Multifunctional dressing for corneal epithelium repair and preparation method thereof
By bifunctional modification of hyaluronic acid and dissolved in phosphate buffer, a self-crosslinked multifunctional dressing was prepared, which solved the problems of corneal epithelial cell proliferation and oxidative stress damage and achieved rapid repair of corneal epithelium and lasting therapeutic effects, avoiding the use of crosslinking agents.
Patent Information
- Application Number
- CN202411306617.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art cannot effectively promote the proliferation and repair of corneal epithelial cells, and lacks transparent, tissue adhesion properties and does not use crosslinking agents, which cannot effectively solve the visual impairment and economic burden caused by corneal epithelial injury.
By bifunctional modification of hyaluronic acid and dissolved in phosphate buffer, a self-crosslinked multifunctional dressing can promote the proliferation of corneal epithelial cells, relieve oxidative stress damage, and have clear and tissue adhesion, and no crosslinking agent is used.
This dressing can significantly accelerate the repair rate of corneal epithelium, improve treatment durability, avoid the potential toxicity of crosslinking agents, significantly increase the proliferation ratio of corneal epithelial cells, reduce oxidative stress damage, and have good tissue adhesion properties.
Smart Images
Figure CN119185637A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of corneal epithelial repair, and in particular to a multifunctional dressing for corneal epithelial repair and a preparation method thereof. Background Art
[0002] The cornea is a non-vascular, scar-free transparent tissue with a highly ordered structure, which is essential for the normal functioning of visual function. The refractive power of the cornea accounts for about two-thirds of the entire eye, and it can refract light through the iris and lens to reach the retina smoothly. From the outside to the inside, the cornea can be divided into three parts: epithelium, stroma, and endothelium. The corneal epithelium is located on the outside. Although it is only 50μm thick, it plays an important role in keeping the cornea moist and transparent and acting as a barrier to prevent pathogenic microorganisms and harmful substances from entering the eye. However, since the corneal epithelium is in direct contact with the external environment, it is susceptible to various physical, chemical or viral damage, resulting in decreased tear film stability and discomfort symptoms such as dry eyes, itching and sandy feeling. In extreme cases, corneal epithelial damage can also induce corneal ulcers and scars, making the cornea opaque, which not only affects vision, but also causes a heavy socioeconomic burden. Many common eye diseases such as dry eyes, keratitis, and alkali burns also cause corneal epithelial damage. The development of eye dressings that promote corneal epithelial repair will help improve the quality of life of related patients.
[0003] The corneal epithelium is composed of five to seven layers of non-keratinized, squamous corneal epithelial cells. When it is damaged, corneal epithelial cells can fill the defect area by proliferation, so that the epithelial structure and function can be restored to normal. Therefore, promoting the proliferation of corneal epithelial cells is the first factor to be considered in repairing the corneal epithelium. Although there are many causes of corneal epithelial damage, the generation of free radicals and the oxidative stress response caused by them are often prevalent in its pathological progression. Oxidative stress can also inhibit the proliferation and migration of corneal epithelial cells, thereby inhibiting repair. The ideal corneal epithelial repair dressing should also be transparent and have tissue adhesion properties, which can avoid visual disturbances and resist the removal of the dressing by tear secretion, thereby prolonging the duration of the therapeutic effect. In addition, the medical dressing should preferably be self-crosslinked, which will avoid the use of crosslinking agents and induce potential toxicity. At present, there is no corneal epithelial repair dressing that can meet the above standards at the same time in clinical practice, so filling this gap has extremely high economic and social value. Summary of the invention
[0004] In order to solve the above problems, the present invention provides a multifunctional dressing for corneal epithelial repair and a preparation method thereof. The technical route adopted by the present invention is as follows:
[0005] In a first aspect, the present invention provides a method for preparing a multifunctional dressing for corneal epithelial repair, comprising performing bifunctional modification on hyaluronic acid and dissolving the product in a phosphate buffer, wherein the solid-to-liquid ratio of the bifunctional modified hyaluronic acid to the phosphate buffer in the multifunctional dressing is 30-50 mg:1 mL;
[0006] The bifunctional modification of hyaluronic acid comprises the following steps:
[0007] S1. Dissolve 398 mg of hyaluronic acid in 40-80 mL of water to obtain solution A.
[0008] S2. Add 230 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 138 mg of N-hydroxysuccinimide to solution A, stir and incubate for 2 h to obtain solution B;
[0009] S3, taking 279 mg of phenylboronic acid compound and adding it to B to obtain solution C;
[0010] S4, take 98.8 mg of 3,4,5-trihydroxybenzoyl hydrazide and add it to C to obtain solution D;
[0011] S5, adjust the pH of D to about 5.0, react at room temperature for 24 hours, and obtain solution E;
[0012] S6 and E were dialyzed for three days and then freeze-dried to obtain the bifunctional modified hyaluronic acid.
[0013] Furthermore, the molecular weight of the hyaluronic acid is 400,000-800,000 Da.
[0014] Furthermore, the phenylboronic acid compound is a hydrochloride formed by m-aminophenylboronic acid.
[0015] Furthermore, the 3,4,5-trihydroxybenzoic acid hydrazide needs to be dissolved in 10 mL of dimethylformamide in advance, and then added dropwise into C.
[0016] Furthermore, the dialysis was performed for three days using a dialysis bag with a molecular weight cutoff of 8000-14000Da, and the solution needed to be changed 14 times, with the first 12 times using acetic acid solution (pH about 5.0) and the last 2 times using water.
[0017] In the second aspect, the multifunctional dressing for corneal epithelial repair prepared by the above-mentioned method for preparing a multifunctional dressing for corneal epithelial repair also falls within the protection scope of the present invention.
[0018] The beneficial effects of the present invention are as follows: the multifunctional dressing for corneal epithelial repair provided by the present invention is a single-component dressing, which is self-crosslinked by a bifunctional modified hyaluronic acid dissolved in a phosphate buffer, and the dressing has a variety of properties that are beneficial to corneal epithelial repair. First, it can promote the proliferation of corneal epithelial cells; second, it can alleviate oxidative stress damage; third, it is transparent and has tissue adhesion properties, which is beneficial for the dressing to resist tear clearance, thereby exerting a more lasting therapeutic effect; finally, it does not use a crosslinking agent, avoiding potential crosslinking agent toxicity. Using the multifunctional dressing provided by the present invention on the ocular surface can accelerate the repair rate of the corneal epithelium. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Before and after hyaluronic acid modification 1 H-NMR spectrum;
[0020] Figure 2 Multifunctional dressing promotes corneal epithelial cell proliferation (EdU labeling method);
[0021] Figure 3 Multifunctional dressing removes oxidative stress damage in corneal epithelial cells (DCFH-DA probe method);
[0022] Figure 4 The multifunctional dressing is transparent in appearance (a) and can adhere to the eye (b), spleen (c) and heart (d).
[0023] Figure 5 Multifunctional dressing accelerates corneal epithelial repair in mice. DETAILED DESCRIPTION
[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0025] Example 1 Synthesis of bifunctional modified hyaluronic acid
[0026] 398 mg of hyaluronic acid with a molecular weight of 400000-800000Da was dissolved in 60 mL of water to obtain solution A; 230 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 138 mg of N-hydroxysuccinimide were added to A, and the mixture was stirred and incubated for 2 hours to obtain solution B; 279 mg of m-aminophenyl boric acid hydrochloride was added to B to obtain solution C; 98.8 mg of 3,4,5-trihydroxybenzoyl hydrazide was dissolved in 10 mL of dimethylformamide, and then dropwise added to C to obtain solution D; the pH of D was adjusted to about 5.0, and the mixture was reacted at room temperature for 24 hours to obtain solution E; E was dialyzed for three days using a dialysis bag with a molecular weight cutoff of 8000-14000Da, during which the liquid was changed 14 times, the first 12 times of which were changed with acetic acid solution (pH about 5.0), and the last 2 times of which were changed with water; the dialyzed product was freeze-dried to obtain a bifunctional modified hyaluronic acid.
[0027] Example 2 Preparation of multifunctional dressing
[0028] Take a bag of phosphate buffer powder (purchased from Regen Biotechnology) and dissolve it in 1L of water; add the bifunctional modified hyaluronic acid to the above solution at a solid-liquid ratio of 40mg:1mL; stir to dissolve; let it stand to obtain a multifunctional dressing.
[0029] Example 3 NMR analysis
[0030] The bifunctional modified hyaluronic acid was dissolved in deuterium oxide and then analyzed using a Bruker 600M NMR. Figure 1 It can be seen that the modified hyaluronic acid has two aromatic hydrogen NMR peaks at 7.96 and 7.03 ppm, proving that the bifunctional modification is successful.
[0031] Example 4 Corneal epithelial cell proliferation analysis
[0032] Corneal epithelial cells were inoculated into 24-well plates at 30,000 cells per well; the plates were placed in an incubator for 24 hours to allow the cells to adhere to the wall; the medium was changed and 0.2 mL of multifunctional dressing was placed in a cell culture chamber; the chamber was placed in a 24-well plate and the cells were incubated for 24 hours; the cell proliferation was analyzed according to the instructions of the EdU-594 cell proliferation detection kit (purchased from Beyotime) and compared with cells not treated with the multifunctional dressing. Figure 2 It can be seen that the multifunctional dressing significantly increases the proportion of proliferating cells.
[0033] Example 5 Analysis of oxidative stress damage in corneal epithelial cells
[0034] Corneal epithelial cells were seeded in 24-well plates at a density of 50,000 cells per well. The plates were placed in an incubator and incubated for 24 hours to allow the cells to adhere to the wall. The culture medium was replaced with DMEM containing 400 μM hydrogen peroxide, and a cell culture chamber containing 0.2 mL of multifunctional dressing was placed in the plate, and the cells were incubated for another 4 hours. The reactive oxygen species detection kit (DCFH-DA probe method; purchased from Beyotime) was used to analyze cell oxidative stress damage and compared with cells not treated with multifunctional dressings. Figure 3 It can be seen that the multifunctional dressing treatment significantly reduced the proportion of DCFH-DA positive areas, that is, reduced the oxidative stress damage caused by hydrogen peroxide.
[0035] Example 6 Analysis of the appearance and tissue adhesion properties of the multifunctional dressing
[0036] Place the multifunctional dressing on paper and judge its transparency by the letters below; apply the multifunctional dressing to the handle of the tweezers so that it contacts the mouse eyes, spleen, and heart, then lift the tweezers and observe the tissue adhesion performance of the multifunctional dressing. Figure 4 As can be seen from a, the multifunctional dressing is a transparent gel-like substance. Figure 4 As shown in b, c, and d, the multifunctional dressing can adhere the mouse eyeball, spleen, and heart to the forceps handle so that they can be hung upside down without falling, thus having good tissue adhesion.
[0037] Example 7 Evaluation of the performance of multifunctional dressing in promoting corneal epithelial repair
[0038] Male C57BL / 6J mice were anesthetized generally and then locally anesthetized with proparacaine hydrochloride eye drops. The mouse cornea was damaged for 30 seconds with a filter paper (2 mm in diameter) soaked in 1M sodium hydroxide. The damaged cornea was thoroughly cleaned with saline and then dripped with clotrimazole eye drops to prevent infection. After modeling, the mice were treated with multifunctional dressings three times a day for five days. The corneal epithelial damage was evaluated by slit lamp and compared with mice treated with saline (i.e., the untreated group). Figure 5 It can be seen that multifunctional dressing can significantly accelerate the repair of corneal epithelium.
[0039] Technical personnel should note that although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention, and any modification using the inventive concept will be included in the scope of protection of this patent.
[0040] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing a multifunctional dressing for corneal epithelial repair, characterized in that: The method comprises performing bifunctional modification on hyaluronic acid and dissolving the product in a phosphate buffer, wherein the solid-liquid ratio of the bifunctional modified hyaluronic acid to the phosphate buffer in the multifunctional dressing is 30-50 mg:1 mL; The bifunctional modification of hyaluronic acid comprises the following steps: S1. Dissolve 398 mg of hyaluronic acid in 40-80 mL of water to obtain solution A. S2. Add 230 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 138 mg of N-hydroxysuccinimide to solution A, stir and incubate for 2 h to obtain solution B; S3, taking 279 mg of phenylboronic acid compound and adding it to B to obtain solution C; S4, take 98.8 mg of 3,4,5-trihydroxybenzoyl hydrazide and add it to C to obtain solution D; S5, adjust the pH of D to about 5.0, react at room temperature for 24 hours, and obtain solution E; S6 and E were dialyzed for three days and then freeze-dried to obtain the bifunctional modified hyaluronic acid.
2. The method for preparing the multifunctional dressing for corneal epithelial repair according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is 400,000-800,000 Da.
3. The method for preparing the multifunctional dressing for corneal epithelial repair according to claim 1, characterized in that: The phenylboronic acid compound is the hydrochloride formed by m-aminophenylboronic acid.
4. The method for preparing the multifunctional dressing for corneal epithelial repair according to claim 1, characterized in that: The 3,4,5-trihydroxybenzoic acid hydrazide needs to be dissolved in 10 mL of dimethylformamide in advance, and then added dropwise into C.
5. The method for preparing the multifunctional dressing for corneal epithelial repair according to claim 1, characterized in that: The dialysis was performed for three days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and the liquid needed to be changed 14 times.
6. The method of claim 5, wherein the liquid is changed 14 times, The first 12 times the solution was replaced with acetic acid solution (pH about 5.0), and the last 2 times the solution was replaced with water.
7. A multifunctional dressing for corneal epithelial repair prepared by the method for preparing a multifunctional dressing for corneal epithelial repair according to any one of claims 1 to 6.