An agarose-based artificial cornea and its preparation method

Abstract

The present invention discloses an agarose-based artificial cornea and its preparation method, belonging to the field of biomedical materials. The method includes the following steps: grafting a hydrophobic chain onto the agarose backbone through an esterification reaction, adding the obtained hydrophobic agarose polymer into dimethyl sulfoxide to obtain a clear and transparent solution. Pouring the solution into a mold by casting, then soaking the mold in water. After partial solvent exchange, tearing off the film on the surface of the solution, and the remaining solution forms a soft and opaque hydrogel at the bottom of the mold. Placing the opaque hydrogel on a plastic petri dish, drying it to obtain a transparent agarose-based film. Swelling the film sufficiently in simulated tear fluid to obtain the agarose-based artificial cornea. This artificial cornea has appropriate transparency, mechanical properties, and excellent biocompatibility, and has broad application prospects in ophthalmic implant materials.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to an agarose-based artificial cornea and a preparation method thereof. Background Art

[0002] Corneal tissue damage is a very common clinical disease and one of the important reasons for vision decline. Physical injury, bacterial infection, chemical drugs, and irregular daily eye use can all cause corneal damage, leading to visual impairment or even blindness. According to statistics, about 23 million people in the world have vision decline or even blindness due to corneal structural damage. The cornea plays an important protective barrier role for the eyeball structure. Due to its highly transparent and avascular characteristics, corneal injury repair remains a major challenge. Patients can choose corneal transplantation to treat corneal diseases. However, the scarcity of corneal donors and the problems of immune rejection and infection after corneal transplantation have discouraged patients. There is a great demand for biomaterials that can efficiently promote corneal tissue regeneration in the ophthalmic market. An artificial cornea is a special refractive device made of artificial synthetic materials, used to replace the cloudy cornea that hinders the optical path of the eyeball after disease, so that patients can obtain a certain degree of vision. Patients can choose the transplantation area and thickness according to their needs, and are no longer limited by donors and can be used for life.

[0003] Regarding artificial corneal stroma materials, the following is disclosed in Chinese patent literature:

[0004] Literature 1: Artificial Corneal Scaffold, Artificial Corneal Stroma and Its Manufacturing Method (CN109395161):

[0005] The artificial corneal stroma material includes methacrylated hyaluronic acid, collagen, and forms a dense network structure under the action of a photoinitiator.

[0006] Literature 2: An Artificial Membrane Skin and Its Preparation Method (CN114618016):

[0007] Assemble biomacromolecule materials through electrochemical deposition technology: design and customize electrodes, improve the EDP technology to form collagen and chemical cross-linking steps, and obtain a collagen-based artificial cornea with the same shape as the cornea on the electrode, with a transparency of more than 80%, controllable thickness and curvature, and can replace and repair the natural cornea.

[0008] Literature 3: Active Regenerative Artificial Corneal Graft and Its Preparation Method (CN101380486):

[0009] Dissolve chitosan, collagen, and chondroitin sulfate in hydrochloric acid, acetic acid, and water respectively, and then inject each component (also including original ethylene glycol, polyvinyl alcohol, and distilled water) into a mold at room temperature according to a ratio, stir, dry, and then soak, wash, and dry with water to obtain the product.

[0010] Currently, the materials commonly used for preparing artificial corneas are mainly polymer materials, such as poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(ethylene glycol), poly(lactic-co-glycolic acid), etc. However, synthetic materials often exhibit poor biocompatibility with ocular tissues and cannot achieve good integration with host tissues. Decellularized corneal stroma has been approved for market because it retains the collagen structure of the natural corneal stroma. However, its low transparency and weak mechanical properties have limited its application to a certain extent. Developing a biocompatible artificial cornea with high transparency, excellent mechanical properties and the ability to bind to corneal tissues for a long time has great clinical significance. Summary of the Invention

[0011] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a preparation method of an agarose-based artificial cornea with high transparency, strong mechanical properties and good biocompatibility.

[0012] To solve the above technical problems, the present invention adopts the following technical solutions:

[0013] An agarose-based artificial cornea and its preparation method, including the following steps:

[0014] (1) Graft a hydrophobic chain onto the agarose backbone through an esterification reaction to obtain a hydrophobic agarose polymer.

[0015] (2) Add the hydrophobic agarose polymer to dimethyl sulfoxide and stir until it is completely dissolved to obtain a clear and transparent solution.

[0016] (3) Pour the above solution into a mold by casting, soak the mold and the solution inside in water, perform partial solvent exchange on the solution, then tear off the film on the surface of the solution, and perform slow solvent exchange on the remaining solution in the mold until a soft and opaque hydrogel is formed at the bottom of the mold.

[0017] (4) Place the above soft and opaque hydrogel on a plastic petri dish and dry it in an oven to obtain a transparent agarose film.

[0018] (5) Swell the agarose film sufficiently in simulated tear fluid to obtain an agarose-based artificial cornea.

[0019] In step (1), the hydrophobic chain is one of butyric anhydride, dodecenyl succinic anhydride, and hexadecenyl succinic anhydride.

[0020] In step (1), the temperature of the esterification reaction is 30-50°C, the reaction time is 2-4 h, and the agarose concentration is 5-8 wt.%.

[0021] In step (2), the concentration of the hydrophobic agarose polymer solution is 4-10 wt.%.

[0022] In step (3), the mold is a circular polytetrafluoroethylene mold with a diameter of 15 mm and a thickness of 0.5 mm.

[0023] In step (3), the time for soaking the mold and the solution inside it in water is 1 - 5 min.

[0024] In step (3), the time for slow solvent exchange of the remaining solution in the mold is 12 - 36 h.

[0025] In step (4), the drying temperature is 40 - 70 °C and the drying time is 12 - 60 h.

[0026] In step (5), the time for the agarose film to fully swell in the simulated tear fluid is 20 - 60 h; the formula of the simulated tear fluid is: 0.008 g of anhydrous calcium chloride, 0.22 g of sodium bicarbonate, 0.14 g of potassium chloride, and 0.68 g of sodium chloride are dissolved in 100 mL of deionized water, and NaOH is added to adjust the pH to 7.4.

[0027] The present invention also provides an agarose - based artificial cornea prepared by the above - mentioned method and its preparation method.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: The artificial cornea of the present invention is prepared from the natural - source marine polysaccharide agarose, with rich sources, simple process, low cost, and excellent biocompatibility. An agarose - based artificial cornea meets the basic performance requirements of ophthalmic implant materials, has a high light transmittance, and the light transmittance in the range of 400 - 800 nm is 80 - 100%. The agarose - based artificial cornea has appropriate mechanical properties, and the elongation ratio at tensile fracture is 1 - 2.6, which can withstand the suture strength of surgical operations. It has broad application prospects in ophthalmic implant materials. Description of the Drawings

[0029] Figure 1 is a physical picture of the agarose - based artificial cornea prepared in Example 1 of the present invention.

[0030] Figure 2 is the light transmittance curve of the agarose - based artificial cornea prepared in Example 1 of the present invention.

[0031] Figure 3 is the elongation ratio at tensile fracture of the agarose - based artificial cornea prepared in Example 1 of the present invention.

[0032] Figure 4 is the cell survival rate after co - culturing the agarose - based artificial cornea prepared in Example 1 of the present invention with human umbilical vein endothelial cells. Detailed Embodiments

[0033] In order to enable those skilled in the art to more clearly understand the solution and principle of the present invention, the following will describe this part of the content in detail:

[0034] The present invention provides an agarose-based artificial cornea and a preparation method thereof, including the following steps:

[0035] (1) Preparation of hydrophobic agarose polymer: Add 1 g of agarose to 15 mL of dimethyl sulfoxide, stir at 90 °C until dissolved, then cool to 40 °C. After the temperature stabilizes, add 0.1 g of 4-dimethylaminopyridine and 0.75 g of hydrophobic chain molecule to the solution, and continue to react at 40 °C for 3.5 h. After the reaction, perform solvent exchange on the reaction solution with 2 L of deionized water. After the solvent exchange is completed, freeze-dry the product to obtain the hydrophobic agarose polymer.

[0036] (2) Preparation of hydrophobic agarose polymer solution: Add the hydrophobic agarose polymer prepared in step (1) to dimethyl sulfoxide at a concentration of 8 wt.%, and stir until completely dissolved to obtain a clear and transparent solution;

[0037] (3) Pour the above solution into a mold by casting. Immerse the mold and the solution inside in water for 3 min to allow partial solvent exchange of the solution, then tear off the film on the surface of the solution. The remaining solution undergoes slow solvent exchange in the mold for 24 h until a soft and opaque hydrogel forms at the bottom of the mold.

[0038] (4) Place the above soft and opaque hydrogel on a plastic petri dish and dry it in an oven at 60 °C for 36 h to obtain a transparent agarose-based film.

[0039] (5) After the agarose-based film is fully swollen in simulated tear fluid for 48 h, an agarose-based artificial cornea is obtained.

[0040] In step (1), the hydrophobic chain is one of butyric anhydride, dodecenyl succinic anhydride, and hexadecenyl succinic anhydride.

[0041] In step (3), the mold is a circular polytetrafluoroethylene mold with a diameter of 15 mm and a thickness of 0.5 mm.

[0042] In step (5), the formula of the simulated tear fluid is: 0.008 g of anhydrous calcium chloride, 0.22 g of sodium bicarbonate, 0.14 g of potassium chloride, and 0.68 g of sodium chloride are dissolved in 100 mL of deionized water, and NaOH is added to adjust the pH to 7.4.

[0043] In step (5), for the agarose-based artificial cornea and the preparation method thereof, it is characterized in that the light transmittance of the agarose-based artificial cornea in the range of 400 - 800 nm is 80 - 100%.

[0044] In step (5), for the agarose-based artificial cornea and its preparation method, it is characterized in that the elongation ratio at break of the agarose-based artificial cornea is 1 to 2.6.

[0045] The following is a detailed description in conjunction with the accompanying drawings and specific embodiments. The content of the present invention is not limited to any specific embodiment, nor does it represent the best embodiment. General substitutions well-known to those skilled in the art are also covered within the protection scope of the present invention.

[0046] Example 1:

[0047] An agarose-based artificial cornea and its preparation method include the following steps:

[0048] (1) Preparation of hydrophobic agarose polymer: Add 1 g of agarose to 15 mL of dimethyl sulfoxide, stir at 90 °C until dissolved, then cool to 40 °C. After the temperature stabilizes, add 0.1 g of 4-dimethylaminopyridine and 0.75 g of butyric anhydride to the solution, and continue the reaction at 40 °C for 3.5 h. After the reaction is completed, perform solvent exchange on the reaction solution with 2 L of deionized water. After the solvent exchange is completed, freeze-dry the product to obtain butyric anhydride-grafted agarose (BAA).

[0049] (2) Preparation of BBA solution: Add the BBA prepared in step (1) to dimethyl sulfoxide at a concentration of 8 wt.%, and stir until completely dissolved to obtain a clear and transparent solution;

[0050] (3) Pour the above solution into a mold by casting. Immerse the mold and the solution inside in water for 3 min to allow partial solvent exchange of the solution, then tear off the film on the surface of the solution. The remaining solution undergoes slow solvent exchange in the mold for 24 h until a soft and opaque hydrogel forms at the bottom of the mold.

[0051] (4) Place the above soft and opaque hydrogel on a customized mold, and dry it in an oven at 60 °C for 36 h to obtain a transparent BBA film.

[0052] (5) After swelling the BBA film in simulated tear fluid for 48 h, a BBA artificial cornea is obtained. The formula of the simulated tear fluid is: 0.008 g of anhydrous calcium chloride, 0.22 g of sodium bicarbonate, 0.14 g of potassium chloride, and 0.68 g of sodium chloride are dissolved in 100 mL of deionized water, and NaOH is added to adjust the pH to 7.4.

[0053] Taking the agarose-based artificial cornea and its preparation method prepared in Example 1 as an example, the light transmittance of the BBA artificial cornea was measured by an ultraviolet-visible spectrophotometer. The test results are as Figure 2 shown. The light transmittance of the BBA artificial cornea only reaches 17.6% at 750 nm.

[0054] Taking an agarose-based artificial cornea prepared in Example 1 and its preparation method as an example, the mechanical properties of the BBA artificial cornea were measured by a universal mechanical testing machine. The test results are as Figure 3 shown, and the elongation ratio at break of the BBA artificial cornea is 2.1.

[0055] Example 2:

[0056] An agarose-based artificial cornea and its preparation method, comprising the following steps:

[0057] (1) Preparation of a hydrophobic agarose polymer: Add 1 g of agarose to 15 mL of dimethyl sulfoxide, stir at 90 °C until dissolved, then cool to 40 °C. After the temperature stabilizes, add 0.1 g of 4-dimethylaminopyridine and 0.75 g of dodecenyl succinic anhydride to the solution, and continue the reaction at 40 °C for 3.5 h. After the reaction is completed, perform solvent exchange on the reaction solution with 2 L of deionized water. After the solvent exchange is completed, freeze-dry the product to obtain dodecenyl succinic anhydride-grafted agarose (DAA).

[0058] (2) Preparation of a DAA solution: Add the DAA prepared in step (1) to dimethyl sulfoxide at a concentration of 8 wt.%, and stir until completely dissolved to obtain a clear and transparent solution;

[0059] (3) Pour the above solution into a mold by casting, soak the mold and the solution inside in water for 3 min, perform partial solvent exchange on the solution, then tear off the film on the surface of the solution, and perform slow solvent exchange on the remaining solution in the mold for 24 h until a soft and opaque hydrogel is formed at the bottom of the mold.

[0060] (4) Place the above soft and opaque hydrogel on a customized mold, dry it in an oven at 60 °C for 36 h to obtain a transparent DAA film.

[0061] (5) After swelling the DAA film in simulated tear fluid for 48 h, a DAA artificial cornea is obtained. The physical object is as Figure 1 shown. The formula of the simulated tear fluid is: 0.008 g of anhydrous calcium chloride, 0.22 g of sodium bicarbonate, 0.14 g of potassium chloride, and 0.68 g of sodium chloride are dissolved in 100 mL of deionized water, and NaOH is added to adjust the pH = 7.4.

[0062] Taking an agarose-based artificial cornea prepared in Example 2 and its preparation method as an example, the light transmittance of the DAA artificial cornea was measured by a UV-visible spectrophotometer. The test results are as Figure 2 shown, and the light transmittance of the DAA artificial cornea reaches 97.4% at 750 nm.

[0063] Taking an agarose-based artificial cornea prepared in Example 2 and its preparation method as an example, the mechanical properties of the DAA artificial cornea were measured by a universal mechanical testing machine. The test results are as Figure 3 shown, and the elongation ratio at break of the DAA artificial cornea is 2.5.

[0064] Taking an agarose-based artificial cornea prepared in Example 2 and its preparation method as an example, the cytocompatibility of the DAA artificial cornea was measured by the cck8 method. The test results are as Figure 4 shown, and the survival rate of cells exceeded 100% after co-culturing the DAA artificial cornea with human umbilical vein endothelial cells for 2 days.

[0065] Example 3:

[0066] An agarose-based artificial cornea and its preparation method, comprising the following steps:

[0067] (1) Preparation of hydrophobic agarose polymer: Add 1 g of agarose to 15 mL of dimethyl sulfoxide, stir at 90 °C until dissolved, then cool to 40 °C. After the temperature stabilizes, add 0.1 g of 4-dimethylaminopyridine and 0.75 g of hexadecene succinic anhydride to the solution, and continue to react at 40 °C for 3.5 h. After the reaction is completed, perform solvent exchange on the reaction solution with 2 L of deionized water. After the solvent exchange is completed, freeze-dry the product to obtain hexadecene succinic anhydride grafted agarose (HAA).

[0068] (2) Preparation of HAA solution: Add the HAA prepared in step (1) to dimethyl sulfoxide at a concentration of 8 wt.%, and stir until completely dissolved to obtain a clear and transparent solution;

[0069] (3) Pour the above solution into a mold by casting, soak the mold and the solution inside in water for 3 min, perform partial solvent exchange on the solution, then tear off the film on the surface of the solution, and perform slow solvent exchange on the remaining solution in the mold for 24 h until a soft and opaque hydrogel is formed at the bottom of the mold.

[0070] (4) Place the above soft and opaque hydrogel on a customized mold, and dry it in an oven at 60 °C for 36 h to obtain a transparent HAA film.

[0071] (5) After swelling the HAA film in simulated tear fluid for 48 h, an HAA artificial cornea is obtained. The formula of the simulated tear fluid is: 0.008 g of anhydrous calcium chloride, 0.22 g of sodium bicarbonate, 0.14 g of potassium chloride, and 0.68 g of sodium chloride are dissolved in 100 mL of deionized water, and NaOH is added to adjust the pH = 7.4.

[0072] Taking an agarose-based artificial cornea prepared in Example 1 and its preparation method as an example, the light transmittance of the HAA artificial cornea was measured by an ultraviolet-visible spectrophotometer. The test results are as Figure 2 shown, and the light transmittance of the HAA artificial cornea is 29.4%.

[0073] Taking an agarose-based artificial cornea prepared in Example 1 and its preparation method as an example, the mechanical properties of the HAA artificial cornea were measured by a universal mechanical testing machine. The test results are as Figure 3 shown, and the elongation ratio at break of the HAA artificial cornea is 1.0.

[0074] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can make many variations without departing from the purpose and scope of protection of the present invention and the claims. These all belong to the protection scope of the present invention.

Claims

1. A method for preparing an agarose-based artificial cornea, characterized in that, It includes the following operation steps: (1) Graft the hydrophobic chain onto the agarose backbone through an esterification reaction to obtain a hydrophobic agarose polymer; (2) Add the hydrophobic agarose polymer into dimethyl sulfoxide and stir until it is completely dissolved to obtain a clear and transparent solution; (3) Pour the above solution into a mold by casting, soak the mold and the solution inside in water, perform partial solvent exchange on the solution, then tear off the film on the surface of the solution, and perform slow solvent exchange on the remaining solution in the mold until a soft and opaque hydrogel is formed at the bottom of the mold; (4) Place the above soft and opaque hydrogel on a plastic petri dish and dry it in an oven to obtain a transparent agarose-based film; (5) Swell the agarose-based film sufficiently in simulated tears to obtain an agarose-based artificial cornea; In step (1), the hydrophobic chain is dodecenyl succinic anhydride; In step (2), the concentration of the hydrophobic agarose polymer solution is 4-10 wt.%.

2. The preparation method of an agarose-based artificial cornea according to claim 1, characterized in that, In step (1), the temperature of the esterification reaction is 30-50 °C and the reaction time is 2-4 h.

3. The preparation method of an agarose-based artificial cornea according to claim 1, wherein In step (3), the time for soaking the mold and the solution inside in water is 1-5 min.

4. The preparation method of an agarose-based artificial cornea according to claim 1, wherein, In step (3), the time for slow solvent exchange of the remaining solution in the mold is 12-36 h.

5. The preparation method of an agarose-based artificial cornea according to claim 1, characterized in that, In step (4), the drying temperature is 40-70 °C and the drying time is 12-60 h.

6. An agarose-based artificial cornea, characterized in that: Prepared according to any one of claims 1-5.

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