Polysaccharide-based composite hydrogel as well as preparation method and application thereof

Abstract

The invention relates to polysaccharide-based composite hydrogel as well as a preparation method and application thereof. The composite hydrogel is prepared from the following raw materials: hydroformylated modified ortho-hydroxyl-containing polysaccharide, an extracellular matrix enzyme digestive juice, tannic acid, cannabidiol, neomycin sulfate and borneol, by compounding multiple components, the use performance of the hydrogel can be remarkably improved, and the hydrogel is particularly suitable for a harsh environment. The composite hydrogel disclosed by the invention has excellent mechanical properties, good biological safety, certain self-repairing performance and relatively high anti-inflammatory, antibacterial and antioxidant properties, and can be applied to skin or mucous membrane tissue wound repairing.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and particularly relates to a polysaccharide-based composite hydrogel dressing, a preparation method thereof, and an application thereof. Background Art

[0002] Polysaccharide-based hydrogels have many unique physical and chemical properties, such as biocompatibility, biodegradability, and no immune response. Therefore, polysaccharides are widely used in various biomedical applications. There are a large number of functional groups available for modification in the polysaccharide structural unit, and polysaccharide-based composite hydrogels can be constructed through physical crosslinking, chemical crosslinking, or enzyme crosslinking. Polysaccharides have many advantages such as relatively low price, wide sources of raw materials, easy industrial extraction, and good biological activity. They have a variety of physiological activities, including antiviral, antioxidant, and anti-inflammatory activities, etc., and have broad application prospects in biomedicine.

[0003] Decellularized extracellular matrix refers to a biomaterial formed from human or animal organs / tissues by removing immunogenic cellular components through decellularization technology. The decellularized extracellular matrix is mainly composed of extracellular matrix, which contains extracellular macromolecules such as collagen, elastin, fibronectin, laminin, and matricellular proteins. The hydrogel prepared by the traditional method of adjusting the decellularized extracellular matrix to neutral after enzymatic digestion alone has disadvantages such as poor mechanical properties and no self-healing ability when used as a dressing, which limits the practical application of decellularized extracellular matrix hydrogels.

[0004] Therefore, it is urgent to develop hydrogels with composite components to improve the mechanical properties of decellularized extracellular matrix gels, endow the hydrogels with excellent application properties, and broaden their use environments. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides a polysaccharide-based composite hydrogel, a preparation method thereof, and an application thereof. Through the design of the raw materials for preparing the hydrogel, the obtained hydrogel has good biocompatibility, analgesic effect, adhesiveness, antioxidant property, self-healing ability, and excellent mechanical properties, and at the same time has good corrosion resistance, and is particularly suitable for harsh environments.

[0006] The specific technical solution of the present invention is as follows: In the first aspect of the present invention, a polysaccharide-based composite hydrogel is provided, and its raw materials include: aldehyde-modified polysaccharide containing ortho-hydroxy groups, decellularized extracellular matrix enzyme digestion solution, tannic acid, cannabidiol, neomycin sulfate, and borneol; the aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxyl groups in the polysaccharide containing ortho-hydroxy groups to aldehyde groups.

[0007] In the composite hydrogel of the present invention, the aldehyde-modified polysaccharide containing ortho-hydroxy groups can react with the acellular extracellular matrix enzyme digestion solution through a Schiff base reaction, that is, the aldehyde groups of the aldehyde-modified polysaccharide containing ortho-hydroxy groups crosslink with the amino groups of the acellular extracellular matrix enzyme digestion solution, and the acetal reaction of the aldehyde-modified polysaccharide containing ortho-hydroxy groups itself forms a gel substrate; combined with tannic acid, it can further cooperate with the polysaccharide and the acellular extracellular matrix to improve the strength of the hydrogel and enhance the adhesion ability; in addition, tannic acid, cannabidiol, borneol and neomycin sulfate cooperate with each other to further improve the antibacterial performance of the system, improve the hemostatic, analgesic and anti-inflammatory effects of the hydrogel, accelerate wound healing, and reduce the risk of infection at the affected area. The obtained hydrogel has good adhesion, strength and corrosion resistance, and is more suitable for harsh environments.

[0008] In one implementation of the present invention, in the aldehyde-modified polysaccharide containing ortho-hydroxy groups, the degree of oxidation of the hydroxy groups is 10-50%.

[0009] In one implementation of the present invention, the polysaccharide containing ortho-hydroxy groups can be at least one of fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose and konjac glucomannan.

[0010] It should be noted that the aldehyde groups of the aldehyde-modified polysaccharide in the hydrogel of the present application invention are formed by the oxidation of the ortho-hydroxy groups of the polysaccharide. Therefore, in principle, as long as it is a polysaccharide containing ortho-hydroxy groups, it can be applicable to the present application, not limited to fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose and konjac glucomannan.

[0011] In one implementation of the present invention, the aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained through an oxidation reaction, reaction termination and purification steps from the polysaccharide containing ortho-hydroxy groups; the oxidation reaction includes adding an oxidant to react in a solution of the polysaccharide containing hydroxy groups; the reaction termination includes adding an oxidation termination reagent to terminate the reaction after the reaction with the oxidant is completed; the purification step includes, after oxidation termination, using dialysis and freeze-drying techniques to obtain the purified aldehyde-modified polysaccharide, that is, the aldehyde-modified polysaccharide containing ortho-hydroxy groups of the present application.

[0012] In one implementation of the present invention, the mass ratio of the polysaccharide and the oxidant in the oxidation reaction is 1:(0.01-1), and the oxidation reaction time is 1-72 h.

[0013] In one implementation of the present invention, the oxidation termination reagent is ethylene glycol, and the reaction termination time is 15-60 min.

[0014] In one implementation of the present invention, the dialysis membrane specification used for dialysis is 3500 kDa.

[0015] In one implementation of the present invention, in the hydrogel, by mass ratio, the aldehyde-modified polysaccharide containing ortho-hydroxy groups: the acellular extracellular matrix in the acellular extracellular matrix enzyme digestion solution = (0.1 to 10):(0.2 to 2).

[0016] In one implementation of the present invention, in the hydrogel, the mass fraction of cannabidiol is 0.01 to 0.1 wt%; the mass fraction of borneol is 0.01 to 0.1 wt%; the mass fraction of neomycin sulfate is 0.01 to 0.1 wt%; the mass fraction of tannic acid is 0.01 to 0.1 wt%.

[0017] In one implementation of the present invention, the acellular extracellular matrix enzyme digestion solution is prepared through the following steps: (1) Mix and stir the animal skin tissue and the degreasing solution to obtain the degreased tissue; (2) Mix and stir the degreased tissue with the PBS mixed solution of Triton X-100 and ethylenediaminetetraacetic acid to obtain the pretreated animal tissue matrix; (3) Mix the pretreated animal tissue matrix and the DNA enzyme solution to carry out a DNA enzymatic hydrolysis reaction to obtain the acellular extracellular matrix of the animal tissue with DNA removed; (4) Freeze-dry the acellular extracellular matrix of the animal tissue with DNA removed, pre-cool it in liquid nitrogen, then grind it into powder and sieve it to obtain the acellular extracellular matrix powder; (5) Mix the acellular extracellular matrix powder and the protease solution to carry out an enzymatic hydrolysis reaction to obtain the acellular extracellular matrix enzyme digestion solution.

[0018] In one implementation of the present invention, in step (1), the animal skin tissue includes one or more of pig skin, cow skin and fish skin.

[0019] In one implementation of the present invention, in step (2), the mass percentage content of Triton X-100 in the PBS mixed solution is 0.1 to 1 wt%, and the mass percentage content of ethylenediaminetetraacetic acid in the PBS mixed solution is 0.05 to 1 wt%; the ratio of the mass of the degreased tissue to the volume of the PBS mixed solution is (0.5 to 3) g:(100 to 500) mL.

[0020] In one implementation of the present invention, in step (3), the concentration of the DNA enzyme solution is 10 to 100 U / mL, and the ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is (0.2 to 2) g:(100 to 500) mL.

[0021] In one implementation of the present invention, in step (4), the protease solution is one or more of pepsin, papain, α-amylase, and collagenase; the mass concentration of the protease solution is 0.1 to 2 mg / mL, the enzymatic hydrolysis reaction time is 1 to 36 h; the volume ratio of the acellular extracellular matrix of animal tissue to the protease solution is (0.2~1) g:(50~1000) mL.

[0022] In the second aspect of the present invention, there is also provided a method for preparing the polysaccharide-based composite hydrogel, including: mixing and stirring an aldehyde-modified ortho-hydroxy polysaccharide solution, an acellular extracellular matrix enzymatic digestion solution, a tannic acid solution, a cannabidiol solution, a borneol solution, and a neomycin sulfate solution, and standing to form a gel to obtain a hydrogel dressing.

[0023] In one implementation of the present invention, in the preparation method, the mass percentage of the solute in the aldehyde-modified ortho-hydroxy polysaccharide solution is 1 to 5%.

[0024] In one implementation of the present invention, in the preparation method, the mass percentage of the solute in the acellular extracellular matrix enzymatic digestion solution is 1 to 5%.

[0025] In one implementation of the present invention, in the preparation method, the concentration of cannabidiol in the cannabidiol solution is 0.5 - 2 mol / L.

[0026] In one implementation of the present invention, in the preparation method, the concentration of borneol in the borneol solution is 0.5 - 2 mol / L.

[0027] In one implementation of the present invention, in the preparation method, the concentration of neomycin sulfate in the neomycin sulfate solution is 0.5 - 2 mol / L.

[0028] The present invention has found through research that selecting the mass percentage of the solute in the solution or the concentration within the above ranges is beneficial to the gelation reaction.

[0029] In one implementation of the present invention, the solvents of the cannabidiol solution and the borneol solution are preferably anhydrous ethanol; the solvents of the aldehyde-modified ortho-hydroxy polysaccharide solution, the acellular extracellular matrix enzymatic digestion solution, the tannic acid solution, and the neomycin sulfate solution are preferably water.

[0030] In one implementation of the present invention, rapid vortexing is used for uniform mixing and stirring until no layering is observed visually.

[0031] In one implementation of the present invention, the standing time for gel formation is preferably 10 to 30 min.

[0032] In a third aspect of the present invention, there is also provided an application of the polysaccharide-based composite hydrogel in the preparation of a dressing for repairing skin or mucosal tissue trauma.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: In the composite hydrogel of the present invention, the acellular extracellular matrix and the aldehyde-modified polysaccharide containing ortho-hydroxy groups form a gel substrate. The introduction of tannic acid, cannabidiol, borneol, and neomycin sulfate, on the one hand, tannic acid synergistically improves the adhesion ability of the hydrogel with the acellular extracellular matrix and the aldehyde-modified polysaccharide containing ortho-hydroxy groups, and improves the strength and corrosion resistance of the gel. On the other hand, tannic acid, cannabidiol, borneol, and neomycin sulfate can synergistically improve the hemostatic, analgesic, anti-inflammatory effects and antibacterial properties of the hydrogel, reduce the risk of infection at the affected area, and at the same time have the effect of promoting wound healing. The multiple components of the hydrogel of the present invention are compounded, which can significantly improve the use performance of the hydrogel. The obtained hydrogel has good mechanical strength, high adhesiveness, and also has good antibacterial, hemostatic, analgesic, anti-inflammatory, and wound healing promoting effects, and is particularly suitable for harsh environments. The hydrogel provided by the present invention has good corrosion resistance and high strength, and can maintain adhesion in seawater immersion for more than 24 h when combined with tissues, which is significantly improved compared with the existing hydrogels. Description of the Drawings

[0034] Figure 1 is an optical photograph of the hydrogel in the embodiment of the present application; Figure 2 is a result diagram of the hydrogel in the embodiment of the present application immersed in seawater for 24 h; Figure 3 is a result diagram of the antibacterial test of the hydrogel in the embodiment of the present application; Figure 4 is a result diagram of the cell compatibility of the hydrogel in the embodiment of the present application; Figure 5 is a result diagram of the blood compatibility of the hydrogel in the embodiment of the present application; Figure 6 is a result diagram of the in vivo wound healing of the hydrogel in the embodiment of the present application. Detailed Embodiments

[0035] The present invention provides a polysaccharide-based composite hydrogel, which includes an aldehyde-modified polysaccharide containing ortho-hydroxy groups, an acellular extracellular matrix enzyme digestion solution of animal tissue, tannic acid, neomycin sulfate, borneol, and cannabidiol. The aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxyl groups in the polysaccharide containing ortho-hydroxy groups to aldehyde groups.

[0036] The composite hydrogel provided by the present invention includes an aldehyde-modified polysaccharide containing ortho-hydroxy groups. The aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxyl groups in the polysaccharide containing ortho-hydroxy groups to aldehyde groups.

[0037] In the present invention, the degree of oxidation of the aldehyde-modified polysaccharide containing ortho-hydroxy groups is preferably 10-50%. For example, it can be 10%, 20%, 30%, 40%, 50%, etc.

[0038] In the present invention, the preparation method of the aldehyde-modified polysaccharide containing ortho-hydroxy groups preferably includes the following steps: dissolving the polysaccharide in water to form a uniform solution with a concentration of 0.5-5 wt%, adding an oxidant to a concentration of 0.01-1 wt% in a light-shielded environment, and continuously stirring and oxidizing for 8-40 h. Subsequently, adding a terminator to a concentration of 0.01-1 wt% and continuously stirring for 20-60 min to terminate the reaction. Then, dialysis is carried out using a dialysis bag for 72 h, and freeze-drying is performed to obtain the aldehyde-modified polysaccharide.

[0039] In the present invention, the polysaccharide is preferably guar gum, and the concentration is preferably 1-3 wt%; the oxidant is preferably sodium periodate, and the final concentration in the reaction system is preferably 0.1-0.5 wt%; the terminator is preferably ethylene glycol, and the concentration is preferably 0.1-0.5 wt%.

[0040] The hydrogel provided by the present invention includes an enzyme digestion solution of animal tissue acellular extracellular matrix.

[0041] In the present invention, the preparation method of the enzyme digestion solution of animal tissue acellular extracellular matrix preferably includes the following steps: Mixing and stirring animal skin tissue and a degreasing solution to obtain degreased tissue; Mixing and stirring the degreased tissue with a PBS mixed solution of Triton X-100 and ethylenediaminetetraacetic acid to obtain a pretreated animal tissue matrix; Mixing the pretreated animal tissue matrix and a DNA enzyme solution to carry out a DNA enzymatic hydrolysis reaction to obtain an animal tissue acellular extracellular matrix with DNA removed; Freeze-drying the animal tissue matrix with DNA removed, pre-cooling it in liquid nitrogen, then grinding it into powder and sieving it to obtain acellular extracellular matrix powder; Mixing the acellular extracellular matrix powder and an enzyme solution to carry out an enzymatic hydrolysis reaction to obtain the enzyme digestion solution of animal tissue acellular extracellular matrix.

[0042] In the present invention, animal skin tissue and a degreasing solution are mixed and continuously stirred for a period of time to obtain degreased tissue. In the present invention, the animal skin preferably includes one or more of pig skin, cow skin, and fish skin, and the degreasing solution is an organic solvent, preferably an equal-proportion mixed solution of ethyl acetate and methanol.

[0043] In the present invention, the defatted tissue is placed in a mixed solution of Triton X-100, ethylenediaminetetraacetic acid and PBS, and mixed for a period of time to obtain a pretreated animal tissue matrix. In the present invention, the mass percentage content of Triton X-100 in the PBS mixed solution is preferably 0.1-1 wt%. In the present invention, the mass percentage content of ethylenediaminetetraacetic acid in the PBS mixed solution is preferably 0.05-1 wt%. The ratio of the mass of the defatted tissue to the volume of the PBS mixed solution is preferably (0.5-3) g:(100-500) mL. In the present invention, the temperature of the stirring is preferably 37 °C, and the stirring time is preferably 6-72 h.

[0044] After obtaining the pretreated animal tissue matrix, in the present invention, the pretreated animal tissue matrix and a DNA enzyme solution are mixed for a DNA enzymatic digestion reaction to obtain an acellular extracellular matrix of animal tissue.

[0045] In the present invention, the concentration of the DNA enzyme solution is preferably 10-100 U / mL. The ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is preferably (0.2-2) g:(100-500) mL. In the present invention, the temperature of the DNA enzymatic digestion reaction is preferably 37 °C, and the DNA enzymatic digestion reaction time is preferably 1-8 h.

[0046] After obtaining the acellular extracellular matrix, in the present invention, the acellular extracellular matrix is freeze-dried, pre-cooled in liquid nitrogen, and then ground into a powder and sieved to obtain an acellular extracellular matrix powder.

[0047] After obtaining the acellular extracellular matrix powder, in the present invention, the acellular extracellular matrix powder and a protease solution are mixed for a proteolytic reaction to obtain the acellular extracellular matrix enzymatic digestion solution of animal tissue.

[0048] In the present invention, the protease solution is one or more of pepsin, papain, α-amylase and collagenase, preferably a pepsin solution. The mass concentration of the protease solution is preferably 0.1-2 mg / mL, and the enzymatic reaction time is 1-36 h. The volume ratio of the acellular extracellular matrix of animal tissue to the protease solution is preferably (0.2-1) g:(50-1000) mL. The temperature of the proteolytic reaction is preferably 37 °C.

[0049] The composite hydrogel provided by the present invention further includes tannic acid, cannabidiol, neomycin sulfate and borneol.

[0050] The present invention provides a preparation method of the hydrogel according to the above technical solution, including the following steps: Mix the aldehyde-modified polysaccharide with adjacent hydroxyl groups evenly with the animal acellular extracellular matrix, and adjust to neutral with a sodium hydroxide solution to obtain a hydrogel matrix. In the present invention, the mixing is preferably carried out at 37 °C.

[0051] Mix the hydrogel matrix evenly with a tannic acid solution to obtain a hydrogel matrix compounded with tannic acid. In the present invention, the mixing is preferably carried out at 37 °C.

[0052] Mix the hydrogel matrix compounded with tannic acid evenly with a cannabidiol solution, a neomycin sulfate solution and a borneol solution to obtain the composite hydrogel of the present invention. In the present invention, the mixing is preferably carried out at 37 °C.

[0053] In the present invention, the concentration of tannic acid in the tannic acid solution is preferably 1 to 0.01 g / mL, and the solvent is preferably water. The concentration of cannabidiol in the cannabidiol solution is preferably 1 to 0.01 g / mL, and the solvent is preferably ethanol. The concentration of borneol in the borneol solution is preferably 1 to 0.01 g / mL, and the solvent is preferably ethanol. The concentration of neomycin sulfate in the neomycin sulfate solution is preferably 1 to 0.01 g / mL, and the solvent is preferably water.

[0054] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Unless otherwise specified, the reagents, methods and equipment used in the implementation of the present invention are conventional reagents, methods and equipment in the technical field.

[0056] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0057] Example 1 1) Preparation of acellular extracellular matrix enzyme digestion solution: Cut fresh pig skin into small pieces of 1 cm × 1 cm in size and wash them repeatedly; place the cut small pieces of pig skin tissue in a blue-capped bottle, add an equal-volume solution with a volume ratio of ethyl acetate to methanol of 1:1 to completely immerse the pig skin tissue, and stir for a period of time to obtain degreased pig skin tissue; Add a mixed solution of PBS containing 1 wt% of Triton X-100 and 0.8 wt% of ethylenediaminetetraacetic acid to the degreased pig skin tissue for 8 h continuously. After filtration, take the solid matter and wash it with water to obtain a pretreated animal tissue matrix; Add a DNA enzyme solution of 50 U / mL to the pretreated animal tissue matrix, where the mass of the pretreated animal tissue matrix and the volume of the DNA enzyme solution are in a ratio of 1 g:200 mL, stir continuously for 8 h, filter, take the solid matter and wash it with water to obtain an acellular extracellular matrix; After freeze-drying the acellular extracellular matrix, it was pre-cooled in liquid nitrogen, ground into particles using a grinder, and passed through a 100-mesh sieve to obtain acellular extracellular matrix powder. The acellular extracellular matrix powder was added to a pepsin solution at a concentration of 0.5 mg / mL. Among them, the mass ratio of the acellular extracellular matrix powder to the volume of the protease solution was 1 g:200 mL to obtain a porcine skin acellular extracellular matrix enzyme digestion solution.

[0058] 2) Preparation of aldehyde-modified polysaccharide containing ortho-hydroxy groups: Weigh 5 g of guar gum powder, stir and dissolve the raw material powder in 250 mL of water to prepare a polysaccharide solution with a concentration of 2%.

[0059] Weigh 1 g of sodium periodate as an oxidant and add it to the polysaccharide solution. React under magnetic stirring at room temperature in the dark for 12 h.

[0060] After the reaction is completed, pipette 2 mL of ethylene glycol as an oxidation termination reagent into the reaction vessel and terminate the reaction under magnetic stirring for 40 min.

[0061] After the reaction is terminated, transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 3500 kDa and dialyze it with ultrapure water for 3 days, changing the water twice a day.

[0062] After dialysis, freeze it at -20 °C for 24 h. Subsequently, freeze-dry it in a freeze-dryer to obtain oxidized guar gum. The oxidation degree of the oxidized guar gum prepared in this example was measured using the hydroxylamine hydrochloride titration method, and the oxidation degree was 15.27%.

[0063] 3) Preparation of hydrogel: Weigh 0.08 g of the oxidized guar gum obtained in step 2) and dissolve it in 2 mL of water to obtain an oxidized guar gum solution with a concentration of 4%. Weigh 0.04 g of tannic acid and dissolve it in 1 mL of water to obtain a tannic acid solution with a concentration of 4%. Weigh 314 mg of cannabidiol and dissolve it in 1 mL of ethanol to obtain a cannabidiol solution with a concentration of 314 mg / mL. Weigh 712 mg of neomycin sulfate and dissolve it in water to obtain a neomycin sulfate solution with a concentration of 712 mg / mL. Weigh 154 mg of borneol and dissolve it in 1 mL of ethanol to obtain a borneol solution with a concentration of 154 mg / mL.

[0064] At 37 °C, mix 1 mL of the oxidized guar gum solution with 1 mL of the acellular extracellular matrix enzyme digestion solution obtained in step 1) evenly, then add 20 μL of the tannic acid solution, 10 μL of the cannabidiol solution, 10 μL of the borneol solution, and 10 μL of the neomycin sulfate solution and mix evenly. Let it stand at 37 °C for 10 minutes to obtain a hydrogel. (In this step, the molar ratio of cannabidiol, borneol, and neomycin sulfate is cannabidiol:borneol:neomycin sulfate = 1:1:1) Example 2 An embodiment of the present invention provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is as follows: 3) During the preparation of the hydrogel, the added volumes of the cannabidiol solution, borneol, and neomycin sulfate solution are changed, while keeping the total volume and total number of moles of the three unchanged. The volume ratio of cannabidiol, borneol, and neomycin sulfate becomes cannabidiol:borneol:neomycin sulfate = 1:3:2.

[0065] Example 3 An embodiment of the present invention provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is as follows: 3) During the preparation of the hydrogel, the added volumes of the cannabidiol solution, borneol, and neomycin sulfate solution are changed, while keeping the total volume and total number of moles of the three unchanged. The volume ratio of cannabidiol, borneol, and neomycin sulfate becomes cannabidiol:borneol:neomycin sulfate = 4:2:1.

[0066] Example 4 This example provides a composite hydrogel. The only difference in the preparation method of the hydrogel dressing from that of Example 1 is as follows: 3) During the preparation of the hydrogel, the volume of the oxidized guar gum solution is changed to 1.5 mL, and the volume of the acellular extracellular matrix enzyme digestion solution is changed to 0.5 mL.

[0067] Example 5 An embodiment of the present invention provides a composite hydrogel. The only difference in the preparation method of the hydrogel dressing from that of Example 1 is that the guar gum in step 2) is changed to konjac glucomannan.

[0068] Example 6 This example provides a composite hydrogel. The only difference in the preparation method of the hydrogel dressing from that of Example 1 is that the guar gum in step 2) is changed to sodium alginate.

[0069] Comparative Example 1 This comparative example provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is as follows: In step 3), tannic acid, neomycin sulfate, the cannabidiol solution, and the borneol solution are not added, and an aqueous solution of the same volume is used as a substitute.

[0070] Comparative Example 2 This comparative example provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is as follows: In step 3), neomycin sulfate, the cannabidiol solution, and the borneol solution are not added, and an aqueous solution of the same volume is used as a substitute.

[0071] Comparative Example 3 This comparative example provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is that in step 3), borneol solution and neomycin sulfate solution are not added, and the same volume of aqueous solution is used as a substitute.

[0072] Comparative Example 4 This comparative example provides a composite hydrogel. The only difference in the preparation method of the hydrogel from that of Example 1 is that in step 3), neomycin sulfate solution is not added, and the same volume of aqueous solution is used as a substitute.

[0073] Effect verification: 1. Comparison of gelation speed: By timing, the gelation times of the hydrogels prepared in Examples 1-6 were compared, and the results are shown in Table 1.

[0074] The results show that the hydrogels of each example can form gels, but both the type of polysaccharide and the volume ratio of the acellular extracellular matrix enzyme digestion solution to the polysaccharide solution have an impact on the gelation speed of the hydrogel.

[0075] Table 1 Summary of hydrogel gelation times

[0076] 2. Adhesion performance test of hydrogel: The hydrogel of Example 1 and the hydrogel of Comparative Example 1 were adhered to the same piece of pigskin. The pigskin was straightened horizontally and the pigskin and the hydrogel were facing downwards. The falling time of the hydrogel was observed by timing. The final result was that the hydrogel of Example 1 did not fall after 24 h of adhesion, and the hydrogel of Comparative Example 1 fell after 5 h of adhesion. This shows that the introduction of tannic acid can further enhance the adhesion performance.

[0077] 3. Seawater erosion experiment: The hydrogel of Example 1 was adhered to pigskin and immersed in seawater for 24 h. The adhesion of the hydrogel at 0 h and 24 h was photographed and observed. The results are as Figure 2 shown.

[0078] Seawater contains a large number of microorganisms and various ions and has certain corrosiveness. After the hydrogel was attached to the pigskin and immersed in seawater for a period of time, due to the oxidation of TA, the color became darker. The results show that the adhesion of this hydrogel can be maintained for 24 h or more in a complex environment, and at the same time, the macroscopic structure of the hydrogel is not damaged, which to a certain extent proves that the hydrogel of Example 1 has good strength and corrosion resistance and can ensure continuous attachment even under harsh conditions during this time period.

[0079] 4. Conventional antibacterial test: The antibacterial effects of the hydrogel dressings of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 on strains were tested by the plate counting method. Two types of strains were selected for testing, namely Gram-positive bacteria (E) and Gram-negative bacteria (S). The hydrogel dressings were co-incubated with each strain for 24 h, and then the strains co-incubated with the hydrogel were diluted and evenly spread on a soy casein agar plate. Finally, the agar plate was incubated in an incubator for 16 h, and the inhibitory effects of different types of hydrogels on Gram-positive bacteria and Gram-negative bacteria were observed and photographed; the antibacterial rates of Gram-positive bacteria and Gram-negative bacteria corresponding to all test groups were calculated respectively, and the average values of the antibacterial rates of Gram-positive bacteria and Gram-negative bacteria were taken respectively; the group without applying the hydrogel was used as the control group.

[0080] Calculation formula for antibacterial rate: (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group × 100% The results are as Figure 3 shown. It can be seen that in Comparative Example 1, when tannic acid, neomycin sulfate, cannabidiol and borneol were not added, the antibacterial property was very poor, especially there was no antibacterial property against Gram-positive bacteria; after adding tannic acid, the antibacterial property was significantly improved, and further combined with neomycin sulfate, cannabidiol and borneol, the antibacterial property was further enhanced. It can be seen that tannic acid, neomycin sulfate, cannabidiol and borneol can act synergistically to improve the antibacterial property of the gel.

[0081] 5. Biocompatibility test The freeze-dried samples of the hydrogels of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were respectively extracted with high-glucose complete medium at 37 °C for 24 h. After the extraction was completed, the concentration of the extract was diluted to 1 mg / mL, 0.1 mg / mL and 0.01 mg / mL. Mouse fibroblast NIH3T3 cells were inoculated on a culture plate containing complete medium and cultured in a cell incubator at 37 °C and 5% carbon dioxide concentration for 24 h. Then, the complete medium was discarded and replaced with the hydrogel extract, and cultured in a cell incubator at 37 °C and 5% carbon dioxide concentration for 24 h. The cells were treated with CCK-8 reagent, and the absorbance at a wavelength of 450 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate was calculated according to the absorbance values of each well.

[0082] The calculation formula is: Cell activity (%) = (A1 - A c / A0 - A c ) × 100%. Where A1 is the absorbance value of the experimental group, A0 is the absorbance value of the control group, and A c is the absorbance of the blank group.

[0083] Figure 4The results showed that the cell survival rate of the hydrogel dressing co-cultured with cells measured by the above method was above 85%, with good cell compatibility, meeting the cell biosafety requirements of GB / T 16886.

[0084] 6. Blood Compatibility Experiment of Hydrogel Dressing The hydrogels of Comparative Example 1, Comparative Example 2 and Example 1 were placed in 2 mL centrifuge tubes, and diluted red blood cells were added. Red blood cells treated with PBS and ultrapure water were used as the negative group and the positive group. After static incubation at 37 °C for 1 h, the supernatant was removed by centrifugation, and the absorbance at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The calculation method of hemolysis rate is as follows: HR (%) = (OD gel - OD blank ) / (OD control - OD blank ) × 100% OD gel : Absorbance at 540 nm of the supernatant of red blood cells treated with hydrogel OD control : Absorbance at 540 nm of the supernatant of red blood cells treated with ultrapure water OD blank : Absorbance at 540 nm of the supernatant of red blood cells treated with PBS The test results are as Figure 3 shown. The hydrogel of Example 1 had good blood compatibility, with a hemolysis rate of less than 5%, meeting the blood biosafety requirements of GB / T 16886. Moreover, the comparison results also reflected that the addition of tannic acid further reduced the blood compatibility of the hydrogel.

[0085] 7. Experiment on the Ability of Hydrogel to Promote Wound Healing: The hair on the back of BALB / c mice was shaved and a wound was created. A 20 μL suspension of Staphylococcus aureus at a concentration of 1 × 10 8 CFU / mL was dropped onto the wound. The hydrogel dressing substrate and the hydrogel dressing of Example 1 were completely applied to the wound surface; mice without adding hydrogel were used as the control group. Finally, all groups were protected with a layer of 3M™ Tegaderm™ transparent dressing. After the anesthesia recovery, the animals were monitored for any discomfort and provided with water and feed. The hydrogel dressing was changed daily, and the body weight was measured.

[0086] The test results are as Figure 6 shown. The hydrogel had a significant antibacterial effect compared with the 3M™ Tegaderm™ transparent dressing group, and at the same time could significantly promote wound healing.

[0087] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A polysaccharide-based composite hydrogel, characterized in that: The raw materials include: hydroformylation-modified polysaccharide containing ortho-hydroxyl groups, decellularized extracellular matrix enzyme digestion solution, tannic acid, cannabidiol, neomycin sulfate and borneol; the hydroformylation-modified polysaccharide containing ortho-hydroxyl groups is obtained by oxidizing the hydroxyl groups in the polysaccharide containing ortho-hydroxyl groups to aldehyde groups.

2. The polysaccharide-based composite hydrogel according to claim 1, characterized in that: In the aldehyde-modified polysaccharide containing ortho-hydroxyl groups, the degree of oxidation of hydroxyl groups is 10-50%.

3. The polysaccharide-based composite hydrogel according to claim 1, characterized in that: In the hydrogel, according to the mass ratio, the aldehyde-modified polysaccharide containing ortho-hydroxyl group: the decellularized extracellular matrix in the decellularized extracellular matrix enzyme digestion solution = (0.1-10): (0.2-2); Preferably, in the hydrogel, the mass fraction of cannabidiol is 0.01-0.1wt%; the mass fraction of borneol is 0.01-0.1wt%; the mass fraction of neomycin sulfate is 0.01-0.1wt%; and the mass fraction of tannic acid is 0.01-0.1wt%.

4. The polysaccharide-based composite hydrogel according to claim 1, characterized in that: The decellularized extracellular matrix enzyme digestion solution is prepared by the following steps: (1) mixing and stirring the animal skin tissue and the defatting solution to obtain defatted tissue; (2) mixing and stirring the defatted tissue with a mixed solution of Triton X-100 and ethylenediaminetetraacetic acid in PBS to obtain a pretreated animal tissue matrix; (3) mixing the pretreated animal tissue matrix and the DNA enzyme solution to perform a DNA enzymatic hydrolysis reaction to obtain an animal tissue decellularized extracellular matrix from which DNA has been removed; (4) freeze-drying the DNA-removed animal tissue decellularized extracellular matrix, precooling it in liquid nitrogen, grinding it into powder, and sieving it to obtain decellularized extracellular matrix powder; (5) The decellularized extracellular matrix powder and the protease solution are mixed to perform an enzymatic hydrolysis reaction to obtain the decellularized extracellular matrix enzyme digestion solution.

5. The polysaccharide-based composite hydrogel according to claim 4, characterized in that: In step (1), the animal skin tissue includes one or more of pig skin, cow skin and fish skin.

6. The polysaccharide-based composite hydrogel according to claim 4, characterized in that: In step (2), the mass percentage of Triton X-100 in the PBS mixed solution is 0.1-1wt%, and the mass percentage of ethylenediaminetetraacetic acid in the PBS mixed solution is 0.05-1wt%; the ratio of the mass of the defatted tissue to the volume of the PBS mixed solution is (0.5-3) g: (100-500) mL.

7. The polysaccharide-based composite hydrogel according to claim 4, characterized in that: In step (3), the concentration of the DNA enzyme solution is 10-100 U / mL, and the ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is (0.2-2) g: (100-500) mL.

8. The polysaccharide-based composite hydrogel according to claim 4, characterized in that: In step (4), the protease solution is one or more of pepsin, papain, α-amylase and collagenase; the mass concentration of the protease solution is 0.1-2 mg / mL, and the enzymatic reaction time is 1-36 h; the volume ratio of the animal tissue decellularized extracellular matrix to the protease solution is (0.2-1) g: (50-1000) mL.

9. The method for preparing the polysaccharide-based composite hydrogel according to any one of claims 1 to 8, characterized in that: include: The hydrogel dressing is prepared by mixing and stirring a solution of a hydroformylation-modified ortho-hydroxyl polysaccharide, a decellularized extracellular matrix enzyme digestion solution, a tannic acid solution, a cannabidiol solution, a borneol solution and a neomycin sulfate solution, and letting the mixture stand to form a gel; Preferably, the mass percentage of the solute in the solution containing the aldehyde-modified ortho-hydroxyl-containing polysaccharide is 1-5%; Preferably, the mass percentage of the solute in the decellularized extracellular matrix enzyme digestion solution is 1-5%; Preferably, the mass percentage of tannic acid in the tannic acid solution is 1-10%; Preferably, the concentration of cannabidiol in the cannabidiol solution is 0.5-2 mol / L; Preferably, the concentration of borneol in the borneol solution is 0.5-2 mol / L; Preferably, the concentration of neomycin sulfate in the neomycin sulfate solution is 0.5-2 mol / L.

10. Use of the polysaccharide-based composite hydrogel according to any one of claims 1 to 8 or the polysaccharide-based composite hydrogel prepared by the preparation method according to claim 9 in preparing a wound repair dressing for skin or mucosal tissue.

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