Biomimetic cornea and method for manufacturing the same

Abstract

The present application relates to a biomimetic cornea and a method for producing the same, which belongs to the field of biotechnology. The present application provides a biomimetic cornea. The biomimetic cornea is made from a polyethylene glycol-modified collagen-like protein, which includes a collagen-like protein and PEG-40k and PEG-20k modified on the collagen-like protein. By modifying with PEG-40k and PEG-20k, the crosslinkability of the collagen-like protein is significantly improved, and the strength of the cornea of ​​the present application is improved by about 20 times compared to the strength of a cornea produced using a collagen-like protein and by about 12 times compared to the strength of a cornea produced using other conventional polyethylene glycol-modified collagen-like proteins, so that the future of the cornea of ​​the present application in the field of corneal transplantation is highly promising. The molar ratio of PEG-40k to PEG-20k is 2 to 3:1. This molar ratio of PEG modification provides a higher effect of improving the crosslinkability of the collagen-like protein, and further improves the strength of the cornea of ​​the present application.

Description

Technical Field

[0005] ,

[0004] , , ,

[0001] [Cross - reference to Related Applications] This application claims the priority of a Chinese patent application with an application number of 202111059840.1 and an invention title of "Bio - mimicking Cornea and Its Manufacturing Method", which was filed with the China National Intellectual Property Administration on September 13, 2021. All the contents of this application are incorporated herein by reference.

[0002] [Technical Field] This application relates to a bio - mimicking cornea and its manufacturing method, belonging to the field of biotechnology.

Background Art

[0003] The cornea is the most anterior convex and highly transparent substance of the eye. It has a horizontally oval shape, covers the iris, pupil, and anterior chamber, and bears most of the refractive power of the eye. Combined with the refractive power of the lens, light can be accurately focused on the retina to form an image. The cornea has very sensitive nerve endings. When a foreign object contacts the cornea, the eyelid closes unconsciously to protect the eye. To maintain transparency, the cornea has no blood vessels and obtains nutrients and oxygen through tears and aqueous humor.

[0004] The cornea is very fragile. Eye trauma, inflammation, allergic reactions, physical damage, chemical burns, intense exercise, overuse of the eyes, etc. can all cause corneal lesions. When the cornea gets sick, obvious eye symptoms such as eye pain, photophobia, tearing, and vision loss will appear, and in severe cases, it can lead to blindness.

[0005] Corneal transplantation is a treatment method that replaces the patient's existing diseased cornea with a normal cornea to restore the vision of the diseased eye, control corneal lesions, improve vision, and treat specific corneal diseases. In the case of some corneal diseases that cause severe visual impairment or blindness in patients, corneal transplantation can achieve complete treatment and relieve the pain of these unfortunate patients. Since the cornea itself has no blood vessels and is in an "immune tolerance" state, the success rate of corneal transplantation is relatively high among other allogeneic organ transplants.

[0006] However, corneal resources are limited and cannot meet the needs of patients. To address this problem, some researchers have proposed artificial corneal transplantation. Artificial corneal transplantation is a surgical method that restores vision by surgically implanting a special optical device made of transparent medical polymer material into the corneal tissue, replacing a portion of the corneal scar tissue. However, problems such as rejection of synthetic materials by corneal tissue have not yet been resolved, and the long-term results are poor, often leading to leakage of aqueous humor at the transplant site or detachment of the graft, so it cannot be widely used at present. Currently, artificial corneas are only suitable for people who have lost sight in both eyes after suffering from various serious corneal diseases, especially those with corneal leukoplakia due to severe chemical burns, and those who have failed multiple corneal transplants and cannot undergo other surgeries.

[0007] Based on artificial corneas, researchers are proposing biomimetic corneas. For example, in the paper "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants," Jangamreddy, Jaganmohan R., et al. propose polyethylene glycolated collagen-like proteins. By chemically crosslinking these polyethylene glycolated collagen-like proteins, biomimetic corneas can be obtained. These biomimetic corneas possess excellent biocompatibility and can effectively solve the problem of strong rejection reactions of corneal tissue to artificial synthetic materials.

[0008] However, the strength of this biomimetic cornea is very low at 0.022 MPa, and if this biomimetic cornea is used for corneal transplantation, problems remain such as difficulty in transplantation and the possibility of causing keratoconus. [Overview of the project]

[0009] To address the problem of the low strength of conventional biomimetic corneas, this invention provides a biomimetic cornea using polyethylene glycolated collagen-like protein as a raw material, wherein the polyethylene glycolated collagen-like protein comprises a collagen-like protein and a polyethylene glycol derivative modified to the collagen-like protein, and the polyethylene glycol derivative includes PEG-40k and PEG-20k.

[0010] In one embodiment of the present invention, the biomimetic cornea is obtained by crosslinking and curing polyethylene glycolated collagen-like protein.

[0011] In one embodiment of the present application, the polyethylene glycol derivative consists of PEG-40k and PEG-20k.

[0012] In one embodiment of the present invention, the amino acid sequence of the collagen-like protein is shown as SEQ ID NO. 1. In SEQ ID NO. 1, X is 4Hyp (4-hydroxyproline), that is, The amino acid sequence of SEQ ID NO.1 is as follows: H-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-OH

[0013] In one embodiment of the present invention, the molar ratio of PEG-40k to PEG-20k is 0.5 to 6:1.

[0014] In one embodiment of the present application, the molar ratio of PEG-40k to PEG-20k is 2 to 3:1. In one embodiment of the present application, the number of active groups of PEG-40k is one or more of 8-arm, 4-arm, 2-arm, or 1-arm, and the number of active groups of PEG-20k is one or more of 8-arm, 4-arm, 2-arm, or 1-arm.

[0015] In one embodiment of the present application, the number of activating groups of PEG-40k is 4-arm or 8-arm, and the number of activating groups of PEG-20k is 4-arm or 8-arm.

[0016] In one embodiment of the present application, the activating group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS, or -EDC, and the activating group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS, or -EDC.

[0017] In one embodiment of the present application, the activating group of PEG-40k is -MAL, and the activating group of PEG-20k is -MAL.

[0018] In one embodiment of the present invention, a linker is attached to one end of the collagen-like protein, and the polyethylene glycol derivative is modified to the linker through an activating group.

[0019] In one embodiment of the present invention, the linker is attached to the N-terminus of a collagen-like protein.

[0020] In one embodiment of the present application, the amino acid sequence of the linker is indicated by SEQ ID NO.2 or SEQ ID NO.3.

[0021] In one embodiment of the present application, the modification site of the polyethylene glycol derivative to the linker is one or more of mercapto, amino, carboxy, or imidazolyl.

[0022] In one embodiment of the present application, when the amino acid sequence of the linker is shown by SEQ ID NO.2, the modification site of the polyethylene glycol derivative to the linker is mercapto; when the amino acid sequence of the linker is shown by SEQ ID NO.3, the modification site of the polyethylene glycol derivative to the linker is amino.

[0023] In one embodiment of the present application, the core conformation of the polyethylene glycolated collagen-like protein is one or more of HG or TP.

[0024] In one embodiment of the present application, the core conformation of the polyethylene glycol derivative in the polyethylene glycolated collagen-like protein is TP.

[0025] In one embodiment of the present application, the amino acid arrangement of the collagen-like protein in the polyethylene glycolated collagen-like protein is one or more of D-type or L-type.

[0026] In one embodiment of the present application, the molecular weight of the polyethylene glycolated collagen-like protein is 15,000 - 75,000 Da.

[0027] In one embodiment of the present application, the molecular weight of the polyethylene glycolated collagen-like protein is 30,000 - 75,000 Da.

[0028] The present application also provides a reaction step of reacting a collagen-like protein and a polyethylene glycol derivative under the conditions of pH 4.0 - 10.0 and temperature 2 - 40°C for 1 - 48 h to obtain a reaction product, a lyophilization step of lyophilizing the reaction product to obtain a lyophilized preparation, a cross-linking step of dissolving the lyophilized preparation in a buffer solution to obtain a dissolved solution, mixing the dissolved solution with an MPC mother solution to obtain a mixture 1, and mixing the mixture 1 with a DMTMM mother solution to obtain a mixture 2, The present invention provides a method for producing a biomimetic cornea, comprising a hardening step of injecting a mixed solution 2 into a corneal mold and allowing it to stand to obtain a crude corneal product.

[0029] In one embodiment of the present invention, the reaction step involves reacting a collagen-like protein with a polyethylene glycol derivative for 5 to 8 hours under conditions of pH 6.0 to 8.0 and a temperature of 2 to 8°C.

[0030] In one embodiment of the present invention, the ratio of the number of moles supplied of collagen-like protein and polyethylene glycol derivative in the reaction step is 1 to 16:1.

[0031] In one embodiment of the present invention, the ratio of the number of moles supplied of collagen-like protein and polyethylene glycol derivative in the reaction step is 8 to 12:1.

[0032] In one embodiment of the present invention, the reaction solvent for the collagen-like protein and polyethylene glycol derivative in the reaction step is water or a dilute hydrochloric acid solution.

[0033] In one embodiment of the present invention, the concentration of the dilute hydrochloric acid solution is 1 to 10 mmol / L, and the pH of the dilute hydrochloric acid solution is adjusted to 6.0 to 8.0 with a dilute alkaline solution.

[0034] In one embodiment of the present invention, the dilute alkaline solution is a sodium hydroxide solution or ammonia water with a pH of 9.0 to 11.0.

[0035] In one embodiment of the present invention, the supply concentration of collagen-like protein in the reaction solvent during the reaction step is 1 to 15 mg / mL.

[0036] In one embodiment of the present invention, the supply concentration of collagen-like protein in the reaction solvent during the reaction step is 8-10 mg / mL.

[0037] In one embodiment of the present invention, the freeze-drying step involves mixing the reaction product with a freeze-drying protective agent and freeze-drying it to obtain a freeze-dried formulation.

[0038] In one embodiment of the present invention, the freeze-drying protective agent is one or more of mannitol, sucrose, or alanine.

[0039] In one embodiment of the present invention, the freeze-drying includes the following steps: Stage 1: Freeze-dry for 6 hours under conditions of -45°C temperature and a vacuum of 500 m Torr. Stage 2: Freeze-dry for 17 hours under conditions of -30°C temperature and a vacuum of 100 m Torr. Stage 3: Freeze-dry for 7 hours under conditions of 25°C temperature and 100 m Torr vacuum.

[0040] In one embodiment of the present invention, the pH of the buffer solution in the crosslinking step is 5.5 to 8.0. In one embodiment of the present invention, the pH of the buffer solution in the crosslinking step is 6.5 to 7.5. In one embodiment of the present invention, the concentration of the buffer solution in the crosslinking step is 0.5 to 0.7 mol / L.

[0041] In one embodiment of the present invention, in the crosslinking step, the buffer is MOPS Buffer, MES Buffer, or PBS Buffer.

[0042] In one embodiment of the present invention, in the crosslinking step, the concentration of polyethylene glycolated collagen-like protein in the buffer solution is 5 to 40 g / mL.

[0043] In one embodiment of the present invention, in the crosslinking step, the concentration of polyethylene glycolated collagen-like protein in the buffer solution is 12 to 18 g / mL.

[0044] In one embodiment of the present invention, in the crosslinking step, the concentration of polyethylene glycolated collagen-like protein in the buffer solution is 15 g / mL.

[0045] In one embodiment of the present invention, in the crosslinking step, the mixing mass ratio of the dissolving solution and the MPC mother liquor is 2:1 to 4:1.

[0046] In one embodiment of the present invention, in the crosslinking step, the mixing mass ratio of the mixed liquid 1 and the DMTMM mother liquor is 5:1 to 7:1.

[0047] In one embodiment of the present invention, the crosslinking step is performed at 25 to 60°C. In one embodiment of the present invention, the crosslinking step is performed at 45 to 55°C.

[0048] In one embodiment of the present invention, the standing temperature in the curing step is 4 to 35°C. In one embodiment of the present invention, the standing temperature in the curing step is 4 to 25°C. In one embodiment of the present invention, the standing time in the curing step is 8 to 20 hours. In one embodiment of the present invention, the components of the MPC mother liquor include MPC (2-methacryloyloxyethyl phosphocholine), PEGDA (poly(ethylene glycol) diacrylate), TEMED (N,N,N',N'-tetramethylethylenediamine) and a solvent.

[0049] In one embodiment of the present application, the concentration of MPC in the MPC mother liquor is 15 to 40 g / mL, the concentration of PEGDA in the MPC mother liquor is 0.6 to 15% by volume, and the concentration of TEMED is 0.05 to 2% by volume.

[0050] In one embodiment of the present invention, the concentration of PEGDA in the MPC mother liquor is 8-12%. In one embodiment of the present invention, the concentration of PEGDA in the MPC mother liquor is 10%. In one embodiment of the present invention, the solvent of the MPC mother liquor is a buffer solution, and the pH of the buffer solution is 6.0-8.0.

[0051] In one embodiment of the present invention, the pH of the buffer solution in the MPC mother liquor is 6.5 to 7.5. In one embodiment of the present invention, the concentration of the buffer solution in the MPC mother liquor is 0.5 to 0.7 mol / L.

[0052] In one embodiment of the present invention, the buffer in the MPC mother liquor is MOPS Buffer, MES Buffer, or PBS Buffer.

[0053] In one embodiment of the present application, the components of the DMTMM mother liquor include DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride), APS (ammonium persulfate), and a solvent.

[0054] In one embodiment of the present invention, the concentration of DMTMM in the DMTMM mother liquor is 5 to 20 g / mL, and the concentration of APS is 0.5 to 5 g / mL.

[0055] In one embodiment of the present invention, the solvent of the DMTMM mother liquor is a buffer solution, and the pH of the buffer solution is 6.0 to 8.0.

[0056] In one embodiment of the present invention, the pH of the buffer solution in the DMTMM mother liquor is 6.5 to 7.5.

[0057] In one embodiment of the present invention, the concentration of the buffer solution in the DMTMM mother liquor is 0.5 to 0.7 mol / L.

[0058] In one embodiment of the present invention, in the DMTMM mother liquor, the buffer is MOPS Buffer, MES Buffer, or PBS Buffer.

[0059] In one embodiment of the present invention, after the hardening step, the method further includes an immersion step in which a crude corneal sample is placed in a buffer solution together with a mold and a first immersion is performed, after the first immersion is completed, the mold is opened and a second immersion is performed, and after the second immersion is completed, the mold is demolded to obtain a finished corneal product.

[0060] In one embodiment of the present invention, the pH of the buffer solution is 5.5 to 8.0 during the immersion step. In another embodiment of the present invention, the pH of the buffer solution is 6.5 to 7.5 during the immersion step.

[0061] In one embodiment of the present invention, the concentration of the buffer solution in the immersion step is 0.05 to 1 mol / L.

[0062] In one embodiment of the present invention, in the immersion step, the buffer is MOPS Buffer, MES Buffer, or PBS Buffer.

[0063] In one embodiment of the present invention, the first immersion is performed at a temperature of 4 to 35°C for a duration of 5 to 24 hours.

[0064] In one embodiment of the present invention, the second immersion is performed at a temperature of 4 to 35°C for a duration of 3 to 10 hours.

[0065] In one embodiment of the present application, after the reaction step and before the freeze-drying step, The above method further includes a purification step of filtering out substances with a molecular weight of 30,000 Da or more from the reaction product to obtain polyethylene glycolated collagen-like protein.

[0066] In one embodiment of the present invention, the filtration is dialysis or ultrafiltration. [Effects of the Invention]

[0067] The technical solution of this application has the following advantages.

[0068] 1. This invention provides a biomimetic cornea using polyethylene glycolated collagen-like protein as a raw material, wherein the polyethylene glycolated collagen-like protein comprises collagen-like protein and PEG-40k and PEG-20k modified to the collagen-like protein. By modifying with PEG-40k and PEG-20k, the crosslinkability of the collagen-like protein is significantly improved, and the strength of the cornea of ​​this invention can be increased by approximately 20 times compared to the strength of a cornea produced using collagen-like protein, and by approximately 12 times compared to the strength of a cornea produced using other conventional polyethylene glycolated collagen-like proteins (in the literature "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants," the strength of a cornea produced using polyethylene glycolated collagen-like protein is only 0.022 MPa). Therefore, the future potential of the cornea of ​​this invention in the field of corneal transplantation is extremely promising.

[0069] Furthermore, the molar ratio of PEG-40k to PEG-20k is 2-3:1. This molar ratio of PEG modification provides a better effect in improving the crosslinkability of collagen-like proteins, thereby further improving the strength of the cornea in this invention.

[0070] Furthermore, the PEG-40k is 8-arm-PEG-40k-MAL, and the PEG-20k is 4-arm-PEG-20k-MAL. This PEG modification configuration provides a better effect in improving the crosslinkability of collagen-like proteins, and can further improve the strength of the cornea in this invention.

[0071] 2. This application provides a method for producing the biomimetic cornea described above, the method comprising four steps: a reaction step, a freeze-drying step, a crosslinking step, and a curing step, wherein the reaction step involves reacting a collagen-like protein and a PEG derivative under conditions of pH 6.0-8.0 and a temperature of 2-8°C to obtain polyethylene glycolated collagen-like protein. The polyethylene glycolated collagen-like protein produced in this reaction step has superior crosslinkability and can further improve the strength of the produced cornea. Furthermore, compared to other conventional methods for producing polyethylene glycolated collagen-like protein, the reaction time in this reaction step is short, taking only 5-8 hours to obtain polyethylene glycolated collagen-like protein that can be made into a hydrogel for corneal formation (in the literature "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants," it takes 4 weeks to prepare polyethylene glycolated collagen-like protein that can be made into a hydrogel for corneal formation), contributing to large-scale industrial production of corneas.

[0072] Furthermore, in the reaction step, the ratio of the number of moles supplied for collagen-like protein to polyethylene glycol derivative is 8 to 12:1. Polyethylene glycolated collagen-like protein produced with this supply molar ratio has superior crosslinkability and can further improve the strength of the produced cornea.

[0073] Furthermore, in the reaction step, the supply concentration of collagen-like protein in the reaction solvent is 8-10 mg / mL. Polyethylene glycolated collagen-like protein produced at this supply concentration has superior crosslinking properties and can further improve the strength of the resulting cornea.

[0074] Furthermore, in the crosslinking step, the lyophilized formulation is crosslinked under conditions of pH 6.5 to 7.5 and temperature 45 to 55°C to produce a biomimetic cornea. The biomimetic cornea produced under these crosslinking conditions has higher strength.

[0075] Furthermore, in the cross-linking step, the concentration of polyethylene glycolated collagen-like protein is controlled to 12-18 g / mL. Biomimetic corneas produced with this concentration of polyethylene glycolated collagen-like protein have higher strength.

[0076] Furthermore, in the cross-linking step, the concentration of PEGDA in the MPC mother liquor is controlled to 8-12%. Biomimetic corneas produced with this PEGDA concentration have higher strength.

[0077] Furthermore, the freeze-drying step involves freeze-drying the polyethylene glycolated collagen-like protein under the protection of a freeze-drying protective agent. The freeze-drying protective agent can protect the polyethylene glycolated collagen-like protein so that its structure is not destroyed during the freeze-drying process, thereby improving the crosslinkability of the freeze-dried formulation and further improving the strength of the cornea produced. [Modes for carrying out the invention]

[0078] The following embodiments are provided to better illustrate the present application and are not limited to the best embodiments described herein, nor do they limit the content or scope of protection of the present application. Any product identical or similar to the present application, obtained by combining the suggestions of the present application or features of the present application with other prior art features, falls within the scope of protection of the present application.

[0079] Unless otherwise specified in the following examples, any specific experimental steps or conditions may be followed according to the procedures or conditions of conventional experimental steps described in the literature of the art. Unless otherwise specified, the manufacturers of the reagents and equipment used are all commercially available conventional reagent products. In the following examples, the synthesis of collagen-like proteins and the linking of collagen-like proteins to linkers are performed by Shanghai AmbioPharm Inc. All PEG derivatives in the following examples are purchased from Xiamen Sinobang Biotechnology Co., Ltd. [Examples]

[0080] Example 1: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea obtained by crosslinking and curing a polyethylene glycolated collagen-like protein. The polyethylene glycolated collagen-like protein consists of a collagen-like protein whose amino acid sequence is indicated by SEQ ID NO. 1, a linker whose amino acid sequence is indicated by SEQ ID NO. 2 linked to the N-terminus of the collagen-like protein, and 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL modified on the mercapto of the linker. Here, the molar ratio of 8-arm-PEG-40k-MAL to 4-arm-PEG-20k-MAL is 2:1, the core conformation of the polyethylene glycol derivative in the polyethylene glycolated collagen-like protein is TP, and the molecular weight of the polyethylene glycolated collagen-like protein is 30,000 to 75,000 Da. This biomimetic cornea is named Biomimetic Cornea 1. The method for manufacturing the biomimetic cornea 1 described above includes the following steps. Reaction steps: Collagen-like protein and polyethylene glycol derivative were dissolved in a 5 mmol / L dilute hydrochloric acid solution (the pH of the dilute hydrochloric acid solution was adjusted to 6.5 with a pH 11.0 sodium hydroxide solution). The molar concentrations of collagen-like protein and polyethylene glycol derivative in the dilute hydrochloric acid solution were adjusted to 2.4 mmol / L and 0.3 mmol / L, respectively, to obtain the reaction system. The reaction system was allowed to react for 12 hours under conditions of pH 6.5 and temperature 5°C to obtain the reaction product. Purification step: The reaction product was ultrafiltered at 5°C using an ultrafiltration membrane with a pore size of 30,000 Da to block substances with a molecular weight of 30,000 Da or more from the reaction product, yielding polyethylene glycolated collagen-like protein. Lyophilization step: Polyethylene glycolated collagen-like protein and mannitol were mixed in a mass ratio of 1:5 to obtain a lyophilized system. The lyophilized system was lyophilized to obtain a lyophilized formulation. Crosslinking step: The lyophilized formulation was dissolved in MES Buffer at a concentration of 0.5 mol / L and pH 5.5 to obtain a solution containing polyethylene glycolated collagen-like protein at a concentration of 12.5 g / mL. The solution and the MPC mother liquor were mixed in a mass ratio of 5:1 to obtain mixture 1. Mixture 1 and the DMTMM mother liquor were mixed in a mass ratio of 7:1 to obtain mixture 2. The entire crosslinking process was carried out at 45°C. Curing step: Mixture 2 was poured into a corneal mold and left to stand at 25°C for 12 hours to obtain a crude corneal sample. Immersion step: The corneal crude material (along with the mold) was added to PBS Buffer with a concentration of 0.1 mol / L and a pH of 5.5, immersed at 4°C for 24 hours, then the mold was opened, and the cornea was immersed again at 4°C for another 4 hours to release it, and a biomimetic cornea 1 was obtained. Here is the formulation of the MPC mother liquor: 30 g / mL MPC, 10% PEGDA (v / v), 1% TEMED (v / v), solvent 0.5 mol / L, pH 5.5 MOPS Buffer. DMTMM mother liquor formulation: 10 g / mL DMTMM, 15 g / mL APS, 0.5 mol / L solvent, pH 5.5 MOPS Buffer Freeze-drying involves the following steps: Stage 1: Freeze-dry for 6 hours under conditions of -45°C temperature and a vacuum of 500 m Torr. Stage 2: Freeze-dry for 17 hours under conditions of -30°C temperature and a vacuum of 100 m Torr. Stage 3: Freeze-dry for 7 hours under conditions of 25°C temperature and 100 m Torr vacuum.

[0081] Example 2: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the PEG formulations used (8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 2:1) replaced with the following: 8-arm-PEG-40kMAL and 4-arm-PEG-20kMAL with a molar ratio of 1:0 8-arm-PEG-40kMAL and 4-arm-PEG-20kMAL with a molar ratio of 0:1 8-arm-PEG-40kMAL and 4-arm-PEG-20kMAL with a molar ratio of 6:1 8-arm-PEG-40kMAL and 4-arm-PEG-20kMAL with a molar ratio of 4:1 8-arm-PEG-40kMAL and 4-arm-PEG-40kMAL with a molar ratio of 2:1 8-arm-PEG-40kMAL, 4-arm-PEG-20kMAL, and 4-arm-PEG-40kMAL with a molar ratio of 4:2:1 8-arm-PEG-40kMAL, 4-arm-PEG-40kMAL, and 4-arm-PEG-20kMAL with a molar ratio of 2:2:1 8-arm-PEG-40kMAL, 4-arm-PEG-40kMAL, and 8-arm-PEG-20kMAL with a molar ratio of 2:2:1 The above biomimetic corneal proteins will be sequentially named Biomimetic Corneal Proteins 2-9. The manufacturing methods for biomimetic corneas 2-9 described above are the same as those for biomimetic cornea 1.

[0082] Example 3: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is obtained by crosslinking and curing a polyethylene glycolated collagen-like protein. The polyethylene glycolated collagen-like protein consists of a collagen-like protein whose amino acid sequence is indicated by SEQ ID NO. 1, a linker whose amino acid sequence is indicated by SEQ ID NO. 3 linked to the N-terminus of the collagen-like protein, and 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL modified on the linker amino, with a molar ratio of 8-arm-PEG-40k-MAL to 4-arm-PEG-20k-MAL being 2:1, the core conformation of the polyethylene glycol derivative in the polyethylene glycolated collagen-like protein being TP, and the molecular weight of the polyethylene glycolated collagen-like protein being 30,000 to 75,000 Da. This biomimetic cornea is named biomimetic cornea 10. The method for manufacturing the biomimetic cornea 10 described above is the same as that for biomimetic cornea 1.

[0083] Example 4: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the reaction temperature (5°C) of the reaction step changed to 2°C, 8°C, 25°C, and 37°C, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 11-14.

[0084] Example 5: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the reaction pH (6.5) of the reaction step changed to pH 2.5, pH 4.5, pH 6.0, pH 6.5, pH 7.0, pH 8.0, pH 8.5, and pH 10.5, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 15-22.

[0085] Example 6: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the molar concentrations of collagen-like protein and polyethylene glycol derivative in the dilute hydrochloric acid solution during the reaction step changed to the following concentrations. In a dilute hydrochloric acid solution, the molar concentrations of collagen-like protein and polyethylene glycol derivative were adjusted to 3.0 mmol / L and 0.375 mmol / L, respectively. In a dilute hydrochloric acid solution, the molar concentrations of collagen-like protein and polyethylene glycol derivative were adjusted to 1.8 mmol / L and 0.225 mmol / L, respectively. In a dilute hydrochloric acid solution, the molar concentrations of collagen-like protein and polyethylene glycol derivative were adjusted to 1.2 mmol / L and 0.15 mmol / L, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 23-25.

[0086] Example 7: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the reaction time (12h) in the reaction step changed to 4h, 8h, 16h, and 24h, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 26-29.

[0087] Example 8: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the freeze-drying protective agent (mannitol) in the freeze-drying step replaced with sucrose and alanine, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 30-31.

[0088] Example 9: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the crosslinking temperature (45°C) in the crosslinking step changed to 20°C, 25°C, 40°C, 50°C, 55°C, 60°C, and 65°C, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 32-38.

[0089] Example 10: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the pH (5.5) of the Buffer in the crosslinking step changed to 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 39-46.

[0090] Example 11: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the concentration (15 g / mL) of polyethylene glycolated collagen-like protein in MES Buffer in the crosslinking step changed to 1 g / mL, 5 g / mL, 10 g / mL, 20 g / mL, 25 g / mL, 30 g / mL, 35 g / mL, 40 g / mL, and 45 g / mL, respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 47-55.

[0091] Example 12: Biomimetic cornea and its manufacture This embodiment provides a biomimetic cornea, which is based on the biomimetic cornea 1 of Example 1, with the concentration (10%, v / v) of PEGDA in the MPC mother liquor during the crosslinking step changed to 0.5%, 0.6%, 1%, 5%, 8%, 12%, and 15% (v / v), respectively. The biomimetic corneas described above will be sequentially named biomimetic corneas 56-62.

[0092] Experiment Example 1: Experiment on the effects of PEG formulation and manufacturing process on the strength of biomimetic corneas. This experimental example provides an experiment on the effects of PEG formulation and manufacturing processes on the strength of biomimetic corneas, and the experimental procedure is as follows. Referring to the manufacturing method of biomimetic cornea 1, a biomimetic cornea was manufactured using the collagen-like protein whose amino acid sequence is indicated by SEQ ID NO. 1. This biomimetic cornea was used as a blank, and the strength of corneas 1 to 62 was detected using a universal tensile machine. The detection results are shown in Table 1.

[0093] Table 1 shows that both the combination of PEG modification formulations and the preparation process affect the strength of the biomimetic cornea. In particular, the combination of PEG modification formulations has a significant impact on corneal strength and elasticity. Reaction temperature and pH are directly related to the binding rate of collagen-like protein and PEG derivatives, and are one of the important factors in determining whether the modified product can be formed into a hydrogel and create a cornea by affecting the formation of the polymer network structure. The composition of the lyophilized excipient provides skeletal support and protection for polyethylene glycolated collagen, thereby affecting the spatial structure and biological activity of the polymer, resulting in differences in hardness after film formation. If the concentration of polyethylene glycolated collagen-like protein is too low, the strength of the cornea decreases, and if the concentration is too high, the hardening time of the cornea becomes very short, making it impossible to complete the subsequent injection step. Since the isoelectric point of polyethylene glycolated collagen-like protein is neutral, if the crosslinking pH is far from the isoelectric point, the groups involved in the reaction cannot approach each other, resulting in decreased corneal strength or failure of the polyethylene glycolated collagen-like protein to gel. Hydrogels produced from polyethylene glycolated collagen-like protein are temperature-sensitive. At low temperatures, solubility is poor, resulting in a low corneal solid content concentration and reduced strength. At high temperatures, the crosslinking rate becomes very fast, making it impossible to pour into molds and causing premature gelation. If the PEGDA concentration in the MPC mother liquor is too low, the corneal strength decreases. If the concentration is too high, the polyethylene glycolated collagen-like protein is excessively crosslinked, increasing the brittleness and toughness of the hydrogel produced from polyethylene glycolated collagen-like protein, decreasing toughness, and consequently reducing corneal strength. In Table 1, corneas 1-13, 16-21, 23-31, 33-37, 39-44, 49-53, and 56-62 exhibit excellent strength and biocompatibility, and are expected to have applications in the field of corneal transplantation.

[0094] [Table 1]

[0095] Clearly, the above embodiments are merely illustrative examples and do not limit the embodiments. Those skilled in the art may make other different forms of variations or modifications based on the above description. It is neither necessary nor possible to list all embodiments exhaustively here. Any obvious variations or modifications derived therefrom are also included within the scope of protection of this application.

Claims

1. A biomimetic cornea obtained by crosslinking and curing a polyethylene glycolated collagen-like protein, wherein the polyethylene glycolated collagen-like protein comprises a collagen-like protein and a polyethylene glycol derivative modified to the collagen-like protein, the polyethylene glycol derivative consists of PEG-40k and PEG-20k, the molar ratio of PEG-40k to PEG-20k is 2 to 6:1, and the amino acid sequence of the collagen-like protein is indicated by SEQ ID NO.

1.

2. The molar ratio of PEG-40k to PEG-20k is 2 to 3:1, and / or The number of activating groups in PEG-40k is one or more of 8-arm, 4-arm, 2-arm, or 1-arm, and / or the number of activating groups in PEG-20k is one or more of 8-arm, 4-arm, 2-arm, or 1-arm, and / or The biomimetic cornea according to claim 1, characterized in that the activating group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS, or -EDC, and the activating group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS, or -EDC.

3. The biomimetic cornea according to claim 2, characterized in that the number of activating groups of PEG-40k is 4-arm or 8-arm, and the number of activating groups of PEG-20k is 4-arm or 8-arm.

4. The biomimetic cornea according to claim 2, characterized in that the activating group of PEG-40k is -MAL, and the activating group of PEG-20k is -MAL.

5. The biomimetic cornea according to claim 1 or 2, characterized in that a linker is linked to one end of the collagen-like protein, and the polyethylene glycol derivative is modified to the linker through an activating group.

6. The biomimetic cornea according to claim 5, characterized in that the linker is linked to the N-terminus of a collagen-like protein.

7. The biomimetic cornea according to claim 1 or 2, characterized in that the amino acid sequence of the linker is indicated by SEQ ID NO. 2 or SEQ ID NO.

3.

8. The biomimetic cornea according to claim 7, characterized in that the modification site of the polyethylene glycol derivative to the linker is one or more of mercapto, amino, carboxy, or imidazolyl.

9. The biomimetic cornea according to claim 8, characterized in that when the amino acid sequence of the linker is indicated by SEQ ID NO. 2, the modification site of the polyethylene glycol derivative to the linker is a mercapto, and when the amino acid sequence of the linker is indicated by SEQ ID NO. 3, the modification site of the polyethylene glycol derivative to the linker is an amino.

10. A reaction step comprising reacting a collagen-like protein with a polyethylene glycol derivative for 1 to 48 hours under conditions of pH 4.0 to 10.0 and temperature 2 to 40°C to obtain a reaction product, wherein the polyethylene glycol derivative consists of PEG-40k and PEG-20k, the molar ratio of PEG-40k to PEG-20k is 2 to 6:1, and the amino acid sequence of the collagen-like protein is shown as SEQ ID NO.

1. A freeze-drying step to freeze-dry the reaction product to obtain a freeze-dried formulation, A crosslinking step is performed by dissolving the lyophilized formulation in a buffer solution to obtain a dissolution, mixing the dissolution with the MPC mother liquor to obtain mixture 1, and mixing mixture 1 with the DMTMM mother liquor to obtain mixture 2. The process includes a hardening step in which the mixed solution 2 is poured into a corneal mold and left to stand to obtain a rough corneal product, The method for producing a biomimetic cornea according to claim 1, characterized in that the components of the MPC mother liquor include MPC, PEGDA, TEMED, and a solvent, and the components of the DMTMM mother liquor include DMTMM, APS, and a solvent.

11. The reaction step involves reacting a collagen-like protein with a polyethylene glycol derivative at a pH of 6.0 to 8.0 and a temperature of 2 to 8°C for 5 to 8 hours, and / or In the reaction step, the ratio of the number of moles supplied of collagen-like protein and polyethylene glycol derivative is 1 to 16:1, and / or In the reaction step described above, the reaction solvent for the collagen-like protein and polyethylene glycol derivative is water or a dilute hydrochloric acid solution, and / or In the above reaction step, the supply concentration of collagen-like protein in the reaction solvent is 1 to 15 mg / mL, and / or The method according to 10, characterized in that the freeze-drying step involves mixing the reaction product with a freeze-drying protective agent and freeze-drying it to obtain a freeze-dried formulation.

12. The method according to 11, characterized in that, in the reaction step, the ratio of the number of moles supplied of collagen-like protein and polyethylene glycol derivative is 8 to 12:

1.

13. The method according to 11, characterized in that the concentration of the dilute hydrochloric acid solution is 1 to 10 mmol / L, and the pH of the dilute hydrochloric acid solution is adjusted to 6.0 to 8.0 with a dilute alkaline solution.

14. The method according to claim 11, characterized in that the supply concentration of collagen-like protein in the reaction solvent is 8 to 10 mg / mL.

15. The method according to 11, characterized in that the freeze-drying protective agent is one or more of mannitol, sucrose, or alanine.

16. In the crosslinking step, the pH of the buffer solution is 5.5 to 8.0, and / or In the crosslinking step, the concentration of polyethylene glycolated collagen-like protein in the buffer is 5 to 40 g / mL, and / or In the crosslinking step, the mixing mass ratio of the dissolving solution and the MPC mother liquor is 2:1 to 4:1, and / or In the crosslinking step, the mass ratio of the mixed liquid 1 and the DMTMM mother liquor is 5:1 to 7:1, and / or The method according to 10 or 11, characterized in that the crosslinking step is performed at 25 to 60°C.

17. The method according to 16, characterized in that the pH of the buffer solution is 6.5 to 7.

5.

18. The method according to 16, characterized in that the concentration of polyethylene glycolated collagen-like protein in the buffer solution is 12 to 18 g / mL.

19. The method according to 16, characterized in that the crosslinking step is performed at 45 to 55°C.

20. The method according to 10 or 11, characterized in that the standing temperature during the curing step is 4 to 35°C.

21. The method according to 10, characterized in that, in the MPC mother liquor, the concentration of MPC is 15 to 40 g / mL, the concentration of PEGDA is 0.6 to 15% by volume, and the concentration of TEMED is 0.05 to 2% by volume.

22. The method according to 21, characterized in that the concentration of PEGDA in the MPC mother liquor is 8 to 12%.

23. The method according to 10 or 11, characterized in that the solvent of the MPC mother liquor is a buffer, and the pH of the buffer is 6.0 to 8.

0.

24. The method according to 10, characterized in that, in the DMTMM mother liquor, the concentration of DMTMM is 5 to 20 g / mL and the concentration of APS is 0.5 to 5 g / mL.

25. The method according to 10 or 11, characterized in that the solvent of the DMTMM mother liquor is a buffer solution, and the pH of the buffer solution is 6.0 to 8.

0.

26. After the curing step, The method according to 10 or 11, further comprising the steps of: placing a crude corneal sample in a buffer solution together with a mold and performing a first immersion; after the first immersion is completed, opening the mold and performing a second immersion; and after the second immersion is completed, demolding to obtain a finished corneal product.

27. The method according to 26, characterized in that the pH of the buffer solution is 5.5 to 8.0 during the immersion step.

28. The first immersion is performed at a temperature of 4 to 35°C for a duration of 5 to 24 hours, and / or The method according to 26, characterized in that the second immersion is performed at a temperature of 4 to 35°C and for a duration of 3 to 10 hours.

29. After the reaction step and before the freeze-drying step, The method according to 10 or 11, further comprising a purification step of filtering out substances with a molecular weight of 30,000 Da or more in the reaction product to obtain polyethylene glycolated collagen-like protein.

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