An artificial cornea stent and its preparation method

Abstract

The present invention provides an artificial cornea stent and a preparation method thereof, belonging to the technical field of new material preparation; the preparation process includes the following steps: preparation of modified krill protein; preparation of polymethyl methacrylate microsphere template; modification of polyacrylic acid with modified krill protein; preparation of N-isopropylacrylamide monomer reaction solution; preparation of artificial cornea stent. In the present invention, the material composition of the double-network hydrogel has low cytotoxicity and immunogenicity, can reduce the irritation and rejection reaction to ocular tissues, is helpful for cell attachment and growth, and the interpenetrating double-network structure endows the stent with sufficient strength and toughness, enabling it to withstand the normal physiological pressure and movement of the eye, while maintaining the structural stability. The porous property is conducive to the exchange of nutrients and metabolic wastes, provides a good microenvironment for cell survival and function maintenance, and can also guide the migration, proliferation and differentiation of corneal cells, contributing to the repair and regeneration of damaged corneal tissues.

Description

Technical Field

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

[0002] 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 restricting the wide application of corneal transplantation. In addition, traditional corneal transplantation surgeries also have problems such as immune rejection reactions and postoperative complications, bringing certain risks and challenges to the rehabilitation of patients.

[0003] With the continuous development of biomedical engineering technology, the research on artificial cornea stents has gradually become an important direction for solving the treatment problems of corneal diseases. Currently, the existing artificial cornea stents have many deficiencies in terms of material selection, structural design, and preparation processes.

[0004] For example, some artificial cornea stents use materials with poor biocompatibility, which are prone to causing inflammatory reactions and tissue rejection; the structural designs of some stents are unreasonable and cannot effectively simulate the physiological structure and functions of natural corneas, resulting in poor mechanical and optical properties; there are also some preparation methods that are complex, costly, and difficult to achieve large-scale production and clinical application.

[0005] Therefore, developing a new type of artificial cornea stent and its preparation method, which have 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 the quality of life of patients. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an artificial cornea stent and a preparation method thereof.

[0007] An artificial cornea stent and a preparation method thereof include the following steps:

[0008] S1: Preparation of modified krill protein

[0009] Perform ultrasonic coupling succinylation modification on krill protein to obtain modified krill protein;

[0010] S2: Preparation of polymethyl methacrylate microsphere template

[0011] Mix methyl methacrylate monomer with deionized water and react to obtain a reaction solution. Add an emulsifier and an initiator to the reaction solution, react, and then inject it into a mold for sintering to obtain a polymethyl methacrylate microsphere template;

[0012] S3: Modify modified polyacrylic acid with modified krill protein

[0013] Acrylic acid was added to deionized water and then to the krill protein solution. Under a nitrogen atmosphere, after the reaction, N,N'-methylenebisacrylamide, tetramethylethylenediamine, D-glucono-δ-lactone, and calcium carbonate were added. After the reaction, it was poured into a mold of a sintered polymethyl methacrylate microsphere template, and vacuum cured to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0014] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0015] The N-isopropylacrylamide monomer was dissolved in a mixed solvent of ultrapure water and ethanol, and a crosslinking agent and an initiator were added. After vortex mixing, an N-isopropylacrylamide monomer reaction solution was obtained;

[0016] S5: Preparation of artificial cornea stent

[0017] The polyacrylic acid composite hydrogel-microsphere template mixture was frozen and then vacuum freeze-dried. The N-isopropylacrylamide monomer reaction solution was injected into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template through a syringe, swollen, irradiated under an ultraviolet lamp, and then subjected to Soxhlet extraction and rinsing to obtain an artificial cornea stent.

[0018] Furthermore, the preparation of the modified krill protein in step S1 specifically includes the following steps:

[0019] S1.1: The krill protein and deionized water were mixed at a weight ratio of 1-2:100, and then the mixture was stirred at room temperature for 1-2 h to obtain a suspension. Under an ice bath condition, a suspension was treated with an ultrasonic probe with a diameter of 10-12 mm at 100-300 W for 30-35 min. The treatment process was to turn on for 5-6 s and turn off for 5-6 s to obtain an ultrasonically treated krill protein mixture;

[0020] S1.2: Succinic anhydride was added to the ultrasonically treated krill protein mixture, and then stirred at room temperature for 3-4 h. During the stirring process, 1-2 M sodium hydroxide solution was used to adjust the pH to keep it at 12.5-13. After the stirring ended, a mixed solution was obtained;

[0021] S1.3: The pH of the mixed solution was adjusted to 4-4.5, centrifuged to obtain the protein, and the protein was dialyzed with distilled water at 4-5 °C for 24-25 h, and then freeze-dried to obtain the modified krill protein.

[0022] Furthermore, the preparation of the polymethyl methacrylate microsphere template in step S2 specifically includes the following steps:

[0023] S2.1: Mix 40 - 50 parts by weight of methyl methacrylate monomer and 350 - 400 parts by weight of deionized water, stir and add them into a four-necked glass flask, introduce nitrogen, turn on the condenser water, heat at 50 - 60 °C for 20 - 23 min, cool to room temperature after the reaction to obtain a reaction solution;

[0024] S2.2: Add 1 - 2 parts by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat it again to 70 - 72 °C, 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 it into the reaction solution, stir and react for 2 - 3 h to obtain a microsphere emulsion;

[0025] S2.3: Inject the microsphere emulsion into a mold using a syringe, ultrasonically vibrate the mold for 15 - 20 min, then sinter at 175 - 180 °C for 24 - 25 h, take out to obtain a polymethyl methacrylate microsphere template mold, and cool it to room temperature for standby.

[0026] Furthermore, step S3 modifies krill protein to modify polyacrylic acid, which specifically includes the following steps:

[0027] S3.1: Dissolve 2 - 3 parts by weight of modified krill protein in 5 - 8 parts by weight of 5% sodium hydroxide solution, use an ultrasonic cell disruptor to accelerate dissolution for 5 - 10 min to obtain a krill protein solution;

[0028] 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 for 1 - 2 h in a nitrogen environment, 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 evenly to obtain a mixed solution;

[0029] S3.3: Add 1 - 2 parts by weight of D-glucono-δ-lactone 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 h to obtain a mixed solution. Pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template, evacuate, cure in an oven at 80 - 85 °C for 1 - 2 h, and then cure at room temperature for 24 - 25 h to obtain a polyacrylic acid composite hydrogel-microsphere template mixture.

[0030] Furthermore, the preparation of the N-isopropylacrylamide monomer reaction solution in step S4 specifically includes the following steps:

[0031] S4.1: Dissolve 10 - 12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2 - 3 parts by weight of ultrapure water and ethanol, ultrasonically vibrate for 10 - 12 min to obtain a mixed solution;

[0032] S4.2: Add 0.002 - 0.003 parts by weight of crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution. After mixing evenly, add 0.05 - 0.07 parts by weight of initiator 2,2 - dimethoxy - benzophenone, and ultrasonically vibrate for 10 - 12 min to obtain a mixed reaction solution;

[0033] S4.3: Vortex - mix the mixed reaction solution to obtain an N - isopropylacrylamide monomer reaction solution. Put the N - isopropylacrylamide monomer reaction solution into a sealed container, fill it with nitrogen for 3 - 5 min. After fully exhausting the air, put it into the central control drying room for standby.

[0034] Furthermore, the preparation of the artificial cornea stent in step S5 specifically includes the following steps:

[0035] S5.1: Put the polyacrylic acid composite hydrogel - microsphere template mixture into a container filled with liquid nitrogen for freezing treatment for 1 - 2 h. Then transfer the frozen polyacrylic acid composite hydrogel - microsphere template mixture to the container, connect it to the interface of the freeze - drying vacuum machine, and freeze - dry it at - 70 °C to - 72 °C under vacuum for 48 - 50 h to obtain an anhydrous polyacrylic acid composite hydrogel - microsphere template mixture;

[0036] S5.2: Inject the 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. Place the mold in a glass vacuum tank and swell it for 72 - 75 h to obtain a swollen composite hydrogel;

[0037] S5.3: Move the swollen composite hydrogel to be irradiated under an ultraviolet lamp, irradiate the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5 - 8 min each to obtain a stable double - network hydrogel mixture;

[0038] 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 - 60 °C, and extract for 72 - 75 h to obtain a stable double - network hydrogel;

[0039] S5.5: Place the stable double - network hydrogel in acetone and pure water successively and shake and rinse for 7 - 8 d, and replace the new solvent every 20 - 24 h. After the rinsing is completed, an artificial cornea stent is obtained.

[0040] Furthermore, in step S1.2, the mass ratio of succinic anhydride to krill protein is 1 - 2:10.

[0041] Furthermore, in step S2.3, the mold is composed of two glass slides clamping a polytetrafluoroethylene gasket with a thickness of 0.5 - 1 mm, and the mold capacity volume is 0.6 - 0.7 mm.

[0042] Further, in step S4.1, the weight ratio of ultrapure water to ethanol is 1-2:1.

[0043] An artificial cornea stent is prepared by the preparation method of an artificial cornea stent described in any one of the above.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] 1. The present invention effectively combines the spherical mold porous stent preparation technology and the double-network hydrogel preparation technology. Based on polyacrylic acid hydrogel and poly(isopropylacrylamide) hydrogel, an artificial cornea stent with a porous structure having uniformly sized controllable pores and highly penetrated internally is successfully prepared. The material composition of the polyacrylic acid-poly(isopropylacrylamide) double-network hydrogel has low cytotoxicity and immunogenicity, can reduce the irritation and rejection reaction to ocular tissues, is conducive to cell attachment and growth. The interpenetrating double-network structure endows the stent with sufficient strength and toughness, enabling it to withstand the normal physiological pressure and movement of the eye, while maintaining the structural stability. The porous characteristics are beneficial to the exchange of nutrients and metabolic wastes, providing a good microenvironment for cell survival and function maintenance, and at the same time can guide the migration, proliferation and differentiation of corneal cells, contributing to the repair and regeneration of damaged corneal tissues.

[0046] 2. The present invention performs ultrasonic coupling succinylation modification on krill protein. Ultrasonic treatment can increase the solubility and dispersibility of krill protein. Succinylation modification can introduce succinyl groups onto krill protein. The introduction of new functional groups can enhance the interaction between krill protein and polyacrylic acid, improve the compatibility, and further improve the mechanical properties and biocompatibility of the artificial cornea stent.

[0047] 3. The present invention modifies polyacrylic acid with modified krill protein. Modified krill protein has good biocompatibility and biological activity. It can better fuse with human tissues, reduce the occurrence of immune rejection reactions, improve the stability and safety after implantation of the artificial cornea stent. After modification, it can improve the mechanical strength of polyacrylic acid, enabling it to better withstand the pressure and tension of the eye, while improving its flexibility and breathability, contributing to the transmission of nutrients and the excretion of metabolic wastes. Modifying polyacrylic acid with modified krill protein helps the attachment, proliferation and migration of corneal cells on the artificial cornea stent, accelerating the regeneration and repair process of corneal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The drawings incorporated herein and constituting a part of the specification illustrate embodiments of the present disclosure and, together with the specification, are further used to explain the principles of the present disclosure and enable those skilled in the relevant art to implement and use the present disclosure.

[0049] Figure 1 Flow chart of a preparation method of an artificial cornea stent adopted in an embodiment of the present invention. Specific implementation mode

[0050] The following will describe in detail a preparation method of an artificial cornea stent provided by the present invention with reference to the accompanying drawings and specific embodiments.

[0051] Embodiment 1

[0052] A preparation method of an artificial cornea stent, as Figure 1 shown, includes the following steps:

[0053] S1: Preparation of modified krill protein

[0054] S1.1: Mix krill protein and deionized water in a weight ratio of 1:100, then stir the mixture at room temperature for 1 h to obtain a suspension. Under ice bath conditions, use an ultrasonic probe with a diameter of 10 mm to treat the suspension at 100 W for 30 min. The treatment process is to turn on for 5 s and turn off for 5 s to obtain the ultrasonically treated krill protein mixture;

[0055] S1.2: Add succinic anhydride to the ultrasonically treated krill protein mixture. The mass ratio of succinic anhydride to krill protein is 1:10. Then stir at room temperature for 3 h. During the stirring process, use 1 M sodium hydroxide solution to adjust the pH to keep it at 12.5. After the stirring ends, obtain a mixed solution;

[0056] S1.3: Adjust the pH of the mixed solution to 4, centrifuge to obtain the protein. Dialyze the protein with distilled water at 4 °C for 24 h, and then freeze-dry to obtain the modified krill protein;

[0057] S2: Preparation of poly(methyl methacrylate) microsphere template

[0058] S2.1: Mix 40 parts by weight of methyl methacrylate monomer and 350 parts by weight of deionized water, stir and add them to a four-necked glass flask, introduce nitrogen, turn on the condenser water, heat at 50 °C for 20 min, and cool to room temperature after the reaction to obtain a reaction solution;

[0059] S2.2: Add 1 part by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat to 70 °C again, then dissolve 0.2 part by weight of initiator ammonium persulfate in 30 parts by weight of deionized water, stir evenly and add it to the reaction solution, and stir and react for 2 h to obtain a microsphere emulsion;

[0060] 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.

[0061] S3: Modified krill protein modified with modified polyacrylic acid

[0062] 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;

[0063] 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;

[0064] 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;

[0065] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0066] 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;

[0067] 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;

[0068] 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;

[0069] S5: Preparation of artificial cornea scaffold

[0070] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture into a container filled with liquid nitrogen for freezing treatment for 1 h. Then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture into a container, connect it to the interface of a freeze-dryer, and freeze-dry it at -70°C under vacuum for 48 h to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0071] S5.2: Inject the 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. Place the mold in a glass vacuum tank and let it swell for 72 h to obtain a swollen composite hydrogel;

[0072] S5.3: Transfer the swollen composite hydrogel to under an ultraviolet lamp and irradiate the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5 min each to obtain a stable double-network hydrogel mixture;

[0073] 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, and extract for 72 h to obtain a stable double-network hydrogel;

[0074] S5.5: Place the stable double-network hydrogel in acetone and pure water successively and shake and rinse for 7 d, replacing the fresh solvent every 20 h. After the rinsing is completed, an artificial cornea stent is obtained.

[0075] Example 2

[0076] A preparation method of an artificial cornea stent, as Figure 1 shown, includes the following steps:

[0077] S1: Preparation of modified krill protein

[0078] S1.1: Mix krill protein and deionized water at a weight ratio of 1:100, then stir the mixture at room temperature for 2 h to obtain a suspension. Under ice bath conditions, use an ultrasonic probe with a diameter of 12 mm to treat the suspension at 300 W for 35 min. The treatment process is to turn on for 6 s and turn off for 6 s to obtain the ultrasonically treated krill protein mixture;

[0079] S1.2: Add succinic anhydride to the ultrasonically treated krill protein mixture. The mass ratio of succinic anhydride to krill protein is 1:10. Then stir at room temperature for 4 h. During the stirring process, use 1 M sodium hydroxide solution to adjust the pH to keep it at 12.5. After the stirring is completed, a mixed solution is obtained;

[0080] 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;

[0081] S2: Preparation of polymethyl methacrylate microsphere template

[0082] 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;

[0083] 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;

[0084] 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.

[0085] S3: Modified krill protein modified with modified polyacrylic acid

[0086] 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;

[0087] 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;

[0088] 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;

[0089] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0090] S4.1: Dissolve 10 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2 parts by weight of ultrapure water and ethanol, with the weight ratio of ultrapure water to ethanol being 1:1. Ultrasonically vibrate for 12 min to obtain a mixed solution;

[0091] S4.2: Add 0.002 parts by weight of the crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution. After mixing evenly, add 0.05 parts by weight of the initiator 2,2-dimethoxy-benzophenone. Ultrasonically vibrate for 12 min to obtain a mixed reaction solution;

[0092] S4.3: Vortex-mix the mixed reaction solution to obtain an N-isopropylacrylamide monomer reaction solution. Place the N-isopropylacrylamide monomer reaction solution in a sealed container, fill it with nitrogen for 5 min. After fully discharging the air, place it in a central control drying room for standby;

[0093] S5: Preparation of the artificial cornea stent

[0094] S5.1: Place the polyacrylic acid composite hydrogel-microsphere template mixture in a container filled with liquid nitrogen for freezing treatment for 2 h. Then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to a container, connect it to the interface of a freeze-drying vacuum machine, and freeze-dry it at -72°C under vacuum for 50 h to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0095] S5.2: Inject the 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. Place the mold in a glass vacuum tank and swell it for 75 h to obtain a swollen composite hydrogel;

[0096] S5.3: Move the swollen composite hydrogel to under an ultraviolet lamp and irradiate the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 8 min each to obtain a stable double-network hydrogel mixture;

[0097] 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 60°C, and extract for 75 h to obtain a stable double-network hydrogel;

[0098] S5.5: Place the stable double-network hydrogel in acetone and pure water successively and shake and rinse for 8 d, replacing the new solvent every 24 h. After the rinsing is completed, obtain the artificial cornea stent.

[0099] Example 3

[0100] A preparation method of an artificial cornea stent, as Figure 1 shown, includes the following steps:

[0101] S1: Preparation of modified krill protein

[0102] 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;

[0103] 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;

[0104] 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;

[0105] S2: Preparation of polymethyl methacrylate microsphere template

[0106] 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;

[0107] 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;

[0108] 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.

[0109] S3: Modified krill protein modified with modified polyacrylic acid

[0110] 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;

[0111] S3.2: Add 5 parts by weight of acrylic acid to 20 parts by weight of deionized water, then add the krill protein solution, stir and react at 75 °C for 1 h under a nitrogen atmosphere, then add 0.04 parts by weight of N,N'-methylenebisacrylamide and 0.00005 parts by weight of tetramethylethylenediamine, and stir and mix evenly to obtain a mixed solution;

[0112] S3.3: Add 2 parts by weight of D-glucono-lactone to the mixed solution, stir and mix, then add 0.08 parts by weight of calcium carbonate, and then react for 2 h to obtain a mixed solution. 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 h, and then cure at room temperature for 24 h to obtain a polyacrylic acid composite hydrogel-microsphere template mixture;

[0113] S4: Preparation of N-isopropylacrylamide monomer reaction solution

[0114] S4.1: Dissolve 12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 3 parts by weight of ultrapure water and ethanol, and the weight ratio of ultrapure water to ethanol is 2:1. Ultrasonically vibrate for 10 min to obtain a mixed solution;

[0115] S4.2: Add 0.003 parts by weight of the crosslinking agent tetraethylene glycol dimethacrylate to the mixed solution, mix evenly, then add 0.07 parts by weight of the initiator 2,2-dimethoxy-benzophenone, and ultrasonically vibrate for 10 min to obtain a mixed reaction solution;

[0116] S4.3: Vortex and mix the mixed reaction solution to obtain an N-isopropylacrylamide monomer reaction solution. Put the N-isopropylacrylamide monomer reaction solution into a sealed container, fill it with nitrogen for 3 min, fully discharge the air, and then put it into the central control drying room for standby;

[0117] S5: Preparation of artificial cornea stent

[0118] S5.1: Put the polyacrylic acid composite hydrogel-microsphere template mixture into a container filled with liquid nitrogen for freezing treatment for 1 h, and then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to the container, connect it to the interface of the freeze-drying vacuum machine, and freeze-dry at -70 °C under vacuum for 48 h to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture;

[0119] S5.2: Inject the 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. Place the mold in a glass vacuum tank and swell for 72 h to obtain a swollen composite hydrogel;

[0120] S5.3: Transfer the swollen composite hydrogel to under an ultraviolet lamp and irradiate both the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5 minutes each to obtain a stable double-network hydrogel mixture;

[0121] S5.4: Put the double-network hydrogel mixture into a Soxhlet extraction apparatus, dissolve the polymethyl methacrylate microsphere template with dichloromethane, heat the round-bottom flask of the Soxhlet extraction apparatus to 50 °C, and extract for 72 hours to obtain a stable double-network hydrogel;

[0122] S5.5: Place the stable double-network hydrogel successively in acetone and pure water, shake and rinse for 7 days, and replace the solvent with a new one every 20 hours. After the rinsing is completed, an artificial cornea scaffold is obtained.

[0123] Comparative Example 1

[0124] Compared with Example 1, the difference in Comparative Example 1 is that in Comparative Example 1, step S1 is removed, and the modified krill protein in step S3.1 is replaced with an equal weight portion of krill protein, and the remaining steps remain unchanged to prepare an artificial cornea scaffold, denoted as Comparative Example 1.

[0125] Comparative Example 2

[0126] Compared with Example 1, the difference in Comparative Example 2 is that in Comparative Example 2, the krill protein solution in step S1, step S3.1, and step S3.2 is removed, and the remaining steps remain unchanged to prepare an artificial cornea scaffold, denoted as Comparative Example 2.

[0127] Perform relevant performance tests on Examples 1-3 and Comparative Examples 1-2, and the test results are shown in Table 1 for reference.

[0128] Table 1. Performance test results of Examples 1-3 and Comparative Examples 1-2

[0129]

[0130] It can be seen from the data in Table 1 that the data of the examples are all 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 cornea scaffold. After adding the modified krill protein, its in vitro cytotoxicity test is ≤ grade 1, meeting the standards for Class III medical devices and satisfying the clinical biocompatibility standards of the product. At the same time, the mechanical properties of the examples are better than those of Comparative Examples 1-2. Therefore, using the modified krill protein to modify polyacrylic acid can improve the mechanical properties and biocompatibility of the artificial cornea scaffold, enhance the stability and safety after implantation of the artificial cornea scaffold. It can be seen from the data of the examples that the artificial cornea 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 treatment effect of corneal diseases and the quality of life of patients.

[0131] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing an artificial cornea stent, characterized in that, It includes the following steps: S1: Preparation of modified krill protein The krill protein is subjected to ultrasonic coupling succinylation modification to obtain modified krill protein; S2: Preparation of polymethyl methacrylate microsphere template Methyl methacrylate monomer is mixed with deionized water and reacted to obtain a reaction solution. An emulsifier and an initiator are added to the reaction solution, and after reaction, it is injected into a mold and sintered to obtain a polymethyl methacrylate microsphere template; S3: Modification of modified polyacrylic acid with modified krill protein S3.1: Dissolve 2-3 parts by weight of modified krill protein in 5-8 parts by weight of 5% sodium hydroxide solution, and use an ultrasonic cell disruptor to accelerate dissolution for 5-10 min 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, and stir and react at 75-80 °C for 1-2 h in a nitrogen environment. 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 evenly to obtain a mixed solution; S3.3: Add 1-2 parts by weight of D-glucono-δ-lactone 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 h to obtain a mixed solution. Pour the mixed solution into the mold of the sintered polymethyl methacrylate microsphere template, evacuate, and cure in an oven at 80-85 °C for 1-2 h, and then cure at room temperature for 24-25 h to obtain a polyacrylic acid composite hydrogel-microsphere template mixture; S4: Preparation of N-isopropylacrylamide monomer reaction solution Dissolve N-isopropylacrylamide monomer in a mixed solvent of ultrapure water and ethanol, add a crosslinking agent and an initiator, and obtain an N-isopropylacrylamide monomer reaction solution by vortex mixing; S5: Preparation of artificial cornea stent Freeze-dry the polyacrylic acid composite hydrogel-microsphere template mixture under vacuum, inject the N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe, swell, irradiate under an ultraviolet lamp, and then perform Soxhlet extraction and rinsing to obtain an artificial cornea stent.

2. The preparation method of an artificial cornea stent according to claim 1, wherein, The preparation of modified krill protein in step S1 specifically includes the following steps: S1.1: Mix krill protein and deionized water according to a weight ratio of 1-2:100, then stir the mixture at room temperature for 1-2 h to obtain a suspension. Under ice bath conditions, use an ultrasonic probe with a diameter of 10-12 mm to treat the suspension at 100-300 W for 30-35 min. The treatment process is to turn on for 5-6 s and turn off for 5-6 s to obtain the ultrasonically treated krill protein mixture; S1.2: Add succinic anhydride to the ultrasonically treated krill protein mixture, and then stir at room temperature for 3-4 h. During the stirring process, use 1-2 M sodium hydroxide solution to adjust the pH to keep it at 12.5-13. After the stirring ends, obtain a mixed solution; S1.3: Adjust the pH of the mixed solution to 4 - 4.5, centrifuge to obtain the protein, dialyze the protein in distilled water at 4 - 5 °C for 24 - 25 h, and then freeze-dry to obtain the modified krill protein.

3. The preparation method of an artificial cornea stent according to claim 2, wherein, Step S2 Preparation of polymethyl methacrylate microsphere templates, which specifically includes the following steps: S2.1: Mix 40 - 50 parts by weight of methyl methacrylate monomer and 350 - 400 parts by weight of deionized water, stir and add them to a four-necked glass flask, introduce nitrogen, turn on the condenser water, heat at 50 - 60 °C for 20 - 23 min, cool to room temperature after the reaction to obtain a reaction solution; S2.2: Add 1 - 2 parts by weight of emulsifier sodium dodecyl sulfate to the reaction solution, heat 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 it to the reaction solution, stir and react for 2 - 3 h to obtain a microsphere emulsion; S2.3: Inject the microsphere emulsion into a mold using a syringe, ultrasonically vibrate the mold for 15 - 20 min, then sinter at 175 - 180 °C for 24 - 25 h, take out to obtain a polymethyl methacrylate microsphere template mold, and cool at room temperature for standby.

4. The preparation method of an artificial cornea stent according to claim 3, characterized in that, Step S4 Preparation of N-isopropylacrylamide monomer reaction solution, which specifically includes the following steps: S4.1: Dissolve 10 - 12 parts by weight of N-isopropylacrylamide monomer in a mixed solvent of 2 - 3 parts by weight of ultrapure water and ethanol, ultrasonically vibrate for 10 - 12 min to obtain a mixed solution; S4.2: Add 0.002 - 0.003 parts by weight of crosslinker tetraethylene glycol dimethacrylate to the mixed solution, mix evenly and then add 0.05 - 0.07 parts by weight of initiator 2,2-dimethoxy-benzophenone, ultrasonically vibrate for 10 - 12 min to obtain a mixed reaction solution; S4.3: Vortex-mix the mixed reaction solution to obtain an N-isopropylacrylamide monomer reaction solution, put the N-isopropylacrylamide monomer reaction solution into a sealed container, fill it with nitrogen for 3 - 5 min, fully discharge the air, and then put it into a central control drying room for standby.

5. The preparation method of an artificial cornea stent according to claim 4, wherein, Step S5 Preparation of artificial cornea scaffolds: S5.1: Put the polyacrylic acid composite hydrogel-microsphere template mixture into a container filled with liquid nitrogen for freezing treatment for 1 - 2 h, then transfer the frozen polyacrylic acid composite hydrogel-microsphere template mixture to the container, connect it to the interface of the freeze-drying vacuum machine, and freeze-dry at -70 °C to -72 °C under vacuum for 48 - 50 h to obtain an anhydrous polyacrylic acid composite hydrogel-microsphere template mixture; S5.2: Inject the N-isopropylacrylamide monomer reaction solution into the mold of the anhydrous polyacrylic acid composite hydrogel-microsphere template mixture through a syringe to keep it full of the mold, place the mold in a glass vacuum tank and swell for 72 - 75 h to obtain a swollen composite hydrogel; S5.3: Move the swollen composite hydrogel to be irradiated under an ultraviolet lamp, irradiate the front and back sides of the swollen composite hydrogel in the mold with ultraviolet light for 5 - 8 min each to obtain a stable double-network hydrogel mixture. S5.4: Pour the double-network hydrogel mixture into a Soxhlet extraction apparatus, dissolve the poly(methyl methacrylate) microsphere template with dichloromethane, heat the round-bottom flask of the Soxhlet extraction apparatus to 50 - 60 °C, and extract for 72 - 75 h to obtain a stable double-network hydrogel; S5.5: Place the stable double-network hydrogel successively in acetone and pure water, shake and rinse for 7 - 8 d, and replace the solvent with fresh solvent every 20 - 24 h. After rinsing, an artificial cornea scaffold is obtained.

6. The preparation method of an artificial cornea stent according to claim 2, wherein, In step S1.2, the mass ratio of succinic anhydride to krill protein is 1 - 2:

10.

7. The preparation method of an artificial cornea stent according to claim 3, characterized in that, In step S2.3, the mold consists of two glass slides clamping a polytetrafluoroethylene gasket with a thickness of 0.5 - 1 mm, and the volume of the mold is 0.6 - 0.7 mm.

8. The preparation method of an artificial cornea stent according to claim 4, wherein, In step S4.1, the weight ratio of ultrapure water to ethanol is 1 - 2:

1.

9. An artificial cornea stent, characterized in that, It is prepared by the preparation method of an artificial cornea scaffold according to any one of claims 1 - 8 above.

Images