CN111150880A

CN111150880A - Antibacterial composite hydrogel and preparation method thereof

Info

Publication number: CN111150880A
Application number: CN202010017199.4A
Authority: CN
Inventors: 蓝咏; 刘玉; 冯龙宝; 翁金亮
Original assignee: Guangzhou Bioscience Co Ltd
Current assignee: Guangzhou Bioscience Co Ltd
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2020-05-15

Abstract

The invention relates to an antibacterial composite hydrogel which comprises the following components in percentage by mass and volume: 2-3.5% of carboxymethyl chitosan, 4-6% of oxidized sodium alginate and 0.3-0.6% of tannic acid, wherein the solution of the hydrogel is PBS buffer solution. The antibacterial composite hydrogel disclosed by the invention is prepared by mixing carboxymethyl chitosan, oxidized sodium alginate and tannic acid on the basis of carboxymethyl chitosan and oxidized sodium alginate under physiological conditions by using a dynamic Schiff base bond method, and has good degradation capability, biocompatibility, antibacterial property and mechanical property, and the wound healing process can be effectively promoted.

Description

Antibacterial composite hydrogel and preparation method thereof

Technical Field

The invention relates to an antibacterial composite hydrogel and a preparation method thereof, belonging to the technical field of medical biomaterials.

Background

Antibacterial wound dressings have gained increasing popularity in recent years, with most commercial suppliers now offering silvered or nanoparticle impregnated dressings. Although such dressings have a broad spectrum of antimicrobial efficacy, several studies have also reported that silver is cytotoxic to mammalian cells and negatively affects the wound healing process. In addition, silver wound dressings are expensive and there continues to be concern about nanoparticle absorption and nanotoxicity. Accordingly, there has been considerable effort directed to identifying alternative antimicrobial agents.

Tannic Acid (TA) is an antioxidant, antimicrobial, antiviral and anti-inflammatory agent and is used as the first treatment of clinical burn injuries in the mid-20 th century. This treatment was abandoned in the 40 s due to concerns about hepatotoxic effects. However, recent re-assessment of earlier studies indicates that natural pathophysiological phenomena are the cause, not TA poisoning. Thus, the use of TA as an adjunct to burn wounds and other therapeutic applications, including as an antimicrobial agent, has been renewed. Given the recent reevaluation of TA toxicity, and the results demonstrating biocompatibility, the local application of TA may be subject to less stringent regulatory conditions in the future. There are studies that demonstrate TA by relying on the putative staphylococcal antigen a, transglycosylase. Biocompatible and biodegradable TA hydrogels with antibacterial and antioxidant properties have recently been reported, synthesizing TA/chitosan/pullulan composite nanofibers for wound healing, although these materials are not pH sensitive. However, the development of pH sensitive hydrogels for TA controlled release has not been reported.

Severely exuding wounds require regular dressing changes to limit bacterial colonization and induce wound healing. The balance between exudate absorption and moisture release can be adjusted using hydrogels, with superabsorbent gels being the most preferred. Among the different hydrogels, responsive hydrogels are of great interest due to their potential to react to external triggers (e.g., pH) and release their payload in a more controlled manner. Among them, the Schiff base crosslinked hydrogel is an ideal pH-responsive hydrogel. Wound fluid pH plays a crucial role in the wound healing process. Although the pH of intact skin is 5.5, the pH of the wound environment can vary significantly depending on the type of wound, the stage of the healing process, or other factors (e.g., infection). The pH of the stage I pressure ulcer is about 5.5-5.6, which will allow it to be treated with a wound dressing. The crosslinking mechanism of the Schiff base crosslinked hydrogel is as follows: the amino and aldehyde group react under neutral or alkaline conditions to generate C ═ N double bonds, and the generated C ═ N double bonds are easily decomposed under acidic conditions, so that the mechanism can provide good conditions for the release of the wound dressing drug.

Carboxymethyl chitosan is a chitosan derivative having biodegradability, biocompatibility and inherent antibacterial activity, and is one of hot spots in research of antibacterial agents. The antibacterial mechanism of carboxymethyl chitosan is that under the condition that the pH value is lower than 6, the positive charge of amino at the C-2 position can interact with the surface of bacteria, so that the bacteria die. Sodium Alginate (SA) is a polysaccharide carbohydrate extracted from brown algae such as herba Zosterae Marinae or Sargassum, and has good biodegradability and biocompatibility. The sodium alginate is easy to dissolve in water, and has good gelatinization performance, strong hygroscopicity and good water retention performance.

The invention adopts a dynamic Schiff base method to prepare the biocompatible hydrogel wound dressing with inherent antibacterial activity. Under physiological conditions, a tannic acid/oxidized sodium alginate (TA/OSA) solution is mixed with a carboxymethyl chitosan (CMC) solution to prepare the hydrogel. In the invention, the antibacterial performance of the OSA-CMC hydrogel is improved by adding the tannic acid.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the antibacterial composite hydrogel and the preparation method thereof, and the hydrogel has excellent biocompatibility and antibacterial performance.

In order to achieve the purpose, the invention adopts the technical scheme that: an antibacterial composite hydrogel comprises the following components in percentage by mass and volume: 2-3.5% of carboxymethyl chitosan, 4-6% of oxidized sodium alginate and 0.3-0.6% of tannic acid, wherein the solution of the hydrogel is PBS buffer solution.

As a preferred embodiment of the hydrogel, the hydrogel comprises the following components in percentage by mass and volume: 3% of carboxymethyl chitosan, 5% of oxidized sodium alginate, 0.5% of tannic acid and the solution of the hydrogel is PBS buffer solution.

As a preferred embodiment of the hydrogel of the present invention, the preparation method of oxidized sodium alginate comprises: dissolving sodium alginate in ethanol, heating in water bath, slowly dropwise adding sodium periodate aqueous solution into sodium alginate solution after sodium alginate is completely dissolved, and stirring for reaction; after the reaction is finished, dropwise adding ethylene glycol, continuously stirring, then adding sodium chloride, pouring the reaction solution into absolute ethyl alcohol, and separating out a precipitate; vacuum filtering, dissolving the precipitate with pure water, dialyzing, and freeze drying to obtain oxidized sodium alginate.

As a preferred embodiment of the hydrogel, the mass ratio of the sodium alginate to the sodium periodate is 1: 1.

As a preferred embodiment of the hydrogel, the temperature of the water bath heating is 60 ℃, the stirring reaction time is 5h, the continuous stirring time is 1h, the cut-off molecular weight of the dialysis bag for dialysis is 8000-15000, and the freeze drying temperature is-80 ℃.

As a preferred embodiment of the hydrogel of the present invention, the preparation method of oxidized sodium alginate comprises: dissolving 10g of sodium alginate in 250mL of 15% (v/v) ethanol solution to prepare 4% w/v solution, heating the solution in a water bath to 60 ℃, weighing 10g of sodium periodate after the sodium alginate is completely dissolved, dissolving the sodium periodate in 20mL of pure water, slowly dropwise adding the solution into the sodium alginate solution under the conditions of light protection and rapid stirring, and reacting for 5 hours; after the reaction is finished, 2mL of ethylene glycol is dropwise added into the reaction solution, and stirring is continued for 1 h; adding 10g of sodium chloride, pouring the reaction solution into 500mL of absolute ethyl alcohol, separating out a precipitate, carrying out vacuum filtration, dissolving the precipitate again by using 50mL of pure water, and dialyzing the precipitate for three days by using a dialysis bag with the molecular weight cutoff of 8000-15000; after dialysis, the solution is freeze-dried at-80 ℃ to obtain oxidized sodium alginate solid.

In a second aspect, the present invention provides a method for preparing the above hydrogel, comprising the steps of:

(1) dissolving carboxymethyl chitosan in a PBS buffer solution to form a solution A;

(2) dissolving oxidized sodium alginate and tannic acid in a PBS buffer solution to form a solution B;

(3) and (3) mixing the solution A obtained in the step (1) and the solution B obtained in the step (2), then placing the mixture in a 48-hole cell pore plate, and uniformly stirring to obtain the antibacterial composite hydrogel.

As a preferred embodiment of the method for producing a hydrogel according to the present invention, the volume ratio of the solution a to the solution B is 1: 1.

in a preferred embodiment of the method for preparing the hydrogel according to the present invention, the carboxymethyl chitosan is present in the solution a in an amount of 4 to 7% by mass/volume. Preferably, the mass volume percentage of the carboxymethyl chitosan is 6%.

As a preferable embodiment of the preparation method of the hydrogel, in the solution B, the mass volume percentage of the oxidized sodium alginate is 8% to 12%. Preferably, the mass volume percentage of the oxidized sodium alginate is 10%.

As a preferable embodiment of the method for producing the hydrogel according to the present invention, the mass volume percentage of tannic acid in the solution B is 0.6% to 1.2%. Preferably, the mass volume percentage of tannic acid is 1%.

Compared with the prior art, the invention has the beneficial effects that:

(1) the antibacterial composite hydrogel disclosed by the invention is prepared by mixing carboxymethyl chitosan, oxidized sodium alginate and tannic acid on the basis of carboxymethyl chitosan and oxidized sodium alginate under physiological conditions by using a dynamic Schiff base bond method, and has good degradation capability, biocompatibility, antibacterial property and mechanical property, and the wound healing process can be effectively promoted.

(2) Compared with the in vitro and in vivo antibacterial hydrogel reported in the past, the hydrogel disclosed by the invention has excellent biocompatibility and antibacterial performance; the hydrogel disclosed by the invention can be slowly degraded under the environment condition of a wound to release tannic acid, so that an antibacterial effect is achieved, and meanwhile, the carboxymethyl chitosan in the hydrogel also has a certain antibacterial property, so that the bacterial number and the inflammation level of the wound can be effectively reduced.

Drawings

FIG. 1 is a graph showing the solubility of chitosan and carboxymethyl chitosan.

FIG. 2 is a scanning electron microscope photograph of the hydrogel prepared in example 1.

FIG. 3 is an infrared spectrum of sodium alginate and oxidized sodium alginate.

FIG. 4 is a graph showing swelling ratios of the hydrogels prepared in examples 1 and 5.

FIG. 5 is a graph showing the degradation performance test of the hydrogel prepared in example 1.

FIG. 6 is a statistical chart of the compressive strength of the hydrogels prepared in examples 1-5.

FIG. 7 is a statistical chart of the water vapor transmission rate of the hydrogels prepared in examples 1-5.

FIG. 8 is a graph showing the water vapor drug release results of the hydrogel prepared in example 5.

FIG. 9 is a statistical graph of cell viability of the hydrogels prepared in example 1 and example 5.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1

An OSA/6% CMC hydrogel comprises the following components in percentage by mass and volume: 3% of carboxymethyl chitosan (CMC) and 5% of Oxidized Sodium Alginate (OSA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (6% w/v in PBS) and 0.2mL of OSA (10% w/v in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a gun head to obtain an OSA/6% CMC hydrogel.

The preparation method of the oxidized sodium alginate comprises the following steps: dissolving 10g of sodium alginate in 250mL of 15% (v/v) ethanol solution to prepare 4% w/v solution, heating the solution in a water bath to 60 ℃, weighing 10g of sodium periodate after the sodium alginate is completely dissolved, dissolving the sodium periodate in 20mL of pure water, slowly dropwise adding the solution into the sodium alginate solution under the conditions of light protection and rapid stirring, and reacting for 5 hours; after the reaction is finished, 2mL of ethylene glycol is dropwise added into the reaction solution, and stirring is continued for 1 h; adding 10g of sodium chloride, pouring the reaction solution into 500mL of absolute ethyl alcohol, separating out a precipitate, carrying out vacuum filtration, dissolving the precipitate again by using 50mL of pure water, and dialyzing the precipitate for three days by using a dialysis bag with the molecular weight cutoff of 8000-15000; after dialysis, the solution is freeze-dried at-80 ℃ to obtain oxidized sodium alginate solid.

Example 2

An OSA/4% CMC hydrogel comprises the following components in percentage by mass and volume: 2% of carboxymethyl chitosan (CMC) and 5% of Oxidized Sodium Alginate (OSA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (4% w/v in PBS) and 0.2mL of OSA (10% w/v in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a gun head to obtain an OSA/4% CMC hydrogel.

Example 3

An OSA/5% CMC hydrogel comprises the following components in percentage by mass and volume: 2.5% of carboxymethyl chitosan (CMC) and 5% of Oxidized Sodium Alginate (OSA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (5% w/v in PBS) and 0.2mL of OSA (10% w/v in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a gun head to obtain an OSA/5% CMC hydrogel.

Example 4

An OSA/7% CMC hydrogel comprises the following components in percentage by mass and volume: 3.5% of carboxymethyl chitosan (CMC) and 5% of Oxidized Sodium Alginate (OSA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (7% w/v in PBS) and 0.2mL of OSA (10% w/v in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a gun tip to obtain an OSA/7% CMC hydrogel.

Example 5

An OSA/6% CMC/TA composite hydrogel comprises the following components in percentage by mass and volume: 3% of carboxymethyl chitosan, 5% of oxidized sodium alginate and 0.5% of Tannic Acid (TA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (6% w/v in PBS) and 0.2mL of OSA/TA (10% w/v OSA and 1% w/v TA in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a lance tip to obtain an OSA/6% CMC/TA composite hydrogel.

Example 6

An OSA/CMC/TA composite hydrogel comprises the following components in percentage by mass and volume: 2% of carboxymethyl chitosan, 4% of oxidized sodium alginate and 0.3% of Tannic Acid (TA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (4% w/v in PBS) and 0.2mL of OSA/TA (8% w/v OSA and 0.6% w/v TA in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a lance tip to obtain an OSA/CMC/TA composite hydrogel.

Example 7

An OSA/CMC/TA composite hydrogel comprises the following components in percentage by mass and volume: 3.5% of carboxymethyl chitosan, 6% of oxidized sodium alginate and 0.6% of Tannic Acid (TA), wherein the solution of the hydrogel is PBS buffer solution.

The preparation method comprises the following steps: 0.2mL of CMC (7% w/v in PBS) and 0.2mL of OSA/TA (12% w/v OSA and 1.2% w/v TA in PBS) were placed in a 48-well cell-well plate, and the mixture was gently stirred using a lance tip to obtain an OSA/CMC/TA composite hydrogel.

Examples of effects

(1) Solubility of carboxymethyl chitosan

0.02g of chitosan is dissolved in neutral water, and 0.02g of carboxymethyl chitosan is dissolved in neutral water. The clarity of the solution was observed and the results of the dissolution are shown in figure 1.

The results show that the water solubility of chitosan in neutral water is turbid, and the sample is insoluble. The carboxymethyl chitosan has good solubility in neutral water, because the carboxyl in the carboxymethyl chitosan obviously improves the solubility of the chitosan.

(2) Scanning electron microscopy

The OSA/6% CMC hydrogel prepared in example 1 was placed in liquid nitrogen, after it had set, it was gently broken with a glass rod, a suitable cross-section was selected, lyophilized, and the appearance of the lyophilized sample was photographed with a scanning electron microscope, the scanning electron microscope image of which is shown in fig. 2.

As can be seen from FIG. 2, the hydrogel has a better pore size, and is suitable for entrapping and releasing tannic acid.

(3) Infrared spectrogram

Preparing a sample to be detected by a potassium bromide tabletting method, and measuring the infrared spectrum by using a Fourier infrared spectrometer. FIG. 3 is an infrared spectrum of Sodium Alginate (SA) and Oxidized Sodium Alginate (OSA), wherein the molecular structure of Sodium Alginate (SA) has more hydroxyl groups, and the hydroxyl groups are mutually influenced to make it be 3410cm^-1Shows a broad peak, and after partial hydroxyl groups are oxidized by sodium periodate, the peak is at 3410cm^-1The absorption peak is weakened, because the molecular structure of the sodium alginate has carboxyl, the absorption peak of the aldehyde group after the sodium periodate oxidation coincides with the carboxyl, but is 1150cm^-1And a new absorption peak appears, which is the absorption peak of the C-O bond in the aldehyde group, and can prove that part of hydroxyl in the sodium alginate is oxidized into the aldehyde group.

(4) Swelling Rate test

The weight of the hydrogel was weighed using balance and recorded as W₀The hydrogel was placed in PBS (0.01M, pH 7.4) and at a specific time point, the surface of the hydrogel was quickly wiped off with a slightly moistened filter paper (moist atraumatic gel), and weighed and reported as W_s. Each sample was run in parallel for three times and averaged, and the swelling ratio of the sample was calculated as follows:

wherein, W₀Weight of hydrogel (g); w_sWeight (g) of hydrogel after water absorption; x is the swelling ratio (%) of the hydrogel.

FIG. 4 is a graph showing the swelling ratios of the samples of example 1(OSA/CMC) and example 5(OSA/CMC/TA) which are not very different from each other, and the swelling ratio of the hydrogel loaded with tannic acid is slightly reduced compared with that of the pure water gel.

(5) Test for degradation Properties

Get water to be condensedThe mass of the gum after it has been released from the mould is designated W₀Then, the mixture was immersed in a PBS solution at pH 7.4 and pH 5.5 and placed on a constant temperature shaker (37 ℃ C., 70 rpm). At the time points of measurement (1, 3, 7, 14, 21 days), the hydrogel was removed, the surface of the hydrogel was quickly wiped off with slightly wetted filter paper, and the mass was accurately weighed and recorded (W)₁). Calculating the weight ratio after degradation by adopting a formula: weight after degradation is W₁/W₀*100％

Fig. 5 is a graph showing the degradation performance of example 1 in PBS buffer solution with PH 7.4 and PH 5.5, and it can be found that the hydrogel degrades in the solution with PH 5.5 significantly faster than in the solution with PH 7.4, because the hydrogel is a schiff base crosslinked hydrogel, and the C ═ N double bond is unstable and easily dissociates under acidic conditions.

(6) Compression modulus test

The diameter and length of the hydrogel were measured using a vernier caliper, and the compression modulus of elasticity of the sample was tested using an electronic universal tester at a deformation rate of 1mm/min within 40% deformation.

FIG. 6 shows the results of the compressive strength tests of examples 1 to 5, from which it can be seen that the compressive strength of the hydrogel is the greatest when the OSA concentration is constant and the final concentration of CMC is 3%; pure hydrogel has a compression strength comparable to that of hydrogel loaded with tannic acid.

(7) Water vapor transmission rate test

The Water Vapor Transmission Rate (WVTR) of the hydrogel was determined according to ASTM E96-00 by the United states Bureau of standards. The method comprises the following specific steps: first, the hydrogel was placed at the mouth of a vial (diameter 9.67mm) already filled with 5mL of deionized water, the gap between the hydrogel and the mouth was sealed with vaseline to prevent water vapor from escaping, and the initial weight was weighed. Next, the hydrogel-covered sample bottle was placed in a constant temperature and humidity incubator (temperature 37 ℃, relative humidity 79%), and a sample bottle containing only 5mL of deionized water was used as a blank control group. After 24h, the mixture was taken out and weighed. The water vapor transmission rate is calculated according to the following formula:

wherein, Deltam/Deltat is the water loss weight loss (g/day) of 24 hours, and A is the surface area (m) of the bottle mouth²)。

Fig. 7 shows the water vapor transmission rate test results of examples 1 to 5, and it can be seen from the figure that examples 1 to 5 all have good water vapor transmission rate, good water and air permeable functions, and can maintain a high humidity environment for the wound surface to promote its rapid healing.

(8) Hydrogel in vitro drug release test

Adding the prepared drug-loaded composite hydrogel into 10mL of PBS solution, placing the solution in a constant-temperature shaking table at 37 ℃, taking out 1mL of aqueous solution at a specific time point, adding 1mL of new PBS to keep the volume of the 10mL of PBS solution unchanged, measuring the concentration of the drug in the taken-out solution by using an ultraviolet spectrophotometer, and continuously measuring the release behavior of the drug-loaded hydrogel for 7 days.

Fig. 8 is a graph of the results of example 5 drug release in PBS solution at PH 7.4 and PH 5.5, from which it can be seen that the drug released most of the tannins on the first day and then slowly released over a subsequent period. In a PBS (phosphate buffer solution) with the pH value of 5.5, tannin is released more quickly than the PBS with the pH value of 7.4, the final release amount is more than the PBS with the pH value of 7.4, and the pH environment of a wound is acidic.

(9) Biocompatibility testing

Cultured L929 cells were digested with 0.25% trypsin and suspended at a density of 2X 10 per well⁴one/mL cell suspension was seeded in 48-well plates. After 12h of culture, the original culture solution was taken out, and 500. mu.L of the leaching solution of the experimental material was added to each well dish, and only 500. mu.L of complete medium was added as a blank control. Each group is provided with at least 5 holes. Liquid is changed every 24h, and two time points of 24h and 48h are set in the experiment. The specific operation method comprises the following steps:

cell survival rate: cell viability was quantified using CCK 8. Taking out corresponding holes at specified time intervalsPlate, 100. mu.L of CCK8 working solution per well, carbon dioxide incubator (containing 5% CO) at constant temperature of 37 deg.C₂) After incubation for 1-2 h, measuring absorbance (OD) at the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to a formula:

cell survival (%) ═ OD_{Short experiment}/OD_{Contrast medium}×100％。

FIG. 9 is a statistical graph of cell viability for examples 1 and 5, and it can be seen that the cell viability of tannin-loaded hydrogels was slightly decreased compared to that of pure hydrogel, but both survivors were over 80%, which confirms that both hydrogel materials and tannin-loaded composite hydrogels have better biocompatibility and can be used for treating skin wounds.

(10) Zone of inhibition test

A round sample of the prepared hydrogel (diameter 8mm) was sterilized by UV irradiation on a clean bench for 30 min. Dripping 100 μ L of the bacterial suspension on a solid LB culture medium, uniformly coating and sticking a sample to be detected on a coating rod, rightly placing for 15min, and placing the culture dish in a 37 ℃ biochemical incubator for inverted culture. After 24h of culture, the growth of bacteria on the culture medium was observed, the diameter (D) of the zone of inhibition was recorded, and the hydrogel prepared in examples 1 to 7 was subjected to zone of inhibition tests, the test results being shown in Table 1.

TABLE 1

As can be seen from the table 1, the hydrogel has a certain antibacterial effect, the concentration of the OSA is controlled to be unchanged, the antibacterial effect of the CMC is enhanced along with the increase of the concentration within the concentration range of 2-3%, and when the final concentration of the CMC is increased to 3.5%, the antibacterial effect is not great as that of 3%; after the tannin is loaded, the antibacterial effect of the hydrogel is obviously enhanced. The antibacterial effect of example 5 is the best, and the hydrogel comprises the following components in percentage by mass and volume: the best antibacterial effect is achieved when the content of the carboxymethyl chitosan is 3%, the content of the oxidized sodium alginate is 5%, and the content of the tannic acid is 0.5%.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antibacterial composite hydrogel is characterized by comprising the following components in percentage by mass and volume: 2-3.5% of carboxymethyl chitosan, 4-6% of oxidized sodium alginate and 0.3-0.6% of tannic acid, wherein the solution of the hydrogel is PBS buffer solution.

2. The hydrogel according to claim 1, comprising the following components in mass volume percent: 3% of carboxymethyl chitosan, 5% of oxidized sodium alginate, 0.5% of tannic acid and the solution of the hydrogel is PBS buffer solution.

3. The hydrogel according to claim 1 or 2, wherein the oxidized sodium alginate is prepared by a method comprising: dissolving sodium alginate in ethanol, heating in water bath, slowly dropwise adding sodium periodate aqueous solution into sodium alginate solution after sodium alginate is completely dissolved, and stirring for reaction; after the reaction is finished, dropwise adding ethylene glycol, continuously stirring, then adding sodium chloride, pouring the reaction solution into absolute ethyl alcohol, and separating out a precipitate; vacuum filtering, dissolving the precipitate with pure water, dialyzing, and freeze drying to obtain oxidized sodium alginate.

4. The hydrogel according to claim 3, wherein the mass ratio of sodium alginate to sodium periodate is 1: 1.

5. The hydrogel according to claim 3, wherein the temperature of the water bath heating is 60 ℃, the time of the stirring reaction is 5 hours, the time of the continuous stirring is 1 hour, the cut-off molecular weight of the dialysis bag for dialysis is 8000-15000, and the temperature of the freeze-drying is-80 ℃.

6. A method of preparing a hydrogel according to any one of claims 1 to 5 comprising the steps of:

7. The method of claim 6, wherein the volume ratio of solution A to solution B is 1: 1.

8. the method for preparing a hydrogel according to claim 6, wherein the carboxymethyl chitosan is present in the solution A in an amount of 4 to 7% by mass.

9. The method for preparing hydrogel according to claim 6, wherein the mass volume percentage of oxidized sodium alginate in the solution B is 8-12%.

10. The method of preparing a hydrogel according to claim 6, wherein the mass volume percentage of tannic acid in the solution B is 0.6 to 1.2%.