Artificial cornea bracket and preparation method thereof

Abstract

The invention provides an artificial cornea scaffold and a preparation method thereof, and belongs to the technical field of new material preparation. The preparation process comprises the following steps: preparing the modified krill protein; preparing a polymethyl methacrylate microsphere template; modifying the modified polyacrylic acid with the modified krill protein; preparing an N, N-isopropylacrylamide monomer reaction solution; and preparing the artificial cornea bracket. The material composition of the double-network hydrogel has low cytotoxicity and immunogenicity, stimulation and rejection reaction to eye tissues can be reduced, attachment and growth of cells are facilitated, and the stent is endowed with sufficient strength and toughness by the interpenetrating double-network structure, so that the stent can bear normal physiological pressure and movement of eyes, and the stent has a good application prospect in the field of ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology, ophthalmology and ophthalmology. Meanwhile, the stability and the porous characteristic of the structure are kept, so that exchange of nutrient substances and metabolic wastes is facilitated, a good microenvironment is provided for survival and function maintenance of cells, migration, proliferation and differentiation of corneal cells can be guided, and repair and regeneration of damaged corneal tissues are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of new material preparation, and in particular to an artificial cornea support and a preparation method thereof. Background Art

[0002] The cornea is a transparent film at the front of the eye, which plays a vital role in the refraction and focusing of light. However, due to various reasons, such as disease and trauma, many people's corneas may be damaged or diseased, resulting in severe vision loss or even blindness.

[0003] In the past, corneal transplantation was the main method for treating corneal diseases. However, the shortage of corneal donors has always been a key factor limiting the widespread application of corneal transplantation. In addition, traditional corneal transplantation surgery also has problems such as immune rejection and postoperative complications, which brings certain risks and challenges to the patient's recovery.

[0004] With the continuous development of biomedical engineering technology, the research of artificial corneal scaffolds has gradually become an important direction to solve the problem of corneal disease treatment. The existing artificial corneal scaffolds have many shortcomings in material selection, structural design and preparation process.

[0005] For example, the materials used in some artificial corneal stents have poor biocompatibility and are prone to cause inflammatory reactions and tissue rejection; the structural design of some stents is unreasonable and cannot effectively simulate the physiological structure and function of the natural cornea, resulting in poor mechanical and optical properties; and some preparation methods are complex and costly, making it difficult to achieve large-scale production and clinical application.

[0006] Therefore, developing a new type of artificial corneal scaffold and its preparation method, which has good biocompatibility, excellent mechanical properties and a simple and feasible preparation process, is of great significance for improving the treatment effect of corneal diseases and improving the quality of life of patients. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention aims to provide an artificial cornea support and a preparation method thereof.

[0008] An artificial cornea support and a preparation method thereof, comprising the following steps:

[0009] S1: Preparation of modified krill protein

[0010] The krill protein is subjected to ultrasound-coupled succinylation modification to obtain modified krill protein;

[0011] S2: Preparation of polymethyl methacrylate microsphere template

[0012] Methyl methacrylate monomer and deionized water are mixed and reacted to obtain a reaction solution, an emulsifier and an initiator are added to the reaction solution, and then the mixture is injected into a mold for sintering to obtain a polymethyl methacrylate microsphere template;

[0013] S3: Modified krill protein modified with modified polyacrylic acid

[0014] Acrylic acid is added to deionized water and krill protein solution, and then N'N-methylenebisacrylamide, tetramethylethylenediamine, D-gluconolactone and calcium carbonate are added to react in a nitrogen atmosphere, and then poured into a mold of a sintered polymethyl methacrylate microsphere template, and vacuum cured to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0015] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0016] Dissolving N-isopropylacrylamide monomer in a mixed solvent of ultrapure water and ethanol, adding a crosslinking agent and an initiator, and mixing by vortexing to obtain an N-isopropylacrylamide monomer reaction solution;

[0017] S5: Preparation of artificial cornea scaffold

[0018] The polyacrylic acid composite hydrogel-microsphere template mixture is frozen and then freeze-dried in vacuo, and N-isopropylacrylamide monomer reaction solution is injected into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe, and the mixture is swollen and irradiated under ultraviolet light, followed by Soxhlet extraction and rinsing to obtain an artificial corneal scaffold.

[0019] Furthermore, step S1 of preparing the modified krill protein specifically comprises the following steps:

[0020] S1.1: krill protein and deionized water are mixed in a weight ratio of 1-2:100, and the mixture is stirred at room temperature for 1-2 hours to obtain a suspension. The suspension is treated at 100-300W for 30-35 minutes using an ultrasonic probe with a diameter of 10-12 mm in an ice bath, with an on time of 5-6 seconds and an off time of 5-6 seconds, to obtain an ultrasonically treated krill protein mixture;

[0021] S1.2: adding succinic anhydride to the krill protein mixture after ultrasonic treatment, and then stirring at room temperature for 3-4 hours. During the stirring process, a 1-2M sodium hydroxide solution is used to adjust the pH to maintain it at 12.5-13, and a mixed solution is obtained after the stirring is completed;

[0022] S1.3: The pH of the mixed solution is adjusted to 4-4.5, and the mixture is centrifuged to obtain protein. The protein is dialyzed with distilled water at 4-5°C for 24-25 hours, and then freeze-dried to obtain modified krill protein.

[0023] Furthermore, step S2 of preparing the polymethyl methacrylate microsphere template specifically comprises the following steps:

[0024] S2.1: 40-50 parts by weight of methyl methacrylate monomer and 350-400 parts by weight of deionized water are mixed and added to a four-necked glass flask with stirring, nitrogen is introduced, condensed water is opened, and the mixture is heated at 50-60° C. for 20-23 min. After the reaction, the mixture is cooled to room temperature to obtain a reaction solution;

[0025] S2.2: Add 1-2 parts by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it to 70-72°C again, then dissolve 0.2-0.3 parts by weight of initiator ammonium persulfate in 30-40 parts by weight of deionized water, stir evenly and add to the reaction solution, stir and react for 2-3 hours to obtain a microsphere emulsion;

[0026] S2.3: Pour the microsphere emulsion into the mold using a syringe, ultrasonically vibrate the mold for 15-20 minutes, and then sinter at 175-180° C. for 24-25 hours. Take out the polymethyl methacrylate microsphere template mold, cool it at room temperature, and set it aside.

[0027] Furthermore, step S3 of modifying krill protein to modify polyacrylic acid specifically comprises the following steps:

[0028] S3.1: dissolving 2-3 parts by weight of modified krill protein in 5-8 parts by weight of 5% sodium hydroxide solution, and using an ultrasonic cell pulverizer to accelerate the dissolution for 5-10 minutes to obtain a krill protein solution;

[0029] S3.2: Add 4-5 parts by weight of acrylic acid to 10-20 parts by weight of deionized water, then add the krill protein solution, stir and react at 75-80° C. under a nitrogen environment for 1-2 hours, then add 0.03-0.04 parts by weight of N'N-methylenebisacrylamide and 0.00004-0.00005 parts by weight of tetramethylethylenediamine, stir and mix well to obtain a mixed solution;

[0030] S3.3: Add 1-2 parts by weight of D-gluconolactone to the mixed solution, stir and mix, then add 0.07-0.08 parts by weight of calcium carbonate, and then react for 2-3 hours to obtain a mixed solution, and pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template. After vacuuming, cure it in an oven at 80-85°C for 1-2 hours, and then cure it at room temperature for 24-25 hours to obtain a polyacrylic acid composite hydrogel-microsphere template mixture.

[0031] Furthermore, the preparation of the N-isopropylacrylamide monomer reaction solution in step S4 specifically comprises the following steps:

[0032] S4.1: dissolving 10-12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2-3 parts by weight of ultrapure water and ethanol, and ultrasonically shaking for 10-12 minutes to obtain a mixed solution;

[0033] S4.2: Add 0.002-0.003 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix well, add 0.05-0.07 parts by weight of initiator 2,2-dimethoxy-phenyl ketone, and perform ultrasonic oscillation for 10-12 minutes to obtain a mixed reaction solution;

[0034] S4.3: Vortex the mixed reaction liquid to obtain N-isopropylacrylamide monomer reaction liquid, put the N-isopropylacrylamide monomer reaction liquid into a sealed container, fill it with nitrogen for 3-5 minutes, and after fully exhausting the air, put it into a central control drying room for standby use.

[0035] Furthermore, step S5 of preparing the artificial cornea scaffold specifically includes the following steps:

[0036] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing for 1-2 hours, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect the freeze-drying vacuum machine interface at -70°C to -72°C, and freeze-dry for 48-50 hours under vacuum to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0037] S5.2: injecting N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep the mold full, placing the mold in a glass vacuum tank and swelling for 72-75 hours to obtain a swollen composite hydrogel;

[0038] S5.3: moving the swollen composite hydrogel under an ultraviolet lamp for irradiation, irradiating the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5-8 minutes each, to obtain a stable double network hydrogel mixture;

[0039] S5.4: putting the double network hydrogel mixture into a Soxhlet extraction apparatus, using dichloromethane to dissolve the polymethyl methacrylate microsphere template, heating the round-bottom flask of the Soxhlet extraction apparatus to 50-60°C, extracting for 72-75 hours, and obtaining a stable double network hydrogel;

[0040] S5.5: The stable double network hydrogel is placed in acetone and pure water respectively, and shaken and rinsed for 7-8 days, and the new solvent is replaced every 20-24 hours. After the rinsing, an artificial cornea scaffold is obtained.

[0041] Furthermore, in step S1.2, the mass ratio of succinic anhydride to krill protein is 1-2:10.

[0042] Furthermore, in step S2.3, the mold is composed of two glass slides sandwiching a polytetrafluoroethylene gasket with a thickness of 0.5-1 mm, and the mold capacity volume is 0.6-0.7 mm.

[0043] Furthermore, in step S4.1, the weight ratio of ultrapure water to ethanol is 1-2:1.

[0044] An artificial cornea scaffold is prepared by any of the above-mentioned methods for preparing an artificial cornea scaffold.

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] 1. The present invention effectively combines the spherical mold porous scaffold preparation technology and the double network hydrogel preparation technology, and successfully prepares an artificial corneal scaffold with a controllable pore size of uniform size and a highly permeable porous structure with polyacrylic acid hydrogel and poly (isopropyl acrylamide) hydrogel as the basic materials. The material composition of the polyacrylic acid poly (isopropyl acrylamide) double network hydrogel has low cytotoxicity and immunogenicity, can reduce the stimulation and rejection reaction to the eye tissue, and is conducive to the attachment and growth of cells. The interspersed double network structure gives the scaffold sufficient strength and toughness to enable it to withstand the normal physiological pressure and movement of the eye while maintaining the stability of the structure. The porous characteristics are conducive to the exchange of nutrients and metabolic wastes, providing a good microenvironment for cell survival and function maintenance, and can guide the migration, proliferation and differentiation of corneal cells, which is conducive to the repair and regeneration of damaged corneal tissue.

[0047] 2. The present invention performs ultrasonic-coupled succinylation modification on krill protein. Ultrasonic treatment can increase the solubility and dispersibility of krill protein. Succinylation modification can introduce succinyl groups into krill protein. The introduction of new functional groups can enhance the interaction between krill protein and polyacrylic acid, improve compatibility, and thus improve the mechanical properties and biocompatibility of the artificial corneal scaffold.

[0048] 3. The present invention modifies modified polyacrylic acid by modified krill protein. The modified krill protein has good biocompatibility and biological activity, can be well integrated with human tissue, reduce the occurrence of immune rejection reaction, and improve the stability and safety of artificial corneal scaffold after implantation. After modification, the mechanical strength of polyacrylic acid can be improved, so that it can better withstand the pressure and tension of the eye, while improving its flexibility and air permeability, which is conducive to the delivery of nutrients and the discharge of metabolic waste. After the modified krill protein is modified, the modified polyacrylic acid is conducive to the attachment, proliferation and migration of corneal cells on the artificial corneal scaffold, and the regeneration and repair process of corneal tissue is accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0050] Figure 1 The present invention is a flowchart of a method for preparing an artificial cornea scaffold used in an embodiment of the present invention. DETAILED DESCRIPTION

[0051] The following is a detailed description of a method for preparing an artificial cornea support provided by the present invention in conjunction with the accompanying drawings and specific embodiments.

[0052] Example 1

[0053] A method for preparing an artificial cornea scaffold, such as Figure 1 As shown, the following steps are included:

[0054] S1: Preparation of modified krill protein

[0055] S1.1: krill protein and deionized water were mixed in a weight ratio of 1:100, and the mixture was stirred at room temperature for 1 hour to obtain a suspension. The suspension was treated at 100 W for 30 minutes using an ultrasonic probe with a diameter of 10 mm in an ice bath, with an on time of 5 seconds and an off time of 5 seconds, to obtain an ultrasonically treated krill protein mixture;

[0056] S1.2: adding succinic anhydride to the krill protein mixture after ultrasonic treatment, the mass ratio of succinic anhydride to krill protein is 1:10, and then stirring at room temperature for 3 hours. During the stirring process, 1M sodium hydroxide solution is used to adjust the pH to keep it at 12.5, and a mixed solution is obtained after the stirring is completed;

[0057] S1.3: adjusting the pH of the mixed solution to 4, centrifuging to obtain protein, dialyzing the protein with distilled water at 4°C for 24 hours, and then freeze-drying to obtain modified krill protein;

[0058] S2: Preparation of polymethyl methacrylate microsphere template

[0059] S2.1: 40 parts by weight of methyl methacrylate monomer and 350 parts by weight of deionized water were mixed and added to a four-necked glass flask with stirring, nitrogen was introduced, condensed water was opened, and the mixture was heated at 50° C. for 20 min. After the reaction, the mixture was cooled to room temperature to obtain a reaction solution;

[0060] S2.2: Add 1 part by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it to 70°C again, then dissolve 0.2 parts by weight of initiator ammonium persulfate in 30 parts by weight of deionized water, stir evenly and add to the reaction solution, stir and react for 2 hours to obtain a microsphere emulsion;

[0061] S2.3: The microsphere emulsion is injected into the mold by a syringe. The mold is composed of two glass slides sandwiching a polytetrafluoroethylene gasket with a thickness of 0.5 mm. The mold capacity is 0.6 mm. The mold is ultrasonically vibrated for 15 minutes, and then sintered at 175°C for 24 hours. The polymethyl methacrylate microsphere template mold is taken out and cooled at room temperature for standby use.

[0062] S3: Modified krill protein modified with modified polyacrylic acid

[0063] S3.1: dissolving 2 parts by weight of modified krill protein in 5 parts by weight of 5% sodium hydroxide solution, and using an ultrasonic cell pulverizer to accelerate the dissolution for 5 minutes to obtain a krill protein solution;

[0064] S3.2: 4 parts by weight of acrylic acid are added to 10 parts by weight of deionized water, and then the krill protein solution is added, and the mixture is stirred at 75° C. for 1 hour under a nitrogen environment, and then 0.03 parts by weight of N'N-methylenebisacrylamide and 0.00004 parts by weight of tetramethylethylenediamine are added, and the mixture is stirred and mixed to obtain a mixed solution;

[0065] S3.3: Add 1 part by weight of D-gluconolactone to the mixed solution, stir and mix, then add 0.07 part by weight of calcium carbonate, and then react for 2 hours to obtain a mixed solution, and pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template, evacuate, and cure in an oven at 80°C for 1 hour, and then cure at room temperature for 24 hours to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0066] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0067] S4.1: dissolving 10 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2 parts by weight of ultrapure water and ethanol, wherein the weight ratio of ultrapure water to ethanol is 1:1, and ultrasonically vibrating for 10 minutes to obtain a mixed solution;

[0068] S4.2: Add 0.002 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix well, add 0.05 parts by weight of initiator 2,2-dimethoxy-phenyl ketone, and perform ultrasonic vibration for 10 minutes to obtain a mixed reaction solution;

[0069] S4.3: The mixed reaction liquid is mixed by vortexing to obtain an N-isopropylacrylamide monomer reaction liquid, and the N-isopropylacrylamide monomer reaction liquid is placed in a sealed container, filled with nitrogen for 3 minutes, and after the air is fully exhausted, the liquid is placed in a central control drying room for standby use;

[0070] S5: Preparation of artificial cornea scaffold

[0071] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing for 1 hour, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect it to a freeze-drying vacuum machine interface and freeze-dry it at -70°C under vacuum for 48 hours to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0072] S5.2: injecting N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep the mold full, placing the mold in a glass vacuum tank and swelling for 72 hours to obtain a swollen composite hydrogel;

[0073] S5.3: moving the swollen composite hydrogel under a UV lamp for irradiation, irradiating the front and back sides of the swollen composite hydrogel in the mold with UV light for 5 min each, to obtain a stable double network hydrogel mixture;

[0074] S5.4: Put the double network hydrogel mixture into a Soxhlet extraction apparatus, use dichloromethane to dissolve the polymethyl methacrylate microsphere template, heat the round bottom flask of the Soxhlet extraction apparatus to 50°C, extract for 72 hours, and obtain a stable double network hydrogel;

[0075] S5.5: The stable double network hydrogel is placed in acetone and pure water respectively, and shaken and rinsed for 7 days, and the new solvent is replaced every 20 hours. After the rinsing, an artificial cornea scaffold is obtained.

[0076] Example 2

[0077] A method for preparing an artificial cornea scaffold, such as Figure 1 As shown, the following steps are included:

[0078] S1: Preparation of modified krill protein

[0079] S1.1: krill protein and deionized water were mixed in a weight ratio of 1:100, and the mixture was stirred at room temperature for 2 hours to obtain a suspension. The suspension was treated at 300 W for 35 minutes using an ultrasonic probe with a diameter of 12 mm in an ice bath, with an on time of 6 seconds and an off time of 6 seconds to obtain an ultrasonically treated krill protein mixture;

[0080] S1.2: adding succinic anhydride to the krill protein mixture after ultrasonic treatment, the mass ratio of succinic anhydride to krill protein is 1:10, and then stirring at room temperature for 4 hours. During the stirring process, 1M sodium hydroxide solution is used to adjust the pH to keep it at 12.5, and a mixed solution is obtained after the stirring is completed;

[0081] S1.3: adjusting the pH of the mixed solution to 4, centrifuging to obtain protein, dialyzing the protein with distilled water at 5°C for 25 hours, and then freeze-drying to obtain modified krill protein;

[0082] S2: Preparation of polymethyl methacrylate microsphere template

[0083] S2.1: 40 parts by weight of methyl methacrylate monomer and 350 parts by weight of deionized water were mixed and added to a four-necked glass flask with stirring, nitrogen was introduced, condensed water was opened, and the mixture was heated at 50° C. for 20 min. After the reaction, the mixture was cooled to room temperature to obtain a reaction solution;

[0084] S2.2: Add 1 part by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it to 72°C again, then dissolve 0.2 parts by weight of initiator ammonium persulfate in 30 parts by weight of deionized water, stir evenly and add to the reaction solution, stir and react for 3 hours to obtain a microsphere emulsion;

[0085] S2.3: The microsphere emulsion is injected into the mold by a syringe. The mold is composed of two glass slides sandwiching a polytetrafluoroethylene gasket with a thickness of 0.5 mm. The mold capacity is 0.6 mm. The mold is ultrasonically vibrated for 20 minutes, and then sintered at 180°C for 25 hours. The polymethyl methacrylate microsphere template mold is taken out and cooled at room temperature for standby use.

[0086] S3: Modified krill protein modified with modified polyacrylic acid

[0087] S3.1: dissolving 2 parts by weight of modified krill protein in 5 parts by weight of 5% sodium hydroxide solution, and using an ultrasonic cell pulverizer to accelerate the dissolution for 10 minutes to obtain a krill protein solution;

[0088] S3.2: 4 parts by weight of acrylic acid are added to 10 parts by weight of deionized water, and then the krill protein solution is added, and the mixture is stirred at 80° C. for 2 h under a nitrogen environment, and then 0.03 parts by weight of N'N-methylenebisacrylamide and 0.00004 parts by weight of tetramethylethylenediamine are added, and the mixture is stirred and mixed to obtain a mixed solution;

[0089] S3.3: Add 1 part by weight of D-gluconolactone to the mixed solution, stir and mix, then add 0.07 part by weight of calcium carbonate, and then react for 3 hours to obtain a mixed solution, and pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template, evacuate, and cure in an oven at 85°C for 2 hours, and then cure at room temperature for 25 hours to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0090] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0091] S4.1: dissolving 10 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2 parts by weight of ultrapure water and ethanol, wherein the weight ratio of ultrapure water to ethanol is 1:1, and ultrasonically vibrating for 12 minutes to obtain a mixed solution;

[0092] S4.2: Add 0.002 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix well, add 0.05 parts by weight of initiator 2,2-dimethoxy-phenyl ketone, and perform ultrasonic vibration for 12 minutes to obtain a mixed reaction solution;

[0093] S4.3: The mixed reaction liquid is mixed by vortexing to obtain an N-isopropylacrylamide monomer reaction liquid, and the N-isopropylacrylamide monomer reaction liquid is placed in a sealed container, filled with nitrogen for 5 minutes, and after the air is fully exhausted, the container is placed in a central control drying room for standby use;

[0094] S5: Preparation of artificial cornea scaffold

[0095] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing for 2 hours, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect it to a freeze-drying vacuum machine interface, and freeze-dry it at -72°C under vacuum for 50 hours to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0096] S5.2: injecting N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep the mold full, placing the mold in a glass vacuum tank and swelling for 75 hours to obtain a swollen composite hydrogel;

[0097] S5.3: moving the swollen composite hydrogel under ultraviolet light for 8 minutes on both the front and back sides of the swollen composite hydrogel in the mold to obtain a stable double network hydrogel mixture;

[0098] S5.4: putting the double network hydrogel mixture into a Soxhlet extraction apparatus, using dichloromethane to dissolve the polymethyl methacrylate microsphere template, heating the round-bottom flask of the Soxhlet extraction apparatus to 60°C, extracting for 75 hours, and obtaining a stable double network hydrogel;

[0099] S5.5: The stable double network hydrogel is placed in acetone and pure water respectively, and shaken and rinsed for 8 days, and the new solvent is replaced every 24 hours. After the rinsing, an artificial cornea scaffold is obtained.

[0100] Example 3

[0101] A method for preparing an artificial cornea scaffold, such as Figure 1 As shown, the following steps are included:

[0102] S1: Preparation of modified krill protein

[0103] S1.1: krill protein and deionized water were mixed in a weight ratio of 2:100, and the mixture was stirred at room temperature for 1 hour to obtain a suspension. The suspension was treated at 100 W for 30 minutes using an ultrasonic probe with a diameter of 10 mm in an ice bath, with an on time of 5 seconds and an off time of 5 seconds, to obtain an ultrasonically treated krill protein mixture;

[0104] S1.2: adding succinic anhydride to the krill protein mixture after ultrasonic treatment, the mass ratio of succinic anhydride to krill protein is 2:10, and then stirring at room temperature for 3 hours. During the stirring process, a 2M sodium hydroxide solution is used to adjust the pH to keep it at 13, and a mixed solution is obtained after the stirring is completed;

[0105] S1.3: adjusting the pH of the mixed solution to 4.5, centrifuging to obtain protein, dialyzing the protein with distilled water at 4°C for 24 hours, and then freeze-drying to obtain modified krill protein;

[0106] S2: Preparation of polymethyl methacrylate microsphere template

[0107] S2.1: 50 parts by weight of methyl methacrylate monomer and 400 parts by weight of deionized water were mixed and added to a four-necked glass flask with stirring, nitrogen was introduced, condensed water was opened, and the mixture was heated at 50° C. for 20 min. After the reaction, the mixture was cooled to room temperature to obtain a reaction solution;

[0108] S2.2: Add 2 parts by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it to 70°C again, then dissolve 0.3 parts by weight of initiator ammonium persulfate in 40 parts by weight of deionized water, stir evenly and add to the reaction solution, stir and react for 2 hours to obtain a microsphere emulsion;

[0109] S2.3: The microsphere emulsion is injected into the mold by a syringe. The mold is composed of two glass slides sandwiching a polytetrafluoroethylene gasket with a thickness of 1 mm. The mold capacity is 0.7 mm. The mold is ultrasonically vibrated for 15 minutes, and then sintered at 175°C for 24 hours. The polymethyl methacrylate microsphere template mold is taken out and cooled at room temperature for standby use.

[0110] S3: Modified krill protein modified with modified polyacrylic acid

[0111] S3.1: dissolving 3 parts by weight of modified krill protein in 8 parts by weight of 5% sodium hydroxide solution, and using an ultrasonic cell pulverizer to accelerate the dissolution for 5 minutes to obtain a krill protein solution;

[0112] S3.2: 5 parts by weight of acrylic acid are added to 20 parts by weight of deionized water, and then the krill protein solution is added, and the mixture is stirred at 75° C. for 1 hour under a nitrogen environment, and then 0.04 parts by weight of N'N-methylenebisacrylamide and 0.00005 parts by weight of tetramethylethylenediamine are added, and the mixture is stirred and mixed to obtain a mixed solution;

[0113] S3.3: Add 2 parts by weight of D-gluconolactone to the mixed solution, stir and mix, then add 0.08 parts by weight of calcium carbonate, and then react for 2 hours to obtain a mixed solution, and pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template, evacuate, and cure in an oven at 80°C for 1 hour, and then cure at room temperature for 24 hours to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0114] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0115] S4.1: dissolving 12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 3 parts by weight of ultrapure water and ethanol, wherein the weight ratio of ultrapure water to ethanol is 2:1, and ultrasonically vibrating for 10 minutes to obtain a mixed solution;

[0116] S4.2: Add 0.003 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix well, add 0.07 parts by weight of initiator 2,2-dimethoxy-phenyl ketone, and perform ultrasonic vibration for 10 minutes to obtain a mixed reaction solution;

[0117] S4.3: The mixed reaction liquid is mixed by vortexing to obtain an N-isopropylacrylamide monomer reaction liquid, and the N-isopropylacrylamide monomer reaction liquid is placed in a sealed container, filled with nitrogen for 3 minutes, and after the air is fully exhausted, the liquid is placed in a central control drying room for standby use;

[0118] S5: Preparation of artificial cornea scaffold

[0119] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing for 1 hour, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect it to a freeze-drying vacuum machine interface and freeze-dry it at -70°C under vacuum for 48 hours to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0120] S5.2: injecting N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep the mold full, placing the mold in a glass vacuum tank and swelling for 72 hours to obtain a swollen composite hydrogel;

[0121] S5.3: moving the swollen composite hydrogel under a UV lamp for irradiation, irradiating the front and back sides of the swollen composite hydrogel in the mold with UV light for 5 min each, to obtain a stable double network hydrogel mixture;

[0122] S5.4: Put the double network hydrogel mixture into a Soxhlet extraction apparatus, use dichloromethane to dissolve the polymethyl methacrylate microsphere template, heat the round bottom flask of the Soxhlet extraction apparatus to 50°C, extract for 72 hours, and obtain a stable double network hydrogel;

[0123] S5.5: The stable double network hydrogel is placed in acetone and pure water respectively, and shaken and rinsed for 7 days, and the new solvent is replaced every 20 hours. After the rinsing, an artificial cornea scaffold is obtained.

[0124] Comparative Example 1

[0125] Compared with Example 1, the difference of Comparative Example 1 is that Comparative Example 1 removes step S1, replaces the modified krill protein in step S3.1 with an equal weight portion of krill protein, and prepares an artificial cornea scaffold without changing the other steps, which is recorded as Comparative Example 1.

[0126] Comparative Example 2

[0127] Compared with Example 1, the difference of Comparative Example 2 is that the krill protein solution in step S1, step S3.1 and step S3.2 is removed in Comparative Example 2, and the artificial cornea scaffold is prepared without changing the other steps, which is recorded as Comparative Example 2.

[0128] Relevant performance tests were performed on Examples 1-3 and Comparative Examples 1-2, and the test results are shown in Table 1.

[0129] Table 1. Performance test results of Examples 1-3 and Comparative Examples 1-2

[0130] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Moisture content (%) 93 92 92 83 79 Tensile strength(Kpa) 678 670 673 597 582 Elongation at break (%) 258 249 251 213 229 In vitro cytotoxicity assay ≤1 ≤1 ≤1 ≤2 ≤2 Light transmittance (%) 95 93 94 90 85

[0131] It can be seen from the data in Table 1 that the data of the embodiments are better than those of the comparative examples. The in vitro cytotoxicity test of comparative examples 1-2 is ≤ Grade 2, which affects the biocompatibility of the artificial corneal scaffold. After adding the modified krill protein, the in vitro cytotoxicity test is ≤ Grade 1, which meets the Class III medical device standard and the product clinical biocompatibility standard. At the same time, the mechanical properties of the embodiments are better than those of comparative examples 1-2. Therefore, the modified polyacrylic acid modified with the modified krill protein can improve the mechanical properties and biocompatibility of the artificial corneal scaffold, and improve the stability and safety of the artificial corneal scaffold after implantation. It can be seen from the data in the embodiments that the artificial corneal scaffold prepared by the present invention has good biocompatibility, excellent mechanical properties and a simple and feasible preparation process, which is of great significance for improving the therapeutic effect of corneal diseases and improving the quality of life of patients.

[0132] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing an artificial cornea scaffold, characterized in that: The steps include: S1: Preparation of modified krill protein The krill protein is subjected to ultrasound-coupled succinylation modification to obtain modified krill protein; S2: Preparation of polymethyl methacrylate microsphere template Methyl methacrylate monomer and deionized water are mixed and reacted to obtain a reaction solution, an emulsifier and an initiator are added to the reaction solution, and then the mixture is injected into a mold for sintering to obtain a polymethyl methacrylate microsphere template; S3: Modified krill protein modified with modified polyacrylic acid Acrylic acid is added to deionized water and krill protein solution, and then N'N-methylenebisacrylamide, tetramethylethylenediamine, D-gluconolactone and calcium carbonate are added to react in a nitrogen atmosphere, and then poured into a mold of a sintered polymethyl methacrylate microsphere template, and vacuum cured to obtain a polyacrylic acid composite hydrogel-microsphere template mixture; S4: Preparation of N-isopropylacrylamide monomer reaction solution Dissolving N-isopropylacrylamide monomer in a mixed solvent of ultrapure water and ethanol, adding a crosslinking agent and an initiator, and mixing by vortexing to obtain an N-isopropylacrylamide monomer reaction solution; S5: Preparation of artificial cornea scaffold The polyacrylic acid composite hydrogel-microsphere template mixture is frozen and then freeze-dried in vacuo, and N-isopropylacrylamide monomer reaction solution is injected into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe, and the mixture is swollen and irradiated under ultraviolet light, followed by Soxhlet extraction and rinsing to obtain an artificial corneal scaffold.

2. The method for preparing an artificial cornea scaffold according to claim 1, characterized in that: Step S1: Preparation of modified krill protein, specifically comprising the following steps: S1.1: krill protein and deionized water are mixed in a weight ratio of 1-2:100, and the mixture is stirred at room temperature for 1-2 hours to obtain a suspension. The suspension is treated at 100-300W for 30-35 minutes using an ultrasonic probe with a diameter of 10-12 mm in an ice bath, with an on time of 5-6 seconds and an off time of 5-6 seconds, to obtain an ultrasonically treated krill protein mixture; S1.2: adding succinic anhydride to the krill protein mixture after ultrasonic treatment, and then stirring at room temperature for 3-4 hours. During the stirring process, a 1-2M sodium hydroxide solution is used to adjust the pH to maintain it at 12.5-13, and a mixed solution is obtained after the stirring is completed; S1.3: The pH of the mixed solution is adjusted to 4-4.5, and the mixture is centrifuged to obtain protein. The protein is dialyzed with distilled water at 4-5°C for 24-25 hours, and then freeze-dried to obtain modified krill protein.

3. The method for preparing an artificial cornea scaffold according to claim 2, characterized in that: Step S2: Preparation of polymethyl methacrylate microsphere template, specifically comprising the following steps: S2.1: 40-50 parts by weight of methyl methacrylate monomer and 350-400 parts by weight of deionized water are mixed and added to a four-necked glass flask with stirring, nitrogen is introduced, condensed water is opened, and the mixture is heated at 50-60° C. for 20-23 min. After the reaction, the mixture is cooled to room temperature to obtain a reaction solution; S2.2: Add 1-2 parts by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it to 70-72°C again, then dissolve 0.2-0.3 parts by weight of initiator ammonium persulfate in 30-40 parts by weight of deionized water, stir evenly and add to the reaction solution, stir and react for 2-3 hours to obtain a microsphere emulsion; S2.3: Pour the microsphere emulsion into the mold using a syringe, ultrasonically vibrate the mold for 15-20 minutes, and then sinter at 175-180° C. for 24-25 hours. Take out the polymethyl methacrylate microsphere template mold, cool it at room temperature, and set it aside.

4. The method for preparing an artificial cornea scaffold according to claim 3, characterized in that: Step S3: modifying krill protein to modify polyacrylic acid, specifically comprising the following steps: S3.1: dissolving 2-3 parts by weight of modified krill protein in 5-8 parts by weight of 5% sodium hydroxide solution, and using an ultrasonic cell pulverizer to accelerate the dissolution for 5-10 minutes to obtain a krill protein solution; S3.2: Add 4-5 parts by weight of acrylic acid to 10-20 parts by weight of deionized water, then add the krill protein solution, stir and react at 75-80° C. under a nitrogen environment for 1-2 hours, then add 0.03-0.04 parts by weight of N'N-methylenebisacrylamide and 0.00004-0.00005 parts by weight of tetramethylethylenediamine, stir and mix well to obtain a mixed solution; S3.3: Add 1-2 parts by weight of D-gluconolactone to the mixed solution, stir and mix, then add 0.07-0.08 parts by weight of calcium carbonate, and then react for 2-3 hours to obtain a mixed solution, and pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template. After vacuuming, cure it in an oven at 80-85°C for 1-2 hours, and then cure it at room temperature for 24-25 hours to obtain a polyacrylic acid composite hydrogel-microsphere template mixture.

5. The method for preparing an artificial cornea scaffold according to claim 4, characterized in that: step The preparation of S4N-isopropylacrylamide monomer reaction solution specifically includes the following steps: S4.1: dissolving 10-12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2-3 parts by weight of ultrapure water and ethanol, and ultrasonically shaking for 10-12 minutes to obtain a mixed solution; S4.2: Add 0.002-0.003 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix well, add 0.05-0.07 parts by weight of initiator 2,2-dimethoxy-phenyl ketone, and perform ultrasonic oscillation for 10-12 minutes to obtain a mixed reaction solution; S4.3: Vortex the mixed reaction liquid to obtain N-isopropylacrylamide monomer reaction liquid, put the N-isopropylacrylamide monomer reaction liquid into a sealed container, fill it with nitrogen for 3-5 minutes, and after fully exhausting the air, put it into a central control drying room for standby use.

6. The method for preparing an artificial cornea scaffold according to claim 5, characterized in that: Step S5: Preparation of artificial cornea scaffold: S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing for 1-2 hours, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect the freeze-drying vacuum machine interface at -70°C to -72°C, and freeze-dry for 48-50 hours under vacuum to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture; S5.2: injecting N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep the mold full, placing the mold in a glass vacuum tank and swelling for 72-75 hours to obtain a swollen composite hydrogel; S5.3: moving the swollen composite hydrogel under an ultraviolet lamp for irradiation, irradiating the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5-8 minutes each, to obtain a stable double network hydrogel mixture; S5.4: putting the double network hydrogel mixture into a Soxhlet extraction apparatus, using dichloromethane to dissolve the polymethyl methacrylate microsphere template, heating the round-bottom flask of the Soxhlet extraction apparatus to 50-60°C, extracting for 72-75 hours, and obtaining a stable double network hydrogel; S5.5: The stable double network hydrogel is placed in acetone and pure water respectively, and shaken and rinsed for 7-8 days, and the new solvent is replaced every 20-24 hours. After the rinsing, an artificial cornea scaffold is obtained.

7. The method for preparing an artificial cornea scaffold according to claim 2, characterized in that: In step S1.2, the mass ratio of succinic anhydride to krill protein is 1-2:

10.

8. The method for preparing an artificial cornea scaffold according to claim 3, characterized in that: In step S2.3, the mold is composed of two glass slides sandwiching a polytetrafluoroethylene gasket with a thickness of 0.5-1 mm, and the mold capacity volume is 0.6-0.7 mm.

9. The method for preparing an artificial cornea scaffold according to claim 5, characterized in that: The weight ratio of ultrapure water to ethanol in step S4.1 is 1-2:

1.

10. An artificial cornea support, characterized in that: It is prepared by the method for preparing an artificial cornea scaffold as described in any one of claims 1 to 9.

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