxidative stress is a biochemical process in which excessive oxidizing substances in cells disrupt redox balance. Specifically, during oxidative stress, free radicals and other active oxidants in cells damage biomolecules such as proteins, lipids, and DNA, leading to cell damage, excessive inflammation, tissue aging, and delayed wound healing. Therefore, scavenging excess ROS or reactive nitrogen is an important strategy to reduce oxidative stress. Antioxidants are divided into synthetic antioxidants and natural antioxidants. Synthetic antioxidants can delay the peroxidation of lipids in food by scavenging free radicals, thereby extending the shelf life. However, due to their certain biological toxicity, synthetic antioxidants 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 the negative effects of plant extracts as antioxidants to resist oxidative stress caused by ROS or reactive nitrogen. The sources of natural antioxidants are mainly phenols and sugars from plants. The antioxidant mechanism of phenols is as follows: phenols contain hydroxyl groups connected to aromatic rings, which have strong electron-donating effects and are easily oxidized by losing electrons, thus having antioxidant activity; the antioxidant mechanism of sugars is more complex, including enhancing the activity of antioxidant enzymes, 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, can safely and effectively remove free radicals in organisms 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. Hydrogels are soft, have strong water-holding capacity, and have good biocompatibility. They are widely used in biomedical fields such as cell culture, drug delivery, tissue engineering, and other medicines. On the one hand, hydrogels can be used as drug delivery carriers 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 substances that make up the hydrogels themselves have antibacterial and antioxidant properties, which reduce the toxic effects of antibiotics or non-natural antioxidants on cells, and have significant advantages in wound treatment. Therefore, hydrogels are increasingly attracting attention as alternative materials for treating infected wounds. Summary of the invention

[0006] The scheme of the present invention aims to prepare a hydrogel dressing capable of treating infected wounds. First, in view of the pus exudation phenomenon of infected wounds, the hydrogel has the property of swelling and can absorb the pus exuded from the wound. In addition, the exudation of pus is accompanied by the fact that the wound is forced to be in a wet environment. The hydrogel with poor viscosity is difficult to adhere to the wound and is easy to fall off from the wound. Therefore, the hydrogel should have a higher adhesion ability and can adhere firmly to the wound surface. Thirdly, in view of the phenomenon of bacterial infection of infected wounds, in order to prevent infection with multi-drug resistant strains, it is best to avoid the use of antibiotics. Therefore, the hydrogel is required to have good antibacterial ability to reduce the inflammation and complications caused by bacterial infection. Finally, in view of 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. The present invention solves the current situation that infected wounds are difficult to heal through the above four factors. Therefore, the present invention aims to prepare a hydrogel dressing with good swelling performance, adhesion performance, antibacterial and antioxidant capabilities to promote the rapid healing of infected wounds.

[0007] Traditional Chinese herbal medicine has 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 famous for its hemostatic, heat-clearing, anti-inflammatory and wound healing properties. Bletilla striata polysaccharide (BSP) is an effective ingredient extracted from the traditional Chinese herbal medicine Bletilla striata. It is a natural glucomannan containing multiple hydroxyls, composed of α-mannose, β-mannose and β-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, antibacterial agent, mucosal protective agent, anti-ulcer agent, antioxidant, anti-aging agent and immunomodulator. Due to the versatility of BSP, the present invention selects BSP as a raw material. However, BSP, a type of glucomannan, has three disadvantages: first, BSP contains a large number of hydroxyl groups and a small number of aldehyde groups, and cannot adhere to the skin through the bonding of multiple groups; second, previous studies have verified that BSP has weak antioxidant activity and is 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 hydrogels formed by hydrogen bonds is easily broken and easily damaged when subjected to stress. Therefore, the present invention 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 to enhance its antioxidant activity; second, the thiol groups in tBSP can form hydrogels by cross-linking reactions with double-bonded substances through photo-click chemistry. Compared with hydrogels formed by hydrogen bonds, hydrogels formed by photo-click chemistry methods often have fast reaction speeds, uniform gel pore sizes, and stronger gel stability. Third, tBSP has four functional groups: hydroxyl, aldehyde, carboxyl, and thiol. It can form hydrogen bonds, carbon-nitrogen double bonds, ionic bonds, disulfide bonds, etc. with various substances on the skin, so that it can adhere firmly to the skin. The preparation of tBSP first grafts the formic acid group of chloroacetic acid onto the hydroxyl group of BSP by etherification to prepare carboxymethylated Bletilla striata polysaccharide (CBSP), and then grafts L-cysteine ​​onto the formic acid group of CBSP by amidation to prepare tBSP. At this time, tBSP has four types of functional groups, including hydroxyl, aldehyde, carboxyl, and thiol, which provide action sites for the cross-linking reaction, antioxidant properties, and skin adhesion of the hydrogel.

[0008] Four-arm PEGNB is a functionalized polymer. Structurally, it has four arms, with polyethylene glycol as the basic structural unit. The four arms are grafted with norbornene groups through amidation, which has good water solubility, biodegradability and biocompatibility. Under the action of photoinitiator and ultraviolet light, PEGNB will produce thiol-ene photoclick chemical reaction with tBSP to form thiolated Bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel (tBP hydrogel). Compared with the traditional chain growth photopolymerization reaction, the polymerization mode of thiol-ene photoclick chemical reaction is free radical-mediated stepwise growth, which is faster, with less free oxygen growth and a more uniform grid. 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 prone to rupture or damage caused by local stress concentration, thereby showing better stability. Second, the uniform grid is conducive to the uniform diffusion and absorption of water molecules inside the hydrogel, making the hydrogel more stable during the swelling process. Third, in a uniform grid, the diffusion path of small molecules such as drug molecules is more regular, and they can be quickly transported inside the hydrogel and accompanied by regular gel degradation. Fourth, the uniform structure may reduce the potential immunogenic sites in the hydrogel, reduce the risk of triggering an immune response, and improve the safety and biocompatibility of the hydrogel.

[0009] TA is a natural polyphenol, a hydrolyzable, amphiphilic tannin derivative of gallic acid, with antibacterial, antioxidant and anti-inflammatory activities. TA was incorporated into tBP hydrogel by soaking to prepare tBP@TA hydrogel. The soaking method of incorporating TA has three advantages. First, TA's own metal chelating ability and phenolic hydroxyl characteristics make TA have antioxidant and antibacterial abilities. Adding TA to tBP hydrogel enhances the antibacterial and antioxidant abilities of the hydrogel. Second, TA contains abundant phenolic hydroxyl groups, which can interact with the hydroxyl, carboxyl, and thiol groups of tBP in tBP hydrogel to form hydrogen bonds, thereby enhancing the cross-linking structure inside tBP@TA hydrogel, so that tBP@TA hydrogel has two network structures: the network generated by cross-linking of tBSP and PEGNB through thiol-ene photoclick chemistry, and the network generated by hydrogen bonds between the phenolic hydroxyl groups on TA and the hydroxyl groups of tBP. The double network structure will enhance the cohesion of the hydrogel, and this cohesion will enhance the adhesion ability of tBP@TA hydrogel in disguise. Third, the surface of the thiolated Bletilla striata polysaccharide hydrogel (tBP@TA hydrogel) formed by immersing TA into tBP hydrogel will have a layer of mucous membrane. The main component of this mucous membrane is TA, and the rich phenolic hydroxyl groups of TA can form hydrogen bonds or hydrophobic interactions with the surface of skin tissue, making the hydrogel have better tissue adhesion. Among them, the network structure mentioned in the second point increases the cohesion of the hydrogel, which is relatively important for improving the adhesion ability of the gel. Simply using tBSP, PEGNB, and TA to treat wounds does not have the above advantages.

[0010] In summary, the scheme of the present invention first prepares tBSP as the raw material of the hydrogel, and then tBSP and PEGNB undergo thiol-ene photoclick chemical reaction under ultraviolet light to form crosslinks, namely tBP hydrogel, and then the tBP hydrogel is immersed in TA solutions of different concentrations to prepare tBP@TA hydrogel. First, tBP@TA hydrogel has swelling and strong adhesion properties, which can firmly adhere to the wound and absorb the pus exuded from the wound; secondly, tBP@TA hydrogel has antibacterial and antioxidant properties, which can prevent the wound from healing delays caused by microbial infection and oxidative stress, and finally promote the healing of infected wounds.

[0011] The present invention is achieved by the following technical means:

[0012] The present invention first discloses a method for preparing a thiolated Bletilla striata polysaccharide hydrogel, comprising:

[0013] (1) Preparation of thiolated Bletilla striata polysaccharide:

[0014] 0.5-5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to the carboxymethylated bletilla polysaccharide solution for activation for 40-70 min, 0.5-3 g of L-cysteine ​​hydrochloride is added for reaction, the pH value is adjusted to 4.0 using 20 wt % NaOH, the mixture is stirred in a 25° C. water bath for 1-5 h, the pH of the reaction mixture is adjusted to 6.0, the mixture is stirred at 25° C. for 0.5-5 h, the pH of the reaction mixture is adjusted to neutral, the mixture is dialyzed with RO water for 72 h using a dialysis bag with a molecular weight of 3500 Da, and then freeze-dried to obtain thiolated bletilla polysaccharide;

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

[0016] 1-3wt% four-arm polyethylene glycol norbornene, 5-25wt% thiolated bletilla striata polysaccharide and 0.11wt% I2959 are added into an EP tube and dissolved in RO water to obtain a hydrogel precursor. The hydrogel precursor solution is irradiated under an ultraviolet lamp to induce cross-linking to form a thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel. Subsequently, 0-2wt% tannic acid solution is added to the thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel. The obtained mixture is placed away from light to obtain a thiolated bletilla striata polysaccharide hydrogel.

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

[0018] Dissolve 0.5-5 g of carboxymethylated Bletilla striata polysaccharide in 100 mL of RO water to obtain a carboxymethylated Bletilla striata polysaccharide solution.

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

[0020] Dissolving 0.5-5 g of Bletilla striata polysaccharide in 20 mL of 20 wt % NaOH in an ice bath, and stirring until completely dissolved, to obtain a first mixture;

[0021] Add 30 mL of isopropanol to the first mixture and stir for 10 to 60 min, then add 0.1 to 2 mL of chloroacetic acid solution to the first mixture in 10 portions and stir in a 55° C. water bath for 30 to 120 min to obtain a second mixture;

[0022] The pH value was adjusted to neutral using 40 wt % glacial acetic acid, dialyzed in RO water using a dialysis bag, and then freeze-dried to obtain carboxymethylated Bletilla striata polysaccharide.

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

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

[0025] Furthermore, 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 0-2%; the volume ratio of the thiolated Bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel to the tannin solution is 0.5-2:1-4; the concentration of the four-arm polyethylene glycol norbornene is 2wt%, and the concentration of the thiolated Bletilla striata polysaccharide is 15wt%.

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

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

[0028] Furthermore, the light-proof storage condition in step (2) is: storage at 37° C. in the dark for 24 to 48 hours.

[0029] The invention also discloses a thiolated bletilla striata polysaccharide hydrogel prepared according to any one of the above preparation methods.

[0030] The invention also discloses an application of the thiolated bletilla striata polysaccharide hydrogel in preparing a medical dressing.

[0031] The beneficial effects of the present invention are:

[0032] The present invention first prepares tBSP as the raw material of the hydrogel, and then tBSP and PEGNB undergo thiol-ene photo-click chemical reaction under ultraviolet light to form crosslinks, namely tBP hydrogel, and then the tBP hydrogel is immersed in TA solutions of different concentrations to prepare tBP@TA hydrogel. The tBP@TA hydrogel prepared using specific raw materials has a far superior effect on the healing of infectious wounds than simple superposition. The details are as follows:

[0033] Improved adhesion performance: The present invention combines tBP hydrogel with TA, and the adhesion performance of tBP@TA hydrogel is improved. tBP hydrogel has four groups, namely hydroxyl, aldehyde, carboxyl and thiol. TA solution is rich in phenolic hydroxyl groups. The present invention incorporates TA into tBP hydrogel by immersion method. The phenolic hydroxyl groups in TA will form hydrogen bonds with the hydroxyl groups in tBP hydrogel, forming a second network structure in the original tBP hydrogel network, so that tBP@TA hydrogel has a double network structure. This double network structure provides stronger cohesion than a single network structure, and this cohesion gives tBP@TA hydrogel stronger adhesion performance.

[0034] Enhanced stability: The present invention combines tBP hydrogel with TA, and the stability of tBP@TA hydrogel is improved. The present invention incorporates TA into tBP hydrogel by immersion method, so that the hydrogel has a double network structure. This double network structure gives tBP@TA hydrogel stronger stability.

[0035] (1) Advantages of material properties: First, after BSP is grafted with thiol groups, tBSP has stronger antioxidant activity than BSP because thiol groups easily react with free radicals. Second, tBSP and TA in the components of tBP@TA hydrogel contain multiple groups such as hydroxyl, aldehyde, carboxyl, thiol, and phenolic hydroxyl groups. These groups can form hydrogen bonds and various covalent bonds with the surface of skin tissue, thereby enhancing the adhesion to the skin. Third, both tBSP and TA in the components of tBP@TA hydrogel have antibacterial and antioxidant activities, which can better treat bacterial infections and oxidative stress in infected wounds.

[0036] (2) Advantages of hydrogel network: tBP@TA hydrogel is prepared in two steps. First, tBSP and PEGNB are polymerized by thiol-ene photoclick chemistry to form tBP hydrogel. Then, tBP hydrogel is immersed in TA solution and TA is slowly loaded into tBP hydrogel to form tBP@TA hydrogel. tBP@TA hydrogel has three advantages. First, thiol-ene photoclick chemistry is a step-by-step growth mediated by free radicals. The reaction is faster, free oxygen growth is less, and the formed grid is more uniform. Second, tBP@TA hydrogel has a double network structure (the first network is the network generated by cross-linking tBSP and PEGNB through thiol-ene photoclick chemistry, and the second network is the network generated by hydrogen bonding between phenolic hydroxyl groups on TA and hydroxyl groups on tBP). This double network structure provides stronger cohesion than a single network structure. The rich functional groups and cohesion provided by the double network give tBP@TA hydrogel good adhesion ability. Third, the uniform network structure of tBP@TA hydrogel provides a good channel for absorbing exudate from infected wounds, and the cohesion provided by the double network also provides good swelling capacity. This combination of strong adhesion and high swelling ensures that while absorbing pus exudate from infected wounds, it can also adhere to the wound surface to exert antibacterial and antioxidant functions, thereby promoting wound healing as soon as possible.

[0037] (3) Biocompatibility advantage: PEGNB, tBSP, and TA in the tBP@TA hydrogel components all have good biocompatibility; the polymerization mode of the thiol-ene photoclick chemistry reaction is a free radical-mediated step-by-step polymerization growth mode at room temperature, with less free oxygen generated, which greatly reduces the oxidative damage of free oxygen to the body's biomacromolecules. Therefore, whether from the perspective of materials or the preparation method of the hydrogel, the hydrogel has good biocompatibility.

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

[0039] Figure 1 H NMR spectra of BSP, CBSP and tBSP;

[0040] Figure 2 is the scavenging rate of BSP and tBSP for DPPH free radicals;

[0041] Figure 3 Comparison of the gelation states of tBP and tBP@2%TA hydrogel;

[0042] Figure 4 is the scavenging rate of Examples 26 to 29 for DPPH free radicals;

[0043] Figure 5is the swelling ratio of Examples 26 to 29;

[0044] Figure 6 Examples 26 to 29 show antibacterial properties against E. coli and S. aureus;

[0045] Figure 7 is the inhibition rate of Examples 26 to 29 against E. coli;

[0046] Figure 8 is the antibacterial rate of Examples 26 to 29 against S. aureus;

[0047] Fig. 9 is the hemolysis rate of Examples 26 to 29;

[0048] Fig.10 Compatibility with mouse mononuclear macrophages (Raw264.7), mouse fibroblasts (L929), and human umbilical vein endothelial cells (EC) of Examples 26 to 29;

[0049] Fig.11 For the 1-3 day cytotoxicity of Examples 26-29 to mouse mononuclear macrophages;

[0050] Fig.12 The cytotoxicity of Examples 26 to 29 to mouse fibroblasts for 1 to 3 days;

[0051] Fig.13 The 1-3 day cytotoxicity of Examples 26-29 to human umbilical vein endothelial cells;

[0052] Fig.14 This is a scanning electron microscope image of the tBSP@2%TA hydrogel of Example 28;

[0053] Fig.15 is the elastic modulus of the tBSP@2%TA hydrogel of Example 28;

[0054] Fig.16 Schematic diagram of the adhesion of tBSP@2%TA hydrogel of Example 28 on pig skin;

[0055] Fig.17 The tBSP@2%TA of Example 28 is used for in vivo wound healing of infected wounds in SD rats. DETAILED DESCRIPTION

[0056] In contrast to the prior art, the present invention provides a method for preparing a thiolated Bletilla striata polysaccharide hydrogel. The specific meanings of the English abbreviations involved in the present invention are as follows:

[0057] BSP: Bletilla striata polysaccharide. The molecular weight of BSP used in the present invention is 6×10 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 average molecular weight of the PEGNB used in the present invention is 5000 Da.

[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 relevant solution of the present invention is prepared by the following method:

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

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

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

[0068] A 0.11 wt% I2959 solution was prepared for use, and the solvent was RO water.

[0069] A TA (tannic acid) solution with a mass fraction of 0 to 3 wt% is prepared for standby use, the solvent is RO water, and the 0 wt% TA solution is an RO water of the same volume.

[0070] A method for preparing a thiolated bletilla striata polysaccharide hydrogel comprises:

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

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

[0073] Example 1

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

[0075] Example 2

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

[0077] Example 3

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

[0079] Example 4

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

[0081] Example 5

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

[0083] Example 6

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

[0085] Example 7

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

[0087] Example 8

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

[0089] Example 9

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

[0091] Example 10

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

[0093] Embodiment 11

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

[0095] Example 12

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

[0097] Example 13

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

[0099] Embodiment 14

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

[0101] Embodiment 15

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

[0103] Example 16

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

[0105] Embodiment 17

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

[0107] Embodiment 18

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

[0109] Embodiment 19

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

[0111] Embodiment 20

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

[0113] Embodiment 21

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

[0115] Embodiment 22

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

[0117] Embodiment 23

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

[0119] Embodiment 24

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

[0121] Embodiment 25

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

[0123] Embodiment 26

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

[0125] Embodiment 27

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

[0127] Embodiment 28

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

[0129] Example 29 Hydrogel

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

[0131] Test Example 1

[0132] Preparation of tBSP

[0133] (1) NMR detection of tBSP

[0134] In order to verify whether L-cysteine ​​hydrochloride is grafted onto the sugar chain of BSP, nuclear magnetic resonance spectrometer (NMR) was used to detect the hydrogen spectrum of BSP, CBSP and tBSP at an analysis frequency of 400 MHz. 2 O), placing the solution in a nuclear magnetic resonance tube and putting it into the instrument for detection to observe whether there is a corresponding characteristic peak.

[0135] like Figure 1 As shown, in the 1HNMR spectrum, the OH peak of deuterated water appears at δ4.79ppm, the sugar backbone (CH2) of BSP appears at 3.1-4.0ppm, and the newly formed peak at 2.9ppm corresponds to the characteristic peak of CH2 on L-cys, indicating that the grafting of the thiol group is successful.

[0136] (2) Antioxidant activity detection of tBSP

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

[0138]

[0139] Where 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 scavenging ability of tBSP and BSP on DPPH free radicals at different concentrations is shown in Figure 2 Figure 2 As shown in the results, the antioxidant activity of tBSP was significantly improved compared with BSP. At low concentration (1 mg / mL), the DPPH free radical scavenging rate of BSP was 47%, and the DPPH free radical scavenging rate of tBSP reached 68%. At high concentration (5 mg / mL), the DPPH free radical scavenging rate of BSP was 52%, and the DPPH free radical scavenging rate of tBSP reached 96%. This proves that compared with BSP, tBSP grafted with thiol groups can give hydrogel wound dressings stronger antioxidant activity.

[0141] Test Example 2

[0142] Screening of tBP hydrogels

[0143] 50 uL of tBP solution in Examples 1 to 25 was dropped onto a coverslip, and the gel state was observed by UV irradiation. The coverslip was flipped every 5 seconds until the hydrogel was completely formed and stopped flowing. The gel time was recorded and the gel state was observed.

[0144] In the hydrogel gelation experiment, the gelation time of the hydrogel increased with the increase of tBSP and PEGNB concentrations, and the gelation speed also accelerated. During the experiment, it was found that when the PEGNB concentration was 3wt% and above, the gel was brittle and difficult to adhere to the wound surface. When the tBSP concentration was above 15wt%, the gel formed was opaque and contained flocs inside, indicating that tBSP was not completely dissolved. Experimental observations found that when the PEGNB concentration was 2wt% and the tBSP concentration was 15wt%, the hydrogel morphology was optimal. Therefore, the concentrations of PEGNB and tBSP in the tBP hydrogel were set to 2wt% and 15wt%, and Examples 26 to 29 were carried out at these concentrations.

[0145] Test Example 3

[0146] Screening of tBP@TA hydrogels

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

[0148] The samples prepared in Examples 26 to 29 were tested for swelling properties. u After the hydrogel was prepared, the dry weight (W d The hydrogel was then immersed in PBS, taken out at a specific time, excess water was wiped off, and the wet weight of the hydrogel (W t ), the swelling rate is calculated according to formula (1-2):

[0149]

[0150] Swelling is a basic property of hydrogels, reflecting their ability to absorb wound exudate. 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 the swelling rate of tBP@2%TA and tBP@3%TA hydrogels reached 90% at 15min and about 100% at 30min, indicating that tBSP@0%TA, tBP@2%TA and tBP@3%TA hydrogels can absorb wound exudate in a short time.

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

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

[0153]

[0154] Among them 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] like Figure 4 As shown, the DPPH radical scavenging ability of tBP@TA hydrogel increases with the increase of 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 the tBSP@2%TA planted in Example 28 and the 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 dressing.

[0156] (3) Antibacterial property testing of tBP@TA hydrogel

[0157] The samples prepared in Examples 26 to 29 were tested for antibacterial properties. E. coli (ATCC 87393) and S. aureus (ATTC 29213) were used to evaluate the antibacterial activity of the hydrogel. First, E. coli and S. aureus were cultured in LB broth until the bacteria grew well.

[0158] 10 μL of bacterial suspension (1×10 5CFU / mL) and 40μL sterile PBS were added to the surface of the hydrogel. Wrap the well plate with tin foil to keep the hydrogel away from light, and then place it in an incubator at 37°C for 6h. Afterwards, 100μL sterile PBS was used to suspend the unkilled bacteria again, and then 20μL of the bacterial suspension was aspirated and spread on the agar plate. 10μL of the bacterial suspension was diluted in 100μL sterile PBS, and 20μL was spread on the agar plate as a control group. The above agar plates were placed at 37°C for 24h and then the colonies were counted. The experiment was repeated three times for each group, and the results were expressed as the lethality rate, and the inhibition rate was calculated according to formula (1-4):

[0159]

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

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

[0162] The samples prepared in Examples 26 to 29 were subjected to hemolysis rate detection. Rat citrate whole blood was used, and the whole blood was diluted with PBS so that the volume ratio of whole blood to PBS was 5:4. The hydrogel was placed in a centrifuge tube, 10 mL of PBS was added and incubated at 37°C for 30 min, and the same volume of PBS and ultrapure water were placed in centrifuge tubes as negative control and positive control, respectively. 200 μL of diluted whole blood was added to each centrifuge tube, and incubated at 37°C for 60 min. The reaction solution after incubation was placed in 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 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 is the absorbance value of the positive control and the bright control.

[0165] Fig. 9 As shown in the figure, with the increase of TA solution concentration, the hemolysis rate of tBP@TA hydrogel 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 relevant national standard (5%). It proves that the three groups of hydrogels tBP@0%TA, tBP@1%TA, and tBP@2%TA have good blood compatibility.

[0166] (5) Cytocompatibility testing of tBP@TA hydrogel

[0167] The samples prepared in Examples 26 to 29 were subjected to cell compatibility testing. After the hydrogel was gelled, it was washed three times with PBS. This step was used to remove the unreacted solution. Then the hydrogel was immersed in high-glucose DMEM containing 15% FBS and 1% penicillin / streptomycin, incubated at 37°C for 24 hours, and filtered to obtain the hydrogel extract. L929 cells were cultured in high-glucose DMEM containing 15% FBS and 1% penicillin / streptomycin. When the cells covered 90% of the bottom of the cell culture bottle, the cells were inoculated in a 48-well cell culture plate at a cell density of 2×10 4 After the cells adhered to the wall, the culture medium was replaced with a culture medium containing hydrogel extract and incubated at 37°C with 5% CO 2After 1, 2, and 3 days of cell incubation, the cell viability was detected using a CCK-8 kit and the absorbance was detected at 450 nm using a microplate reader. The cell viability of RAW264.7 cells was determined in the same way. The culture medium of EC cells was DMEM / F-12 culture medium. The rest of the cell viability was sequenced in the same way. Cell viability was calculated according to formula (1-6):

[0168]

[0169] Where V s is the absorbance of the hydrogel group, V c is the absorbance of the blank control group, V 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, rinse with PBS three times, add 2.5% glutaraldehyde to fix the cells, remove the glutaraldehyde after 24 hours, rinse with PBS three times, then add rhodamine dye to the cell culture wells, remove it after 30 minutes, rinse with PBS three times, and place the well plate under a fluorescence microscope to observe the cell morphology.

[0171] Cytocompatibility is crucial for the clinical application of wound dressings. Fig.10 As shown in the figure, under a fluorescence microscope, cells in different groups have similar morphology. RAW264.7 cells are round, L929 cells are spindle-shaped, and EC cells are irregular polygons. Compared with the control group, there was no significant change in the cell morphology of the hydrogel extract group. Figures 11 to 13 As shown, tBSP@0%TA, tBSP@1%TA, tBSP@2%TA, tBSP@3%TA, L929 and EC cell activities decreased with the increase of TA concentration, but were still higher than 80% of the Control group. For RAW264.7 cells, they had a certain effect of promoting proliferation. The results show that the four groups of hydrogels prepared by the scheme of the present invention, tBP@0%TA, tBP@1%TA, tBP@2%TA, tBP@3%TA, have good cell compatibility.

[0172] After the tBP@0%TA, tBP@1%TA, tBP@2%TA, and tBP@3%TA hydrogels prepared in Examples 26 to 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 properties, low antioxidant activity, monoclonal antibody S.aureus, and good blood compatibility and cell compatibility; tBP@1%TA exhibited higher swelling properties, low antioxidant activity, monoclonal antibody S.aureus, and good blood compatibility and cell compatibility; tBP@2%TA hydrogel exhibited good swelling properties, high antioxidant activity, antibacterial ability against E.coli and S.aureus, good hemolysis rate, and cell compatibility; tBP@3%TA hydrogel exhibited good swelling properties, high antioxidant activity, good antibacterial ability, and good 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) Microstructure 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℃ for 24 hours, broken, and vacuum dried for 48 hours. The freeze-dried sample was attached to a conductive adhesive and sprayed with gold for 20 minutes in a vacuum environment. The cross-sectional microstructure was observed by scanning electron microscopy at a voltage of 3.0KV.

[0177] like Fig.14 As shown in Figure 2, tBP@2%TA presents a connected porous structure with a small and uniform pore size. This uniform and interconnected porous microstructure can provide an inlet for water vapor, facilitate nutrient and oxygen supply, and remove waste required for cell survival.

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

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

[0180] like Fig.15As shown in the figure, as the angular velocity continues to increase, the storage modulus (G') of the tBP@2%TA hydrogel is greater than the loss modulus (G"), indicating that the tBP@2%TA hydrogel is always in a relatively stable state. Mechanical strength plays a key role in hydrogel wound dressings. Too strong mechanical properties cannot guarantee the softness of biomaterials, making them prone to brittle fracture, while poor mechanical properties may cause tearing, peeling and deformation of hydrogel dressings.

[0181] The storage modulus of tBP@2%TA hydrogel is similar to that of skin (300-2000Pa), showing its potential as a wound dressing.

[0182] (3) Tissue adhesion performance testing of tBP@2%TA hydrogel

[0183] The tissue adhesion properties of the hydrogels were evaluated by stretching and adhesion to porcine skin.

[0184] like Fig.16 As shown, after the tBP@2%TA hydrogel prepared in Example 28 is adhered to the pig skin, it can still fit the pig skin well when the main pig skin is stretched or twisted. tBSP contains functional groups such as carboxyl, hydroxyl, and thiol, and TA contains abundant phenolic hydroxyl groups. These abundant functional groups can form physical and chemical bonds with various groups on the skin, which helps to improve the interfacial adhesion ability of tBSP@2%TA hydrogel. The sticky hydrogel dressing that can fully adhere to and combine with the tissue can prevent wound infection, make the hydrogel fit closely to the skin wound site, and provide a good microenvironment for wound healing.

[0185] (4) Wound healing experiment of full-thickness skin defect infection in rats

[0186] Compared with ordinary wounds, contaminated skin wounds with a high bacterial infection rate will greatly delay wound healing time. 10 CFU / mL) and E. coli (10 10 An infected full-thickness skin defect model was established at the animal wound site infected with a mixed bacterial solution of (400 μg / mL) and (250 μg / mL) to evaluate the healing ability of the hydrogel on bacterial-contaminated wounds.

[0187] First, SD rats (200-250g, male) that had adapted to the living environment for one week were anesthetized and the back hair was removed. A circular full-thickness skin defect with a diameter of 8mm was formed on the back of the rat using a biopsy punch. Next, 20μL of mixed bacterial solution was dropped onto the wound, and the wound was covered with a sterile closed PU film for 24 hours before the PU film was removed to obtain a bacterially infected wound model. At this point, the prepared hydrogel samples were used to treat the full-thickness skin wounds infected with bacteria. The wound treatment groups for each mouse were designed as follows:

[0188] ①PBS group;

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

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

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

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

[0193] Afterwards, the rats' wounds were regularly monitored for any discomfort and inflammation. The wound healing was also observed, and photos and samples were taken at 3, 7, 14, and 21 days.

[0194] The experimental results are as follows Fig.17 As shown, the successfully modeled wounds showed obvious pathogen infection. On the third day, the wounds in the Control group and the commercial 3M dressing group were still inflamed and had yellowish exudate, but the wounds treated with tBP@2%TA hydrogel and the wounds treated with tBP hydrogel had obviously no inflammation. Fig.16 It also shows that all wounds treated with the hydrogel method showed significant contraction on the 3rd, 7th, and 14th 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 groups, the wounds treated with tBP@2%TA hydrogel contracted the fastest, which is mainly due to tBSP and TA, as materials for forming hydrogels, which have been proven to have good antibacterial and tissue repair capabilities. In summary, tBSP@2%TA hydrogel can eliminate bacterial infections, relieve oxidative stress, and promote the healing of infected wounds.

[0195] The above description of the disclosed embodiments enables professionals in the field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals in the field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the scope consistent with the principles and novel features disclosed herein. For example, it can be obtained by changing the molecular weight of Bletilla striata polysaccharide, the molecular weight / structure of four-arm polyethylene glycol norbornene, and the volume ratio of tBP hydrogel to tannic acid.

Claims

1. A method for preparing a thiolated Bletilla striata polysaccharide hydrogel, comprising: (1) Preparation of thiolated Bletilla striata polysaccharide: 0.5-5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to the carboxymethylated bletilla polysaccharide solution for activation for 40-70 min, 0.5-3 g of L-cysteine ​​hydrochloride is added for reaction, the pH value is adjusted to 4.0 using 20 wt % NaOH, the mixture is stirred in a 25° C. water bath for 1-5 h, the pH of the reaction mixture is adjusted to 6.0, the mixture is stirred at 25° C. for 0.5-5 h, the pH of the reaction mixture is adjusted to neutral, the mixture is dialyzed with RO water for 72 h using a dialysis bag with a molecular weight of 3500 Da, and then freeze-dried to obtain thiolated bletilla polysaccharide; (2) Preparation of thiolated Bletilla striata polysaccharide hydrogel: 1-3wt% four-arm polyethylene glycol norbornene, 5-25wt% thiolated bletilla striata polysaccharide and 0.11wt% I2959 are added into an EP tube and dissolved in RO water to obtain a hydrogel precursor. The hydrogel precursor solution is irradiated under an ultraviolet lamp to induce cross-linking to form a thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel. Subsequently, a tannic acid solution is added to the thiolated bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel. The obtained mixture is placed away from light to obtain a 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 is prepared by the following method: Dissolving 0.5-5 g of Bletilla striata polysaccharide in 20 mL of 20 wt % NaOH in an ice bath, and stirring until completely dissolved, to obtain a first mixture; Add 30 mL of isopropanol to the first mixture and stir for 10 to 60 min, then add 0.1 to 2 mL of chloroacetic acid solution to the first mixture in 10 portions and stir in a 55° C. water bath for 30 to 120 min to obtain a second mixture; The pH value was adjusted to neutral using 40 wt % glacial acetic acid, dialyzed in RO water using a dialysis bag, and then freeze-dried to obtain carboxymethylated Bletilla striata polysaccharide.

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

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 Exposure to ultraviolet light; The concentration of the tannin solution is 0-2%; The volume ratio of the thiolated Bletilla striata polysaccharide-four-arm polyethylene glycol norbornene hydrogel to the tannin solution is 0.5-2:1-4; The concentration of the four-arm polyethylene glycol norbornene is 2 wt %, and the concentration of the thiolated Bletilla striata polysaccharide is 15 wt %.

6. The preparation method according to claim 5, wherein: The concentration of the tannin solution is 2%; The volume ratio of the thiolated Bletilla striata polysaccharide-four-arm 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, thiolated bletilla striata polysaccharide and I2959 is 10:39:

1.

8. The preparation method according to claim 1, wherein: The light-proof storage condition of step (2) is: storage at 37° C. in the dark for 24 to 48 hours.

9. A thiolated Bletilla striata polysaccharide hydrogel prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the thiolated Bletilla striata polysaccharide hydrogel according to claim 9 in preparing medical dressings.

Images