Disclosure of Invention
The invention aims to provide a self-crosslinking hydrogel with synergistic action of sugar and enzyme and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for preparing a self-crosslinking hydrogel, comprising the steps of:
mixing carboxymethyl chitosan, glucose oxidase and peroxidase in water, and standing to obtain the self-crosslinking hydrogel.
Further, the method comprises the following steps:
dissolving carboxymethyl chitosan in water to obtain solution A; and (3) performing enzymolysis on glucose, glucose oxidase and peroxide in water to obtain a solution B, mixing the solution A and the solution B, and standing to obtain the crosslinked hydrogel.
Further, the method comprises the following steps:
(1) mixing glucose and carboxymethyl chitosan to obtain solution C;
(2) dissolving glucose oxidase and peroxidase in water to obtain solution D;
(3) the solutions C and D were mixed and left to stand to obtain a crosslinked hydrogel.
Further, the peroxidase is at least one of catalase and horseradish peroxidase.
Further, the mass concentration ratio of the glucose to the carboxymethyl chitosan in the step (1) is (0.5-1.7): (0.6-1.8)%.
Further, the mass ratio of the glucose oxidase to the peroxidase in the step (2) is 0.0025-0.0030: 0.0025-0.0030 g.
Further, the mass concentration of the carboxymethyl chitosan in the solution A is 0.6-1.8%; standing for 4-5 min.
Further, the enzyme in the solution B is glucose oxidase and/or peroxidase, and the mass concentration of the enzyme is 0.5-1.7%.
A self-crosslinking hydrogel prepared by the method of any one of the preceding claims, wherein the hydrogel comprises glucose, chitosan, glucose oxidase and peroxidase.
Further, the chitosan is carboxymethyl chitosan; the peroxidase is at least one of catalase and horseradish peroxidase.
Further, the hydrogel comprises the following components in parts by weight: 0.5-1.7% of glucose, 0.6-1.8% of carboxymethyl chitosan, 0.1-1% of glucose oxidase and 0.1-1% of catalase.
And further, standing for 20-25 minutes.
When the inventor carries out early experiments, the hydrogel is formed for too long time, and the experimental result is influenced. It was discovered occasionally later that mixing glucose with the enzyme followed by the carboxymethyl chitosan solution significantly increased the gel formation time of the gel. The compression is carried out from more than ten minutes to several minutes, so that the working efficiency is greatly improved. And no pH regulator is needed to be added additionally, the reaction condition is mild, and the biocompatibility is good.
The Glucose Oxidase (GOD) can catalyze and oxidize glucose into gluconic acid, plays a role in regulating the pH of a system, and does not need to add an additional pH regulator; the byproduct hydrogen peroxide generated by enzyme-catalyzed oxidation of glucose has an inhibiting effect on the activity of enzyme, the response time of hydrogel to the change of glucose concentration in the environment is influenced, catalase (POD) and GOD are jointly fixed in the hydrogel, the POD can catalyze and decompose the hydrogen peroxide to generate oxygen, the oxygen consumed by GOD reaction in the gel is made up, and the activity utilization rate of the enzyme is improved.
The invention has the beneficial effects that:
the self-crosslinking hydrogel with the synergistic effect of the carboxymethyl chitosan and the enzyme does not need to add a crosslinking agent or an initiator with biological/cell toxicity or an additional pH regulator, has mild reaction conditions, good biocompatibility and strong bacteriostatic effect. The raw materials are all green and environment-friendly, the resource is rich, the price is low and the biocompatibility is good.
Detailed Description
A method for preparing a self-crosslinking hydrogel, comprising the steps of:
mixing carboxymethyl chitosan, glucose oxidase and peroxidase in water, and standing to obtain the self-crosslinking hydrogel.
Preferably, the method comprises the following steps:
dissolving carboxymethyl chitosan in water to obtain solution A; and (3) performing enzymolysis on glucose, glucose oxidase and peroxide in water to obtain a solution B, mixing the solution A and the solution B, and standing to obtain the crosslinked hydrogel.
Preferably, the method comprises the following steps:
(1) mixing glucose and carboxymethyl chitosan to obtain solution C;
(2) dissolving glucose oxidase and peroxidase in water to obtain solution D;
(3) the solutions C and D were mixed and left to stand to obtain a crosslinked hydrogel.
Preferably, the peroxidase is at least one of catalase and horseradish peroxidase.
Preferably, the mass concentration ratio of the glucose to the carboxymethyl chitosan in the step (1) is (0.5-1.7): (0.6-1.8)%.
Preferably, the mass ratio of the glucose oxidase to the peroxidase in the step (2) is 0.0025-0.0030: 0.0025-0.0030 g.
Preferably, the mass concentration of the carboxymethyl chitosan in the solution A is 0.6-1.8%; standing for 4-5 min; standing for 4-5 min.
A self-crosslinking hydrogel prepared by the method of any one of the preceding claims, wherein the hydrogel comprises glucose, chitosan, glucose oxidase and peroxidase.
Preferably, the chitosan is carboxymethyl chitosan; the peroxidase is at least one of catalase and horseradish peroxidase.
Preferably, the hydrogel consists of the following components in parts by weight: 0.5-1.7% of glucose, 0.6-1.8% of carboxymethyl chitosan, 0.1-1% of glucose oxidase and 0.1-1% of catalase.
A method for preparing a self-crosslinking hydrogel, comprising the steps of: mixing carboxymethyl chitosan, glucose oxidase and peroxidase in water, and standing to obtain the self-crosslinking hydrogel.
Example 1
Solution A: 1ml 1.8% carboxymethyl chitosan solution
Solution B: 0.1514g glucose, 0.5mL ultrapure water, 0.0025g glucose oxidase, 0.0025g catalase
Gel time: 4 to 5 minutes
The preparation method comprises the following steps: dissolving 1 mL1.8% carboxymethyl chitosan in water to obtain solution A; 0.1514g glucose, 0.0025g glucose oxidase and 0.0025g hydrogen peroxide are enzymolyzed in 0.5mL water to obtain solution B; and mixing the solution A and the solution B, and standing for 4-5 minutes to obtain the crosslinked hydrogel.
Example 2:
solution A: 1ml 1.4% carboxymethyl chitosan solution
Solution B: 0.1514g glucose, 0.5mL ultrapure water, 0.0025g glucose oxidase, 0.0025g catalase
Gel time: 4 to 5 minutes
The preparation method comprises the following steps: dissolving 1 mL1.4% carboxymethyl chitosan in water to obtain solution A; 0.1514g glucose, 0.0025g glucose oxidase and 0.0025g hydrogen peroxide are enzymolyzed in 0.5mL water to obtain solution B; and mixing the solution A and the solution B, and standing for 4-5 minutes to obtain the crosslinked hydrogel.
Example 3:
solution A: 1mL 1% carboxymethyl chitosan solution
Solution B: 0.1514g glucose, 0.5mL ultrapure water, 0.0025g glucose oxidase, 0.0025g catalase
Gel time: 4 to 5 minutes
The preparation method comprises the following steps: dissolving 1mL of 1% carboxymethyl chitosan in water to obtain solution A; 0.1514g glucose, 0.0025g glucose oxidase and 0.0025g hydrogen peroxide are enzymolyzed in 0.5mL water to obtain solution B; and mixing the solution A and the solution B, and standing for 4-5 minutes to obtain the crosslinked hydrogel.
Comparative example 1:
solution A: 0.1514g glucose, 1 mL1.8% carboxymethyl chitosan solution
Solution B: 0.0025g of glucose oxidase, 0.0025g of catalase and 0.3mL of ultrapure water were dissolved
Gel time: 10 to 20 minutes
The preparation method comprises the following steps: (1) 0.1514g glucose and 1ml 1.8% carboxymethyl chitosan are mixed to obtain solution C, 0.0025g glucose oxidase and 0.0025g peroxidase are dissolved in water to obtain solution D; (2) and mixing the solutions C and D, keeping the initial pH value at 6-7, and standing for 10-20 minutes to obtain the hydrogel.
Comparative example 2:
solution A: 0.1514g glucose, 1 mL1.4% carboxymethyl chitosan solution
Solution B: 0.0025g of glucose oxidase, 0.0025g of catalase and 0.5mL of ultrapure water were dissolved
Gel time: 10 to 20 minutes
The preparation method is the same as that of comparative example 1.
Comparative example 3:
solution A: 0.1514g glucose, 1mL 1% carboxymethyl chitosan solution
Solution B: 0.0025g of glucose oxidase, 0.0025g of catalase and 0.3mL of ultrapure water were dissolved
Gel time: 10 to 20 minutes
The preparation method is the same as that of comparative example 1.
The hydrogel of the above example was further tested for its effectiveness.
Gel collapsibility:
after the hydrogel prepared in the example is added into water and soaked for 24 hours, 48 hours and 72 hours, the shape of the gel is basically unchanged by visual observation. The gel did not collapse, only turning yellow in color.
The main key performance indexes are as follows:
appearance: colorless transparent gel, and turns yellow transparent gel after standing for a period of time. FIG. 1 is a state before the hydrogel of the present invention is gelled, and FIG. 2 is a state after the hydrogel is gelled.
Cytotoxicity: the cytotoxic response was less than grade 1.
Sensitization test: no delayed hypersensitivity reaction.
And (3) gelling time: less than 20min and short time.
Bacteriostasis: the composite gel prepared by the invention has stronger antibacterial effect on staphylococcus aureus, escherichia coli and candida albicans, can be used as a wound dressing, and plays roles in protecting and obstructing external bacteria and preventing infection.
And (3) determination of antibacterial activity:
the antibacterial effect of the composite gel on the strains to be detected (staphylococcus aureus, escherichia coli and candida albicans) is determined by adopting an agar diffusion paper sheet method and a colony counting method.
The method comprises the following steps: preparation of a catalyst containing 1X 108Each/mL of the bacterial suspension of the strain to be detected is evenly coated on the surface of a solid nutrient agar medium plate, and each plate is coated with 100 mu L. Under the aseptic condition, the composite gel prepared by the invention is uniformly coated on the surface of a filter paper sheet which is sterilized and dried to be used as a composite gel paper sheet. The prepared composite gel paper sheet (with the sterilized filter paper sheet as a blank control) was attached to the surface of the plate. Placing four pieces of paper on each plate for biological repetition, and culturing at constant temperature of 37 deg.C for 24 timesh. And (3) gradient dilution of the contact surface culture (4-5 gradients of Candida albicans dilution and 5-6 gradients of staphylococcus aureus and Escherichia coli), putting 1mL of diluted bacterial suspension on a flat plate, pouring agar into the flat plate, uniformly mixing, after solidification, inversely putting the flat plate in a 37 ℃ incubator for culture for 24 hours, and calculating the total number of colonies by referring to GB 47892-2016.
The inhibition ratio R/% (total number of blank colonies-total number of gel colonies)/total number of blank colonies × 100.
TABLE 1 bacteriostatic ratio (%) of hydrogel of carboxymethyl chitosan at various concentrations
As can be seen from table 1, the hydrogels of example 1 (1.8%), example 2 (1.4%) and example 3 (1%) all have strong bacteriostatic effects on staphylococcus aureus, escherichia coli and candida albicans, wherein the hydrogel bacteriostatic rates of example 1 (1.8%) reach 98%, 95% and 96%, respectively.
In vitro cytotoxicity assay
In vitro cytotoxicity tests were performed on the composite hydrogels prepared in example 1 (1.8%), example 2 (1.4%), and example 3 (1%). The specific test method is as follows:
3T3(L929) cells were passaged more than 2 times and stably grown. After the pancreatin digestion, human cell culture solution is added, and counting is carried out under a microscope after the cell culture solution is uniformly blown. According to the appropriate cell concentration, the cells were diluted to 250000/mL by adding culture solution and then inoculated into 10mL dishes. Rotating to uniformly disperse cells on the surface of the culture dish, placing at 37 ℃ and 5% CO2Culturing in an incubator.
After the cells grew into a monolayer, the original culture solution was aspirated, and 1.2% agar medium and l 0% MEM medium were preheated to 39 ℃ respectively, and then mixed uniformly at a volume ratio of 1:1 and coagulated. The amount of the cell layer to be added is preferably just enough to cover the cell layer. 5mL of 0. l% neutral red staining solution was added to each dish, and the mixture was placed in an incubator and incubated in the dark for 20min, and then the excess staining solution was discarded.
The test sample was gently placed on a solid nutrient agar medium plate, with the sample covering about one-tenth of the cell layer surface. Negative and positive controls were prepared in the same manner, and the dishes were placed in an incubator for further 24 h. After the culture was completed, the outline of the test sample was marked on the bottom of the petri dish, and the sample was removed. The culture dish was placed under a microscope to observe the discoloration of the cell area.
As a result: the results show that the gels of example 1 (1.8%), example 2 (1.4%) and example 3 (1%) had a grade 1 cytotoxicity response.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.