WO2025246313A1 - Solid or liquid biological repair material and preparation method therefor - Google Patents

Abstract

The present application relates to a solid or liquid biological material and a preparation method therefor, pertaining to the technical field of biology. The present application provides a method for preparing a biological material. The method comprises: dissolving collagen, a collagen-like protein, a collagen freeze-dried powder, and/or a collagen-like protein freeze-dried powder in a buffer solution, and dissolving an auxiliary material in the buffer solution to obtain a dissolution solution; and subjecting the dissolution solution to pre-crosslinking, followed by crosslinking to obtain the biological material. When the biological material is a solid biological material, the method can properly improve the biomechanical strength and light transmittance of the solid biological material without significantly affecting the water content of the solid biological material. When the biological material is a liquid biological material, the method can properly improve the rupture pressure of the liquid biological material while maintaining high light transmittance of the graft bed and graft, so that the liquid biological material can withstand intraocular pressure and play a role in promoting wound healing in the eye.

Description

A solid or liquid bioremediation material and its preparation method

Cross-references to related applications

This application claims priority to Chinese Patent Application No. 202410702771.9, filed on May 31, 2024, entitled "A Solid or Liquid Biomaterial and a Method for Preparing the Same", the entire contents of which are incorporated herein by reference.

Technical Field

This application relates to a solid or liquid biomaterial and its preparation method, belonging to the field of biotechnology.

Background Technology

The cornea is the frontmost convex, highly transparent material of the eye, horizontally oval in shape. It covers the iris, pupil, and anterior chamber, and provides most of the eye's refractive power. Combined with the refractive power of the lens, light is accurately focused onto the retina to form an image. The cornea has highly sensitive nerve endings; if a foreign object touches the cornea, the eyelids will involuntarily close to protect the eye. To maintain its transparency, the cornea lacks blood vessels, obtaining nutrients and oxygen through tears and aqueous humor. The cornea is very fragile; eye injuries, inflammation, allergic reactions, physical damage, chemical burns, strenuous exercise, and excessive eye strain can all lead to corneal diseases. Once the cornea becomes diseased, it causes obvious eye symptoms such as eye pain, photophobia, tearing, and decreased vision; in severe cases, it can lead to blindness.

Corneal transplantation is the procedure of replacing a patient's existing diseased cornea with a healthy cornea to restore sight or control corneal disease, thereby improving vision or treating certain corneal disorders. Some corneal diseases that cause severe vision loss or even blindness can be completely cured through corneal transplantation, helping these unfortunate patients escape suffering. Because the cornea itself does not contain blood vessels and is in an "immune-privileged" state, the success rate of corneal transplantation is among the highest of other allogeneic organ transplants. However, corneal resources are limited and far from meeting the needs of patients. To address this issue, researchers have proposed artificial corneal transplantation, which uses a special optical device made of transparent synthetic polymer materials or biomass materials (such as hydrolyzed collagen or decellularized natural cornea). This device is surgically implanted into the corneal tissue to replace part of the corneal scar tissue, allowing patients to regain their vision. The special optical device used in artificial corneal transplantation is called an artificial cornea.

Acellular corneal grafts (also known as acellular corneal stroma) are materials obtained by decellularizing and processing the anterior elastic layer and part of the corneal stroma of animal corneas. While removing cells, they retain as much of the natural multi-layered network structure of the cornea as possible. They have excellent optical properties, and their composition and structure are similar to those of human corneas. After implantation, the cornea has good transparency and stable mechanical structure, exhibiting mechanical properties similar to those of human corneas, which can meet the needs of clinical surgery. Furthermore, since they are derived from animals, their biocompatibility is significantly better than that of traditional heterogeneous artificial corneas. It is evident that acellular corneal grafts demonstrate good scaffold potential in terms of diameter, thickness, refractive state, and biocompatibility, and are highly promising for use as artificial corneas in artificial corneal transplantation.

However, decellularized corneal grafts still have some drawbacks. For example, studies have found that corneal calcification occurs after lamellar keratoplasty using decellularized corneal stromal grafts (see: Li, S., Deng, Y., Tian, B., Huang, H., Zhang, H., Yang, R., et al. (2020) Healing Characteristics of Acellular Porcine Corneal Stroma Following Therapeutic Keratoplasty. Xenotransplantation). The calcified portion obstructs the pupil in the central cornea, which may affect vision. Furthermore, studies have shown that applying decellularized corneal grafts in peripheral corneal transplantation may lead to persistent epithelial defects postoperatively (see reference: Shi, W., Zhou, Q., Gao, H., Li, S., Dong, M., Wang, T., et al. (2019) Protectively Decellularized Porcine Cornea versus Human Donor Cornea for Lamellar Transplantation. Advanced Functional Materials, 29.). These drawbacks are mainly caused by two reasons. First, although acellular corneal grafts have similar elasticity to natural corneas, their hardness is lower (see: Li, H., Dong, M., Zhou, Q., Zhao, L., Wang, F., Wang, X., et al. (2020) Corneal Calcification of Acellular Porcine Corneal Stroma Following Lamellar Keratoplasty. Acta Ophthalmologica.). This makes it difficult for the implanted acellular corneal graft to maintain its original shape after undergoing protein water absorption and edema, as well as endothelial cell water absorption and dehydration. Second, although acellular corneal grafts have the advantage of low immunogenicity, the implanted collagen is still prone to conflict with host proteins, leading to immune rejection and ultimately protein-level scarring. Therefore, there is an urgent need to find acellular corneal grafts with higher hardness and less likely to induce immune rejection for use as artificial corneas in artificial corneal transplantation.

Bio-adhesive is a biomaterial that can bond wounds. Since ophthalmic surgery, especially ophthalmic surgery involving the optical zone (e.g., corneal transplantation), does not involve blood vessels and there is no large amount of bleeding or tissue fluid exudation, compared with suture surgery, using bio-adhesive to bond the wound can better fit the implant bed and the graft, which is more conducive to wound healing. In addition, using bio-adhesive to bond the wound can reduce the stimulation and damage of the wound by suture needle holes, reduce the formation of corneal vascularization, and at the same time, using bio-adhesive to bond the wound can significantly reduce the operation time (see reference: Yan B, Peng L, Peng H, et al. Modified sutureless and glue-free method versus conventional sutures for conjunctival autograft fixation in primary pterygium surgery: a randomized controlled trial[J]. Cornea, 2019, 38(11): 1351-1357.).

Currently, common biological adhesives used in ophthalmic surgery mainly include cyanoacrylate products, fibrinogen + coagulation products (see Sugioka K, Fukuda K, Nishida T, et al. The fibrinolytic system in the cornea: A key regulator of corneal wound healing and biological defense[J]. Experimental Eye Research, 2021, 204: 108459.) and PEG-based products (see Mah F S. Effect on gel formation time of adding topical ophthalmic medications to resure sealant, an in situ hydrogel[J]. Journal of Ocular Pharmacology and Therapeutics, 2016, 32(6): 396-399.). However, currently, cyanoacrylate products suffer from insufficient biocompatibility, fibrinogen + coagulation products have drawbacks such as insufficient adhesive strength and the risk of viral infection, while pure PEG products, due to the rapid degradation of PEG in vivo, exhibit a problem where the adhesive effect gradually decreases and eventually disappears within a short period (7 days to 1 month) (see FDA-PMA: Ocular Therapeutix, Inc.). Sealant and Baxter Healthcare Corporation's CoSeal Surgical Sealant). Furthermore, existing bioadhesives used in ophthalmic surgery generally suffer from insufficient light transmittance, severely limiting their application in ophthalmic surgeries involving the optical zone. Therefore, there is an urgent need to find bioadhesives with controllable degradation time, good biocompatibility, and high light transmittance for use in ophthalmic surgeries, especially those involving the optical zone.

Summary of the Invention

To address the aforementioned problems, this application provides a method for preparing biomaterials, the method comprising: dissolving collagen, collagen-like substances, lyophilized collagen powder and/or lyophilized collagen-like substances in a buffer solution, and dissolving excipients in the buffer solution to obtain a solution; and pre-crosslinking the solution before crosslinking to obtain biomaterials.

In one embodiment of this application, the biomaterial is a solid biomaterial or a liquid biomaterial;

When the biomaterial is a solid biomaterial, the excipients include one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride (CMC);

When the biomaterial is a liquid biomaterial, the excipients include hyaluronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

In one embodiment of this application, the biomaterial is a solid biomaterial or a liquid biomaterial;

When the biomaterial is a solid biomaterial, the excipients include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the excipients include N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride and N-hydroxysuccinimide;

When the biomaterial is a liquid biomaterial, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride; or, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide; or, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, N-cyclohexyl-N′-(2-morpholineethyl)carbodiimide hydrochloride, and N-hydroxysuccinimide. High molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight greater than 1000 kDa. Medium molecular weight hyaluronic acid refers to hyaluronic acid with a molecular weight of 100 kDa to 500 kDa.

In one embodiment of this application, the solid biomaterial includes a composite cornea; the liquid biomaterial includes a bio-adhesive.

In one embodiment of this application, the bio-adhesive includes ophthalmic bio-adhesive.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the method includes the following steps:

Dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and/or lyophilized collagen-like substances are dissolved in a buffer solution, and the excipients are dissolved in the buffer solution to obtain a solution; the excipients include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the excipients include N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride and N-hydroxysuccinimide;

Pre-crosslinking step: Stir the solution. After stirring, adjust the pH with an alkaline solution to obtain a pre-crosslinking solution.

Crosslinking step: The decellularized corneal graft is immersed in the pre-crosslinking solution and stirred to obtain solid biomaterial.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 1 to 100 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 30-70 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the buffer solution is 4.5 to 45 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the buffer solution is 4.5 to 18 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the buffer solution is 1:4 to 10:1.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the buffer solution is 2 to 4:1.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the buffer solution is 5-100 mg/mL, and the concentration of N-hydroxysuccinimide is 2-4 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the buffer solution is 20-60 mg/mL, and the concentration of N-hydroxysuccinimide is 2.7 mg/mL.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the pH of the buffer solution in the dissolution step is 2.6 to 6.5.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the pH of the buffer solution in the dissolution step is 4 to 5.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the concentration of the buffer solution in the dissolution step is 0.05 to 0.65 mol/L.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the concentration of the buffer solution in the dissolution step is 0.2 to 0.65 mol/L.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the buffer solution in the dissolution step is a MES buffer solution.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the concentration of the alkaline solution in the pre-crosslinking step is 0.1 to 10 mol/L.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the concentration of the alkaline solution in the pre-crosslinking step is 1 to 5 mol/L.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the alkaline solution in the pre-crosslinking step is a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the pH adjustment using an alkaline solution is to adjust the pH to 4-14 using an alkaline solution.

In one embodiment of this application, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the pH adjustment using an alkaline solution is to adjust the pH to 7-8 using an alkaline solution.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring temperature in the pre-crosslinking step is 15-70°C, the stirring time is 5-240 min, and the stirring speed is 200-700 rpm.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring temperature in the pre-crosslinking step is 25-55°C, the time is 60-240 min, and the rotation speed is 200-700 rpm.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring temperature in the crosslinking step is 15-70°C, the time is 1-48h, and the rotation speed is 200-700rpm.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring temperature in the crosslinking step is 25-45°C, the time is 1-48h, and the rotation speed is 200-700rpm.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring includes mechanical stirring, magnetic stirring, or shaking table stirring.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring in the pre-crosslinking step is magnetic stirring.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the stirring in the crosslinking step is magnetic stirring.

In one embodiment of this application, when the biomaterial is a solid biomaterial, the method includes the following steps:

Dissolution Steps: Collagen, collagen-like substances, lyophilized collagen powder, and/or lyophilized collagen-like substances are dissolved in a buffer solution. Medium-molecular-weight hyaluronic acid and high-molecular-weight hyaluronic acid are dissolved in the buffer solution and then mixed to obtain a mixture. A cross-linking agent solution is added dropwise to the mixture and stirred to obtain a solution. The cross-linking agent in the cross-linking agent solution includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, or the cross-linking agent in the cross-linking agent solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the cross-linking agent in the cross-linking agent solution includes N-cyclohexyl-N′-(2-morpholineethyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

Pre-crosslinking step: Stir the solution, and after stirring, dialyze the product to obtain a pre-crosslinking solution; concentrate the pre-crosslinking solution to obtain glue A;

Crosslinking step: Prepare a crosslinking agent solution for secondary crosslinking to obtain glue B; mix glue A and glue B to obtain liquid biomaterial; the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and urea, or the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and ethylenediamine, or the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and hexamethylenediamine dihydrazide.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 3-50 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 15-30 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of medium molecular weight hyaluronic acid in the buffer solution is 10-75 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of medium molecular weight hyaluronic acid in the buffer solution is 50-75 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of high molecular weight hyaluronic acid in the buffer solution is 1-10 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of high molecular weight hyaluronic acid in the buffer solution is 3-10 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the pH of the buffer solution in the dissolution step is 4.5 to 7.5.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the pH of the buffer solution in the dissolution step is 5.5 to 7.5.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the concentration of the buffer solution in the dissolution step is 0.05 to 0.65 mol/L.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the concentration of the buffer solution in the dissolution step is 0.1 to 0.65 mol/L.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the buffer solution in the dissolution step is a MES buffer solution.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring in the dissolution step includes mechanical stirring, magnetic stirring, shaking table stirring, or T-type mixer stirring.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring temperature in the dissolution step is 15-55°C, the time is 30-120 min, and the rotation speed is 500-1500 rpm.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring temperature in the dissolution step is 25-55°C, the time is 30-120 min, and the rotation speed is 750-1250 rpm.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring in the dissolution step is a shaker stirring or mechanical stirring.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the mixture is 2-10 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the mixture is 3 to 10 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the mixture is 4.5-45 mg/mL, and the concentration of N-hydroxysuccinimide is 4-6 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the mixture is 4.5-18 mg/mL, and the concentration of N-hydroxysuccinimide is 5 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the mixture is 5-100 mg/mL, and the concentration of N-hydroxysuccinimide is 4-6 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the mixture is 20-60 mg/mL, and the concentration of N-hydroxysuccinimide is 5 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring in the pre-crosslinking step includes mechanical stirring, magnetic stirring, shaking table stirring, or T-type mixer stirring.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring temperature in the pre-crosslinking step is 15-55°C, the stirring time is 60-600s, and the stirring speed is 500-1500rpm.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring temperature in the pre-crosslinking step is 25-55°C, the stirring time is 60-600s, and the stirring speed is 500-1250rpm.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the stirring in the pre-crosslinking step is magnetic stirring or T-type mixer stirring.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the dialysis in the pre-crosslinking step includes dialysis through a dialysis bag.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the concentration in the pre-crosslinking step includes: concentrating the pre-crosslinking solution using a dialysis bag until the concentration of high molecular weight hyaluronic acid in the pre-crosslinking solution is 10-100 mg/mL.

In one embodiment of this application, the dialysis bag has a specification of ≥8000D.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the crosslinking step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the crosslinking agent solution for secondary crosslinking is 15-75 mg/mL, and the concentration of urea, ethylenediamine or hexamethylenediamine dihydrazide is 3.3-16.5 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the crosslinking step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the crosslinking agent solution for secondary crosslinking is 45-75 mg/mL, and the concentration of urea, ethylenediamine or hexamethylenediamine dihydrazide is 10-16.5 mg/mL.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the crosslinking step includes mixing A glue and B glue respectively at both ends of a T-type mixer.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the crosslinking step involves 10 to 40 extrusions of the mixture at a temperature of 15 to 45°C.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, the crosslinking step involves 10 to 30 extrusions of the mixture at a temperature of 15 to 45°C.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the crosslinking step, the volume ratio of A glue to B glue in the ophthalmic bio-adhesive is 2 to 4:1.

In one embodiment of this application, when the biomaterial is a liquid biomaterial, in the crosslinking step, the volume ratio of glue A to glue B in the ophthalmic bio-adhesive is 3:1.

In one embodiment of this application, the collagen-like protein is a polyethylene glycol-modified collagen-like protein.

In one embodiment of this application, the method for preparing the polyethylene glycol-based collagen includes the following steps:

Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

In one embodiment of this application, the collagen-like lyophilized powder is a polyethylene glycol-modified collagen-like lyophilized powder.

In one embodiment of this application, the preparation method of the polyethylene glycol-modified collagen lyophilized powder includes the following steps:

Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein;

Freeze-drying step: The reaction product containing polyethylene glycol-modified collagen is freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

In one embodiment of this application, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 4.0 to 10.0 and a temperature of 2 to 40°C for 1 to 48 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

In one embodiment of this application, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 6.0-8.0 and a temperature of 2-8°C for 5-8 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

In one embodiment of this application, the molar ratio of collagen-like protein to polyethylene glycol derivative in the reaction step is 1 to 16:1.

In one embodiment of this application, the molar ratio of collagen-like protein to polyethylene glycol derivative in the reaction step is 8 to 12:1.

In one embodiment of this application, the reaction solvent for the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.

In one embodiment of this application, the concentration of the dilute hydrochloric acid solution is 1-10 mmol/L; the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 using a dilute alkaline solution.

In one embodiment of this application, the dilute alkaline solution is a sodium hydroxide solution or ammonia solution with a pH of 9.0 to 11.0.

In one embodiment of this application, the concentration of collagen-like protein in the reaction solvent during the reaction step is 1–15 mg/mL.

In one embodiment of this application, the concentration of collagen-like protein in the reaction solvent during the reaction step is 8-10 mg/mL.

In one embodiment of this application, the freeze-drying step is as follows: the reaction product containing polyethylene glycol-modified collagen and the freeze-drying protectant are mixed and then freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

In one embodiment of this application, in the freeze-drying step, the mass ratio of the reaction product containing polyethylene glycol-based collagen and the freeze-drying protectant is 1:1 to 10.

In one embodiment of this application, the freeze-drying protectant is one or more of mannitol, sucrose, or alanine.

In one embodiment of this application, after the reaction step and before the freeze-drying step, the method further includes a purification step; the purification step is: by filtration, at a temperature of 2-8°C, substances with a molecular weight greater than or equal to 30,000 Da in the reaction product containing polyethylene glycol-modified collagen are retained to obtain polyethylene glycol-modified collagen.

In one embodiment of this application, the filtration is dialysis or ultrafiltration.

In one embodiment of this application, the polyethylene glycol derivative comprises PEG-40k and PEG-20k.

In one embodiment of this application, the polyethylene glycol derivative is composed of PEG-40k and PEG-20k.

In one embodiment of this application, the molar ratio of PEG-40k to PEG-20k is 0.5 to 6:1.

In one embodiment of this application, the molar ratio of PEG-40k and PEG-20k is 2 to 3:1.

In one embodiment of this application, the PEG-40k has one or more of eight arms, four arms, two arms, or one arm as its activating groups; the PEG-20k has one or more of eight arms, four arms, two arms, or one arm as its activating groups.

In one embodiment of this application, the PEG-40k has four or eight activating groups; the PEG-20k has four or eight activating groups.

In one embodiment of this application, the activating group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; the activating group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.

In one embodiment of this application, the activating group of PEG-40k is -MAL; the activating group of PEG-20k is -MAL.

In one embodiment of this application, a linker peptide is attached to one end of the collagen-like protein; in the reaction step, the polyethylene glycol derivative is modified onto the linker peptide by an activating group to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

In one embodiment of this application, the linker peptide is attached to the N-terminus of a collagen-like protein.

In one embodiment of this application, the modification site of the polyethylene glycol derivative on the linker peptide is one or more of thiol, amino, carboxyl or imidazole groups.

In one embodiment of this application, the parent nucleus conformation of the polyethylene glycol-based collagen is one or more of HG or TP.

In one embodiment of this application, the parent nucleus conformation of the polyethylene glycol derivative in the polyethylene glycol-based collagen is TP.

In one embodiment of this application, the collagen-like amino acid configuration of the polyethylene glycol-modified collagen is one or more of the D-type or L-type.

In one embodiment of this application, the molecular weight of the polyethylene glycol-modified collagen is 15,000 to 75,000 Da.

In one embodiment of this application, the molecular weight of the polyethylene glycol-modified collagen is 30,000 to 75,000 Da.

This application also provides a composite cornea, wherein the biomaterial is prepared using the above-described method for preparing biomaterials.

In one embodiment of this application, the biomaterial is a solid biomaterial or a liquid biomaterial.

In one embodiment of this application, the solid biomaterial includes a composite cornea; the liquid biomaterial includes a bio-adhesive.

In one embodiment of this application, the bio-adhesive includes ophthalmic bio-adhesive.

The technical solution of this application has the following advantages:

1. This application provides a method for preparing solid biomaterials, comprising three steps: dissolution, pre-crosslinking, and crosslinking. In the dissolution step, lyophilized powder and a crosslinking agent are thoroughly and uniformly dissolved in a MES buffer solution. Simultaneously, the crosslinking agent is activated to form a relatively stable NHS ester intermediate under acidic conditions with the carboxyl groups on the lyophilized powder. In the pre-crosslinking step, an alkaline solution is added to activate the NHS ester intermediate. In the crosslinking step, a graft is added to the crosslinking mother liquor, allowing the activated polyethylene glycol-modified collagen NHS ester intermediate in the mother liquor to slowly and uniformly react with the amino groups on the graft, thereby enabling the polyethylene glycol-modified collagen to firmly modify the decellularized corneal graft, forming a surface coating. This method can appropriately improve the biomechanical strength and light transmittance of the solid biomaterial without significantly affecting its water content (the solid biomaterial prepared by this method has a water content of 60.7–91.8%, a light transmittance of 60.3–93.5%, and a Shore hardness of 17.2–40.7). Furthermore, the polyethylene glycol-modified collagen used in this method has excellent biocompatibility. After modification of decellularized corneal grafts, it can shield their immunogenicity, improve corneal biocompatibility, and reduce the possibility of corneal graft whitening and dissolution (see reference: Simpson, Fiona C., et al. "Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs." Communications Biology 4.1(2021):608.). Based on this, animal experiments verified that the solid biomaterial prepared using the described method has an inducing effect on epithelial cell growth. Furthermore, the solid biomaterial prepared using this method exhibits good biocompatibility. Applying the solid biomaterial prepared using this method to corneal transplantation can significantly improve the clinical postoperative healing rate and reduce the incidence of graft dissolution and graft whitening with long-term use. In addition, the surface coating of the solid biomaterial prepared using this method can penetrate into the corneal interior, increasing the cross-linking density of the cornea and further enhancing its strength. In conclusion, the solid biomaterial prepared using this method can be used as a composite cornea in corneal transplantation and has great application potential in corneal transplantation.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 1–100 mg/mL. Solid biomaterials prepared under this setting have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 30–70 mg/mL. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the buffer solution is 4.5–45 mg/mL. Solid biomaterials prepared under this setting have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the buffer solution is 4.5–18 mg/mL. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the buffer solution is 1:4 to 10:1. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the buffer solution is 2–4:1. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the buffer solution is 5–100 mg/mL, and the concentration of N-hydroxysuccinimide is 2–4 mg/mL. Solid biomaterials prepared under these conditions exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the buffer solution is 20–60 mg/mL, and the concentration of N-hydroxysuccinimide is 2.7 mg/mL. The solid biomaterial prepared under these conditions exhibits better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the pH of the buffer solution in the dissolution step is 2.6–6.5. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the pH of the buffer solution in the dissolution step is 4-5. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the concentration of the buffer solution in the dissolution step is 0.05–0.65 mol/L. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the concentration of the buffer solution in the dissolution step is 0.2–0.65 mol/L. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the concentration of the alkaline solution in the pre-crosslinking step is 0.1–10 mol/L. The solid biomaterial prepared under this setting has better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the concentration of the alkaline solution in the pre-crosslinking step is 1–5 mol/L. Solid biomaterials prepared under this setting exhibit better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the pH adjustment using an alkaline solution is changed to adjust the pH to 4-14. The solid biomaterial prepared under this setting has better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the pH adjustment using an alkaline solution is changed to adjust the pH to 7-8. The solid biomaterial prepared under this setting has better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the stirring temperature is 15–70°C, the time is 5–240 min, and the rotation speed is 200–700 rpm. Solid biomaterials prepared under these settings have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the stirring temperature is 25–55°C, the time is 60–240 min, and the rotation speed is 200–700 rpm. Solid biomaterials prepared under these settings have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the crosslinking step, the stirring temperature is 15–70°C, the time is 1–48 h, and the rotation speed is 200–700 rpm. Solid biomaterials prepared under these settings have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, in the crosslinking step, the stirring temperature is 25–45°C, the time is 1–48 h, and the rotation speed is 200–700 rpm. Solid biomaterials prepared under these settings have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the stirring includes mechanical stirring, magnetic stirring, or shaking. Solid biomaterials prepared under this setting have better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the stirring in the pre-crosslinking step is magnetic stirring. The solid biomaterial prepared under this setting has better water content, light transmittance, and biomechanical strength.

Furthermore, when the biomaterial is a solid biomaterial, the stirring in the crosslinking step is magnetic stirring. The solid biomaterial prepared under this setting has better water content, light transmittance, and biomechanical strength.

2. This application provides a method for preparing liquid biomaterials, comprising three steps: dissolution, pre-crosslinking, and crosslinking. In the dissolution step, lyophilized powder and hyaluronic acid are first thoroughly and uniformly dissolved in a MES buffer solution, and then a crosslinking agent is added to react the hyaluronic acid with the amino groups of polyethylene glycol-modified collagen to form a solution with a certain viscosity. In the pre-crosslinking step, the solution is concentrated by dialysis using a dialysis bag to remove byproducts generated during the crosslinking agent reaction in the dissolution step, yielding gel A. In the crosslinking step, a crosslinking agent and urea are first formulated into gel B, and then gel A and gel B are mixed to obtain the liquid biomaterial. Applying the liquid biomaterial prepared by this method to the surface of a wound can help close the wound. Furthermore, the method can appropriately increase the rupture pressure of the liquid biomaterial while maintaining the high light transmittance of the implant in the planting bed, enabling the liquid biomaterial to withstand intraocular pressure and promote wound healing in the eye (the light transmittance of the liquid biomaterial prepared by the method is 55.0–96.7%, and the rupture pressure is 56.8–160.8 mmHg). Simultaneously, the polyethylene glycol-based collagen used in the method has excellent biocompatibility, and the liquid biomaterial prepared from it also exhibits correspondingly excellent biocompatibility (see reference: Simpson, Fiona C., et al. "Collagen analogs with phosphorylcholine are inflation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs." Communications Biology 4.1(2021):608.). Furthermore, the polyethylene glycol-based collagen used in this method is chemically synthesized and lacks corresponding enzyme recognition sites in vivo, allowing for slow degradation within the body. This results in a liquid biomaterial with a longer-lasting adhesive effect. Animal experiments have verified that the liquid biomaterial prepared using this method induces epithelial cell growth. Applying this liquid biomaterial to corneal transplantation and other non-vascularized ophthalmic surgical incisions significantly improves postoperative healing rates and accelerates wound healing. Additionally, the liquid biomaterial can penetrate the cornea and cross-link with it, further increasing wound closure strength. In conclusion, the liquid biomaterial prepared using this method can be used as an ophthalmic bio-adhesive in ophthalmic surgery, especially in procedures involving the optical zone, demonstrating significant application potential.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 3–50 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 15–30 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of medium molecular weight hyaluronic acid in the buffer solution is 10–75 mg/mL. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of medium molecular weight hyaluronic acid in the buffer solution is 50–75 mg/mL. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of high molecular weight hyaluronic acid in the buffer solution is 1–10 mg/mL. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of high molecular weight hyaluronic acid in the buffer solution is 3–10 mg/mL. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the pH of the buffer solution in the dissolution step is 4.5–7.5. Liquid biomaterials prepared under this setting exhibit better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the pH of the buffer solution in the dissolution step is 5.5–7.5. Liquid biomaterials prepared under this setting exhibit better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the concentration of the buffer solution in the dissolution step is 0.05–0.65 mol/L. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the concentration of the buffer solution in the dissolution step is 0.1–0.65 mol/L. Under this setting, the liquid biomaterial prepared exhibits better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the buffer solution in the dissolution step is a MES buffer. This setup results in liquid biomaterials with better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the stirring in the dissolution step includes mechanical stirring, magnetic stirring, shaking stirring, or T-mixer stirring. This setup results in liquid biomaterials with better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the stirring temperature is 15–55°C, the time is 30–120 min, and the rotation speed is 500–1500 rpm. Under these settings, the liquid biomaterial obtained has better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the stirring temperature is 25–55°C, the time is 30–120 min, and the rotation speed is 750–1250 rpm. Under these settings, the liquid biomaterial obtained has better light transmittance and rupture pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the stirring in the dissolution step is performed using a shaker or mechanical stirring. This setup results in liquid biomaterials with better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the mixture is 2–10 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the mixture is 3–10 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the mixture is 4.5–45 mg/mL, and the concentration of N-hydroxysuccinimide is 4–6 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the mixture is 4.5–18 mg/mL, and the concentration of N-hydroxysuccinimide is 5 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the mixture is 5–100 mg/mL, and the concentration of N-hydroxysuccinimide is 4–6 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride in the mixture is 20–60 mg/mL, and the concentration of N-hydroxysuccinimide is 5 mg/mL. Liquid biomaterials prepared under this setting exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the stirring in the pre-crosslinking step includes mechanical stirring, magnetic stirring, shaking table stirring, or T-mixer stirring. Liquid biomaterials prepared under this setup exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the pre-crosslinking step, the stirring temperature is 15–55°C, the time is 60–600 s, and the rotation speed is 500–1500 rpm. Liquid biomaterials prepared under these settings exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the pre-crosslinking step, the stirring temperature is 25–55°C, the time is 60–600 s, and the rotation speed is 500–1250 rpm. Liquid biomaterials prepared under these settings exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the stirring in the pre-crosslinking step is performed using magnetic stirring or a T-mixer. This setup results in liquid biomaterials with better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the crosslinking step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the crosslinking agent solution for the secondary crosslinking is 15–75 mg/mL, and the concentration of urea, ethylenediamine, or hexamethylenediamine dihydrazide is 3.3–16.5 mg/mL. The liquid biomaterial prepared under this setting has better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, in the crosslinking step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the crosslinking agent solution for the secondary crosslinking is 45–75 mg/mL, and the concentration of urea, ethylenediamine, or hexamethylenediamine dihydrazide is 10–16.5 mg/mL. Liquid biomaterials prepared under these conditions exhibit better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the mixing step in the crosslinking process includes: mixing glue A and glue B respectively at both ends of a T-type mixer. This setup results in liquid biomaterials with better light transmittance and bursting pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the crosslinking step involves 10–40 extrusion cycles of mixing at a temperature of 15–45°C. This setup results in liquid biomaterials with better light transmittance and burst pressure.

Furthermore, when the biomaterial is a liquid biomaterial, the crosslinking step involves 10–30 extrusion cycles at a temperature of 15–45°C. This setup results in liquid biomaterials with better light transmittance and burst pressure.

Attached Figure Description

Figure 1: The completion of epithelialization after composite corneal implantation into a rabbit eye.

Figure 2: Ophthalmic bio-adhesive before it solidifies into a gel.

Figure 3: Ophthalmic biological adhesive after solidification.

Detailed Implementation

The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

Unless otherwise specified, the experimental steps or conditions described in the following examples can be performed according to the conventional experimental procedures and conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagents. The synthesis of collagen-like proteins and the connection between collagen-like proteins and linker peptides in the following examples were performed by Jiangsu Nuotai Ausino Biopharmaceutical Co., Ltd. The PEG derivatives in the following examples were purchased from Xiamen Sinobang Biotechnology Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride (CMC) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholino hydrochloride (DMTMM) in the following examples were purchased from SIGMA; the medium molecular weight hyaluronic acid in the following examples was purchased from Bloomage Biotechnology (molecular weight 100-500 kDa); the high molecular weight hyaluronic acid in the following examples was purchased from Bloomage Biotechnology (molecular weight 1000-). 2000kda); the dialysis bags in the following examples were purchased from Solarbio (specification ≥8000D); the T-type mixers in the following examples were purchased from Guangdong Baihe Medical Co., Ltd.; the New Zealand rabbits involved in the following examples were purchased from Air Force Medical University; the pig eyes involved in the following examples were purchased from Changshu Slaughterhouse; the decellularized corneal grafts in the following examples were purchased from Air Force Medical University of the Chinese People's Liberation Army; the magnetic stirring in the following examples was performed by a magnetic stirrer purchased from Shanghai Titan Technology Co., Ltd.; the shaking agitation in the following examples was performed by a stirrer purchased from Shanghai Yiheng Co., Ltd.; the mechanical stirring in the following examples was performed by a stirrer purchased from Shanghai Lichen Bangxi Co., Ltd.

Example 1-1: A composite cornea and its preparation

This embodiment provides a composite cornea, and the preparation method of the composite cornea includes the following steps:

Dissolution step: Polyethylene glycol-modified collagen lyophilized powder (for the preparation of polyethylene glycol-modified collagen lyophilized powder, please refer to Example 1 of the patent application text with publication number CN115990969A), EDC, and NHS are dissolved in MES buffer with a concentration of 0.4 mol/L and pH 5, so that the concentration of polyethylene glycol-modified collagen in the MES buffer is 50 mg/mL, the concentration of EDC is 9 mg/mL, and the concentration of NHS is 2.7 mg/mL, to obtain a solution;

Pre-crosslinking step: The solution is stirred. After stirring, the pH is adjusted to 7.5 with a 5 mol/L sodium hydroxide aqueous solution to obtain a pre-crosslinking solution. The stirring is done by magnetic stirring. The stirring temperature is 45℃, the time is 120 min, and the speed is 250 rpm.

Crosslinking step: The decellularized corneal graft is immersed in the pre-crosslinking solution and stirred to obtain a composite cornea; the stirring is magnetic stirring; the stirring temperature is 45℃, the time is 24h, and the speed is 250rpm (this composite cornea is named composite cornea 1).

Examples 1-2: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Example 1, except that the concentration of polyethylene glycol-modified collagen (50 mg/mL) in the dissolution step is replaced with: 1 mg/mL, 10 mg/mL, 30 mg/mL, 70 mg/mL, and 100 mg/mL respectively.

The above-mentioned composite corneas are named composite corneas 2 to 6 in sequence.

Examples 1-3: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Example 1, except that the concentration of EDC (9 mg/mL) in the dissolution step is replaced with: 4.5 mg/mL, 6.3 mg/mL, 13.5 mg/mL, 18 mg/mL, 36 mg/mL, and 45 mg/mL respectively.

The above-mentioned composite corneas are named Composite Corneas 7 to 12 in sequence.

Examples 1-4: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Example 1, except that the molar ratio (2:1) of EDC and NHS in the dissolution step is replaced with: 1:4, 1:2, 1:1, 3:1, 4:1, 10:1, respectively, that is, the concentration of NHS (2.7 mg/mL) is replaced with: 21.6 mg/mL, 10.8 mg/mL, 5.4 mg/mL, 1.8 mg/mL, 1.4 mg/mL, 0.54 mg/mL, respectively.

The above-mentioned composite corneas are named Composite Corneas 13 to 18 in sequence.

Examples 1-5: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 in Example 1, except that the concentration of MES buffer (0.4 mol/L) in the dissolution step is replaced with 0.05 mol/L, 0.2 mol/L, and 0.65 mol/L, respectively.

The above-mentioned composite corneas are named Composite Cornea 19 to 21 in sequence.

Examples 1-6: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 in Example 1, except that the pH (5) of the MES buffer in the dissolution step is replaced with 2.6, 4, and 6.5 respectively.

The above-mentioned composite corneas are named composite corneas 22 to 24 in sequence.

Examples 1-7: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 in Example 1, except that the concentration (5 mol/L) of the sodium hydroxide aqueous solution in the pre-crosslinking step is replaced with: 0.1 mol/L, 0.5 mol/L, 1 mol/L, 2 mol/L, 7 mol/mL, and 10 mol/L, respectively.

The above-mentioned composite corneas are named Composite Cornea 25 to 30 in sequence.

Examples 1-8: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Embodiment 1, except that the pH (7.5) of the pre-crosslinking solution in the pre-crosslinking step is replaced with 4, 6, 7, 8, 10, and 14 respectively.

The above-mentioned composite corneas are named composite corneas 31 to 36 in sequence.

Examples 1-9: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 in Embodiment 1, except that the stirring temperature (45°C) in the pre-crosslinking step is replaced with: 4°C, 15°C, 25°C, 35°C, 55°C, and 70°C respectively.

The above-mentioned composite corneas are named composite corneas 37 to 42 in sequence.

Examples 1-10: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 in Embodiment 1, except that the stirring time (120 min) in the pre-crosslinking step is replaced with: 5 min, 30 min, 60 min, 180 min, and 240 min respectively.

The above-mentioned composite corneas are named composite corneas 43 to 47 in sequence.

Examples 1-11: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Embodiment 1, except that the stirring time (24h) in the crosslinking step is replaced with: 1h, 4h, 12h, 18h, 30h, and 48h respectively.

The above-mentioned composite corneas are named composite corneas 48 to 53 in sequence.

Examples 1-12: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Embodiment 1, except that the stirring temperature (45°C) in the crosslinking step is replaced with: 4°C, 15°C, 25°C, 35°C, 55°C, and 70°C respectively.

The above-mentioned composite corneas are named composite corneas 54 to 59 in sequence.

Examples 1-13: A composite cornea and its preparation

This embodiment provides a composite cornea. Based on the composite cornea 1 in Embodiment 1, the dissolution step is replaced by: dissolving polyethylene glycol-modified collagen lyophilized powder and EDC in a MES buffer solution with a concentration of 0.4 mol/L and pH 5, so that the concentration of polyethylene glycol-modified collagen in the MES buffer solution is 50 mg/mL and the concentration of EDC is 9 mg/mL, to obtain a solution.

The above composite corneas are named Composite Cornea 60.

Examples 1-14: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Example 1, except that the dissolution step is replaced by: dissolving polyethylene glycol-modified collagen lyophilized powder, CMC, and NHS in a MES buffer solution with a concentration of 0.4 mol/L and pH 5, so that the concentration of polyethylene glycol-modified collagen in the MES buffer solution is 50 mg/mL, the concentrations of CMC are 5 mg/mL, 20 mg/mL, 40 mg/mL, 60 mg/mL, and 100 mg/mL, and the concentration of NHS is 2.7 mg/mL, to obtain a solution.

The above-mentioned composite corneas are named composite corneas 61 to 65 in sequence.

Examples 1-15: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Embodiment 1, except that the stirring speed (250 rpm) in the crosslinking step is replaced with 200 rpm, 300 rpm, 500 rpm, and 700 rpm respectively.

The above-mentioned composite corneas are named Composite Corneas 66 to 68 in sequence.

Examples 1-16: A composite cornea and its preparation

This embodiment provides a composite cornea, which is based on the composite cornea 1 of Embodiment 1, except that the stirring method (magnetic stirring) is replaced by shaker stirring and mechanical stirring respectively.

The above-mentioned composite corneas are named Composite Corneas 69 to 71 in sequence.

Experiment Example 1: The Influence of Manufacturing Process on the Performance of Composite Corneas

This experimental example demonstrates the effect of the manufacturing process on the performance of composite corneas. The experimental procedure is as follows:

Experiment 1: Using untreated original decellularized porcine corneas (i.e., the decellularized corneal grafts used in Example 1, with a water content of 80%) as a control, the water content of composite corneas 1 to 71 was measured using a UV-Vis spectrophotometer at room temperature (25°C) within the wavelength range of 300–1100 nm. The results are shown in Table 1.

Experiment 2: Using untreated, untreated decellularized porcine corneas (Shore hardness 22.9) as a control, the hardness of composite corneas 1-71 was tested at room temperature (25℃) using a Shore hardness tester. The test results are shown in Table 1.

Experiment 3: Using untreated, untreated decellularized porcine corneas (78% transmittance) as a control, the transmittance of composite corneas 1-71 was measured using a UV-Vis spectrophotometer at room temperature (25℃) within the wavelength range of 380-780nm. The results are shown in Table 1.

Experiment 4: A 6mm diameter incision was made at the central pupil of a New Zealand rabbit eye to serve as the implantation bed, with a thickness of 250μm. After the incision was completed, composite corneas 1 and 4 were implanted into the rabbit eye, respectively. After implantation, the completion of epithelialization of composite corneas 1 and 3 was continuously observed. The observation results are shown in Figure 1.

Table 1 shows that the preparation process affects the water content of the composite cornea. Specifically, in the dissolution step, the concentration of polyethylene glycol-modified collagen affects the degree of cross-linking in the composite cornea; a higher concentration results in a higher degree of cross-linking and a lower water content. The concentration and type of cross-linking agent (EDC/NHS) affect the looseness of the composite cornea's microstructure; a higher cross-linking agent concentration results in a denser cross-linking and a lower water content. Too low a cross-linking agent concentration leads to insufficient cross-linking, a poorer coating effect, and a higher water content. In the pre-cross-linking step, a low final pH leads to insufficient cross-linking and a higher water content, while a high pH leads to rapid hydrolysis of the cross-linking agent, also resulting in insufficient reaction and a higher water content. In the cross-linking step, a low reaction temperature leads to insufficient cross-linking between the product and the coating, resulting in a higher water content, while a high reaction temperature leads to over-cross-linking, reducing the water content but significantly increasing the hardness. In Table 1, except for composite corneas 2, 3, 6, 11, 12, 18, 31, 32, 36, 37, 42, 43, 44, 54, 58, 59, 60, 61, 65, 70, and 71, the remaining composite corneas have good water content, close to that of the original decellularized porcine cornea, and good biocompatibility, making them highly promising for the treatment of presbyopia.

Table 1 shows that the preparation process affects the hardness of the composite cornea. Specifically, in the dissolution step, the concentration and type of crosslinking agent (EDC/NHS) affect the looseness of the composite cornea's microstructure. Higher crosslinking agent concentrations result in denser crosslinking and higher hardness, while excessively low concentrations lead to insufficient crosslinking and inadequate coating modification. In the pre-crosslinking step, excessively low final pH results in insufficient crosslinking and higher product water content, while excessively high pH leads to rapid hydrolysis of the crosslinking agent, also resulting in insufficient reaction and higher product water content. In the crosslinking step, excessively low reaction temperature leads to insufficient crosslinking between the product and coating, resulting in higher product water content, while excessively high reaction temperature leads to over-crosslinking and excessively low water content. In Table 1, except for composite corneas 2, 10, 11, 12, 13, 14, 15, 18, 26, 31, 36, 37, 38, 41, 42, 43, 44, 45, 54, 59, 65, and 71, the hardness of the other composite corneas is similar to or higher than that of the original decellularized porcine corneas, provided that the corneal stromal structure is not affected. They have great application prospects in the field of corneal transplantation.

Table 1 shows that the preparation process affects the light transmittance of the composite cornea. Specifically, in the dissolution step, the concentration of polyethylene glycol-modified collagen affects the degree of cross-linking in the composite cornea; higher concentrations result in higher cross-linking and higher light transmittance. The concentration and type of cross-linking agent (EDC/NHS) affect the looseness of the composite cornea's microstructure; higher cross-linking agent concentrations lead to denser cross-linking and higher light transmittance. Insufficient cross-linking agent concentration results in incomplete cross-linking, poor coating performance, and lower light transmittance. In the pre-cross-linking step, an excessively low final pH leads to insufficient cross-linking and lower light transmittance; an excessively high pH causes rapid hydrolysis of the cross-linking agent, also resulting in insufficient reaction and lower light transmittance. In the cross-linking step, an excessively low reaction temperature leads to insufficient cross-linking between the product and the coating, resulting in lower light transmittance; an excessively high reaction temperature leads to over-cross-linking, resulting in higher light transmittance but significantly increased hardness. In Table 1, except for composite corneas 2, 13, 14, 15, 18, 19, 22, 25, 26, 29, 30 (composite cornea 30, in addition to having slightly poor light transmittance, also used high-concentration sodium hydroxide, which can damage the internal structure of the cornea, leading to composite corneal damage), 31, 32, 35, 36, 38, 43, 44, 55, 60, 61, 62, 70, and 71, the remaining composite corneas have good light transmittance, which is close to or higher than that of the original decellularized porcine cornea, and have good biocompatibility, making them highly promising for the treatment of presbyopia.

As shown in Figure 1, composite corneas 1 and 4 began to undergo epithelialization at day 7 and completed epithelialization at day 28. This result indicates that composite corneas 1 and 4 have an inducing effect on epithelial cell growth. Furthermore, composite corneas 1 and 4 exhibit good biocompatibility. Applying composite corneas 1 and 4 to corneal transplantation can significantly improve the clinical postoperative healing rate and reduce the incidence of graft dissolution and graft whitening with long-term use.

Table 1. Water content, hardness, and light transmittance of composite corneas 1-71

Example 2-1: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, the preparation method of which includes the following steps:

Dissolution Steps: Polyethylene glycol-modified collagen lyophilized powder (for details on the preparation of the lyophilized collagen lyophilized powder, please refer to Example 1 of the patent application text with publication number CN115990969A), medium molecular weight hyaluronic acid, and high molecular weight hyaluronic acid were dissolved in a 0.5 mol/L MES buffer solution with a pH of 5.5, resulting in a concentration of 30 mg/mL for polyethylene glycol-modified collagen, 50 mg/mL for medium molecular weight hyaluronic acid, and 10 mg/mL for high molecular weight hyaluronic acid in the MES buffer solution. The mixture was then stirred to obtain a solution. A 20 mg/mL DMTMM solution (water as solvent) was added dropwise to the solution, resulting in a DMTMM concentration of 4 mg/mL in the solution, thus obtaining a solution. The stirring was performed on a shaker at a temperature of 35°C for 60 minutes and a rotation speed of 1000 rpm.

Pre-crosslinking step: The solution is stirred. After stirring, the product is dialyzed using a dialysis bag to remove excess small molecules, resulting in a pre-crosslinking solution. The pre-crosslinking solution is concentrated using a dialysis bag until the concentration of high molecular weight hyaluronic acid in the pre-crosslinking solution is 67 mg/mL, resulting in gel A. The stirring is performed using magnetic stirring. The stirring temperature is 35℃, the stirring time is 300s, and the stirring speed is 1000 rpm.

Crosslinking step: A crosslinking agent solution (solvent is water) with a DMTMM concentration of 45 mg/mL and a urea concentration of 10 mg/mL was prepared to obtain glue B; glue A and glue B were respectively placed at both ends of a T-type mixer and mixed so that the volume ratio of glue A to glue B in the mixed product was 3:1, to obtain ophthalmic bio-adhesive; the mixing was performed by extrusion 20 times at a temperature of 35°C (this ophthalmic bio-adhesive was named ophthalmic bio-adhesive 1).

Example 2-2: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the pH (5.5) of the MES buffer solution in the dissolution step is replaced with 4.5, 6.5, and 7.5 respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 2 to 4 in sequence.

Examples 2-3: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on ophthalmic bio-adhesive 1 in Example 2-1, except that the concentration of MES buffer (0.5 mol/L) in the dissolution step is replaced with 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, and 0.65 mol/L, respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 5 to 8 in sequence.

Examples 2-4: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the concentration of medium molecular weight hyaluronic acid (50 mg/mL) in the dissolution step is replaced with: 5 mg/mL, 10 mg/mL, 15 mg/mL, 25 mg/mL, 75 mg/mL, and 100 mg/mL respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 9 to 14 in sequence.

Examples 2-5: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the concentration of high molecular weight hyaluronic acid (5mg/mL) in the dissolution step is replaced with: 1mg/mL, 3mg/mL, 7mg/mL, 10mg/mL, 25mg/mL, and 50mg/mL respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 15 to 20 in sequence.

Examples 2-6: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the concentration of polyethylene glycol collagen (30mg/mL) in the dissolution step is replaced with: 3mg/mL, 10mg/mL, 15mg/mL, 50mg/mL, 75mg/mL, and 100mg/mL respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 21 to 26 in sequence.

Examples 2-7: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring temperature (35°C) in the dissolution step is replaced with: 4°C, 15°C, 25°C, 45°C, 55°C, and 70°C respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 27 to 32 in sequence.

Examples 2-8: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring time (60 min) in the dissolution step is replaced with 30 min and 120 min respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 33-34 in sequence.

Examples 2-9: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring method (shaking table stirring) in the dissolution step is replaced by mechanical stirring.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesive 35.

Examples 2-10: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring speed (1000 rpm) in the dissolution step is replaced with: 500 rpm, 750 rpm, 1250 rpm, or 1500 rpm.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 36 to 39 in sequence.

Examples 2-11: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the DMTMM concentration (4mg/mL) in the dissolution step is replaced with: 0.4mg/mL, 2mg/mL, 3mg/mL, 6mg/mL, 8mg/mL, and 10mg/mL respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 40-45 in sequence.

Examples 2-12: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on ophthalmic bio-adhesive 1 in Example 2-1, except that the crosslinking agent (DMTMM solution with a concentration of 20 mg/mL) in the dissolution step is replaced with: a solution containing 14 mg/mL EDC and 5 mg/mL NHS, and a solution containing 30 mg/mL CMC and 5 mg/mL NHS.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 46-47 in sequence.

Examples 2-13: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring method (magnetic stirring) in the pre-crosslinking step is replaced by: T-type mixer stirring.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesive 48.

Examples 2-14: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring temperature (35°C) in the pre-crosslinking step is replaced with: 4°C, 15°C, 25°C, 45°C, 55°C, and 70°C respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 49 to 54 in sequence.

Examples 2-15: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring time (300s) in the pre-crosslinking step is replaced with: 10s, 60s, 120s, 360s, 480s, and 600s respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 55-60 in sequence.

Examples 2-16: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the stirring speed (1000 rpm) in the pre-crosslinking step is replaced with 500 rpm, 750 rpm, 1250 rpm, and 1500 rpm respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 61 to 64 in sequence.

Examples 2-17: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on ophthalmic bio-adhesive 1 of Example 2-1, except that the B glue (a secondary crosslinking agent solution with a DMTMM concentration of 45 mg/mL and a urea concentration of 10 mg/mL) in the crosslinking step is replaced with: a secondary crosslinking agent solution with a DMTMM concentration of 5 mg/mL and a urea concentration of 1.1 mg/mL; and a secondary crosslinking agent solution with a DMTMM concentration of 15 mg/mL and a urea concentration of 3.3 mg/mL. Combined crosslinking agent solutions: crosslinking agent solutions for secondary crosslinking with DMTMM concentration of 30 mg/mL and urea concentration of 6.6 mg/mL; crosslinking agent solutions for secondary crosslinking with DMTMM concentration of 60 mg/mL and urea concentration of 13.2 mg/mL; crosslinking agent solutions for secondary crosslinking with DMTMM concentration of 75 mg/mL and urea concentration of 16.5 mg/mL; crosslinking agent solutions for secondary crosslinking with DMTMM concentration of 105 mg/mL and urea concentration of 23.1 mg/mL.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 65-70 in sequence.

Examples 2-18: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the urea in the B glue in the crosslinking step is replaced with CLP-PEG, ethylenediamine, and hexamethylenediamine dihydrazide.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 71 to 73 in sequence.

Examples 2-19: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the mixing temperature (35°C) in the crosslinking step is replaced with: 15°C, 20°C, 25°C, 30°C, 40°C, and 45°C respectively.

The above-mentioned ophthalmic biological adhesives are named ophthalmic biological adhesives 74 to 79 in sequence.

Examples 2-20: An ophthalmic bio-adhesive and its preparation

This embodiment provides an ophthalmic bio-adhesive, which is based on the ophthalmic bio-adhesive 1 of Example 2-1, except that the number of extrusions (20 times) in the crosslinking step is replaced with 10 times, 30 times, and 40 times respectively.

The above-mentioned composite corneas were named ophthalmic bio-adhesives 80-82 in sequence.

Experiment Example 2: Effect of Preparation Process on the Properties of Ophthalmic Bioadhesives

This experimental example demonstrates the effect of preparation process on the properties of ophthalmic bioadhesives. The experimental procedure is as follows:

Experiment 1: Using the light transmittance of human cornea under normal conditions (90%) as a control (Reference: SPeris-Martínez C, García-Domene M C, Penadés M, et al. Spectral transmission of the human corneal layers[J]. Journal of Clinical Medicine, 2021, 10(19):4490.), ophthalmic bio-adhesive was injected into a 500μm mold, and the eye... The ophthalmic bio-adhesive solidified (it solidified after standing for 30 seconds; before injecting it into the mold, the ophthalmic bio-adhesive was placed in a centrifuge tube to observe its state and obtain its solidification time; the ophthalmic bio-adhesive before solidification is shown in Figure 2, and the ophthalmic bio-adhesive after solidification is shown in Figure 3), and then demolded to prepare a sheet with the same thickness (500 μm) as the decellularized porcine cornea. The average transmittance of the ophthalmic bio-adhesive sheets 1-82 prepared were measured using a UV-Vis spectrophotometer at room temperature (25℃) within the wavelength range of 400-1100 nm. The test results are shown in Table 2.

Experiment 2: Using the rupture pressure of fibrinogen + thrombin products as a reference (78.3 mmHg) (Reference: Zhao X, Li S, Du X, et al. Natural polymer-derived photocurable bioadhesive hydrogels for sutureless keratoplasty[J]. Bioactive Materials, 2022, 8: 196-209.), a pig eye was used, and a 4 mm diameter and 450 μm thick implant bed was cut at the center of the cornea. 50 μL of ophthalmic bio-adhesive was evenly applied to the implant bed, and the cut implant was placed back on the implant bed. After the ophthalmic bio-adhesive solidified (it will solidify after standing for 30 seconds), pressure was applied to the eyeball, and the pressure at which the implant detached due to pressure was measured and named the rupture pressure. The rupture pressure of ophthalmic bio-adhesive 1 to 82 was tested, and the cut implant was placed back on the implant bed for testing. The results are shown in Table 2.

Table 2 shows that the preparation process affects the light transmittance of ophthalmic bio-adhesives. Specifically, in the dissolution step, the concentrations of polyethylene glycol-based collagen and hyaluronic acid affect the degree of cross-linking in the ophthalmic bio-adhesive; higher concentrations result in higher cross-linking and higher light transmittance. During dissolution and pre-cross-linking, excessive amounts of hyaluronic acid, polyethylene glycol-based collagen, and cross-linking agents can lead to excessively high product viscosity. Air bubbles introduced during stirring cannot be eliminated, resulting in abnormally low light transmittance. Insufficient stirring speed leads to uneven product mixing and inconsistent cross-linking density, resulting in lower light transmittance. Excessive stirring speed introduces a large number of air bubbles that cannot be eliminated, also reducing light transmittance. During cross-linking, higher stirring speed and cross-linking agent concentration result in denser cross-linking and higher light transmittance; conversely, insufficient cross-linking leads to incomplete cross-linking. In the cross-linking step, excessively low reaction temperature leads to insufficient cross-linking and lower light transmittance. While higher cross-linking agent concentration increases light transmittance, excessively high cross-linking can lead to an overly dense network, hindering nutrient flow and introducing more byproducts for in-situ cross-linking. In Table 2, except for ophthalmic biological adhesives 2, 9, 10, 11, 12, 13, 14, 15, 19, 20, 21, 24, 25, 26, 27, 28, 32, 35, 36, 39, 40, 41, 49, 50, 54, 55, 64, 65, 66, 67, 70, 71, and 82, the light transmittance of all other ophthalmic biological adhesives is higher than that of the human eye under normal conditions (90%), and they have great application prospects in ophthalmology, which does not involve blood vessels.

Table 2 shows that the preparation process affects the burst pressure of ophthalmic bio-adhesives. Specifically, in the dissolution step, the concentrations of polyethylene glycol-based collagen and hyaluronic acid affect the degree of cross-linking in the composite cornea; higher concentrations result in higher cross-linking and higher burst pressure. During dissolution and pre-cross-linking, excessive amounts of hyaluronic acid, polyethylene glycol-based collagen, and cross-linking agents can lead to excessively high product viscosity. This can cause air bubbles to be trapped during stirring, resulting in an abnormally low burst pressure. Insufficient stirring speed and uneven product mixing, along with inconsistent cross-linking density, also lead to lower burst pressure. Conversely, excessively fast stirring speeds, which introduce a large number of air bubbles, also reduce burst pressure. During the cross-linking process, higher cross-linking agent concentrations result in higher product viscosity. The denser the crosslinking, the higher the bursting pressure. Too low a concentration of crosslinking agent will lead to insufficient crosslinking of the product. In the crosslinking process, if the reaction temperature is too low, the product will not be crosslinked sufficiently, resulting in a lower bursting pressure. However, excessively high temperatures may damage the planting bed. The number of mixing steps in the crosslinking process is bidirectional. Too few mixing steps will result in uneven mixing and uneven crosslinking density, leading to a low bursting pressure. Too many mixing steps will result in over-crosslinking, which will prevent the glue from having enough active groups to crosslink with the planting bed and plantlets, resulting in a low bursting pressure. Furthermore, the higher the concentration of crosslinking agent, the higher the bursting pressure. However, excessively high crosslinking will lead to an overly dense network, making it difficult for nutrients to flow and bringing more byproducts to in-situ crosslinking. In Table 2, except for ophthalmic bio-adhesives 5, 9, 10, 11, 14, 15, 19, 20, 27, 33, 34, 40, 41, 49, 55, 65, 66, and 82, the breaking strength of other ophthalmic bio-adhesives is higher than that of fibrinogen + thrombin products. The breaking pressure is 78.3 mmHg, indicating that they have great application potential in ophthalmic fields that do not involve blood vessels.

Table 2. Light transmittance and bursting pressure of ophthalmic bio-adhesives 1-82

Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims (25)

A method for preparing biomaterials, characterized in that the method comprises: dissolving collagen, collagen-like substances, lyophilized collagen powder and/or lyophilized collagen-like substances in a buffer solution, and dissolving excipients in the buffer solution to obtain a solution; pre-crosslinking the solution and then crosslinking it to obtain biomaterials. The method for preparing biomaterials according to claim 1, wherein the biomaterial is a solid biomaterial or a liquid biomaterial; When the biomaterial is a solid biomaterial, the excipients include one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride; When the biomaterial is a liquid biomaterial, the excipients include hyaluronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The method for preparing biomaterials according to claim 2, wherein the biomaterial is a solid biomaterial or a liquid biomaterial; When the biomaterial is a solid biomaterial, the excipients include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the excipients include N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride and N-hydroxysuccinimide; When the biomaterial is a liquid biomaterial, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride; or, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide; or, the excipients include medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, N-cyclohexyl-N′-(2-morpholineethyl)carbodiimide hydrochloride, and N-hydroxysuccinimide. The method for preparing biomaterials according to claim 2 or 3, wherein the solid biomaterial comprises a composite cornea; and the liquid biomaterial comprises a bio-adhesive. The method for preparing biomaterials according to any one of claims 2 to 4, characterized in that, when the biomaterial is a solid biomaterial, the method includes the following steps: Dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and/or lyophilized collagen-like substances are dissolved in a buffer solution, and the excipients are dissolved in the buffer solution to obtain a solution; the excipients include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the excipients include N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide hydrochloride and N-hydroxysuccinimide; Pre-crosslinking step: Stir the solution. After stirring, adjust the pH with an alkaline solution to obtain a pre-crosslinking solution. Crosslinking step: The decellularized corneal graft is immersed in the pre-crosslinking solution and stirred to obtain solid biomaterial. The method for preparing biomaterials according to claim 5 is characterized in that, when the biomaterial is a solid biomaterial, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 1-100 mg/mL, and the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 4.5-45 mg/mL. The method for preparing biomaterials as described in claim 5 or 6 is characterized in that, when the biomaterial is a solid biomaterial, in the dissolution step, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the buffer solution is 1:4 to 10:1. The method for preparing biomaterials according to any one of claims 5 to 7 is characterized in that, when the biomaterial is a solid biomaterial, in the dissolution step, the pH of the buffer solution is 2.6 to 6.5, and the concentration of the buffer solution is 0.05 to 0.65 mol/L. The method for preparing biomaterials according to any one of claims 5 to 8 is characterized in that, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the concentration of the alkaline solution is 0.1 to 10 mol/L, and the pH adjustment using the alkaline solution is to adjust the pH to 4 to 14 using the alkaline solution. The method for preparing biomaterials according to any one of claims 5 to 9 is characterized in that, when the biomaterial is a solid biomaterial, in the pre-crosslinking step, the stirring temperature is 15 to 70°C, the stirring time is 5 to 240 min, and the stirring speed is 200 to 700 rpm. The method for preparing biomaterials according to any one of claims 5 to 10 is characterized in that, when the biomaterial is a solid biomaterial, in the crosslinking step, the stirring temperature is 15 to 70°C, the stirring time is 1 to 48 hours, and the stirring speed is 200 to 700 rpm. The method for preparing biomaterials according to any one of claims 5 to 11 is characterized in that, when the biomaterial is a solid biomaterial, the stirring includes mechanical stirring, magnetic stirring or shaking table stirring. The method for preparing biomaterials according to any one of claims 2 to 4, characterized in that, when the biomaterial is a liquid biomaterial, the method includes the following steps: Dissolution Steps: Collagen, collagen-like substances, lyophilized collagen powder, and/or lyophilized collagen-like substances are dissolved in a buffer solution. Medium-molecular-weight hyaluronic acid and high-molecular-weight hyaluronic acid are dissolved in the buffer solution and then mixed to obtain a mixture. A cross-linking agent solution is added dropwise to the mixture and stirred to obtain a solution. The cross-linking agent in the cross-linking agent solution includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, or the cross-linking agent in the cross-linking agent solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or the cross-linking agent in the cross-linking agent solution includes N-cyclohexyl-N′-(2-morpholineethyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Pre-crosslinking step: Stir the solution, and after stirring, dialyze the product to obtain a pre-crosslinking solution; concentrate the pre-crosslinking solution to obtain glue A; Crosslinking step: Prepare a crosslinking agent solution for secondary crosslinking to obtain glue B; mix glue A and glue B to obtain liquid biomaterial; the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and urea, or the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and ethylenediamine, or the crosslinking agent in the crosslinking agent solution for secondary crosslinking includes 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and hexamethylenediamine dihydrazide. The method for preparing biomaterials according to claim 13 is characterized in that, in the dissolution step, the concentration of collagen or collagen-like protein in the buffer solution is 3-50 mg/mL, the concentration of medium molecular weight hyaluronic acid is 10-75 mg/mL, and the concentration of high molecular weight hyaluronic acid is 1-10 mg/mL. The method for preparing biomaterials as described in claim 13 or 14 is characterized in that, when the biomaterial is a liquid biomaterial, in the dissolution step, the pH of the buffer solution is 4.5 to 7.5, and the concentration of the buffer solution is 0.05 to 0.65 mol/L. The method for preparing biomaterials according to any one of claims 13 to 15 is characterized in that, when the biomaterial is a liquid biomaterial, in the dissolution step, the stirring temperature is 15 to 55°C, the stirring time is 30 to 120 min, and the stirring speed is 500 to 1500 rpm. The method for preparing biomaterials according to any one of claims 13 to 16 is characterized in that, when the biomaterial is a liquid biomaterial, in the dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the mixture is 2 to 10 mg/mL. The method for preparing biomaterials according to any one of claims 13 to 17 is characterized in that, when the biomaterial is a liquid biomaterial, in the pre-crosslinking step, the stirring temperature is 15 to 55°C, the stirring time is 60 to 600 s, and the stirring speed is 500 to 1500 rpm. The method for preparing biomaterials according to any one of claims 13 to 18 is characterized in that, when the biomaterial is a liquid biomaterial, in the pre-crosslinking step, the dialysis includes dialysis through a dialysis bag, and the concentration includes: concentrating the pre-crosslinked solution using a dialysis bag until the concentration of high molecular weight hyaluronic acid in the pre-crosslinked solution is 10 to 100 mg/mL. The method for preparing biomaterials according to any one of claims 13 to 19 is characterized in that, when the biomaterial is a liquid biomaterial, in the crosslinking step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the crosslinking agent solution for secondary crosslinking is 15 to 75 mg/mL, and the concentration of urea, ethylenediamine or hexamethylenediamine dihydrazide is 3.3 to 16.5 mg/mL. The method for preparing biomaterials according to any one of claims 13 to 20 is characterized in that, when the biomaterial is a liquid biomaterial, the mixing step includes: mixing A glue and B glue respectively at both ends of a T-type mixer. The method for preparing biomaterials according to any one of claims 13 to 21 is characterized in that, when the biomaterial is a liquid biomaterial, in the crosslinking step, the number of extrusions of the mixture is 10 to 40 times and the temperature is 15 to 45°C. The method for preparing biomaterials according to any one of claims 13 to 22 is characterized in that, when the biomaterial is a liquid biomaterial, the liquid biomaterial is an ophthalmic bioadhesive, and in the crosslinking step, the volume ratio of A glue to B glue in the ophthalmic bioadhesive is 2 to 4:1. A biomaterial, characterized in that the biomaterial is prepared using the method for preparing biomaterials according to any one of claims 1 to 23. Use of the biomaterial of claim 24 in ophthalmic surgery.