Hyaluronic acid hydrogel as well as preparation method and application thereof in preparation of corneal injury repair material

Abstract

The invention relates to the technical field of biological material preparation, and particularly discloses hyaluronic acid hydrogel as well as a preparation method and application thereof in preparation of a corneal injury repair material. The preparation method of the hyaluronic acid hydrogel comprises the following steps: S1, preparing a hyaluronic acid solution; s2, adding a cross-linking agent into the hyaluronic acid solution, and adjusting to acidic pH to obtain a solution A; s3, adding a carboxyl activator into the solution A, and stirring to obtain hyaluronic acid hydrogel; a preparation method of the hyaluronic acid solution in the step S1 comprises the following steps: taking hyaluronic acid, adding the hyaluronic acid into the traditional Chinese medicine extracting solution, and uniformly stirring to obtain the hyaluronic acid solution. Researches show that the hyaluronic acid hydrogel prepared by the method disclosed by the invention has the effect of promoting proliferation of corneal epithelial cells; therefore, the material has important application value when being used as a corneal injury repair material.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial preparation, and in particular to a hyaluronic acid hydrogel and a preparation method thereof, and application thereof in the preparation of corneal damage repair materials. Background Art

[0002] The cornea is a blood-free, transparent fibrous membrane located in the front 1 / 6 of the eyeball wall. It contains abundant sensory nerve endings, and any slight stimulation, damage or inflammation can cause pain and tears. Corneal injury refers to damage to the cornea caused by external factors or body factors, including corneal abrasions, deep corneal tissue loss, corneal contusions, corneal interlayer tears, corneal penetration and rupture injuries. The specific method of corneal injury repair depends on the specific degree of corneal injury; if the corneal injury is large and symptoms such as corneal lacerations or tears appear, surgical repair is required. In the process of corneal injury surgical repair, corneal injury repair materials are essential materials.

[0003] Chinese invention patent CN200910237605.1 discloses a hyaluronic acid and sodium alginate composite hydrogel and its preparation method. The composite hydrogel is prepared according to a method comprising the following steps: 1) dissolving hyaluronic acid and sodium alginate in water to obtain solution 1, 2) adding a cross-linking agent to the solution 1, and adjusting the pH value to 4.0-5.5 with a buffer solution to obtain a solution 2; 3) adding a carboxyl activator to the solution 2 and stirring, and regulating the pH value of the solution 2 to 2.0-5.5 with a buffer solution during stirring until a gel is formed, that is, a hyaluronic acid and sodium alginate composite hydrogel is obtained. The composite modified material prepared by this method has the advantages of high mechanical properties, resistance to hyaluronidase degradation, biocompatibility, etc., and has potential uses in surgery and tissue engineering. However, the inventor found in the study that the corneal damage repair material prepared by this method has a corneal epithelial cell proliferation effect that needs to be further improved. Summary of the invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a hyaluronic acid hydrogel and a preparation method thereof and an application thereof in preparing a corneal damage repair material.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention first provides a method for preparing a hyaluronic acid hydrogel, comprising the following steps:

[0007] S1. preparing hyaluronic acid solution;

[0008] S2. adding a crosslinking agent to the hyaluronic acid solution and adjusting the pH to an acidic pH to obtain solution A;

[0009] S3. Adding a carboxyl activator to solution A and stirring to obtain a hyaluronic acid hydrogel;

[0010] The preparation method of the hyaluronic acid solution in step S1 is: taking hyaluronic acid, adding it to the traditional Chinese medicine extract, and stirring evenly to obtain the hyaluronic acid solution.

[0011] The inventors found in their research that in the preparation process of hyaluronic acid hydrogel, the hyaluronic acid is dissolved to obtain the hyaluronic acid hydrogel prepared in the traditional Chinese medicine extract, which has the effect of promoting the proliferation of corneal epithelial cells. This is because the traditional Chinese medicine extract contains a variety of active ingredients that have the effect of promoting the proliferation of corneal epithelial cells; they participate in the cross-linking of hyaluronic acid, so that the prepared hyaluronic acid hydrogel is cross-linked with the active ingredients that promote the proliferation of corneal epithelial cells, thereby making the prepared hyaluronic acid hydrogel have the effect of promoting the proliferation of corneal epithelial cells.

[0012] Preferably, in step S1, the dosage ratio of hyaluronic acid to the traditional Chinese medicine extract is 0.2-2 g:100 mL.

[0013] Most preferably, in step S1, the dosage ratio of hyaluronic acid to the traditional Chinese medicine extract is 1 g:100 mL.

[0014] Preferably, the Chinese medicine extract is prepared by the following method:

[0015] The mixed Chinese medicine raw material is obtained by mixing the Chinese medicines, and then added into water for heating and reflux extraction. After the extraction is completed, the mixed Chinese medicine raw material is filtered to obtain the Chinese medicine extract.

[0016] Preferably, the traditional Chinese medicine comprises Cassia seed, Chrysanthemum and Honeysuckle.

[0017] The inventors found in their research that the hyaluronic acid hydrogel prepared by dissolving hyaluronic acid in a Chinese medicinal extract obtained by extracting Chinese medicinal materials such as cassia seed, chrysanthemum and honeysuckle has a good effect in promoting the proliferation of corneal epithelial cells.

[0018] Preferably, the weight ratio of Cassia seed, chrysanthemum and honeysuckle is 2-4:1-3:1-2;

[0019] Most preferably, the weight ratio of Cassia seed, chrysanthemum and honeysuckle is 3:2:1.

[0020] Preferably, the ratio of the mixed Chinese medicinal material to water is 1 g: 10-20 mL;

[0021] Most preferably, the ratio of the mixed Chinese medicinal materials to water is 1 g:15 mL.

[0022] Preferably, the heating reflux extraction refers to heating reflux extraction at 80-100° C. for 1-2 hours;

[0023] Most preferably, the heating reflux extraction refers to heating reflux extraction at 100° C. for 1.5 h.

[0024] More preferably, the traditional Chinese medicine comprises Cassia seed, Chrysanthemum, Honeysuckle, Ligustrum lucidum and Codonopsis pilosula.

[0025] The inventors found in further research that the composition of the Chinese medicinal raw materials in the extraction process of the Chinese medicinal extract of the present invention plays an important influence on the effect of the prepared hyaluronic acid hydrogel on promoting the proliferation of corneal epithelial cells; the study showed that on the basis of cassia seed, chrysanthemum and honeysuckle, Ligustrum lucidum and Codonopsis pilosula were further added; the Chinese medicinal extract obtained by extracting cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula as raw materials can greatly improve the effect of the prepared hyaluronic acid hydrogel on promoting the proliferation of corneal epithelial cells compared to the Chinese medicinal extract obtained by extracting cassia seed, chrysanthemum, honeysuckle and other Chinese medicinal raw materials.

[0026] More preferably, the weight ratio of Cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula is 2-4:1-3:1-2:1-3:1-3;

[0027] Most preferably, the weight ratio of Cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula is 3:2:1:2:2.

[0028] Preferably, in step S2, the ratio of the cross-linking agent to the hyaluronic acid solution is 0.1-1 g: 100 mL;

[0029] Most preferably, in step S2, the ratio of the cross-linking agent to the hyaluronic acid solution is 0.5 g:100 mL.

[0030] Preferably, in step S3, the ratio of the carboxyl activator to solution A is 1.5-3 g:100 mL;

[0031] Most preferably, in step S3, the ratio of the carboxyl activator to solution A is 2 g:100 mL.

[0032] Preferably, the cross-linking agent is adipic acid dihydrazide;

[0033] The carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethyl carbon.

[0034] The present invention also provides a hyaluronic acid hydrogel prepared by the above preparation method.

[0035] The present invention also provides an application of the hyaluronic acid hydrogel in preparing corneal damage repair materials.

[0036] Beneficial effects: The present invention provides a novel method for preparing hyaluronic acid hydrogel; studies have shown that the hyaluronic acid hydrogel prepared by the method of the present invention has the effect of promoting the proliferation of corneal epithelial cells; therefore, it has important application value as a corneal damage repair material. DETAILED DESCRIPTION

[0037] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0038] Example 1 Preparation of hyaluronic acid hydrogel

[0039] S1. preparing hyaluronic acid solution;

[0040] S2. Add a crosslinking agent to the hyaluronic acid solution and adjust the pH to 5.0 to obtain solution A; wherein the amount ratio of the crosslinking agent to the hyaluronic acid solution is 0.5 g: 100 mL; the crosslinking agent is adipic acid dihydrazide;

[0041] S3. Add a carboxyl activator to solution A and stir for 4 hours. During the stirring process, the pH is controlled to be 5.0 to obtain a gel. The gel is washed to obtain the hyaluronic acid hydrogel; wherein the amount ratio of the carboxyl activator to solution A is 2 g: 100 mL; the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethyl carbonyl;

[0042] The preparation method of the hyaluronic acid solution in step S1 is: take 1.5×10 6 Da of hyaluronic acid is added to the Chinese medicine extract, and the mixture is stirred evenly to obtain a hyaluronic acid solution; wherein the ratio of hyaluronic acid to the Chinese medicine extract is 1 g: 100 mL;

[0043] The Chinese medicine extract is prepared by the following method:

[0044] A mixed Chinese medicinal raw material is obtained by mixing Cassia seed, chrysanthemum and honeysuckle in a weight ratio of 3:2:1, and then adding the mixture into water and heating reflux extraction at 100°C for 1.5 hours. After the extraction, the mixture is filtered to obtain the Chinese medicinal extract; wherein the dosage ratio of the mixed Chinese medicinal raw material to water is 1g:15mL.

[0045] Example 2 Preparation of hyaluronic acid hydrogel

[0046] The difference between Example 2 and Example 1 is that the preparation method of the Chinese medicine extract is different; the rest is the same as Example 1.

[0047] The Chinese medicine extract is prepared by the following method:

[0048] A mixed Chinese medicinal raw material is obtained by mixing Cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula in a weight ratio of 3:2:1:2:2, and then adding the mixture into water for heating reflux extraction at 100°C for 1.5h. After the extraction, the mixture is filtered to obtain the Chinese medicinal extract; wherein the dosage ratio of the mixed Chinese medicinal raw material to water is 1g:15mL.

[0049] Example 3 Preparation of hyaluronic acid hydrogel

[0050] The difference between Example 3 and Example 2 is that the preparation method of the Chinese medicine extract is different; the rest is the same as Example 2.

[0051] The Chinese medicine extract is prepared by the following method:

[0052] A mixed Chinese medicinal raw material is obtained by mixing Cassia seed, chrysanthemum, honeysuckle and Ligustrum lucidum in a weight ratio of 3:2:1:2, and then adding the mixture into water for heating reflux extraction at 100°C for 1.5h. After the extraction, the mixture is filtered to obtain the Chinese medicinal extract; wherein the dosage ratio of the mixed Chinese medicinal raw material to water is 1g:15mL.

[0053] Example 4 Preparation of hyaluronic acid hydrogel

[0054] The difference between Example 4 and Example 2 is that the preparation method of the Chinese medicine extract is different; the rest is the same as Example 2.

[0055] The Chinese medicine extract is prepared by the following method:

[0056] A mixed Chinese medicinal raw material is obtained by mixing Cassia seed, chrysanthemum, honeysuckle and Codonopsis pilosula in a weight ratio of 3:2:1:2, and then adding the mixture into water for heating reflux extraction at 100°C for 1.5 hours. After the extraction, the mixture is filtered to obtain the Chinese medicinal extract; wherein the dosage ratio of the mixed Chinese medicinal raw material to water is 1g:15mL.

[0057] Experimental Example 1 Experiment on promoting corneal epithelial cell proliferation

[0058] The hyaluronic acid hydrogels prepared in Examples 1 to 4 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.

[0059] Corneal epithelial cell proliferation rate (%) = [(OD 实验 -OD 空白 ) / OD 空白 ]*100%.

[0060] Table 1. Results of corneal epithelial cell proliferation assay

[0061] Corneal epithelial cell proliferation rate Hyaluronic acid hydrogel prepared in Example 1 35.4％ Hyaluronic acid hydrogel prepared in Example 2 60.5％ Example 3 Hyaluronic acid hydrogel prepared 44.1％ Example 4 Hyaluronic acid hydrogel prepared 42.3％

[0062] From the experimental results in Table 1, it can be seen that the corneal epithelial cell proliferation rate of the hyaluronic acid hydrogel prepared in Example 1 reached 35.4%, which shows that the hyaluronic acid hydrogel prepared in the traditional Chinese medicine extract by dissolving hyaluronic acid has the effect of promoting the proliferation of corneal epithelial cells.

[0063] It can be seen from the experimental results in Table 1 that the corneal epithelial cell proliferation rate of the hyaluronic acid hydrogel prepared in Example 2 is much higher than that of the hyaluronic acid hydrogel prepared in Example 1; this shows that: in the extraction process of the traditional Chinese medicine extract of the present invention, the composition of the traditional Chinese medicine raw materials plays an important role in promoting the proliferation of corneal epithelial cells of the prepared hyaluronic acid hydrogel; studies have shown that on the basis of Cassia seed, chrysanthemum and honeysuckle, Ligustrum lucidum and Codonopsis pilosula are further added; the traditional Chinese medicine extract obtained by extracting Cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula as raw materials can greatly improve the corneal epithelial cell proliferation effect of the prepared hyaluronic acid hydrogel compared to the traditional Chinese medicine extract extracted from traditional Chinese medicine raw materials such as Cassia seed, chrysanthemum and honeysuckle.

[0064] It can be seen from the experimental results in Table 1 that the corneal epithelial cell proliferation rate of the hyaluronic acid hydrogels prepared in Examples 3 and 4 is improved compared with that of the hyaluronic acid hydrogel prepared in Example 1, but the increase is much smaller than that of the hyaluronic acid hydrogel prepared in Example 2; this shows that the composition of the Chinese medicine raw materials plays an important role in promoting the proliferation of corneal epithelial cells in the prepared hyaluronic acid hydrogel; the study shows that on the basis of Cassia seed, chrysanthemum and honeysuckle, only by further adding Ligustrum lucidum and Codonopsis pilosula at the same time; using the Chinese medicine extract obtained by extracting Cassia seed, chrysanthemum, honeysuckle, Ligustrum lucidum and Codonopsis pilosula as raw materials, can the corneal epithelial cell proliferation effect of the prepared hyaluronic acid hydrogel be further greatly improved; however, on the basis of Cassia seed, chrysanthemum and honeysuckle, only by adding Ligustrum lucidum or Codonopsis pilosula to extract the Chinese medicine extract, it cannot further greatly improve the corneal epithelial cell proliferation effect of the prepared hyaluronic acid hydrogel.

Claims

1. A method for preparing a hyaluronic acid hydrogel, comprising the following steps: S1. preparing hyaluronic acid solution; S2. adding a crosslinking agent to the hyaluronic acid solution and adjusting the pH to an acidic pH to obtain solution A; S3. Adding a carboxyl activator to solution A and stirring to obtain a hyaluronic acid hydrogel; It is characterized in that The preparation method of the hyaluronic acid solution in step S1 is: taking hyaluronic acid, adding it to the traditional Chinese medicine extract, and stirring evenly to obtain the hyaluronic acid solution.

2. The preparation method according to claim 1, characterized in that: In step S1, the ratio of hyaluronic acid to Chinese medicine extract is 0.2-2 g:100 mL; Most preferably, in step S1, the dosage ratio of hyaluronic acid to the traditional Chinese medicine extract is 1 g:100 mL.

3. The preparation method according to claim 1, characterized in that: The Chinese medicine extract is prepared by the following method: The mixed Chinese medicine raw material is obtained by mixing the Chinese medicines, and then added into water for heating and reflux extraction. After the extraction is completed, the mixed Chinese medicine raw material is filtered to obtain the Chinese medicine extract.

4. The preparation method according to claim 3, characterized in that: The traditional Chinese medicine comprises cassia seed, chrysanthemum and honeysuckle.

5. The preparation method according to claim 4, characterized in that: The weight ratio of cassia seed, chrysanthemum and honeysuckle is 2-4:1-3:1-2; Most preferably, the weight ratio of Cassia seed, chrysanthemum and honeysuckle is 3:2:

1.

6. The preparation method according to claim 3, characterized in that: The dosage ratio of mixed Chinese medicine raw materials to water is 1g: 10-20mL; Most preferably, the ratio of the mixed Chinese medicinal materials to water is 1 g:15 mL.

7. The preparation method according to claim 1, characterized in that: In step S2, the ratio of the cross-linking agent to the hyaluronic acid solution is 0.1-1 g: 100 mL; Most preferably, in step S2, the ratio of the cross-linking agent to the hyaluronic acid solution is 0.5 g:100 mL.

8. The preparation method according to claim 1, characterized in that: In step S3, the usage ratio of the carboxyl activator to solution A is 1.5-3 g:100 mL.

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

10. Use of the hyaluronic acid hydrogel according to claim 9 in preparing corneal damage repair materials.