A polysaccharide-based composite hydrogel, and a preparation method and application thereof

Abstract

The present application relates to a polysaccharide-based composite hydrogel and a preparation method and application thereof. The raw material of the composite hydrogel comprises: an aldehyde-modified polysaccharide containing ortho-hydroxyl groups, a decellularized extracellular matrix enzyme digestion solution, tannic acid, cannabidiol, neomycin sulfate and borneol; the multiple components are compounded, the service performance of the hydrogel can be significantly improved, and the hydrogel is particularly suitable for severe environments. The composite hydrogel has excellent mechanical properties, good biological safety, certain self-repairing performance, and higher anti-inflammatory, antibacterial and antioxidant properties, and can be applied to skin or mucosal tissue wound repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of hydrogel, and particularly relates to a polysaccharide-based composite hydrogel dressing as well as a preparation method and application thereof. BACKGROUND

[0002] Polysaccharide-based hydrogel has many unique physical and chemical properties, such as biocompatibility, biodegradability and no immune response, so polysaccharides are widely used in various biomedical applications. There are a large number of functional groups in the structural unit of polysaccharide that can be modified, and polysaccharide-based composite hydrogel can be constructed by physical crosslinking, chemical crosslinking or enzyme crosslinking. Polysaccharides have many advantages such as low price, wide raw material sources, easy industrial extraction and good biological activity, and have various physiological activities, including antiviral, antioxidant and anti-inflammatory activities, and have broad application prospects in biological medicine.

[0003] Decellularized extracellular matrix refers to a biomaterial formed by human or animal organs / tissues by removing immunogenic cell 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 matrix cell proteins. The hydrogel prepared by traditional enzyme digestion and neutral gelation of the decellularized extracellular matrix alone has the disadvantages of poor mechanical properties and no self-healing when used as a dressing, which limits the application of the decellularized extracellular matrix hydrogel in practice.

[0004] Therefore, it is urgent to develop a composite component hydrogel to improve the mechanical properties of the decellularized extracellular matrix gel, and to endow the hydrogel with excellent application performance and broaden its use environment. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a polysaccharide-based composite hydrogel, a preparation method and application thereof. By designing the raw materials of the hydrogel, the obtained hydrogel has good biocompatibility, analgesic, adhesive, antioxidant, self-healing ability and excellent mechanical properties, and is also good in corrosion resistance, and is particularly suitable for harsh environments.

[0006] The specific technical solutions of the present application are as follows:

[0007] In the first aspect of the present application, a polysaccharide-based composite hydrogel is provided, and the raw materials thereof include: aldehyde-modified polysaccharide containing ortho-hydroxyl, decellularized extracellular matrix enzyme digestion solution, tannic acid, cannabadiene, neomycin sulfate and borneol; the aldehyde-modified polysaccharide containing ortho-hydroxyl is obtained by oxidizing the hydroxyl in the polysaccharide containing ortho-hydroxyl to aldehyde group.

[0008] In the composite hydrogel of the present application, the aldehyde-modified polysaccharide containing ortho-hydroxyl groups can be reacted with the decellularized extracellular matrix enzyme digestion solution through Schiff base reaction, that is, the aldehyde group of the aldehyde-modified polysaccharide containing ortho-hydroxyl groups is crosslinked with the amino group of the decellularized extracellular matrix enzyme digestion solution, and the acetal reaction of the aldehyde-modified polysaccharide containing ortho-hydroxyl groups itself forms a gel base material; in combination with tannic acid, the polysaccharide and the decellularized extracellular matrix can be further improved to improve the strength of the hydrogel and improve the adhesion capacity; in addition, the tannic acid, cannabidiol, borneol and neomycin sulfate can 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. The obtained hydrogel has good adhesion, strength and corrosion resistance, and is more suitable for harsh environments.

[0009] In an implementation manner of the present application, the degree of oxidation of the hydroxyl group in the aldehyde-modified polysaccharide containing ortho-hydroxyl groups is 10-50%.

[0010] In an implementation manner of the present application, the polysaccharide containing ortho-hydroxyl groups can be at least one of fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose and konjac glucomannan.

[0011] It should be noted that the aldehyde group of the aldehyde-modified polysaccharide in the hydrogel of the present application is formed by oxidation of the ortho-hydroxyl group of the polysaccharide. Therefore, in principle, as long as the polysaccharide contains ortho-hydroxyl groups, it can be applied to the present application, and is not limited to fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose and konjac glucomannan.

[0012] In an implementation manner of the present application, the aldehyde-modified polysaccharide containing ortho-hydroxyl groups is obtained through the steps of oxidation reaction, reaction termination and purification from the polysaccharide containing ortho-hydroxyl groups; the oxidation reaction comprises adding an oxidizing agent in a solution of the polysaccharide containing hydroxyl groups to react; the reaction termination comprises adding an oxidation termination reagent to terminate the reaction after the reaction with the oxidizing agent is completed; and the purification step comprises adopting dialysis and freeze-drying technologies to obtain the purified aldehyde-modified polysaccharide after the oxidation termination, that is, the aldehyde-modified polysaccharide containing ortho-hydroxyl groups of the present application.

[0013] In an implementation manner of the present application, the mass ratio of the polysaccharide and the oxidizing agent in the oxidation reaction is 1: (0.01-1), and the time of the oxidation reaction is 1-72h.

[0014] In an implementation manner of the present application, the oxidation termination reagent is ethylene glycol, and the time of the reaction termination is 15-60min.

[0015] In an implementation manner of the present application, the dialysis membrane used in the dialysis has a specification of 3500kDa.

[0016] In one implementation of the present application, in the hydrogel, the mass ratio of the aldehyde-modified polysaccharide containing ortho-hydroxyl group to the decellularized extracellular matrix in the decellularized extracellular matrix enzyme digestion solution is (0.1-10):(0.2-2).

[0017] In one implementation of the present application, 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%.

[0018] In one implementation of the present application, the decellularized extracellular matrix enzyme digestion solution is prepared by the following steps:

[0019] (1) mixing and stirring animal skin tissue and a defatting solution to obtain defatted tissue;

[0020] (2) mixing and stirring the defatted tissue with a mixed solution of Triton X-100 and ethylenediaminetetraacetic acid in PBS to obtain pretreated animal tissue matrix;

[0021] (3) mixing the pretreated animal tissue matrix and a DNA enzyme solution to perform DNA enzyme reaction to obtain DNA-removed animal tissue decellularized extracellular matrix;

[0022] (4) freeze-drying the DNA-removed animal tissue decellularized extracellular matrix, pre-cooling in liquid nitrogen, grinding, and sieving to obtain decellularized extracellular matrix powder;

[0023] (5) mixing the decellularized extracellular matrix powder and a protease solution to perform enzyme reaction to obtain the decellularized extracellular matrix enzyme digestion solution.

[0024] In one implementation of the present application, in step (1), the animal skin tissue includes one or more of pig skin, cow skin, and fish skin.

[0025] In one implementation of the present application, in step (2), the mass percentage content of Triton X-100 in the mixed solution of PBS is 0.1-1wt%, the mass percentage content of ethylenediaminetetraacetic acid in the mixed solution of PBS is 0.05-1wt%, and the mass ratio of the defatted tissue to the volume of the mixed solution of PBS is (0.5-3)g:(100-500)mL.

[0026] In one implementation of the present application, in step (3), the concentration of the DNA enzyme solution is 10-100U / mL, and the mass ratio of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is (0.2-2)g:(100-500)mL.

[0027] In one implementation form of the present application, in step (4), the protease solution is one or more of pepsin, papain, alpha-amylase and collagenase; the mass concentration of the protease solution is 0.1-2 mg / mL; the enzymatic reaction time is 1-36 h; and the ratio of the volume of the animal tissue decellularized extracellular matrix to the volume of the protease solution is (0.2-1) g:(50-1000) mL.

[0028] In a second aspect, the present application also provides a preparation method of the polysaccharide-based composite hydrogel, which comprises: mixing and stirring a solution containing an aldehyde-modified polysaccharide with adjacent hydroxyl groups, a decellularized extracellular matrix enzyme 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 the hydrogel dressing.

[0029] In one implementation form of the present application, in the preparation method, the mass percentage of the solute in the solution containing the aldehyde-modified polysaccharide with adjacent hydroxyl groups is 1-5%.

[0030] In one implementation form of the present application, in the preparation method, the mass percentage of the solute in the decellularized extracellular matrix enzyme digestion solution is 1-5%.

[0031] In one implementation form of the present application, in the preparation method, the concentration of cannabidiol in the cannabidiol solution is 0.5-2 mol / L.

[0032] In one implementation form of the present application, in the preparation method, the concentration of borneol in the borneol solution is 0.5-2 mol / L.

[0033] In one implementation form of the present application, in the preparation method, the concentration of neomycin sulfate in the neomycin sulfate solution is 0.5-2 mol / L.

[0034] The present application researches and finds that selecting the mass percentage or the concentration of the solute in the solution within the above range is beneficial to the gelation reaction.

[0035] In one implementation form of the present application, the solvents of the cannabidiol solution and the borneol solution are preferably anhydrous ethanol; and the solvents of the solution containing the aldehyde-modified polysaccharide with adjacent hydroxyl groups, the decellularized extracellular matrix enzyme digestion solution, the tannic acid solution and the neomycin sulfate solution are preferably water.

[0036] In one implementation form of the present application, the mixing and stirring are uniformly performed by using a rapid vortex method, and the mixing is performed until there is no stratification observed by naked eyes.

[0037] In one implementation form of the present application, the standing time to form a gel is preferably 10-30 min.

[0038] The third aspect of the present application also provides the use of the polysaccharide-based composite hydrogel in the preparation of a skin or mucosal tissue wound repair dressing.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] The composite hydrogel of the present application, in which the decellularized extracellular matrix and the aldehyde-modified ortho-hydroxyl-containing polysaccharide form a gel base material, the introduction of tannic acid, cannabidiol, borneol and neomycin sulfate, on the one hand, tannic acid cooperates with the decellularized extracellular matrix and the aldehyde-modified ortho-hydroxyl-containing polysaccharide to improve the adhesion of the hydrogel, and on the other hand, tannic acid, cannabidiol, borneol and neomycin sulfate can cooperatively improve the hemostatic, analgesic, anti-inflammatory effect and antibacterial performance of the hydrogel, reduce the risk of infection at the affected site, and also have the effect of promoting wound healing. The composite hydrogel of the present application can significantly improve the performance of the hydrogel, and the obtained hydrogel has good mechanical strength, high adhesion, and also has good bacteriostatic, hemostatic, analgesic, anti-inflammatory and wound healing promoting effects, and is particularly suitable for use in harsh environments. The hydrogel provided by the present application has good corrosion resistance and high strength, and can be soaked in seawater for more than 24 h while maintaining adhesion to the tissue, which is significantly improved compared with existing hydrogels. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is an optical photograph of the hydrogel in the present application;

[0042] Figure 2 is a 24h result graph of the hydrogel in the present application soaked in seawater;

[0043] Figure 3 is an antibacterial test result graph of the hydrogel in the present application;

[0044] Figure 4 is a cell compatibility result graph of the hydrogel in the present application;

[0045] Figure 5 is a blood compatibility result graph of the hydrogel in the present application;

[0046] Figure 6 is a wound healing result graph of the hydrogel in the present application in vivo. DETAILED DESCRIPTION

[0047] The present application provides a polysaccharide-based composite hydrogel, which comprises aldehyde-modified ortho-hydroxyl-containing polysaccharide and animal tissue decellularized extracellular matrix enzyme digestion solution, tannic acid, neomycin sulfate, borneol and cannabidiol. The aldehyde-modified ortho-hydroxyl-containing polysaccharide is obtained by oxidizing the hydroxyl group in the ortho-hydroxyl-containing polysaccharide to an aldehyde group.

[0048] The aldehyde-modified polysaccharide with adjacent hydroxyl groups is obtained by oxidizing the hydroxyl groups in the polysaccharide with adjacent hydroxyl groups into aldehyde groups.

[0049] In the present application, the oxidation degree of the aldehyde-modified polysaccharide with adjacent hydroxyl groups is preferably 10-50%. For example, it can be 10%, 20%, 30%, 40%, 50%, etc.

[0050] In the present application, the preparation method of the aldehyde-modified polysaccharide with adjacent hydroxyl groups preferably comprises the following steps: dissolving the polysaccharide in water to prepare a uniform solution with a concentration of 0.5-5wt%, adding an oxidizing agent to an oxidizing agent concentration of 0.01-1wt% in a light-proof environment, and continuously stirring for 8-40h for oxidation, then adding a terminating agent to a terminating agent concentration of 0.01-1wt% and continuously stirring for 20-60min to terminate the reaction. Then, the solution is dialyzed using a dialysis bag for 72h, and freeze-dried to obtain the aldehyde-modified polysaccharide.

[0051] In the present application, the polysaccharide is preferably guar gum, and the concentration is preferably 1-3wt%; the oxidizing agent is preferably sodium periodate, and the final concentration in the reaction system is preferably 0.1-0.5wt%; and the terminating agent is preferably ethylene glycol, and the concentration is preferably 0.1-0.5wt%.

[0052] The hydrogel provided by the present application comprises an animal tissue decellularized extracellular matrix enzyme digestion solution.

[0053] In the present application, the preparation method of the animal tissue decellularized extracellular matrix enzyme digestion solution preferably comprises the following steps:

[0054] Mixing and stirring animal skin tissue and defatting liquid to obtain defatted tissue;

[0055] Stirring the defatted tissue with a mixed solution of Triton X-100, ethylenediaminetetraacetic acid and PBS to obtain a pretreated animal tissue matrix;

[0056] Mixing the pretreated animal tissue matrix and a DNAase solution to perform DNAase digestion reaction to obtain a DNA-removed animal tissue decellularized extracellular matrix;

[0057] Freeze-drying the DNA-removed animal tissue matrix, pre-cooling in liquid nitrogen, and then grinding into powder and sieving to obtain a decellularized extracellular matrix powder;

[0058] Mixing the decellularized extracellular matrix powder and an enzyme solution to perform enzyme digestion reaction to obtain the animal tissue decellularized extracellular matrix enzyme digestion solution.

[0059] In the present application, animal skin tissue and a defatting solution are mixed and continuously stirred for a period of time to obtain defatted tissue. In the present application, the animal skin preferably includes one or more of pig skin, cow skin and fish skin, and the defatting solution is an organic solvent, preferably an equal proportion mixed solution of ethyl acetate and methanol.

[0060] In the present application, 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 application, the mass percentage content of Triton X-100 in the PBS mixed solution is preferably 0.1-1wt%. In the present application, the mass percentage content of ethylenediaminetetraacetic acid in the PBS mixed solution is preferably 0.05-1wt%. 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 application, the temperature of the stirring is preferably 37℃, and the time of the stirring is preferably 6-72h.

[0061] After obtaining the pretreated animal tissue matrix, the present application mixes the pretreated animal tissue matrix and a DNase solution to perform a DNase digestion reaction to obtain an animal tissue decellularized extracellular matrix.

[0062] In the present application, the concentration of the DNase solution is preferably 10-100U / mL. The ratio of the mass of the pretreated animal tissue matrix to the volume of the DNase solution is preferably (0.2-2)g:(100-500)mL. In the present application, the temperature of the DNase digestion reaction is preferably 37℃, and the time of the DNase digestion reaction is preferably 1-8h.

[0063] After obtaining the decellularized extracellular matrix, the present application freeze-dries the decellularized extracellular matrix, pre-cools it in liquid nitrogen, grinds it into a powder, and sieves it to obtain a decellularized extracellular matrix powder.

[0064] After obtaining the decellularized extracellular matrix powder, the present application mixes the decellularized extracellular matrix powder and a protease solution to perform a protease digestion reaction to obtain the animal tissue decellularized extracellular matrix enzyme digestion solution.

[0065] In the present application, the protease solution is one or more of pepsin, papain, alpha-amylase and collagenase, and is preferably a pepsin solution. The mass concentration of the protease solution is preferably 0.1-2mg / mL, and the time of the enzyme digestion reaction is 1-36h. The ratio of the volume of the animal tissue decellularized extracellular matrix to the volume of the protease solution is preferably (0.2-1)g:(50-1000)mL. The temperature of the protease digestion reaction is preferably 37℃.

[0066] The composite hydrogel provided by the application further comprises tannic acid, cannabidiol, neomycin sulfate and borneol.

[0067] The application provides a preparation method of the hydrogel.

[0068] The aldehyde-modified ortho-hydroxyl-containing polysaccharide is mixed with the animal decellularized extracellular matrix uniformly, and is adjusted to neutral by using a sodium hydroxide solution to obtain a hydrogel matrix. In the application, the mixing is preferably performed at 37 DEG C.

[0069] The hydrogel matrix is mixed with a tannic acid solution uniformly to obtain a tannic acid-composite hydrogel matrix. In the application, the mixing is preferably performed at 37 DEG C.

[0070] The tannic acid-composite hydrogel matrix is mixed with a cannabidiol solution, a neomycin sulfate solution and a borneol solution uniformly to obtain the composite hydrogel of the application. In the application, the mixing is preferably performed at 37 DEG C.

[0071] In the application, the concentration of tannic acid in the tannic acid solution is preferably 1-0.01 g / mL, and the solvent is preferably water. The concentration of cannabidiol in the cannabidiol solution is preferably 1-0.01 g / mL, and the solvent is preferably ethanol. The concentration of borneol in the borneol solution is preferably 1-0.01 g / mL, and the solvent is preferably ethanol. The concentration of neomycin sulfate in the neomycin sulfate solution is preferably 1-0.01 g / mL, and the solvent is preferably water.

[0072] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0073] Unless specifically stated, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the technical field.

[0074] Unless specifically stated, the reagents and materials used in the following examples are commercially available.

[0075] Example 1

[0076] 1) Preparation of decellularized extracellular matrix enzyme digestion solution:

[0077] The fresh pigskin is cut into small pieces of 1 cm x 1 cm in size and washed multiple times; the cut small pieces of pigskin tissue are placed in a blue cap bottle, and an equal volume of a solution of ethyl acetate and methanol with a volume ratio of 1:1 is added to completely immerse the pigskin tissue, and the pigskin tissue is stirred for a period of time to obtain defatted pigskin tissue.

[0078] The mixture solution containing 1wt% Triton X-100 and 0.8wt% ethylenediaminetetraacetic acid in PBS was added into the defatted pigskin tissue, and the mixture was continuously stirred for 8 hours. After filtration, the solid was washed with water to obtain a pretreated animal tissue matrix;

[0079] The DNA enzyme solution was added into the pretreated animal tissue matrix, and the ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution was 1g:200mL. The mixture was continuously stirred for 8 hours. After filtration, the solid was washed with water to obtain the acellular extracellular matrix;

[0080] The acellular extracellular matrix was freeze-dried, pre-cooled in liquid nitrogen, and ground into particles using a grinder. The particles were sieved through a 100-mesh sieve to obtain the acellular extracellular matrix powder.

[0081] The acellular extracellular matrix powder was added into the pepsin solution with a concentration of 0.5mg / mL, and the ratio of the mass of the acellular extracellular matrix powder to the volume of the pepsin solution was 1g:200mL to obtain the pigskin acellular extracellular matrix enzyme digestion solution.

[0082] 2) Preparation of an aldehyde-modified polysaccharide containing an ortho-hydroxyl group:

[0083] 5g of guar gum powder was weighed, and the raw material powder was dissolved in 250mL of water by stirring to prepare a polysaccharide solution with a concentration of 2%.

[0084] 1g of sodium periodate was weighed as an oxidizing agent and added into the polysaccharide solution. The reaction was carried out under magnetic stirring at room temperature in the dark for 12 hours.

[0085] After the reaction was completed, 2mL of ethylene glycol was taken as an oxidation termination reagent and added into the reaction container. The reaction was terminated under magnetic stirring for 40 minutes.

[0086] After the reaction was terminated, the reaction solution was transferred into a dialysis bag with a molecular weight cut-off of 3500kDa, and dialyzed against ultrapure water for 3 days, with water changed twice a day.

[0087] After dialysis, the solution was frozen at -20℃ for 24 hours, and then freeze-dried in a freeze dryer to obtain oxidized guar gum. The degree of oxidation of the oxidized guar gum prepared in this example was determined by the hydroxylamine hydrochloride titration method, and the degree of oxidation was 15.27%.

[0088] 3) Preparation of a hydrogel:

[0089] Take 0.08 g of the oxidized guar gum obtained in step 2) and dissolve it in 2 mL of water to obtain a 4% oxidized guar gum solution. Take 0.04 g of tannic acid and dissolve it in 1 mL of water to obtain a 4% tannic acid solution. Take 314 mg of cannabidiol and dissolve it in 1 mL of ethanol to obtain a 314 mg / mL cannabidiol solution. Take 712 mg of neomycin sulfate and dissolve it in water to obtain a 712 mg / mL neomycin sulfate solution. Take 154 mg of borneol and dissolve it in 1 mL of ethanol to obtain a 154 mg / mL borneol solution.

[0090] Mix 1 mL of the oxidized guar gum solution obtained in step 1) with 1 mL of the decellularized extracellular matrix enzyme digestion solution obtained in step 1) at 37°C, 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 well, and 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)

[0091] Example 2

[0092] The example of the present application provides a composite hydrogel, and the only difference between the preparation method of the hydrogel and example 1 is that: 3) In the preparation process of the hydrogel, the volumes of the cannabidiol solution, the borneol solution, and the neomycin sulfate solution are changed, and the total volume and the total number of moles of the three are kept unchanged, and the volume ratio of cannabidiol, borneol, and neomycin sulfate is changed to cannabidiol: borneol: neomycin sulfate = 1:3:2.

[0093] Example 3

[0094] The example of the present application provides a composite hydrogel, and the only difference between the preparation method of the hydrogel and example 1 is that: 3) In the preparation process of the hydrogel, the volumes of the cannabidiol solution, the borneol solution, and the neomycin sulfate solution are changed, and the total volume and the total number of moles of the three are kept unchanged, and the volume ratio of cannabidiol, borneol, and neomycin sulfate is changed to cannabidiol: borneol: neomycin sulfate = 4:2:1.

[0095] Example 4

[0096] The example provides a composite hydrogel, and the only difference between the preparation method of the hydrogel and example 1 is that: 3) In the preparation process of the hydrogel, the volume of the oxidized guar gum solution is changed to 1.5 mL, and the volume of the decellularized extracellular matrix enzyme digestion solution is changed to 0.5 mL.

[0097] Example 5

[0098] The embodiment of the present application provides a kind of composite hydrogel, the preparation method of the hydrogel dressing is only different from the embodiment 1 in that: guar gum is changed to konjac glucomannan in step 2).

[0099] Example 6

[0100] The embodiment provides a kind of composite hydrogel, the preparation method of the hydrogel dressing is only different from the embodiment 1 in that: guar gum is changed to sodium alginate in step 2).

[0101] Comparative Example 1

[0102] The comparative example provides a kind of composite hydrogel, the preparation method of the hydrogel is only different from the embodiment 1 in that: in step 3), no tannic acid, neomycin sulfate, cannabidiol solution and menthol solution are added, and the same volume of aqueous solution is used as a replacement.

[0103] Comparative Example 2

[0104] The comparative example provides a kind of composite hydrogel, the preparation method of the hydrogel is only different from the embodiment 1 in that: in step 3), no neomycin sulfate, cannabidiol solution and menthol solution are added, and the same volume of aqueous solution is used as a replacement.

[0105] Comparative Example 3

[0106] The comparative example provides a kind of composite hydrogel, the preparation method of the hydrogel is only different from the embodiment 1 in that: in step 3), no menthol solution and neomycin sulfate solution are added, and the same volume of aqueous solution is used as a replacement.

[0107] Comparative Example 4

[0108] The comparative example provides a kind of composite hydrogel, the preparation method of the hydrogel is only different from the embodiment 1 in that: in step 3), no neomycin sulfate solution is added, and the same volume of aqueous solution is used as a replacement.

[0109] Effect verification:

[0110] 1, gelation speed comparison: the gelation time of the hydrogel prepared in examples 1-6 is compared by timing, and the results are shown in table 1.

[0111] The results show that the hydrogel of each example can be gelled, but the type of polysaccharide and the volume ratio of decellularized extracellular matrix enzyme digestion solution to polysaccharide solution both affect the gelation speed of the hydrogel.

[0112] Table 1 Summary of hydrogel gelation time

[0113]

[0114] 2, hydrogel adhesion performance test:

[0115] The hydrogel of Example 1 and the hydrogel of Comparative Example 1 were adhered to the same piece of pigskin, the pigskin was stretched horizontally and the pigskin was faced down with the hydrogel, and the time for the hydrogel to fall off was observed. The final result was that the hydrogel of Example 1 did not fall off after 24 h of adhesion, and the hydrogel of Comparative Example 1 fell off after 5 h of adhesion. This indicated that the introduction of tannic acid can further enhance the adhesion performance.

[0116] 3. Seawater erosion experiment:

[0117] The hydrogel of Example 1 was adhered to pigskin and soaked in seawater for 24 h, and the adhesion of the hydrogel at 0 h and 24 h was observed by taking pictures, and the results are shown in Figure 2 .

[0118] Seawater contains a large number of microorganisms and various ions, and has a certain corrosive property. After the hydrogel is attached to the pigskin and soaked in seawater for a period of time, the color becomes darker due to the oxidation of TA. The results show that the adhesion of the hydrogel can be maintained for 24 h or more in a complex environment, and the macrostructure of the hydrogel is not damaged, which to some extent proves that the hydrogel of Example 1 has good strength and corrosion resistance, and can ensure continuous adhesion within this time period even in harsh conditions.

[0119] 4. Conventional antibacterial test:

[0120] The antibacterial effect of the hydrogel dressings of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 on bacterial strains was tested by plate counting method. Two types of bacterial strains were selected for testing, namely Gram-positive bacteria (E) and Gram-negative bacteria (S). The hydrogel dressing was incubated with each bacterial strain for 24 h, and then the bacterial strain incubated with the hydrogel was diluted and evenly spread on soybean casein agar plates. Finally, the agar plates were incubated in an incubator for 16 h, and the inhibition effect of different types of hydrogel on Gram-positive bacteria and Gram-negative bacteria was observed and photographed; the antibacterial rates of Gram-positive bacteria and Gram-negative bacteria corresponding to all test groups were calculated, and the average values of the antibacterial rates of Gram-positive bacteria and Gram-negative bacteria were taken respectively; the group without hydrogel was taken as the control group.

[0121] The calculation formula of the antibacterial rate is: (the number of colonies in the control group - the number of colonies in the experimental group) / the number of colonies in the control group x 100%

[0122] The results are shown in Figure 3The results show that the cell survival rate of the hydrogel dressing and the cells co-cultured according to the above method is distributed above 85%, which has good cell compatibility and meets the cell biological safety requirements of GB / T 16886.

[0123] 5. Biocompatibility test

[0124] The hydrogel lyophilized samples of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were respectively immersed with high-sugar complete culture medium at 37℃ for 24h. After the immersion was completed, the concentration of the immersion liquid was diluted to 1 mg / mL, 0.1 mg / mL and 0.01 mg / mL. Mouse fibroblast cells NIH3T3 were inoculated on culture plates containing complete culture medium and cultured in a cell incubator at 37℃ and a carbon dioxide concentration of 5% for 24. Then, the complete culture medium was discarded and replaced with the hydrogel immersion liquid, and cultured in a cell incubator at 37℃ and a carbon dioxide concentration of 5% for 24h. The cells were treated with CCK-8 reagent, and the absorbance at 450nm was detected by a microplate reader. The cell survival rate was calculated according to the absorbance of each well.

[0125] The calculation formula is: Cell activity (%) = (A1-A c / A0-A c ) × 100%. Wherein A1 is the absorbance of the experimental group, A0 is the absorbance of the control group, and A c is the absorbance of the blank group.

[0126] Figure 4 The results show that the cell survival rate of the hydrogel dressing and the cells co-cultured according to the above method is distributed above 85%, which has good cell compatibility and meets the cell biological safety requirements of GB / T 16886.

[0127] 6. Blood compatibility experiment of hydrogel dressing

[0128] 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. PBS and ultrapure water treated red blood cells were used as negative and positive groups. After incubation at 37℃ for 1h, the supernatant was removed by centrifugation, and the absorbance at 540nm was measured by a microplate reader. The calculation method of hemolysis rate is as follows:

[0129] HR (%) = (OD gel -OD blank ) / (OD control - OD blank ) × 100%

[0130] OD gel : Absorbance at 540 nm of hydrogel-treated red blood cell supernatant

[0131] OD control : Absorbance at 540 nm of red blood cell supernatant treated with ultrapure water

[0132] OD blank : Absorbance at 540 nm of PBS-treated red blood cell supernatant

[0133] Test results are as follows Figure 3 As shown. The hydrogel in Example 1 exhibits good blood compatibility, with a hemolysis rate of less than 5%, meeting the blood biocompatibility requirements of GB / T 16886. Furthermore, the comparative results also reflect that the addition of tannic acid further reduces the blood compatibility of the hydrogel.

[0134] 7. Experiment on the wound healing ability of hydrogels:

[0135] Shave the back of BALB / c mice to create a wound, and insert 1×10 8 A 20 μL drop of CFU / mL Staphylococcus aureus suspension was added to the wound. The hydrogel dressing substrate and the hydrogel dressing from Example 1 were then completely applied to the wound; mice without hydrogel served as a control group. Finally, all groups were protected with a layer of 3M™ Tegaderm™ clear dressing. After recovery from anesthesia, animals were monitored for any discomfort and provided with water and food. The hydrogel dressing was changed daily, and body weight was measured.

[0136] Test results are as follows Figure 6 As shown, the hydrogel exhibits a significant antibacterial effect compared to the 3M™ Tegaderm™ transparent dressing group, while also significantly promoting wound healing.

[0137] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A polysaccharide-based composite hydrogel, characterized in that: The raw material comprises: aldehyde-modified polysaccharide containing adjacent hydroxyl, decellularized extracellular matrix enzyme digestion solution, tannic acid, cannabidiol, neomycin sulfate and borneol; the aldehyde-modified polysaccharide containing adjacent hydroxyl is obtained by oxidizing the hydroxyl in the polysaccharide containing adjacent hydroxyl into aldehyde group; In the hydrogel, the mass ratio of the aldehyde-modified polysaccharide containing adjacent hydroxyl to the decellularized extracellular matrix in the decellularized extracellular matrix enzyme digestion solution is (0.1-10):(0.2-2); 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%; The decellularized extracellular matrix enzyme digestion solution is prepared by the following steps: (1) mixing and stirring animal skin tissue and 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 pretreated animal tissue matrix; (3) mixing the pretreated animal tissue matrix and DNA enzyme solution to carry out DNA enzyme reaction to obtain DNA-removed animal tissue decellularized extracellular matrix; (4) freeze-drying the DNA-removed animal tissue decellularized extracellular matrix, pre-cooling in liquid nitrogen, grinding, sieving to obtain decellularized extracellular matrix powder; (5) mixing the decellularized extracellular matrix powder and protease solution to carry out enzyme reaction to obtain the decellularized extracellular matrix enzyme digestion solution.

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

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

4. The polysaccharide-based composite hydrogel according to claim 1, characterized in that: In step (2), the mass percentage content of Triton X-100 in the PBS mixed solution is 0.1-1wt%, and the mass percentage content 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.

5. The polysaccharide-based composite hydrogel according to claim 1, characterized in that: In step (3), the concentration of the DNA enzyme solution is 10-100U / 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.

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

7. Process for the preparation of a polysaccharide-based composite hydrogel according to any one of claims 1-6, characterized in that, Comprise: Mixing and stirring the aldehyde-modified polysaccharide containing adjacent hydroxyl solution, the decellularized extracellular matrix enzyme digestion solution, the tannic acid solution, the cannabidiol solution, the borneol solution and the neomycin sulfate solution, and standing into a gel to obtain a hydrogel dressing; The mass percentage of solute in the aldehyde-modified solution of the polysaccharide containing ortho-hydroxyl groups is 1-5%; The mass percentage of solute in the decellularized extracellular matrix enzyme digestion solution is 1-5%; The mass percentage of tannic acid in the tannic acid solution is 1-10%; The concentration of cannabidiol in the cannabidiol solution is 0.5-2 mol / L; The concentration of borneol in the borneol solution is 0.5-2 mol / L; The concentration of neomycin sulfate in the neomycin sulfate solution is 0.5-2 mol / L.

8. Use of the polysaccharide-based composite hydrogel according to any one of claims 1-6 or prepared by the preparation method of claim 7 in the preparation of a skin or mucosal tissue wound repair dressing.

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