Chitosan hydrogel, preparation method thereof and application of chitosan hydrogel as corneal injury repair material

Abstract

The invention relates to the technical field of ophthalmic material preparation, and particularly discloses chitosan hydrogel, a preparation method thereof and application of the chitosan hydrogel as a corneal injury repair material. The preparation method of the chitosan hydrogel comprises the following steps: adding urea and urease into a chitosan solution for crosslinking to obtain the chitosan hydrogel; an active component is also added into the chitosan solution; the active component is hydrated catechin and / or gentian xanthol. The chitosan hydrogel prepared by adding hydrated catechin or gentiangxanthol has the effect of promoting the growth of corneal epithelial cells, and can obviously promote the growth of the corneal epithelial cells; therefore, the chitosan hydrogel prepared by the method disclosed by the invention has an important application value when being used as a corneal injury repair material.

Description

Technical Field

[0001] The invention relates to the technical field of ophthalmic material preparation, and in particular to a chitosan hydrogel and a preparation method thereof and application thereof as a corneal damage repair material. Background Art

[0002] The cornea is a transparent fiber membrane without blood vessels located in the front 1 / 6 of the eyeball wall. Corneal injury refers to the damage to the cornea caused by external factors or body factors, including corneal interlayer tear, corneal penetration and rupture, corneal abrasion, corneal contusion, deep corneal tissue loss, etc. If the cornea is seriously injured, surgical repair is required. Corneal injury repair materials are indispensable materials in the process of corneal injury surgical repair.

[0003] For example, Chinese invention patent CN201510413137.4 discloses a method for preparing a hydroxyethyl chitosan in-situ hydrogel; the method first dissolves sodium periodate powder in deionized water to obtain a sodium periodate aqueous solution, then stirs the sodium alginate aqueous solution and the sodium periodate aqueous solution to form a mixed solution to make it undergo an oxidation reaction, then adds ethylene glycol to terminate the reaction, filtrates the mixed solution and uses anhydrous ethanol to dehydrate and precipitate, then low-temperature vacuum drying to obtain a crude oxidized sodium alginate product, then dissolves it in distilled water to form a crude oxidized sodium alginate aqueous solution, dialyzes it completely, centrifuges it, and freeze-dries the supernatant to obtain oxidized sodium alginate, finally mixes hydroxyethyl chitosan and oxidized sodium alginate, and squeezes and pushes them into a test tube through a double syringe cross-linking reaction to obtain a hydroxyethyl chitosan in-situ hydrogel for rapid encapsulation and transplantation of limbal stem cells. However, the method requires the use of sodium periodate to oxidize sodium alginate to prepare oxidized sodium alginate, and then the oxidized sodium alginate reacts with hydroxyethyl chitosan to prepare a hydrogel; its preparation method is relatively complicated.

[0004] Chinese invention patent CN201910934544.8 discloses a method for preparing chitosan hydrogel; the method is specifically: adding urea and urease to a chitosan solution, urease catalyzing urea hydrolysis, uniformly increasing the pH value of the chitosan solution to gel it, and obtaining a chitosan hydrogel; the concentration of the chitosan solution is 1-5g / 100ml; the amount of urea added to the chitosan solution is 30-60mM; the amount of urease added to the chitosan solution is 3-60U / ml. This method is simpler than CN201510413137.4. However, the inventors found in their research that when the chitosan hydrogel prepared by this method is used as a corneal damage repair material, its effect of promoting the growth of corneal epithelial cells needs to be further improved. Summary of the invention

[0005] In order to solve at least one of the technical problems existing in the prior art, the present invention provides a chitosan hydrogel and a preparation method thereof and an application thereof as a corneal damage repair material.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention first provides a method for preparing chitosan hydrogel, which comprises the following steps: adding urea and urease to a chitosan solution for cross-linking to obtain a chitosan hydrogel; further adding an active ingredient to the chitosan solution;

[0008] The active ingredients are hydrated catechins and / or gentiananthol.

[0009] The inventors surprisingly found in the research that in the preparation process of the chitosan hydrogel of the present invention, the chitosan hydrogel prepared by adding hydrated catechins or gentiana xanthophylls has the effect of promoting the growth of corneal epithelial cells, and can significantly promote the growth of corneal epithelial cells. Among them, the chitosan hydrogel prepared by adding hydrated catechins has a higher effect of promoting the growth of corneal epithelial cells than the chitosan hydrogel prepared by adding gentiana xanthophylls.

[0010] Preferably, the ratio of the amount of active ingredient added to the amount of chitosan solution is 10-30 g:1L.

[0011] Most preferably, the ratio of the amount of active ingredient added to the amount of chitosan solution is 20g:1L.

[0012] Preferably, the active ingredients consist of hydrated catechins and gentiananthol.

[0013] Preferably, the mass ratio of hydrated catechins to gentiananthol in the active ingredient is 2 to 4:1.

[0014] Most preferably, the mass ratio of hydrated catechins to gentiananthol in the active ingredient is 3:1.

[0015] The inventors further surprisingly found in the study that in the preparation process of the chitosan hydrogel of the present invention, the chitosan hydrogel prepared by adding hydrated catechins and gentiana xanthophenol at the same time can greatly promote the growth of corneal epithelial cells; its effect of promoting the growth of corneal epithelial cells is further significantly higher than that of the chitosan hydrogel prepared by adding hydrated catechins or gentiana xanthophenol alone. In the preparation process of the chitosan hydrogel of the present invention, hydrated catechins and gentiana xanthophenol are added at the same time, which can synergistically improve the effect of the prepared chitosan hydrogel on promoting the growth of corneal epithelial cells.

[0016] Preferably, the chitosan solution is a chitosan acetic acid solution.

[0017] Preferably, the concentration of chitosan in the chitosan solution acetic acid solution is 30-100 g / L, and the concentration of acetic acid is 0.1-0.5 mol / L.

[0018] Most preferably, the concentration of chitosan in the chitosan solution acetic acid solution is 50 g / L, and the concentration of acetic acid is 0.2 mol / L.

[0019] Preferably, the usage ratio of urea to chitosan solution is 0.1-1 mol:1 L; the usage ratio of urease to chitosan solution is 30-100 U:1 mL.

[0020] Most preferably, the usage ratio of urea to chitosan solution is 0.5 mol:1 L; the usage ratio of urease to chitosan solution is 50 U:1 mL.

[0021] Preferably, the cross-linking specifically refers to cross-linking at 30-50° C. for 2-4 hours.

[0022] Most preferably, the cross-linking specifically refers to cross-linking at 45° C. for 3 hours.

[0023] The invention also provides a chitosan hydrogel prepared by the preparation method.

[0024] The present invention also provides an application of the chitosan hydrogel as a corneal damage repair material.

[0025] Beneficial effects: The present invention provides a novel method for preparing chitosan hydrogel; the chitosan hydrogel prepared by adding hydrated catechin or gentiananthone has the effect of promoting the growth of corneal epithelial cells, and can significantly promote the growth of corneal epithelial cells; therefore, the chitosan hydrogel prepared by the method of the present invention is used as a corneal damage repair material, which has important application value. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the protection scope of the present invention.

[0027] Example 1 Preparation of chitosan hydrogel

[0028] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 45°C for 3 hours;

[0029] The chitosan solution is a chitosan solution acetic acid solution; the chitosan concentration in the chitosan solution acetic acid solution is 50 g / L, and the acetic acid concentration is 0.2 mol / L;

[0030] The dosage ratio of urea to chitosan solution is 0.5 mol:1 L; the dosage ratio of urease to chitosan solution is 50 U:1 mL;

[0031] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 20g:1L; the active ingredient is hydrated catechin.

[0032] Example 2 Preparation of chitosan hydrogel

[0033] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 45°C for 3 hours;

[0034] The chitosan solution is a chitosan solution acetic acid solution; the chitosan concentration in the chitosan solution acetic acid solution is 50 g / L, and the acetic acid concentration is 0.2 mol / L;

[0035] The dosage ratio of urea to chitosan solution is 0.5 mol:1 L; the dosage ratio of urease to chitosan solution is 50 U:1 mL;

[0036] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 20g:1L; the active ingredient is gentiananthone.

[0037] Example 3 Preparation of chitosan hydrogel

[0038] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 45°C for 3 hours;

[0039] The chitosan solution is a chitosan solution acetic acid solution; the chitosan concentration in the chitosan solution acetic acid solution is 50 g / L, and the acetic acid concentration is 0.2 mol / L;

[0040] The dosage ratio of urea to chitosan solution is 0.5 mol:1 L; the dosage ratio of urease to chitosan solution is 50 U:1 mL;

[0041] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 20g:1L; the active ingredient is composed of hydrated catechin and gentiananthone in a mass ratio of 3:1.

[0042] Example 4 Preparation of chitosan hydrogel

[0043] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 50°C for 2 hours;

[0044] The chitosan solution is a chitosan solution acetic acid solution; the concentration of chitosan in the chitosan solution acetic acid solution is 100 g / L, and the concentration of acetic acid is 0.1 mol / L;

[0045] The dosage ratio of urea to chitosan solution is 0.1 mol:1 L; the dosage ratio of urease to chitosan solution is 30 U:1 mL;

[0046] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 30g:1L; the active ingredient is hydrated catechin.

[0047] Example 5 Preparation of chitosan hydrogel

[0048] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 30°C for 4 hours;

[0049] The chitosan solution is a chitosan solution acetic acid solution; the concentration of chitosan in the chitosan solution acetic acid solution is 30 g / L, and the concentration of acetic acid is 0.5 mol / L;

[0050] The dosage ratio of urea to chitosan solution is 1 mol:1 L; the dosage ratio of urease to chitosan solution is 100 U:1 mL;

[0051] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 10g:1L; the active ingredient is hydrated catechin.

[0052] Example 6 Preparation of chitosan hydrogel

[0053] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 50°C for 2 hours;

[0054] The chitosan solution is a chitosan solution acetic acid solution; the concentration of chitosan in the chitosan solution acetic acid solution is 100 g / L, and the concentration of acetic acid is 0.1 mol / L;

[0055] The dosage ratio of urea to chitosan solution is 0.1 mol:1 L; the dosage ratio of urease to chitosan solution is 30 U:1 mL;

[0056] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 20g:1L; the active ingredient consists of hydrated catechin and gentiananthone in a mass ratio of 2:1.

[0057] Example 7 Preparation of chitosan hydrogel

[0058] Urea, urease and active ingredients were added to the chitosan solution, and chitosan hydrogel was obtained after cross-linking at 30°C for 4 hours;

[0059] The chitosan solution is a chitosan solution acetic acid solution; the concentration of chitosan in the chitosan solution acetic acid solution is 30 g / L, and the concentration of acetic acid is 0.5 mol / L;

[0060] The dosage ratio of urea to chitosan solution is 1 mol:1 L; the dosage ratio of urease to chitosan solution is 100 U:1 mL;

[0061] The ratio of the amount of the active ingredient added to the amount of the chitosan solution is 10g:1L; the active ingredient is composed of hydrated catechin and gentiananthone in a mass ratio of 4:1.

[0062] Experimental Example 1 Experiment on promoting corneal epithelial cell proliferation

[0063] The chitosan hydrogels prepared in Examples 1 to 3 were crushed and added to the DMEM medium at a weight ratio of 10%, and stirred evenly to obtain the DMEM medium containing the sample to be tested; 1×10 4 Then, 200 μL of DMEM medium containing the sample to be tested was added to each well in the experimental group; 200 μL of DMEM medium was added to the blank control group; after culturing for 24 h, the OD value of each well at 450 nm was tested with a calibration instrument; the proliferation rate of corneal epithelial cells was calculated according to the OD value; the test results are shown in Table 1.

[0064] Corneal epithelial cell proliferation rate (%) = [(OD 实验 -OD 空白 ) / OD 空白 ]*100%.

[0065] Table 1. Results of corneal epithelial cell proliferation assay

[0066] Corneal epithelial cell proliferation rate Chitosan hydrogel prepared in Example 1 58.7％ Chitosan hydrogel prepared in Example 2 45.6％ Chitosan hydrogel prepared in Example 3 86.3％

[0067] It can be seen from the experiment in Table 1 that the chitosan hydrogels prepared in Examples 1 and 2 both have a certain corneal epithelial cell proliferation rate; and the corneal epithelial cell proliferation rate of Example 1 is significantly higher than that of Example 2; this shows that: in the preparation process of the chitosan hydrogel of the present invention, the chitosan hydrogel prepared by adding hydrated catechins or gentiana xanthophylls has the effect of promoting the growth of corneal epithelial cells, and can significantly promote the growth of corneal epithelial cells. Among them, the chitosan hydrogel prepared by adding hydrated catechins has a higher effect of promoting the growth of corneal epithelial cells than the chitosan hydrogel prepared by adding gentiana xanthophylls.

[0068] It can be seen from the experiment in Table 1 that the corneal epithelial cell proliferation rate of the chitosan hydrogel prepared in Example 3 is much higher than that of the chitosan hydrogel prepared in Examples 1 and 2. This shows that: in the preparation process of the chitosan hydrogel of the present invention, the chitosan hydrogel prepared by adding hydrated catechins and gentiana xanthophenol at the same time can greatly promote the growth of corneal epithelial cells; its effect of promoting the growth of corneal epithelial cells is further much higher than that of the chitosan hydrogel prepared by adding hydrated catechins or gentiana xanthophenol alone. In the preparation process of the chitosan hydrogel of the present invention, hydrated catechins and gentiana xanthophenol are added at the same time, which can synergistically improve the effect of the prepared chitosan hydrogel in promoting the growth of corneal epithelial cells.

Claims

1. A method for preparing chitosan hydrogel, comprising the following steps: adding urea and urease to a chitosan solution for cross-linking to obtain a chitosan hydrogel; characterized in that: Active ingredients are also added to the chitosan solution; The active ingredients are hydrated catechins and / or gentiananthol.

2. The method for preparing chitosan hydrogel according to claim 1, characterized in that: The ratio of the amount of active ingredient added to the amount of chitosan solution is 10-30g:1L.

3. The method for preparing chitosan hydrogel according to claim 1, characterized in that: The active ingredients consist of hydrated catechin and gentiananthone.

4. The method for preparing chitosan hydrogel according to claim 3, characterized in that: The mass ratio of hydrated catechin to gentiananthone in the active ingredients is 2-4:

1.

5. The method for preparing chitosan hydrogel according to claim 1, characterized in that: The chitosan solution is chitosan solution acetic acid solution.

6. The method for preparing chitosan hydrogel according to claim 5, characterized in that: The concentration of chitosan in the chitosan solution acetic acid solution is 30-100 g / L, and the concentration of acetic acid is 0.1-0.5 mol / L.

7. The method for preparing chitosan hydrogel according to claim 1, characterized in that: The dosage ratio of urea to chitosan solution is 0.1-1 mol:1L; the dosage ratio of urease to chitosan solution is 30-100 U:1mL.

8. The method for preparing chitosan hydrogel according to claim 1, characterized in that: The cross-linking specifically refers to cross-linking at 30-50° C. for 2-4 hours.

9. The chitosan hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the chitosan hydrogel according to claim 9 as a corneal damage repair material.