Bioadhesives for corneal repair

Abstract

To provide a novel adhesive for corneal repair which mitigates drawbacks of the prior art, such as: low biocompatibility, cytotoxic effects on the cornea and other ocular tissues; lack of transparency, precluding good vision and impairing view of retrocorneal structures; and risk of secondary infection due to high porosity.SOLUTION: A composition for corneal reconstruction comprises a methacryloyl-substituted gelatin, a visible light activated photoinitiator, and a pharmaceutically acceptable carrier.SELECTED DRAWING: Figure 20

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 62 / 292,752, filed February 8, 2016, the entire contents of which are incorporated herein by reference.

[0002] Disclosure Fields The field of the disclosure relates to improved tissue adhesives for use in repairing corneal injuries and defects. These tissue adhesives include elastic biopolymers that are biocompatible, biodegradable, permeable, strongly adhere to corneal tissue, and have a smooth surface and biochemical properties similar to the cornea. [Background technology]

[0003] background Ocular trauma is well known and accounts for approximately 5% of blindness in the general population. 1 Current standard treatments for the repair of corneal damage, including lacerations, structural defects, and thinning, often require suturing, tissue / patch grafting, and / or glue application. However, these standard techniques are associated with significant drawbacks, including: (1) corneal sutures are foreign materials that can be risk factors for microbial invasion and infection, inflammation, and neovascularization; (2) corneal sutures often induce regular or irregular astigmatism, thereby reducing visual acuity; (3) corneal and patch transplants require donor tissue, which may not be readily available; and (4) the use of allogeneic tissue for transplantation increases the risk of immune reactions in acutely damaged and inflamed eyes. Thus, even if structural repair is adequate with the current standard treatments, visual outcomes are often unsatisfactory. 2,3 In addition, (5) the application of any of the currently available glue / adhesive technologies to temporarily treat an injured eye until more definitive treatment can be provided has its own inherent limitations.

[0004] To circumvent some of the limitations of current surgical approaches to corneal repair, the use of adhesives for rapid repair of corneal damage may be considered. However, at present, no adhesives are approved for filling corneal defects. The only sealant approved in the United States, ReSure®, is intended to seal corneal incisions in cataract surgery, is not designed to fill corneal defects, and falls off quickly (usually within 3 days). 4 A sealant used in Europe, OcuSeal®, is also utilized to protect corneal incisions, not to fill corneal defects, and it also falls off quickly. For this reason, cyanoacrylate glues approved for skin wound repair are currently used "off-label" to treat many ophthalmic conditions, such as corneal perforations, impending perforations, and progressive corneal thinning disorders. 5,6 However, cyanoacrylate glues have a number of significant drawbacks, including:

[0005] (1) Poor biocompatibility with cytotoxicity to the cornea and other ocular tissues (if it comes into contact with the eye, there is a risk of cataract formation and retinopathy) 7~11 (2) lack of transparency, obstructing good vision and compromising visibility of posterior corneal structures; (3) risk of secondary infection due to high porosity. 12 (4) difficulty in controlling its application and the possibility of the glue peeling off unexpectedly; (5) a rough surface that requires contact lens wear which adds an additional risk of infection; and (6) failure to fuse with corneal tissue.

[0006] Because existing adhesives for corneal repair have significant drawbacks, there is an unmet need for an adhesive for the repair and regeneration of corneal damage that can meet the following requirements: (1) ease of application, (2) biocompatibility without any toxicity, inflammation or neovascularization, (3) permeability to allow vision to be restored as quickly as possible, (4) ability to quickly seal corneal wounds, (5) ability of corneal cells to integrate with the bioadhesive to promote tissue regeneration, (6) biomechanical properties (rigidity and elasticity) similar to the cornea, (7) strong adhesion to corneal tissue, including good stability and high retention, and (8) a smooth surface to reduce the need for a bandage contact lens and minimize the surface area for microbial adhesion.

[0007] Photopolymerization of methacryloyl-substituted gelatin is an inexpensive and technically simple approach to fabricate hydrogels for biomedical applications 14,38~40 The cytocompatibility of methacryloyl-substituted gelatin has already been demonstrated, suggesting that it may be possible to implant it into living organisms. 41~42 However, its actual function as a corneal repair material has not yet been evaluated. Moreover, the mechanical properties of methacryloyl-substituted gelatin have not been fully investigated, and it is unclear whether it is suitable for the role as a bioadhesive for corneal repair. Summary of the Invention

[0008] Abstract Certain aspects of the present invention relate to a corneal reconstruction composition comprising a methacryloyl gelatin (GelMA) prepolymer, a visible light activated photoinitiator, and a pharma- ceutically acceptable carrier. In some embodiments, the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30%-85%, 60%-85%, or 70%-80%. Methacryloyl gelatin is also referred to herein as methacryloyl-substituted gelatin.

[0009] In some embodiments, the methacryloyl substituted gelatin comprises methacrylamide substitution and methacrylate substitution, wherein the ratio of methacrylamide substitution to methacrylate substitution is 80:20 to 99:1, 90:10 to 98:2, or 93:7 to 97:3.

[0010] In some embodiments, the methacryloyl substituted gelatin is present at a concentration of 5%-25% (w / v), 17%-55% (w / v), 17%-23% (w / v), 5%-15% (w / v), 8%-12% (w / v), about 20% (w / v), or about 10% (w / v).

[0011] In some embodiments, the visible light activated photoinitiator is Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane (Ivocerin®), derivatives thereof, and any combination thereof.

[0012] Preferably, the visible light activated photoinitiator is a mixture including Eosin Y, triethanolamine, and vinylcaprolactam. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.0125-0.5 mM, and / or the concentration of triethanolamine is 0.1-2% w / v, and / or the concentration of vinylcaprolactam is 0.05-1.5% w / v.

[0013] In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09-0.8% w / v. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09-0.8% w / v. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.05-0.08 mM, and / or the concentration of triethanolamine is 0.4-0.8% w / v, and / or the concentration of vinylcaprolactam is 0.18-0.4% w / v. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is about 0.05 mM, and / or the concentration of triethanolamine is about 0.4% w / v, and / or the concentration of vinylcaprolactam is about 0.4% w / v. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.5-0.5 mM, and / or the concentration of triethanolamine is 0.5-2% w / v, and / or the concentration of vinylcaprolactam is 0.5-1.5% w / v. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is about 0.1 mM, and / or the concentration of triethanolamine is about 0.5% w / v, and / or the concentration of vinylcaprolactam is about 0.5% w / v.

[0014] In some embodiments, the composition further comprises corneal cells. Exemplary corneal cells include, but are not limited to, epithelial cells, endothelial cells, keratocytes, and any combination thereof.

[0015] In some embodiments, the composition further comprises a therapeutic agent. Exemplary therapeutic agents for inclusion in the composition include, but are not limited to, antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoeba agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, or any combination thereof.

[0016] Certain aspects of the present invention relate to compositions for corneal reconstruction comprising a crosslinked methacryloyl-substituted gelatin hydrogel and a pharma- ceutically acceptable carrier, the crosslinked methacryloyl-substituted gelatin hydrogel having a degree of methacryloyl substitution of 30%-85% and a concentration of 5%-25% (w / v) in the pharma-ceutically acceptable carrier. These compositions are also referred to herein as crosslinked compositions. Furthermore, such compositions are also referred to herein as Gel-CORE.

[0017] In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel has a degree of methacryloyl substitution of 60%-85% and a concentration of 8%-12% (w / v), or a degree of methacryloyl substitution of 70%-80% and a concentration of about 10% (w / v). In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel has a degree of methacryloyl substitution of 60%-85% and a concentration of 17%-25% (w / v), or a degree of methacryloyl substitution of 70%-80% and a concentration of about 20% (w / v).

[0018] In some embodiments, the crosslinked composition has a Young's modulus of 190-260 kPa. In some embodiments, the crosslinked composition has a Young's modulus of 110-140 kPa.

[0019] In some embodiments, the crosslinked composition has an elastic modulus of 5 to 50 kPa.

[0020] In some embodiments, the crosslinked composition has a compression modulus of 5 to 320 kPa. In some embodiments, the composition has a compression modulus of 5 to 160 kPa. In yet some other embodiments, the composition has a compression modulus of 125 to 175 kPa.

[0021] In some embodiments, the crosslinked composition has a wound closure strength of ≧40 kPa.

[0022] In some embodiments, the crosslinked composition has a shear resistance strength of > 10 kPa. In some embodiments, the crosslinked composition has a shear resistance strength of > 100 kPa.

[0023] In some embodiments, the crosslinked composition has a burst pressure of ≧15 kPa.

[0024] In some embodiments, the crosslinking composition further comprises a therapeutic agent. Some exemplary therapeutic agents are antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoeba agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, or any combination thereof.

[0025] In some embodiments, the crosslinking composition further comprises corneal cells. Preferred corneal cells include endothelial cells, keratocytes, or a combination thereof.

[0026] In some embodiments, the crosslinked composition is substantially permeable.

[0027] In some embodiments, the crosslinked composition comprises a substantially smooth surface.

[0028] Certain aspects of the present invention relate to a method for corneal reconstruction comprising applying to a corneal defect a composition comprising methacryloyl-substituted gelatin, a visible light-activated photoinitiator, and a pharma- ceutically acceptable carrier, and exposing the composition to visible light. In some embodiments of the method, the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30%-85%, 60%-85%, or 70%-80%.

[0029] In some embodiments of this method, the methacryloyl-substituted gelatin comprises methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is 80:20 to 99:1, 90:10 to 98:2, or 92:8 to 97:3.

[0030] In some embodiments of this method, the methacryloyl substituted gelatin is present in a concentration of 5%-25% (w / v), 17%-55% (w / v), 17%-23% (w / v), 5%-15% (w / v), 8%-12% (w / v), about 20% (w / v), or about 10% (w / v).

[0031] In some embodiments of this method, the visible light activated photoinitiator is Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane (Ivocerin®), derivatives thereof, and any combination thereof.

[0032] In some embodiments of this method, the visible light activated photoinitiator comprises a mixture of Eosin Y, triethanolamine, and vinylcaprolactam. In some embodiments of the photoinitiator mixture, the concentration of Eosin Y is 0.0125-0.5 mM, and / or the concentration of triethanolamine is 0.1-2% (w / v), and / or the concentration of vinylcaprolactam is 0.05-1.5% (w / v). In some embodiments of this method, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% (w / v), and / or the concentration of vinylcaprolactam is 0.09-0.8% w / v. In some embodiments of this method, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09-0.8% (w / v). In some embodiments of this method, the concentration of Eosin Y is 0.05-0.08 mM, and / or the concentration of triethanolamine is 0.4-0.8% w / v, and / or the concentration of vinylcaprolactam is 0.18-0.4% (w / v). In some embodiments of this method, the concentration of Eosin Y is about 0.05 mM, and / or the concentration of triethanolamine is about 0.4% (w / v), and / or the concentration of vinylcaprolactam is about 0.4% (w / v). In some embodiments of this method, the concentration of Eosin Y is 0.5-0.5 mM, and / or the concentration of triethanolamine is 0.5-2% (w / v), and / or the concentration of vinylcaprolactam is 0.5-1.5% (w / v). In some embodiments of this method, the concentration of Eosin Y is about 0.1 mM, and / or the concentration of triethanolamine is about 0.5% (w / v), and / or the concentration of vinylcaprolactam is about 0.5% (w / v).

[0033] Generally, any suitable wavelength of light can be used in the method of the present invention. For example, the composition can be exposed to visible light having a wavelength in the range of 450-550 nm. For example, exposure to light can be for any desired period of time. For example, the composition can be exposed to visible light for a period of 10-300 seconds. In some embodiments, the composition can be exposed to visible light for a period of 20-120 seconds or 30-60 seconds. In some embodiments, the composition can be exposed to visible light for a period of 60 seconds to 240 seconds. In some embodiments, the composition can be exposed to visible light for a period of about 60 seconds, about 120 seconds, about 180 seconds, or about 240 seconds. In some preferred embodiments, the composition can be exposed to visible light for a period of about 240 seconds.

[0034] In some embodiments of this method, the composition further comprises a therapeutic agent, preferably an antibacterial agent, an antifungal agent, an antiviral agent, an anti-acanthamoeba agent, an anti-inflammatory agent, an immunosuppressant, an anti-glaucoma agent, an anti-VEGF, a growth factor, or any combination thereof. In some embodiments, the composition further comprises corneal cells, preferably epithelial cells, endothelial cells, keratocyte cells, and combinations thereof. In some embodiments, the composition is substantially permeable. In some embodiments, the method does not include the step of suturing the cornea.

[0035] In some aspects of this method, the composition is a corneal reconstruction composition described herein. [The present invention 1001] A composition for corneal reconstruction comprising methacryloyl-substituted gelatin, a visible light activated photoinitiator, and a pharma- ceutically acceptable carrier. [The present invention 1002] The composition of claim 1001, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30% to 85%. [The present invention 1003] 1001. The composition of claim 1001, wherein the methacryloyl-substituted gelatin comprises methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is from 80:20 to 99:1. [The present invention 1004] The composition of claim 1001, wherein the methacryloyl-substituted gelatin is present in a concentration of 5% to 25% (w / v). [The present invention 1005] The composition of claim 1004, wherein the methacryloyl-substituted gelatin is present in a concentration of 8% to 12% (w / v) or 17% to 23% (w / v). [The present invention 1006] The composition of the present invention 1005, wherein the methacryloyl-substituted gelatin is present in a concentration of about 10% (w / v) or about 20% (w / v). [The present invention 1007] Visible light activated photoinitiators include Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 The composition of the present invention 1001 is selected from the group consisting of (trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, and any combination thereof. [The present invention 1008] The composition of claim 1001, wherein the visible light activated photoinitiator is a mixture comprising Eosin Y, triethanolamine, and vinylcaprolactam. [The present invention 1009] The composition of the present invention 1008, wherein the concentration of eosin Y is 0.0125-0.5 mM, the concentration of triethanolamine is 0.1-2% w / v, and the concentration of vinylcaprolactam is 0.05-1.5% w / v. [The present invention 1010] The composition of the present invention 1009, wherein (i) the concentration of Eosin Y is about 0.1 mM, the concentration of triethanolamine is about 0.5% w / v, and the concentration of vinylcaprolactam is about 0.5% w / v, or (ii) the concentration of Eosin Y is about 0.05 mM, the concentration of triethanolamine is about 0.4% w / v, and the concentration of vinylcaprolactam is about 0.4% w / v. [The present invention 1011] The composition of any one of claims 1 to 10, further comprising corneal cells. [The present invention 1012] The composition of any one of claims 1 to 10, further comprising a therapeutic agent. [The present invention 1013] A composition for corneal reconstruction comprising a cross-linked methacryloyl-substituted gelatin hydrogel and a pharma- ceutically acceptable carrier, wherein the cross-linked methacryloyl-substituted gelatin hydrogel has a degree of methacryloyl substitution of 30%-85% and a concentration of 5%-15% (w / v) in the pharma- ceutically acceptable carrier. [The present invention 1014] The composition of the present invention 1013, wherein the crosslinked methacryloyl-substituted gelatin hydrogel has (i) a degree of methacryloyl substitution of 60% to 85% and a concentration of 8% to 12% (w / v), or (ii) a degree of methacryloyl substitution of 60% to 85% and a concentration of 17% to 25% (w / v). [The present invention 1015] The composition of the present invention 1013, wherein the crosslinked methacryloyl-substituted gelatin hydrogel has (i) a degree of methacryloyl substitution of 70% to 80% and a concentration of about 10% (w / v), or (ii) a degree of methacryloyl substitution of 70% to 80% and a concentration of about 20% (w / v). [The present invention 1016] Features include: (i) Young's modulus between 250 and 350 kPa or between 100 and 150 kPa; (ii) Elastic modulus between 5 and 50 kPa; (iii) a compressibility coefficient between 10 and 250 kPa; (iv) wound closure strength of ≥40 kPa; (v) a shear resistance strength of ≥ 100 kPa; and (vi) A burst pressure of ≥ 15 kPa The composition of the present invention 1013, comprising at least one of the following: [The present invention 1017] The composition of the present invention 1013 further comprising a therapeutic agent. [The present invention 1018] The composition of claim 1013, further comprising corneal cells. [The present invention 1019] The composition of the present invention 1013, which is substantially transparent. [The present invention 1020] The composition of claim 1013, further comprising a substantially smooth surface. [The present invention 1021] (a) applying to a corneal defect a composition comprising methacryloyl-substituted gelatin, a visible light-activated photoinitiator, and a pharma- ceutically acceptable carrier; and (b) exposing the composition to visible light 16. A method for corneal reconstruction comprising: [The present invention 1022] The method of claim 1021, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30% to 85%. [The present invention 1023] 1021. The method of claim 1021, wherein the methacryloyl substituted gelatin comprises methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is from 80:20 to 99:1. [The present invention 1024] The method of claim 1021, wherein the methacryloyl-substituted gelatin is present in a concentration of 5% to 25% (w / v). [The present invention 1025] Methacryloyl-substituted gelatin, 8%-12% (w / v) or 17%-23% (w / v) The method of claim 1021, wherein [The present invention 1026] The method of claim 10, wherein the methacryloyl-substituted gelatin is present in a concentration of about 10% (w / v) or about 20% (w / v). [The present invention 1027] Visible light activated photoinitiators include Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 The method of the present invention 1021, wherein the compound is selected from the group consisting of (trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, and any combination thereof. [The present invention 1028] The method of claim 1021, wherein the visible light activated photoinitiator is a mixture comprising eosin Y, triethanolamine, and vinylcaprolactam. [The present invention 1029] 1028. The method of claim 1028, wherein the concentration of eosin Y is 0.0125-0.5 mM, the concentration of triethanolamine is 0.1-2% w / v, and the concentration of vinylcaprolactam is 0.05-1.5% w / v. [The present invention 1030] The method of the present invention 1028, wherein (i) the concentration of Eosin Y is about 0.1 mM, the concentration of triethanolamine is about 0.5% w / v, and the concentration of vinylcaprolactam is about 0.5% w / v, or (ii) the concentration of Eosin Y is about 0.05 mM, the concentration of triethanolamine is about 0.4% w / v, and the concentration of vinylcaprolactam is about 0.4% w / v. [The present invention 1031] The method of claim 1021, wherein the composition is exposed to visible light having a wavelength in the range of 450 to 550 nm. [The present invention 1032] The method of claim 1021, wherein the composition is exposed to visible light for a period of from 20 to 120 seconds. [The present invention 1033] The method of claim 1021, wherein the composition further comprises a therapeutic agent. [The present invention 1034] The method of claim 1021, wherein the composition further comprises corneal cells. [The present invention 1035] The method of claim 1021, wherein said composition is substantially permeable. [The present invention 1036] The method of claim 1021, which does not include a step of suturing the cornea. [Brief description of the drawings]

[0036] [Figure 1] Figure 1A is an optical coherence tomography (OCT) image of the cyanoacrylate glue on a patient's cornea showing its rough surface and high porosity. Figure 1B is a slit lamp photograph of the cyanoacrylate glue showing its opacity. Figures 1C-1D are slit lamp photographs and Figure 1E is an OCT image of Gel-CORE after ex vivo application to a rabbit corneal defect showing its clarity and smoothness. [Diagram 2] Figure 2A is a reaction scheme showing the fluoroaldehyde assay to determine the conversion of amine groups in GelMA. The reaction between unmodified lysine residues in methacrylic anhydride (MA) modified materials and the fluoroaldehyde reagent produces blue fluorescent derivatives that exhibit excitation and emission maxima at wavelengths of 340 nm and 455 nm, respectively. Figure 2B is a bar graph showing the conversion of amine groups in GelMA samples. Gelatin is used as a control. [Diagram 3]Figures 3A-3E show the Fe(III)-hydroxamic acid assay for the quantification of methacrylate groups in GelMA. Figure 3A is a schematic diagram showing the reaction used for the analysis of methacrylate groups. Step (i) shows that the methacrylate groups in GelMA react with hydroxylamine under basic conditions to produce an equimolar amount of N-hydroxymethacrylamide. Step (ii) shows that N-hydroxymethacrylamide forms a colored complex with Fe(III) ions under acidic conditions. Figure 3B shows a photograph of the Fe(III) solution before and after the addition of acetohydroxamic acid. The color change indicates the formation of the Fe(III)-acetohydroxamic acid complex. Figure 3C shows the normalized UV-Vis absorption spectrum of the Fe(III)-acetohydroxamic acid complex (FeAHA). The absorption peak is centered at 500 nm. Figure 3D shows the calibration curve between absorbance and FeAHA concentration for a series of standard FeAHA solutions. Figure 3E shows the calculated amounts of methacrylamide and methacrylate groups in the GelMA samples. SuperGelMA, HighGelMA, MidGelMA and LowGelMA represent samples prepared with 20% (v / v), 8% (v / v), 5% (v / v) and 0.5% (v / v) MA in the reaction, respectively. [Figure 4] 4A-4C show an exemplary Eosin Y-based photopolymerization system of the present invention. FIG. 4A is a summary of the radical generation reaction initiated by light-induced activation of Eosin Y assisted by TEA. FIG. 4B shows the UV-Vis spectrum of Eosin Y at pH 7.4 in aqueous solution. FIG. 4C shows images of color change upon exposure to visible light upon hydrogel formation, indicating the activation state of Eosin Y. [Diagram 5] Figures 5A-5B show visible light crosslinked GelMA hydrogels with tunable (Figures 5A and 5B) mechanical properties - stress vs. strain (Figure 5A) and tensile modulus vs. TEA concentration (Figure 5B). (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001) [Figure 6] Figures 6A-6B show visible light crosslinked GelMA hydrogels with tunable swelling ratios with different VC (Figure 6A) and TEA (Figure 6B) concentrations. [Figure 7] Figure 7A shows an overview of sample preparation for burst pressure testing. Figure 7B shows a top view of the burst pressure test setup (pig intestine is placed between metal plates). Figure 7C is a bar graph showing burst pressure data for GelMA hydrogels compared to CoSeal and Evicel. GelMA was prepared as in Examples 2 and 3. [Figure 8] Figure 8A outlines the revised standard test method for wound closure strength (ASTM F2458-05). Figure 8B is a bar graph showing the adhesive strength of this sealant using the wound closure test at different GelMA concentrations and exposure times. GelMA was prepared as in Examples 2 and 3. [Figure 9] 9A-9D show slit lamp photographs (FIGS. 9A and 9C) and OCT images (FIGS. 9B and 9D) immediately after (FIGS. 9A and 9B) and 11 days after (FIGS. 9C and 9D) ex vivo application of GelMA to rabbit corneas. As can be seen, GelMA shows excellent retention. [Figure 10] Figure 10A shows line scans obtained at different angles for OCT imaging, and Figure 10B shows images showing the thickness of the bioadhesive measured at the center and 1 mm from the center. [Figure 11] Figures 11A-11G show in vitro evaluation of cytocompatibility and spread of GelMA with keratocytes. Figures 11A-11C show representative live / dead images of keratocytes attached to the surface of Gel-CORE at days 1 (Figure 11A), 3 (Figure 11B) and 7 (Figure 11C). Figures 11D-11F show representative images of F-actin / DAPI stained GEL-CORE containing keratocytes at days 1 (Figure 11D), 3 (Figure 11E) and 7 (Figure 11F) after encapsulation. Figure 11G shows quantification of cell viability in GEL-CORE over 7 days of culture. [Figure 12] 12A-12F are photographs showing IVCM images of different corneal layers in rabbits: superficial epithelium (FIG. 12A), basal epithelium (FIG. 12B), subbasal nerves (FIG. 12C), stromal anterior keratocytes (FIG. 12D), stromal posterior keratocytes (FIG. 12E) and endothelium (FIG. 12F). [Figure 13A] Figure 13A shows slit lamp and OCT images following application of GelMA to partial thickness corneal defects in two rabbit eyes ex vivo. 3 mm partial perforations filled with porcine GelMA crosslinked with blue light for 120 seconds. [Figure 13B] Figure 13B shows slit lamp and OCT images following application of GelMA to partial thickness corneal defects in two rabbit eyes ex vivo. 3 mm partial perforations filled with porcine GelMA crosslinked with blue light for 120 seconds. [Figure 14] Figures 14A-14C show the properties of several exemplary GelMA hydrogels of the invention. Figure 14A is a line graph showing a representative tensile strain / stress curve. Figure 14B is a bar graph showing the tensile modulus of GelMA hydrogels made with various light exposure times and GelMA concentrations. Figure 14C is a bar graph showing the breaking strength of GelMA hydrogels crosslinked with various visible light exposure times and GelMA concentrations. [Figure 15] Figures 15A-15B show the properties of several exemplary GelMA hydrogels of the invention. Figure 15A is a line graph showing representative compressive strain / stress curves. Figure 15B is a bar graph showing the compressive modulus of GelMA hydrogels made with various light exposure times and GelMA concentrations. [Figure 16] 16A-16B show the setup (FIG. 16A) and results (FIG. 16B) for ex vivo burst pressure of corneas sealed with visible light crosslinked GelMA hydrogels formed under different crosslinking conditions. FIG. 16A is a schematic showing the burst pressure setup for measuring the leak pressure of rabbit corneas explanted through a 2 mm diameter incision punch after bioadhesive application and photocrosslinking. By adjusting the visible light exposure time (i.e., 1, 2 and 4 min), hydrogels with different burst pressures are generated (FIG. 16B). The concentration of GelMA was 20% (w / v). [Figure 17] FIG. 17 is a photograph showing the ex vivo evaluation of smoothness, permeability and retention. [Figure 18]FIG. 18 is a photograph showing slit lamp and OCT images immediately and 14 and 28 days after ex vivo application of GEL-CORE to rabbit cornea, demonstrating excellent retention. [Figure 19] 19A-19F are images showing the in vivo application of the bioadhesive to a rabbit corneal defect. One day after application, the bioadhesive was permeable, had a smooth surface, and did not cause corneal inflammation (FIG. 19A). There was an epithelial defect on the bioadhesive (FIG. 19B), and AS-OCT showed complete adhesion of the implant to the cornea (FIG. 19C). One week after application, the bioadhesive was still permeable with no stromal infiltration based on slit lamp examination (FIG. 19D). Epithelial migration on the bioadhesive was evidenced by fluorescein staining (FIG. 19E) and AS-OCT (arrow, FIG. 19F), which also showed no gaps between the bioadhesive and the stroma. [Figure 20] Figures 20A-20C are representative H&E histopathology images (200x) of a rabbit cornea two weeks after creation of a 50% depth stromal defect (Figure 20A, normal uninjured cornea). When the corneal defect was left to heal without bioadhesive, significant epithelial hyperplasia was observed filling the stromal defect (Figure 20B). In contrast, when the stromal defect was filled with GelMA bioadhesive, the biomaterial was covered by epithelial cells and maintained by the cornea, filling the entire defect (Figure 20C). [Figure 21] FIG. 21 is a graph showing 1H NMR spectra of GelMA prepolymer and GelMA hydrogels (20% (w / v)) formed with different visible light exposure times, including 0, 1, 2, and 4 minutes. [Figure 22] FIG. 22 is a bar graph showing quantification of the crosslinking degree of GelMA hydrogels made by using 10 and 20% (w / v) prepolymer concentrations at different visible light exposure times (1, 2, and 4 min) based on 1H NMR spectra. [Figure 23] FIG. 23 is a bar graph showing the ex vivo retention time of GelMA in explanted rabbit eyes after 18 days of incubation in PBS at 4° C. [Figure 24]24A-24D show schematic diagrams illustrating the use of adhesives for the immediate and long-term repair of corneal damage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description The present inventors have developed and optimized a special formulation of a bioadhesive hydrogel for corneal applications, Methacryloyl Gelatin (GelMA) Hydrogel for Corneal Reconstruction (hereafter referred to as Gel-CORE). To form Gel-CORE, gelatin, a natural polymer derived from hydrolyzed collagen and maintaining similar bioactivity to collagen, was used. Gelatin was chemically functionalized with methacryloyl groups to form GelMA, a light-activated adhesive hydrogel with tunable mechanical properties. This hydrogel can be applied to the cornea and photopolymerized with visible light within seconds to form a highly adhesive hydrogel. A special formulation was developed with the desired bioadhesion, bioactivity and degradation profile suitable for corneal applications.

[0038] Although popular for biomedical purposes, UV photocrosslinking poses potential biosafety concerns as it can result in undesirable DNA damage and ocular toxicity. GelMA contains modified natural extracellular matrix components that can be crosslinked through exposure to visible light to generate elastic, biodegradable hydrogels (Gel-CORE) for corneal reconstruction and repair. The natural extracellular matrix components can include gelatin from animals, including but not limited to porcine, bovine, equine, chicken, fish, etc. Gelatin is advantageous in that it can be collected under sterile conditions from animals in a sterile facility to eliminate the risk of disease transmission (e.g., Hepatitis C, Human Immunodeficiency Virus, etc.).

[0039] The in situ photopolymerization of GelMA aids in easy delivery to technically challenging locations, e.g., the cornea, and allows the bioadhesive to cure to precisely follow the shape required by the tissue to be sealed, an advantage over pre-made products, e.g., scaffolds or sheets. In addition to the physical interconnection of the bioadhesive with the tissue surface during curing, gelatin offers an additional option to interact with tissue in the defect area. Gelatin contains multiple domains that bind to cell surface receptors and extracellular matrix proteins, facilitating the initial linkage of the bioadhesive to the corneal tissue as well as subsequent cell attachment and growth on the bioadhesive.

[0040] As used herein, "methacryloyl gelatin" is defined as gelatin having free amines and / or free hydroxyls substituted with at least one methacrylamide group and / or at least one methacrylate group. Gelatin contains amino acids, some of which have side chains that terminate with amines (e.g., lysine, arginine, asparagine, glutamine) or hydroxyls (e.g., serine, threonine, aspartic acid, glutamic acid). One or more of these terminal amines and hydroxyls can be substituted with methacryloyl groups to produce methacryloyl gelatin containing methacrylamide and / or methacrylate groups, respectively. In some embodiments, by exposure to visible light in the presence of a photoinitiator, the methacryloyl group on one gelatin molecule can react with the methacryloyl group on another gelatin molecule to crosslink the methacryloyl gelatin and produce a hydrogel. In some embodiments, gelatin can be functionalized with methacryloyl groups by reacting the gelatin with an appropriate reagent, including but not limited to methacrylic anhydride, methacryloyl chloride, and the like.

[0041] Certain exemplary embodiments of the present invention include a photoinitiator. "Photoinitiator," as used herein, refers to any compound or mixture of compounds that decomposes into free radicals when exposed to light. Preferably, the photoinitiator generates free radicals when exposed to visible light. Exemplary ranges of visible light useful for exciting visible light photoinitiators include green, blue, indigo, and violet. Preferably, the visible light has a wavelength in the range of 450 nm to 550 nm. Examples of photoinitiators are eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 (trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane (Ivocerin®), derivatives thereof, combinations thereof, and the like.

[0042] In some embodiments, the photoinitiator is a mixture comprising Eosin Y, triethanolamine, and vinylcaprolactam. In some embodiments, the concentration of Eosin Y is 0.0125-0.5 mM, and / or the concentration of triethanolamine is 0.1-2% (w / v), and / or the concentration of vinylcaprolactam is 0.05-1.5% (w / v). In some embodiments, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% (w / v), and / or the concentration of vinylcaprolactam is 0.09-0.8% (w / v). In some embodiments, the concentration of Eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09-0.8% (w / v). In some embodiments, the concentration of Eosin Y is 0.05-0.08 mM, and / or the concentration of triethanolamine is 0.4-0.8% (w / v), and / or the concentration of vinylcaprolactam is 0.18-0.4% (w / v). In some embodiments, the concentration of Eosin Y is about 0.05 mM, and / or the concentration of triethanolamine is about 0.4% (w / v), and / or the concentration of vinylcaprolactam is about 0.4% (w / v). In some embodiments, the concentration of Eosin Y is 0.5-0.5 mM, and / or the concentration of triethanolamine is 0.5-2% (w / v), and / or the concentration of vinylcaprolactam is 0.5-1.5% (w / v). In some embodiments, the concentration of Eosin Y is about 0.1 mM, and / or the concentration of triethanolamine is about 0.5% (w / v), and / or the concentration of vinylcaprolactam is about 0.5% (w / v).

[0043] The mechanical properties of Gel-CORE can be tailored for different applications by varying the degree of methacryloyl substitution, GelMA concentration, amount of photoinitiator and light exposure time. As used herein, the degree of methacryloyl substitution is defined as the percentage of free amines or hydroxyls in gelatin that are substituted with methacryloyl groups. In some embodiments, the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 20%-90%, 30%-85%, 50%-90%, 60%-85%, 65%-75% or 70%-80%. In some embodiments, the methacryloyl-substituted gelatin includes methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is 80:20-99:1, 90:10-98:2 or 93:7-97:3.

[0044] As used herein, the concentration of methacryloyl-substituted gelatin is defined as the weight of methacryloyl-substituted gelatin divided by the volume of solvent (w / v) and expressed as a ratio. The solvent can be a pharma-ceutically acceptable carrier. In some embodiments, the methacryloyl-substituted gelatin is present at a concentration of 5%-25% (w / v), 17%-25% (w / v), 17%-23% (w / v), or about 20% (w / v). In some embodiments, the methacryloyl-substituted gelatin is present at a concentration of 5%-15% (w / v), 8%-12% (w / v), or about 10% (w / v). In some embodiments, the methacryloyl substituted gelatin is present at a concentration of 10-40% (w / v), 15%-35% (w / v), 20%-30% (w / v), or about 5%, 10%, 15%, 20% or 25% (w / v).

[0045] In some embodiments, the methacryloyl-substituted gelatin has any of the above methacryloyl degrees of substitution and any of the above concentrations, for example, a methacryloyl degree of substitution of 50% to 90% and a concentration of 10% to 40% (w / v), a methacryloyl degree of substitution of 60% to 85% and a concentration of 20% to 30% (w / v), a methacryloyl degree of substitution of 70% to 80% and a concentration of 25% (w / v), a methacryloyl degree of substitution of 30% to 85% and a concentration of 5% to 15% (w / v), a methacryloyl degree of substitution of 60% to 85% and a concentration of 8% to 12% (w / v), or a methacryloyl degree of substitution of 70% to 80% and a concentration of about 10% (w / v).

[0046] Certain exemplary aspects of the present invention include a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable carrier," as used herein, refers to a pharma- ceutically acceptable substance, composition, or vehicle involved in the transport or transfer of a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation and suitable for administration to a subject, e.g., a human. It must also be suitable for use in contact with any tissue or organ with which it may come into contact, meaning that it must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that significantly outweighs its therapeutic benefit. Examples of pharma- ceutically acceptable carriers include, but are not limited to, solvents or dispersion media containing, for example, water, a pH buffered solution (e.g., phosphate buffered saline (PBS), HEPES, TES, MOPS, etc.), isotonic saline, Ringer's solution, polyol (e.g., glycerol, polypropylene glycol, liquid polyethylene glycol, etc.), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the pharma-ceutically acceptable carrier may be a pH buffered solution (e.g., PBS) or water.

[0047] Corneal cells can be incorporated into the bioadhesive or on the surface of the bioadhesive to promote corneal tissue formation and healing. Thus, in some embodiments, the GelMA or Gel-CORE composition further comprises corneal cells, preferably epithelial cells, endothelial cells, keratocytes, or a combination thereof. The epithelial and / or endothelial cells are preferably seeded on the surface of the composition, and the keratocytes are preferably mixed into the composition prior to photopolymerization.

[0048] The compositions of the present invention may further comprise a therapeutic agent to promote corneal healing and regrowth, to prevent or treat infection or immune responses, to prevent or treat corneal angiogenesis, to treat elevated intraocular pressure, or to promote general ocular health. Non-limiting examples of therapeutic agents include antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoeba agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, or any combination thereof. Non-limiting examples of antibacterial agents include penicillins, cephalosporins, penems, carbapenems, monobactams, aminoglycosides, sulfonamides, macrolides, tetracyclines, lincosides, quinolones, chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid, spectinomycin, trimethoprim, sulfamethoxazole, chitosan, ansamycins, daptomycin, nitrofurans, oxazolidinones, bacitracin, colistin, polymyxin B, and clindamycin. Non-limiting examples of anti-fungal agents include amphotericin B, natamycin, candicin, filipin, hamycin, nystatin, rimocidin, voriconazole, imidazole, triazole, thiazole, allylamine, echinocandin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, tolnaftate, undecylenic acid, and povidone-iodine. Non-limiting examples of anti-viral agents include acyclovir, valacyclovir, famciclovir, penciclovir, trifluridine, and vidarabine. Non-limiting examples of anti-acanthamoeba agents include chlorhexidine, polyhexamethylene biguanide, propamidine, and hexamidine. Non-limiting examples of anti-inflammatory agents include corticosteroids; non-steroidal anti-inflammatory agents including salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, anthranilic acid derivatives, selective cox-2 inhibitors, and sulfonanilides; biologics including antibodies (e.g., tumor necrosis factor alpha inhibitors) and dominant negative ligands (e.g., interleukin 1 receptor antagonists). Non-limiting examples of immunosuppressants include alkylating agents, antimetabolites, mycophenolates, cyclosporine, tacrolimus, and rapamycin.Non-limiting examples of anti-glaucoma agents include prostaglandin analogs, beta blockers, adrenergic agonists, carbonic anhydrase inhibitors, and parasympathomimetic (miotic) agents. Non-limiting examples of anti-vascular endothelial growth factor (anti-VEGF) agents include bevacizumab, ranibizumab, and aflibercept. Non-limiting examples of growth factors include epidermal growth factor, platelet-derived growth factor, vitamin A, fibronectin, annexin a5, albumin, alpha 2 macroglobulin, fibroblast growth factor b, insulin-like growth factor I, nerve growth factor, and hepatocyte growth factor.

[0049] Certain aspects of the present invention relate to a corneal reconstruction composition comprising a crosslinked methacryloyl-substituted gelatin hydrogel and a pharma- ceutically acceptable carrier. As used herein, a "hydrogel" is a hydrophilic polymer chain network that forms a colloidal gel. In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel has a degree of methacryloyl substitution of 20%-90%, 40%-90%, 30-85%, 60%-85%, 65%-75%, or 70%-80%. In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel is present in a pharma-ceutically acceptable carrier at a concentration of 5%-15% (w / v), 8%-12% (w / v), 10%-40% (w / v), 15%-35% (w / v), 20%-30% (w / v), or about 5%, 10%, 15%, 20%, or 25% (w / v). In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel has a combination of any of the above methacryloyl degrees of substitution and any of the above concentrations. In some embodiments, the crosslinked methacryloyl-substituted gelatin hydrogel has a degree of methacryloyl substitution of 60%-80% and a concentration of 10%-40% (w / v) in a pharma- ceutically acceptable carrier, a degree of methacryloyl substitution of 65%-75% and a concentration of 20%-30% (w / v), a degree of methacryloyl substitution of 68%-72% and a concentration of 25% (w / v), a degree of methacryloyl substitution of 30%-85% and a concentration of 5%-15% (w / v), a degree of methacryloyl substitution of 60%-85% and a concentration of 8%-12% (w / v), or a degree of methacryloyl substitution of 70%-80% and a concentration of about 10% (w / v).

[0050] The physical properties of Gel-CORE (such as degradation and mechanical properties) can be tailored so that different bioadhesive compositions for different purposes can be made, e.g., bioadhesives with short or long retention times suitable for different clinical situations. For example, in the case of corneal trauma where intraocular material, e.g., the iris, has been extruded, it may be desirable to apply Gel-CORE for temporary sealing of the damaged eye. Gel-CORE with short retention times can also be used to cover epithelial defects in patients with corneal epithelial defects. In contrast, in the case of corneas that exhibit structural defects or severe thinning, Gel-CORE can be formulated to be retained for long periods of time. Currently available sealant technologies (e.g., cyanoacrylates) do not offer such control over the characteristics of the final product. The following are desirable physical properties for a bioadhesive composition suitable for corneal repair, either alone or in combination. In some embodiments, the composition has a Young's modulus of 95-100 kPa, 110-140 kPa, or 190-260 kPa. In some embodiments, the composition has an elastic modulus of 5-10 kPa, 10-20 kPa, 25-80 kPa, 5-50 kPa, 5-28 kPa, 10-22 kPa, or 14-18 kPa. In some embodiments, the composition has a compressive modulus of 1-55 kPa, 3-160 kPa, 5-320 kPa, 10-250 kPa, 25-200 kPa, 50-175 kPa, or 75-150 kPa. In some embodiments, the composition has a wound closure strength of ≧40 kPa, ≧50 kPa, ≧60 kPa, ≧70 kPa, ≧80 kPa, ≧90 kPa, or ≧100 kPa, as measured using the Wound Closure Test (ASTM F2458-05). In some embodiments, the composition has a shear resistance strength of ≧100 kPa, ≧150 kPa, or ≧200 kPa, as measured using the Lap Shear Test (ASTM F2255-05). In some embodiments, the composition has a burst pressure of ≧15 kPa, ≧17 kPa, or ≧20 kPa, as measured using the Burst Pressure Test (ASTM F2392-04).

[0051] A particular aspect of the present invention is (a) applying to a corneal defect a composition comprising methacryloyl-substituted gelatin, a visible light-activated photoinitiator, and a pharma- ceutically acceptable carrier; and (b) exposing the composition to visible light The present invention relates to a method for corneal reconstruction, comprising:

[0052] The mechanical properties of Gel-CORE can be tailored for various applications by varying the exposure time to visible light. Without being bound by theory, increasing the visible light exposure time increases crosslinking in the methacryloyl substituted gelatin, providing hydrogels with improved mechanical properties, such as adhesive strength, shear strength, compressive strength, tensile strength, etc. In some embodiments, the composition is exposed to visible light for a period of 30 seconds to 6 minutes, 1 minute to 5 minutes, 2 minutes to 4 minutes, or 3 minutes. In some embodiments, the composition is exposed to visible light for a period of less than 1 minute, 10 to 60 seconds or less, 15 to 45 seconds, 20 seconds, or 30 seconds. In some embodiments, the composition is exposed to visible light for a period of 20 to 120 seconds, or 30 to 60 seconds. In some embodiments, the composition can be exposed to visible light for a period of 60 seconds to 240 seconds. In some embodiments, the composition can be exposed to visible light for a period of about 60 seconds, about 120 seconds, about 180 seconds, or about 240 seconds.

[0053] In some embodiments, the method does not include suturing the cornea. Exemplary ranges of visible light useful for crosslinking the Gel-MA composition include green, blue, indigo, and violet. Preferably, the visible light has a wavelength in the range of 450-550 nm.

[0054] definition For convenience, certain terms used in the detailed description, examples and appended claims of this specification are collected here. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. Unless otherwise indicated or clear from the context, the following terms and phrases do not exclude the meanings they have acquired in the field to which they belong. The definitions are provided to aid in the description of certain embodiments and are not intended to limit the invention as set forth in the claims, the scope of the present invention being limited only by the claims. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include the singular.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, the methods, devices, and materials are described herein in this regard.

[0056] Other than in the working examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used to describe the invention using percentages, means ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5% or ±5%.

[0057] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and," unless the context clearly dictates otherwise.

[0058] As used herein, the terms "comprising" or "comprises" mean "including" or "includes" and are used to refer to compositions, methods, systems and each element thereof that are useful to the present invention, leaving room for the inclusion of unspecified elements, whether or not they are useful.

[0059] As used herein, the term "consisting essentially of" describes elements required for a particular embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0060] The term "consisting of" refers to compositions, methods, systems, and each element thereof described herein, exclusive of any element not mentioned in the description of that embodiment.

[0061] The abbreviation "eg" is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0062] Exemplary aspects of the invention may be represented by one or more of the following subparagraphs. 1. A composition for corneal reconstruction comprising methacryloyl-substituted gelatin (gelatin methacryloyl), a visible light activated photoinitiator, and a pharma- ceutically acceptable carrier. 2. The composition of paragraph 1, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30% to 85%. 3. The composition of paragraphs 1 or 2, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 60% to 85%. 4. The composition of any one of paragraphs 1 to 3, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 70% to 80%. 5. The composition of any one of paragraphs 1-4, wherein the methacryloyl-substituted gelatin contains methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is from 80:20 to 99:1. 6. The composition of paragraph 5, wherein the ratio of methacrylamide substitution to methacrylate substitution is from 90:10 to 98:2. 7. The composition of paragraphs 5 or 6, wherein the ratio of methacrylamide substitution to methacrylate substitution is from 92:8 to 97:3. 8. The composition of any one of paragraphs 1 to 7, wherein the gelatin methacryloyl is present in a concentration of 5% to 25% (w / v). 9. The composition of any one of paragraphs 1 to 8, wherein the gelatin methacryloyl is present in a concentration of 17% to 25% (w / v). 10. The composition of any one of paragraphs 1 to 9, wherein the gelatin methacryloyl is present in a concentration of 17% to 23% (w / v). 11. The composition of any one of paragraphs 1 to 10, wherein the gelatin methacryloyl is present in a concentration of about 20%. 12. The composition of any one of paragraphs 1 to 8, wherein the gelatin methacryloyl is present in a concentration of 8% to 12% (w / v). 13. The composition of paragraph 12, wherein the gelatin methacryloyl is present in a concentration of about 10% (w / v). 14. Visible light activated photoinitiators include Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 14. The composition of any one of paragraphs 1-13, wherein the compound is selected from the group consisting of (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, and any combination thereof. 15. The composition of any one of paragraphs 1-14, wherein the visible light activated photoinitiator comprises a mixture comprising eosin Y, triethanolamine, and vinylcaprolactam. 16. The composition of paragraph 15, wherein the concentration of eosin Y is 0.0125-0.5 mM, and / or the concentration of triethanolamine is 0.1-2% (w / v), and / or the concentration of vinylcaprolactam is 0.05-1.5% (w / v). 17. The composition of paragraphs 15 or 16, wherein the concentration of eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% (w / v), and / or the concentration of vinylcaprolactam is 0.09-0.8% (w / v). 18. Any of the compositions of paragraphs 15 to 17, wherein the concentration of eosin Y is 0.025 to 0.15 mM, and / or the concentration of triethanolamine is 0.2 to 1.6% (w / v), and / or the concentration of vinylcaprolactam is 0.09 to 0.8% (w / v). 19. Any of the compositions of paragraphs 15 to 18, wherein the concentration of eosin Y is 0.05 to 0.08 mM, and / or the concentration of triethanolamine is 0.4 to 0.8% (w / v), and / or the concentration of vinylcaprolactam is 0.18 to 0.4% (w / v). 20. Any of the compositions of paragraphs 15 to 19, wherein the concentration of eosin Y is about 0.05 mM, and / or the concentration of triethanolamine is about 0.4% (w / v), and / or the concentration of vinylcaprolactam is about 0.4% (w / v). 21. The composition of paragraphs 15 or 16, wherein the concentration of eosin Y is 0.5 to 0.5 mM, and / or the concentration of triethanolamine is 0.5 to 2% (w / v), and / or the concentration of vinylcaprolactam is 0.5 to 1.5% (w / v). 22. The composition of any of paragraphs 15, 16 or 21, wherein the concentration of eosin Y is about 0.1 mM, the concentration of triethanolamine is about 0.5% (w / v), and the concentration of vinylcaprolactam is about 0.5% (w / v). 23. The composition of any one of paragraphs 1 to 12, further comprising corneal cells. 24. The composition of paragraph 23, wherein the corneal cells comprise epithelial cells, endothelial cells, keratocytes, or a combination thereof. 25. The composition of any one of paragraphs 1-24, further comprising a therapeutic agent. 26. The composition of paragraph 25, wherein the therapeutic agent is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoeba agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, and any combination thereof. 27. A composition for corneal reconstruction comprising a cross-linked gelatin methacryloyl hydrogel and a pharma- ceutically acceptable carrier, wherein the cross-linked methacryloyl-substituted gelatin (gelatin methacryloyl) hydrogel has a degree of methacryloyl substitution of 30%-85% and a concentration of 5%-25% (w / v) in the pharma-ceutically acceptable carrier. 28. The composition of paragraph 27, wherein the concentration is 5% to 15% (w / v). 29. The composition of paragraphs 27 or 28, wherein the crosslinked gelatin methacryloyl hydrogel has a degree of methacryloyl substitution of 60% to 85% and a concentration of 8% to 12% (w / v). 30. The composition of any of paragraphs 27 to 29, wherein the crosslinked gelatin methacryloyl hydrogel has a degree of methacryloyl substitution of 70% to 8% and a concentration of about 10% (w / v). 31. The composition of paragraph 27, wherein the concentration is 17% to 25%. 32. The composition of paragraphs 27 or 28, wherein the crosslinked gelatin methacryloyl hydrogel has a degree of methacryloyl substitution of 60% to 85% and / or a concentration of 17% to 23% (w / v). 33. The composition of any of paragraphs 27 to 29, wherein the crosslinked gelatin methacryloyl hydrogel has a degree of methacryloyl substitution of 70% to 80% and / or a concentration of about 20% (w / v). 34. The composition of any one of paragraphs 27-33, having a Young's modulus of 190 to 260 kPa or 250 to 350 kPa. 35. The composition of any one of paragraphs 27-33, having a Young's modulus of 110 to 140 kPa or 100 to 150 kPa. 36. The composition of any one of paragraphs 27 to 35, having an elastic modulus of 5 to 50 kPa. 37. The composition of any one of paragraphs 27 to 36, having a compressibility modulus of 5 to 320 kPa or 10 to 250 kPa. 38. The composition of any one of paragraphs 27 to 37, having a compression modulus of 5 to 160 kPa or 125 to 175 kPa. 39. The composition of any one of paragraphs 27-38, having a wound closure strength of ≧40 kPa. 40. The composition of any one of paragraphs 27-38, having a wound closure strength of ≧45 kPa. 41. The composition of any one of paragraphs 27 to 40, having a burst pressure of ≥ 10 kPa or ≥ 15 kPa. 42. The composition of any one of paragraphs 27-41, further comprising a therapeutic agent. 43. The composition of paragraph 42, wherein the therapeutic agent is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoeba agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, and any combination thereof. 44. The composition of any one of paragraphs 27 to 43, further comprising corneal cells. 45. The composition of paragraph 44, wherein the corneal cells comprise epithelial cells, endothelial cells, keratocytes, or a combination thereof. 46. ​​The composition of any one of paragraphs 27 to 45, which is substantially transparent. 47. The composition of any one of paragraphs 27 to 46, having a substantially smooth surface. 48. (a) applying to a corneal defect a composition comprising methacryloyl-substituted gelatin, a visible light-activated photoinitiator, and a pharma- ceutically acceptable carrier; (b) exposing the composition to visible light 16. A method for corneal reconstruction comprising: 49. The method of paragraph 48, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30% to 85%. 50. The method of paragraph 48 or 49, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 60% to 85%. 51. The method of any one of paragraphs 48 to 50, wherein the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 70% to 80%. 52. The method of any one of paragraphs 48-51, wherein the methacryloyl-substituted gelatin contains methacrylamide substitution and methacrylate substitution, and the ratio of methacrylamide substitution to methacrylate substitution is from 80:20 to 99:1. 53. The method of paragraph 52, wherein the ratio of methacrylamide substitution to methacrylate substitution is from 90:10 to 98:2. 54. The method of paragraphs 52 or 53, wherein the ratio of methacrylamide substitution to methacrylate substitution is from 92:8 to 97:3. 55. The method of any one of paragraphs 48 to 54, wherein the methacryloyl-substituted gelatin is present in a concentration of 5% to 25% (w / v). 56. The method of any one of paragraphs 48 to 55, wherein the methacryloyl-substituted gelatin is present in a concentration of 17% to 25% (w / v). 57. The method of any one of paragraphs 48 to 56, wherein the methacryloyl-substituted gelatin is present in a concentration of 17% to 23% (w / v). 58. The method of any one of paragraphs 48 to 57, wherein the methacryloyl-substituted gelatin is present at a concentration of about 20%. 59. The method of any one of paragraphs 48 to 55, wherein the methacryloyl-substituted gelatin is present in a concentration of 8% to 12% (w / v). 60. The method of paragraph 59, wherein the methacryloyl-substituted gelatin is present in a concentration of about 10% (w / v). 61. Visible light activated photoinitiators include Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 61. The method of any one of paragraphs 48-60, wherein the compound is selected from the group consisting of (trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, and any combination thereof. 62. The method of any one of paragraphs 48-61, wherein the visible light activated photoinitiator comprises a mixture of eosin Y, triethanolamine, and vinylcaprolactam. 63. The method of paragraph 62, wherein the concentration of eosin Y is 0.0125-0.5 mM, and / or the concentration of triethanolamine is 0.1-2% w / v, and / or the concentration of vinylcaprolactam is 0.05-1.5% w / v. 64. The method of paragraph 62 or 63, wherein the concentration of eosin Y is 0.025-0.15 mM, and / or the concentration of triethanolamine is 0.2-1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09-0.8% w / v. 65. The method of any of paragraphs 62 to 64, wherein the concentration of eosin Y is 0.025 to 0.15 mM, and / or the concentration of triethanolamine is 0.2 to 1.6% w / v, and / or the concentration of vinylcaprolactam is 0.09 to 0.8% w / v. 66. The method of any of paragraphs 62 to 65, wherein the concentration of eosin Y is 0.05 to 0.08 mM, and / or the concentration of triethanolamine is 0.4 to 0.8% w / v, and / or the concentration of vinylcaprolactam is 0.18 to 0.4% w / v. 67. Any of the compositions of paragraphs 62-66, wherein the concentration of eosin Y is about 0.05 mM, and / or the concentration of triethanolamine is about 0.4% w / v, and / or the concentration of vinylcaprolactam is about 0.4% w / v. 68. The method of paragraph 62 or 63, wherein the concentration of eosin Y is 0.5 to 0.5 mM, and / or the concentration of triethanolamine is 0.5 to 2% w / v, and / or the concentration of vinylcaprolactam is 0.5 to 1.5% w / v. 69. The method of any of paragraphs 62, 63 or 68, wherein the concentration of eosin Y is about 0.1 mM, and / or the concentration of triethanolamine is about 0.5% w / v, and / or the concentration of vinylcaprolactam is about 0.5% w / v. 70. The method of any one of paragraphs 48 to 69, wherein the composition is exposed to visible light having a wavelength in the range of 450 to 550 nm. 71. The method of any one of paragraphs 48 to 70, wherein the composition is exposed to visible light for a period of 20 to 120 seconds. 72. The method of any one of paragraphs 48 to 71, wherein the composition is exposed to visible light for a period of 30 to 60 seconds. 73. The method of any one of paragraphs 48-72, wherein the composition further comprises a therapeutic agent. 74. The method of paragraph 73, wherein the therapeutic agent is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, anti-acanthamoebic agents, anti-inflammatory agents, immunosuppressants, anti-glaucoma agents, anti-VEGF, growth factors, or any combination thereof. 75. The method of any one of paragraphs 48-74, wherein the composition further comprises corneal cells. 76. The method of paragraph 75, wherein the corneal cells comprise epithelial cells, endothelial cells, keratocytes, or a combination thereof. 77. The method of any one of paragraphs 48-76, wherein the composition is substantially permeable. 78. The method of any one of paragraphs 48 to 77, which does not include a step of suturing the cornea.

[0063] It is noted that the present invention provides an improved bioadhesive for the repair and reconstruction of corneal defects and injuries. According to ASTM standard tests, the crosslinked methacryloyl-substituted gelatin hydrogel (Gel-CORE) of the present invention was shown to exhibit adhesive properties suitable for application to the cornea, i.e., wound closure strength, shear resistance and burst pressure. In vitro experiments showed that Gel-CORE is cytocompatible with corneal cells and promotes fusion with the cells after application. In vivo experiments in rabbits showed that Gel-CORE can effectively seal corneal defects. The bioadhesive of the present invention is advantageous in that it is low cost, easy to manufacture, and easy to use, making it a promising material for corneal repair, and an easily tunable platform for further optimizing adhesive properties.

[0064] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and are therefore deemed to be within the scope of the invention as defined in the appended claims. Moreover, to the extent not already indicated, it will be understood by those skilled in the art that any one of the various embodiments described and illustrated herein may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[0065] It is to be understood that the invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. EXAMPLES

[0066] The present disclosure is further illustrated by the following examples, which should not be considered limiting. The examples are for illustrative purposes only and are not intended to limit any aspect described herein in any manner. The following examples are not intended to limit the present invention in any manner.

[0067] Example 1: Benefits and Uses of Gel-CORE Advantageously, Gel-CORE bioadhesive can be used in the following applications:

[0068] Filling corneal defects: Gel-CORE can fill corneal defects and corneal thinning defects that occur not only due to physical damage, but also after various corneal inflammatory disorders, such as bacterial keratitis and immune-mediated corneal melting. In such cases, the bioadhesive is used to provide structural support in critical situations. In addition, in the long term, it allows for the regeneration of corneal cells within the bioadhesive, and serves as a stromal replacement technique that does not require the use of traditional treatment modalities, such as corneal transplants and tissue patch transplants.

[0069] The current standard of care for repair of corneal stromal defects and thinning involves tissue / patch grafting or glue application. Corneal transplants and patch grafts require donor tissue, which may not be available. In addition, the use of allogeneic tissue for transplantation carries the risk of immune reactions.

[0070] Currently, there are no adhesives approved for filling corneal defects. Although cyanoacrylate glues are currently used "off-label" to treat many impending ophthalmic situations, such as corneal perforation, impending perforation, and progressive corneal thinning disorders, they suffer from a variety of significant drawbacks, including:

[0071] i. Poor biocompatibility with cytotoxicity towards the cornea and other ocular tissues (risk of cataract formation and retinopathy if it enters the eye); ii. Lack of transparency, obstruction of good vision and impaired visibility of posterior corneal structures; iii. Risk of secondary infection due to high porosity; iv. Difficulty in controlling its application, possibility of unexpected peeling of the glue; v. Rough surfaces that require contact lens wear, which adds an additional risk of infection, and vi. Does not fuse with the corneal tissue.

[0072] In contrast, Gel-CORE has several advantages over currently available adhesives for corneal repair and sealing.

[0073] 1. Biosafety :Gel-CORE has excellent biocompatibility and poses few safety concerns compared to other materials (e.g., cyanoacrylate products) since its raw material is gelatin, a naturally occurring biopolymer derived from collagen that is used in a variety of medical applications. In addition, the selection of a visible light-activated photoinitiator will avoid potential damage associated with UV exposure in previous formulations.

[0074] 2. Adjustable characteristics : The physical properties of Gel-CORE (such as degradation and mechanical properties) can be tailored so that different bioadhesive compositions for different purposes - bioadhesives with short or long retention times suitable for different clinical situations can be produced. For example, in the case of corneal trauma where intraocular material, such as the iris, has been extruded, it may be desirable to apply Gel-CORE for temporary sealing of the damaged eye. In contrast, in the case of corneas that exhibit structural defects or severe thinning, Gel-CORE can be formulated to be retained for long periods of time. Currently available sealant technologies (e.g., cyanoacrylates) do not offer such control over the characteristics of the final product.

[0075] 3. Transparency In ex vivo experiments using rabbit corneas, Gel-CORE was shown to be permeable after administration and photopolymerization into a 50% depth corneal injury, and to have a smooth and convex surface capable of retaining the contours of a normal cornea (Figures 1A-1E).

[0076] 4. Reversibility Unlike cyanoacrylates which cure immediately upon application, Gel-CORE requires the application of light to cure, so incorrect application can be redone if necessary.

[0077] 5. High adhesion and retentionGel-CORE is highly adhesive to tissue based on wound closure, lap shear, burst pressure and ex vivo adhesion testing. In addition, ex vivo data consistently shows that Gel-CORE is retained for several days and remains native and fully attached to the cornea.

[0078] 6. Corneal tissue regeneration Unlike other adhesives used to seal the cornea (e.g. cyanoacrylates), Gel-CORE achieves both tissue sealing and regeneration. Human keratocytes can grow within Gel-CORE. Gel-CORE will strongly attach to the cornea and remain there while the tissue undergoes physiological repair / regeneration.

[0079] Corneal, limbal or scleral wound closure: The traditional treatment for such wounds involves suturing. However, suturing is associated with major drawbacks, including: 1. sutures are foreign materials that can be a risk factor for microbial invasion and infection, inflammation and neovascularization; 2. corneal sutures often induce regular or irregular astigmatism, which impairs vision. To avoid these drawbacks, sealants have been used to close wounds. The only approved sealants, ReSure® (USA) and OcuSeal® (Europe), are for sealing corneal incisions in cataract surgery. However, they fall off quickly. In contrast, Gel-CORE, which provides a firm seal to corneal, limbal or scleral wounds, can be tailored to provide a longer period of closure as desired. Gel-CORE can provide adhesion for corneal wound closure. Under such conditions, it can seal the wound without the need for sutures. Gel-CORE can provide adhesion for closure of limbal or scleral wounds. Under such conditions, it can seal the wound without the need for sutures.

[0080] Covering corneal epithelial defects: Traditional treatments for patients with corneal epithelial defects include eye patches, bandage contact lenses, and sometimes invasive procedures. However, these options are limited by the fact that they can be inconvenient for the patient and can increase the risk of corneal infection. In contrast, for these instances, we can use the fast-degrading Gel-CORE formulation, which protects the cornea while regenerating the corneal epithelium itself.

[0081] Temporary protection of intraocular structures in cases of corneal or scleral lacerations and intraocular structure deviations: The only available treatment option for such cases is to surgically reposition the intraocular structures while suturing the wound, which should be done by a skilled surgeon in an equipped facility. However, this would delay the surgical procedure, which would predispose the patient to intraocular infection. In contrast, the use of Gel-CORE provides temporary support for the cornea / sclera and intraocular structures while preventing infection. Permanent repair can then be performed without exposing the patient to a high risk of intraocular infection. Gel-CORE can be used in cases of large corneal / scleral injuries with iris / choroid-retinal deviations, as the biologic patch containing the therapeutic agent (e.g., antibiotics, etc.) covers the intraocular structures. In such cases, it protects the intraocular structures and prevents infection before the permanent surgical procedure is performed.

[0082] Corneal infection with or without severe thinning: The current standard treatment for corneal infection involves frequent instillation of eye drops, which is troublesome for patients. To avoid this drawback, some research is currently being conducted with contact lenses containing sustained release antibiotics. However, such technology does not provide structural support to the cornea. In contrast, Gel-CORE can provide not only long-term release of antibiotics but also structural support to the cornea with infectious keratitis. Gel-CORE can be used in cases of corneal infection with or without severe thinning as an antibiotic-containing patch that provides long-term release of antibiotics in addition to structural support to the cornea.

[0083] Inflammatory corneal thinning: The current standard of care for inflammatory corneal thinning involves the use of topical or systemic anti-inflammatory drugs. In cases of severe thinning, surgical procedures are performed as described above for corneal stromal defects. In contrast, Gel-CORE can function as a drug reservoir that slowly releases anti-inflammatory drugs, as well as providing structural support as described above for corneal defects, thereby eliminating or reducing the need for additional topical or systemic therapeutic agents. Thus, Gel-CORE can be used as a platform for drug delivery, as this bioadhesive is transparent and can be retained for several weeks with therapeutic use. Gel-CORE can be used in the case of inflammatory corneal thinning as an anti-inflammatory drug-containing patch that provides long-term release of anti-inflammatory drugs in addition to providing structural support to the cornea.

[0084] Refractive Corneal Modeling: For refractive modeling of the cornea, various intracorneal implants have been used in the past, including PermaVision (ReVision Optics), Kamra (AcoFocus), Flexivue Microlens (Presbia) and Raindrop inlay (Revision Optics), but all of them are accompanied by deposit formation or haze generation due to lack of complete biocompatibility with corneal tissue. In contrast, Gel-CORE has a high degree of biocompatibility that prevents these complications. In addition, with Gel-CORE, fusion of the corneal cells with this biomaterial occurs, which is not seen with other inlays. Gel-CORE can be used as an intracorneal implant for corneal modeling to change the refractive power of the cornea in patients with refractive errors (myopia, hyperopia, astigmatism and presbyopia).

[0085] Substitute for corneal tissue in transplantation: Various artificial corneas, including Boston Keratoprosthesis, osteodentokeratoprosthesis, and AlphaCor, have been used previously to replace corneal tissue by transplantation, but they suffer from the fact that corneal cells do not fuse to these artificial corneas. In contrast, Gel-CORE shows a high degree of migration and fusion of native corneal cells to this biomaterial. Gel-CORE can be used as a substitute for corneal grafts in superficial corneal transplantation, replacing the use of donor corneal tissue. Gel-CORE can also be used (similar to artificial corneas) as a substitute for corneal tissue in full-thickness corneal transplantation.

[0086] Example 2: Synthesis of gelatin methacryloyl (GelMA) prepolymer GelMA was synthesized as previously described. 14The concentrations of gelatin and methacrylic anhydride can be varied to produce GelMA with methacryloyl substitution in the ranges disclosed herein. For example, 10% (w / v) porcine gelatin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) and heated at 60°C for 20 min. 8% (v / v) methacrylic anhydride (Sigma-Aldrich, St. Louis, MO, USA) was added dropwise under continuous stirring at 50°C for 3 h, followed by dilution with PBS and dialysis against deionized water at 40-50°C for 7 days. After sterile filtration and lyophilization for 4 days, GelMA was stored at -80°C until use in experiments.

[0087] Quantification of methacrylamide groups. The conversion of amine groups in biomaterials, e.g., GelMA, is conventionally measured by proton nuclear magnetic resonance ( 1 H NMR) spectrum was used to determine 13~14 However, gelatin is a mixture of polypeptides with a complex composition. 1 It may not be feasible to detect and distinguish the resonance peaks of methacrylamide and methacrylate groups from the H NMR spectrum. As an alternative, the fluoroaldehyde assay 43 allows for easier and more accurate determination of the conversion of amine groups. When a modified protein / peptide sample is mixed with an assay solution containing o-phthalaldehyde and 2-mercaptoethanol, all remaining primary amine groups in the material are converted to blue-emitting fluorescent species (Figure 2A). Four different GelMA formulations were prepared that varied in their degree of methacryloyl substitution by adding different amounts of MA to the reaction (Table 1). 14Depending on the degree of modification, the obtained GelMA formulations are referred to as SuperGelMA, HighGelMA, MidGelMA and LowGelMA, respectively. Using gelatin as a standard, the amount of remaining primary amine groups can be easily obtained. The conversion of amine groups of the obtained GelMA samples was determined to be about 93% for SuperGelMA, about 84% for HighGelMA, about 65% for MidGelMA and about 24% for LowGelMA, respectively, using fluoroaldehyde assay (Figure 2B). It is clear that the conversion of amine groups is positively correlated with the amount of MA added under constant reaction temperature and reaction time.

[0088] Table 1. Overview of molecular parameters of different GelMA samples. TIFF0007626793000001.tif52162

[0089] Quantification of methacrylate groups. Our previous publication on the preparation of GelMA 14 Based on the integral area of ​​the resonance peak derived from the amine group, 1 H NMR spectroscopy was used to determine the conversion of amine groups. Quantification of methacrylate groups was performed using the 1H NMR spectroscopy of modified peptide or protein samples. 1 This could not be done due to the absence of discernible hydroxyl resonance peaks in the 1 H NMR spectrum.

[0090] Here, an Fe(III)-hydroxamic acid based assay was used to determine the amount of methacrylate groups in different GelMA samples (Figures 3A-3E). Hydroxamic acids form red-brown complexes with Fe(III) ions, which can serve as a quantitative test for hydroxamic acid species (Figure 3A). This class of complexes has an absorption peak centered around 500 nm (Figures 3B and 3C). The formation of Fe(III)-hydroxamic acid complexes may be useful for other analytical applications, such as the quantification of ester group residues in poly(vinyl alcohol). 45 has been used to quantitate the reactions of lysozyme's amine and hydroxyl groups with a variety of different carboxylic acid anhydrides. 44Acetohydroxamic acid (AHA) was used as a standard to construct a standard curve, and it was hypothesized that the complex of acetohydroxamic acid with Fe(III) ion (FeAHA) has similar spectroscopic properties to that of N-hydroxymethacrylamide with Fe(III) ion (FeHMA). 44 Iron(III) perchlorate was dissolved in dilute hydrochloric acid to prepare an Fe(III) ion solution, which was added in excess to the acetohydroxamic acid solution to form a 1:1 complex. It has been reported that the apparent extinction coefficient reaches its maximum when the molar ratio of Fe(III) and hydroxamic acid is greater than 20, and this will remain independent of the ratio. 46 . 1.3 x 10 -4 ~2.5 x 10 -3 The UV-Vis absorption spectra of a series of standard FeAHA solutions covering mol / L concentrations were recorded on a UV-transparent microplate, and indeed, when the absorbance as a function of AHA concentration was plotted and analyzed using a linear least-squares fit, excellent linearity was achieved (Figure 3D).

[0091] To determine the amount of methacrylate groups in the GelMA samples, an aminolysis reaction was used to convert the methacrylate groups to detectable N-hydroxymethacrylamide compounds. In particular, 50 mg / mL GelMA samples were treated with hydroxylamine solution at room temperature for 10 min to generate N-hydroxymethacrylamide. The resulting solution was acidified with hydrochloric acid followed by the addition of excess Fe(III) ions. A color change upon the addition of Fe(III) ions indicated the formation of FeHMA complexes, thereby confirming the presence of methacrylate groups. The concentration of the FeHMA complexes formed in situ was determined from UV-Vis absorption spectra and could be used to calculate the amount of methacrylate groups in the GelMA samples (Figure 3E). In all tested GelMA samples, methacrylate groups were found to be less than 10% of the total methacryloyl substitutions. These results suggested that amine groups are much more reactive than hydroxyl groups and that methacrylamide groups are the predominant form in GelMA (Figure 3E).

[0092] Example 3: Preparation and material properties of Gel-CORE hydrogel Visible light crosslinkable Gel-CORE was prepared by using Eosin Y (Sigma-Aldrich) as a visible light activated initiator, triethanolamine (TEOA) (Sigma-Aldrich) as a coinitiator, and vinylcaprolactam (VC) (Sigma-Aldrich) as a catalyst. 15、16 Using this crosslinking system, the polymerization of methacryloyl groups in GelMA was performed at 100 mW / cm 2 The imaging was initiated through exposure to blue light (450-550 nm, xenon source) (Figure 4A-4C). Eosin Y is a water-soluble xanthene dye and is a common stain for collagen, the main component of the cornea and sclera. This visible light system has a well-established track record of biocompatibility in a wide range of applications. 15~19 A polyethylene glycol (PEG)-based sealant, FocalSeal® (Genzyme Biosurgical, Cambridge, MA), has been FDA approved for use in the human body. Visible light crosslinkable Gel-CORE hydrogels exhibited tunable physical properties (Figures 5A-5B), and the elastic modulus of Gel-CORE hydrogels could be tuned from 5 to 28 kPa using visible light crosslinking (Figures 5A-5B). In addition, their swelling ratios could be altered from 7% to 13% (w / w) (Figure 6B).

[0093] GelMA with different degrees of methacryloyl modification (30%-85%) can be used to form hydrogels. 14Next, different concentrations of GelMA prepolymer solutions (5-15% w / v) can be prepared in phosphate-buffered saline (PBS) containing Eosin Y (0.1-0.5 mM), TEOA (0.5-2% w / v) and VC (0.5-1.5% w / v). The formulated GelMA prepolymer solutions can be photocrosslinked by exposure for 20-120 s to blue light, which matches the absorption spectrum of Eosin Y (Figure 4B). By altering the GelMA concentration, Eosin Y / TEOA / VC concentration and light exposure time, the physical properties of the resulting hydrogels are altered.

[0094] Since this formulation is to be used for corneal repair, the Gel-CORE hydrogel should have similar elasticity and stiffness (Young's modulus: 250-350 kPa) as the native cornea. The swelling ratio of the hydrogel should be optimized to have a swelling ratio of <20% to ensure that the adhesive maintains its shape after application to the corneal defect. In particular, the swelling ratio can affect the shape, curvature, and smoothness of the defect to be sealed.

[0095] For example, lyophilized GelMA prepared according to Example 2 was dissolved in PBS at a concentration of 10% (w / v). After addition of a photoinitiator mixture of 0.1% (w / v) eosin Y, 0.5% (w / v) triethanolamine and 0.5% (w / v) vinylcaprolactam and dissolution at 80 °C, the prepolymer solution was subjected to visible light irradiation (450-550 nm, xenon source, 100 mW / cm 2 ) to form a hydrogel (Gel-CORE).

[0096] In another example, different concentrations of GelMA (5, 10, 15, 20% (w / v)) were tested for material properties. Lyophilized GelMA (prepared in Example 2) was dissolved in PBS containing 1.875% (w / v) triethanolamine (TEA) and 1.25% (w / v) N-vinylcaprolactam (VC) to concentrations of 5, 10, 15, and 20% (w / v). Eosin Y was separately dissolved in fresh DPBS to a concentration of 0.5 mM. To prepare the hydrogel, 8 μL of GelMA solution was mixed with 2 μL of Eosin Y solution, and the mixture was then placed between two glass cover slips separated by a 150 μm spacer, followed by exposure to blue light (100 mW / cm) in the range of 450–550 nm. 2 The sample was exposed to a xenon source (Genzyme Biosurgery) for 20 seconds.

[0097] Mechanical testing of Gel-CORE samples was performed as previously published. 14 Briefly, prepolymer solutions were photocrosslinked to produce the following shapes: disks for compression tests (n=3–5; diameter 6 mm and height 1.5 mm) and cubes for tensile tests (n=7–10; width 3 mm, length 14 mm and thickness 1.5 mm). The hydrogels were either analyzed directly or stored in PBS for 24 h at 4 °C before testing in a mechanical testing system 5542 (Instron, Norwood, MA, USA). The strain rate in compression and tensile tests was set at 1 mm / min. The compressive and ultimate tensile strengths of the samples were determined at the time of rupture or tearing of the hydrogels. The compressive and elastic moduli were obtained by measuring the slope of the stress-strain curves at strain rates of 0–0.5%.

[0098] To analyze the swelling properties, Gel-CORE hydrogel samples (n=5) were allowed to swell in PBS for 1, 2 or 3 days. At the end of the experiment, excess liquid was carefully removed with tissue paper and the wet weight was measured. After lyophilization, the dry weight of the samples was measured and the swelling ratio was calculated as (wet weight - dry weight) / dry weight (Figures 6A and 6B).

[0099] Ex-vivo testing of Gel-CORE hydrogels used explanted rabbit corneal tissue. First, GelMA prepolymer was applied to an incision made on the explanted cornea, followed by photocrosslinking by exposure to visible light with an optimized light exposure time. The burst pressure was then measured using a pressure sensor after air inflation into the cornea. For example, a rabbit cornea was sealed with Gel-CORE (10% (w / v), prepolymer concentration, 5 mM Eosin Y, and an exposure time of 120 seconds. The incision made on the rabbit cornea was completely sealed with Gel-CORE without any gaps, and the tissue could be pressurized to approximately 3.5 kPa (26 mmHg), which is twice the pressure of a healthy eye. Preferably, the Gel-CORE sample should have a burst pressure higher than 15 kPa (>110 mmHg). 20~21 , lap shear strength of >100 kPa, adhesive strength of >40 kPa and photopolymerization within a light exposure time of <60 seconds.

[0100] Example 4: ASTM Standard In Vitro Testing of Mechanical Properties of Gel-CORE Burst Pressure The burst pressure test of the sealants was adapted from ASTM standard F2392-04 (Standard Test Method for Burst Strength of Surgical Sealants). Prior to sample preparation, porcine skin sheets (40 mm * Two Teflon sheets (35 mm) were soaked in PBS. * A circular defect (3 mm in diameter) was created in the center of a pig skin sheet placed between two Teflon sheets (35 mm in diameter). The top Teflon sheet had a hole (10 mm in diameter) to allow the application of the desired adhesive over the circular defect in the pig skin sheet (Figure 7A). For GelMA, the prepolymer was exposed to visible light. The collagen sheet was then removed and placed in a burst pressure testing system consisting of a pressure detection and recording unit and a syringe pump that applied air to gradually increase the pressure on the sample (Figure 7B). Each tested adhesive group contained five samples.

[0101] The sealants covering standardized defects on porcine skin were tested for their burst pressure resistance by applying increasing air pressure, and each GelMA concentration exhibited higher burst pressure values ​​than Coseal™ (Figure 7C).

[0102] wound closure The wound closure strength of GelMA and the clinically established surgical sealants Evicel® (Ethicon, Somerville, NJ, USA), Coseal™ (Baxter, Deerfield, IL, USA) and Progel™ were tested according to ASTM standard test F2458-05 (Standard Test Method for Wound Closure Strength of Tissue Adhesives and Sealants) with some modifications to accommodate smaller sample sizes. Briefly, fresh porcine skin obtained from a local slaughterhouse was removed from the fatty tissue layer and samples were cut into 5 mm * The skin was prepared by cutting into 15 mm rectangular pieces. During intermittent use, the skin was kept moist with gauze soaked in PBS. Prior to use, the skin was blotted dry to remove excess liquid, and each edge of the skin piece was glued to two poly(methyl methacrylate) slides (30 mm) using Krazy glue (Westerville, OH, USA). * The skin was then cut using a razor blade (Figure 8A) and petrolatum was applied using a syringe to the edges of the desired adhesive application area. * 40 μl of adhesive was applied across a 5 mm skin section and, in the case of GelMA, illuminated with visible light (Figure 8A). After 1 h of incubation in PBS, two plastic slides were clamped in the grips of an Instron system for tensile testing (Figure 8A). The adhesive strength of the sealant samples was determined at the time of tear. Each tested adhesive group contained 4 to 7 samples, and the results are summarized in Figure 8B. The tensile tests measured the elastic modulus (in the range of 5 to 50 kPa) and ultimate tensile strength (stress at the time of breakage after stretching the sample).

[0103] Example 5: Degradability and Retention of Gel-CORE Gel-CORE was applied to a corneal defect of 3 mm >50% depth (using a 10% (w / v) Gel-CORE solution containing 0.01% (w / v) Eosin Y, 0.5% (w / v) TEA and 0.5% (w / v) VC). The solution was exposed to blue light for 120 seconds to form a hydrogel layer on the injured cornea. After this procedure, the eyes were kept in PBS at 4°C. Changes in Gel-CORE over time were evaluated using slit lamp biomicroscopy and serial evaluations by OCT. For at least 11 days, the bioadhesive was found to remain intact (full thickness and spreading was retained) and fully attached to the cornea in all tested eyes. Slit lamp biomicroscopy showed that the bioadhesive remained clear with a smooth surface over this period and did not show any biomicroscopic changes in shape or contour (Figures 9A and 9C). In addition, OCT confirmed that there was no change in the thickness or shape of the Gel-CORE (Figures 9B and 9D). After 11 days, the corneal tissue in PBS began to degrade (as expected from necrosis due to long-term storage under PBS), and at this point, the attachment of the Gel-CORE to the cornea began to be lost.

[0104] A corneal injury model in New Zealand white rabbits was used by creating a 50% depth corneal defect. After general anesthesia of the rabbits using intramuscular injection of ketamine and xylazine, a circular 50% depth corneal defect was created in the right eye with a 3 mm biopsy punch. A superficial keratectomy was then performed using a surgical crescent knife. After removing the superficial layer, the defect surface was dried using a surgical microsponge. The corneal defect was then filled with 10 μl of bioadhesive solution. Excess solution was then removed using a microsponge. This was immediately followed by a corneal biopsy (FocalSeal Xenon Light Source, Genzyme, 100 w / cm 2A 120 second application of blue light (using a 120 second light source) was performed to crosslink the bioadhesive. The degradation and retention of Gel-CORE was evaluated using slit lamp biomicroscopy and OCT at 1, 2 and 4 weeks as described below.

[0105] Two outcome measures were assessed during the 4-week follow-up period: (i) (Fantes grading scale, based on visibility of iris details) 22 (ii) the transparency of the bioadhesive, which is a measure of optical degradation (assessed by slit-lamp biomicroscopy using a CT scanner); and (ii) the thickness of the bioadhesive (measured by OCT, as described below).

[0106] Retention is a function of two parameters: degradability and adhesion. Degradation and / or suboptimal adhesion can both cause the gel to lose its retention. To measure retention, OCT techniques were used to assess (i) the presence of bioadhesive covering the corneal defect, and (ii) any gap spacing between the bioadhesive and the corneal epithelium or stroma during a 4-week follow-up period, as described below.

[0107] Slit-lamp biomicroscopy and OCT imaging were performed under general anesthesia on both rabbit eyes during the 1-week follow-up period and then on the operated eye only. For slit-lamp examination, a Topcon Slit Lamp system was used. The permeability of the bioadhesive was assessed (using the Fantes grading scale) at 25x magnification and with a slit and broad beam. Slit-lamp photographs were also taken during the examination. Optical coherence tomography (OCT) was also used: it is a non-contact imaging method that provides high-resolution cross-sectional images of the cornea in vivo. In this experiment, a spectral domain OCT (Spectralis, Heidelberg Engineering, GmbH, Germany) with an axial resolution of 3.9-7 μm was used. Line scans (8 mm long) were performed on the central cornea at 0, 45, 90 and 135 degrees (Figure 10A). Using proprietary OCT software, the thickness (in microns) of the bioadhesive (in the operated eye) and the cornea (in the non-operated fellow eye) were measured at the center of the cornea and 1 mm away from the center in both directions (Figure 10B). In addition, any gap spacing between the bioadhesive and the corneal tissue was measured in microns. Slit lamp and OCT results were compared between the two eyes and at different time points to determine the degradation of Gel-CORE over time and its retention in the corneal defect. Based on preliminary data, Gel-CORE can remain intact for at least 30 days in vitro.

[0108] Example 6: Biocompatibility and fusion of Gel-CORE The optimal bioadhesive for corneal repair should not only be non-toxic to corneal cells, but also allow cells to integrate with the biomaterial in the long term and prevent extrusion. The in vitro cytocompatibility and fusion ability of Gel-CORE was determined by using the two most abundant cell types in the cornea, including keratocytes and corneal epithelial cells. Keratocytes and corneal epithelial cells were cultured using 2D and 3D culture systems. The biocompatibility and fusion ability of Gel-CORE was evaluated in vivo by investigating the effect of the bioadhesive on corneal cells and the migration of corneal cells into the bioadhesive over time.

[0109] In preliminary experiments, the compatibility of Gel-CORE with keratocytes was demonstrated (FIGS. 11A-11G). Keratocytes were incorporated into a representative Gel-CORE composition and showed ≧95% cell viability when using a 2D culture system, as well as proliferation and migration of keratocytes when grown either on or within the Gel-CORE construct (FIG. 11G).

[0110] In vitro evaluation of Gel-CORE cytocompatibility and cell fusion. To evaluate the in vitro cytocompatibility of Gel-CORE to the cornea, the following experiment was performed. Corneal cells were cultured in culture medium (Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamic acid) in a humidified incubator at 37°C with 5% CO2. A 2D culture system was used to seed epithelial cells onto Gel-CORE to form an epithelial monolayer. In addition, a 3D culture system was used to encapsulate keratocytes within Gel-CORE to form corneal tissue.

[0111] For 2D culture, the Gel-CORE was constructed by exposure to visible light as detailed herein. The gel was then inoculated with 1 x 10 6 ~1 x 10 8 Epithelial cells were seeded at cell densities ranging from 1000 to 10000 cells / mL and incubated for 14 days. Medium was changed every other day. Cell viability was assessed at days 1, 4, 7 and 14 by using a calcein-AM / ethidium homodimer live / dead assay. 23 Actin / DAPI staining was used to assess cell adhesion and spreading as previously described. 13,24-26In addition, the metabolic activity of the cells was assessed on days 1, 4, 7, and 14 using the PrestoBlue assay followed by absorbance readings on a microplate spectrophotometer. In addition, on day 14, cell invasion into and growth within the hydrogels was investigated by histological analysis. 27~29 Furthermore, K12 expression in corneal epithelial cells was analyzed due to its confirmed role in maintaining corneal epithelial function. It is important that epithelial cells grow on the surface of the hydrogel (without penetrating into the gel) to form a dense cell layer required for ocular protection. Based on these in vitro experiments, Gel-CORE was shown to be non-cytotoxic (cell viability >90%), promotes cell metabolic activity and adhesion, and has limited penetration into the gel.

[0112] For 3D culture, keratocytes were cultured at 1 x 10 6 ~1 x 10 8 The gels were mixed with GelMA prepolymer solution at concentrations ranging from 1000 to 15000 cells / mL. The mixture was then exposed to light to form cell-containing Gel-CORE adhesive. The gels were then washed 3 times with PBS and incubated in culture medium for 14 days in a 37°C culture incubator. Cell viability (live / dead assay), cell adhesion and spreading (actin / DAPI), proliferation (Picogreen assay), collagen deposition (Picrosirius Red), and corneal tissue formation (hematoxylin and eosin staining) were assessed at days 1, 4, 7, and 14. Based on these 3D studies, Gel-CORE was shown to be cytocompatible and would promote corneal tissue formation.

[0113] In vivo biocompatibility and fusion ability of Gel-CORE in the cornea. A corneal injury model in New Zealand white rabbits was used by creating a 50% depth corneal defect as described herein. The rabbits were divided into three groups: (i) Gel-CORE group, in which the corneal defect was filled using a bioadhesive as described herein; (ii) Cyanoacrylate group, in which cyanoacrylate glue was used, which is the standard treatment (albeit unapproved) in filling corneal defects to prevent perforation. For this, 10 μl of cyanoacrylate glue (MSI-EpiDermGlu + Flex, Medisav Services, Canada) was applied to fill the corneal defect, after which a soft bandage contact lens was immediately placed on the cornea. (iii) Control group, in which the corneal defect was not filled with adhesive and prophylactic antibiotic (erythromycin) ointment was given for 1 week. The rabbits were then followed for 12 weeks. At weeks 1, 2, 4, and 12, the biocompatibility and fusion potential of Gel-CORE, as well as the extent of corneal inflammation and neovascularization, were evaluated using slit-lamp biomicroscopy and IVCM (as described below) and compared with other groups. In addition, at each time point, six rabbits per group were sacrificed for collection of corneas for histological (n=3) and immunohistochemical evaluation (n=3).

[0114] For biocompatibility evaluation, the following were considered: (i) adhesive / defect periphery (assessed by slit-lamp biomicroscopy using the Fantes grading scale); Corneal permeability , and (ii) the density of epithelial cells, stromal keratocytes, inflammatory cells and blood vessels in the cornea surrounding the bioadhesive (measured by IVCM, histological staining and / or immunohistochemical staining, as described in more detail below).

[0115] For the evaluation of fusion potential, the following were considered as outcome measures: (i) (assessed by slit-lamp biomicroscopy using the Fantes grading scale); Bioadhesive permeability(ii) the extent of corneal epithelial cell migration on the bioadhesive (as measured by slit lamp biomicroscopy, IVCM and histological staining, as described in more detail below), and (iii) the density of stromal keratocytes, corneal nerves, inflammatory cells and blood vessels within the bioadhesive / corneal defect area (as measured by IVCM, histological staining and / or immunohistochemical staining, as described in more detail below).

[0116] Slit lamp biomicroscopy and IVCM were performed under general anesthesia. Slit lamp examination and photography were used to evaluate the permeability of the cornea and bioadhesive as described herein. In addition, to evaluate epithelial migration on the bioadhesive, slit lamp photography was performed with fluorescein staining, and the corneal epithelial defect area on the bioadhesive was evaluated at each time point using Image J's Measure Area tool. In vivo confocal microscopy (IVCM) was used to evaluate cellular changes and migration over time in the same rabbits without sacrificing the animals. This is a non-invasive imaging method that provides high-resolution images at the cellular level from the cornea in a living animal (Figures 12A-12F). In this experiment, a laser scanning IVCM (Heidelberg Retina Tomograph 3 with Rostock Cornea Module, Heidelberg, Germany) was used, using a 670 nm diode laser and with a resolution of 1 μm. It provides images of a corneal area of ​​400 x 400 μm. For IVCM-based readings, the following were scanned and examined: (i) 1.5 mm central cornea (over the bioadhesive, cyanoacrylate glue or original defect in the control group) and (ii) corneal tissue surrounding the bioadhesive in quadrants (superior, nasal, inferior, lateral) 1 mm around the bioadhesive. For scanning, Sequence Mode was used, which automatically acquires 100 consecutive images per sequence. All corneal layers (epithelium, stroma and endothelium) were imaged in each scan by manual navigation. Two Sequence Mode scans were performed in each of the five locations (central, superior, nasal, inferior and lateral). For image analysis in the central cornea, five images were randomly selected from different depths in each scan (total of 10 images). For image analysis of the cornea around the adhesive, one randomly selected image from each corneal layer (epithelium, subbasal layer, and stroma) was selected from each individual sequence scan (a total of eight images per layer). For analysis, the density of epithelial cells, subbasal inflammatory cells, and stromal keratocytes were measured by a blinded observer using Image J software as previously described. 30~35In addition, corneal nerve density was assessed by a blinded observer using NeuronJ software as previously reported. 30,36,37 .

[0117] Histological evaluation using hematoxylin and eosin (H&E) staining was performed on frozen sections of collected corneas. For each cornea, five sections were obtained from the central cornea, including both the defect / bioadhesive location and the surrounding corneal tissue. For image analysis, all sections were evaluated by a blinded observer. Corneal epithelial cell migration on the adhesive was determined. In addition, stromal keratocyte and inflammatory cell density was determined in 10 randomly selected areas of the peripheral cornea 200 μm from the adhesive edge, as well as in 10 randomly selected areas within the adhesive (both in the center and at the edge of the adhesive).

[0118] Immunohistochemical staining was also performed on collected corneal frozen sections with antibodies against: β-tubulin III (2G10 Ab; Abcam), beta2 (CD18) integrin (L13 / 64 for inflammatory cells; GeneTex), and CD31 (polyclonal anti-CD31 for blood vessels; Abcam). The density of these cells was determined by a blinded observer in both the adhesive application and the surrounding corneal matrix as described for H&E staining. For this, serial sections from 10 randomly selected areas in the adhesive and 10 randomly selected areas of the surrounding cornea within 200 μm of the adhesive were used for analysis. For each measurement, the mean and standard deviation (SD) were determined.

[0119] Slit lamp, IVCM, histology and immunohistochemistry results were compared between the three groups at each time point to determine the biocompatibility and fusion ability of Gel-CORE in filling the corneal defect. These comparisons between the Gel-CORE and control groups helped determine whether Gel-CORE caused more or less inflammation and tissue damage than expected from secondary healing (also included as a control). In addition, comparisons between the Gel-CORE and cyanoacrylate groups showed whether potential tissue damage was less in the Gel-CORE group compared to the current standard of care adhesive. Comparisons between the different time points within each group showed whether corneal cell fusion to Gel-CORE occurred over time and whether potential tissue damage caused by the adhesive weakened or worsened over time.

[0120] Example 7: A new bioadhesive for rapid and long-term repair of corneal stromal defects Use of the tunable properties of the bioadhesive to optimize its physical and adhesive properties. The data show that GelMA prepolymers with a methacryloyl functionalization degree of 80-90% can be effectively crosslinked to form stable hydrogels with tunable physical properties by using Eosin Y as the photosensitizer, triethanolamine (TEOA) as the initiator, and vinylcaprolactam (VC) as the catalyst. As the crosslinking efficiency depends on the concentrations of the photosensitizer, initiator, and catalyst, systematic optimization of these conditions is mandatory. By adjusting the concentrations of Eosin Y, TEOA, and VC, the key mechanical properties of the hydrogel can be precisely controlled such that formulations with tensile and compressive moduli comparable to those of the native cornea are produced (Figures 14A-14C, 15A, and 15B). Based on the data, the optimal concentrations of Eosin Y (0.05 mM), TEOA (0.4% w / v), and VC (0.4% w / v) are used in the following experiments.

[0121] Adhesive properties of the engineered bioadhesives. A standard burst pressure test was used to comprehensively evaluate the sealing ability of 20% w / v visible light crosslinked GelMA hydrogels formed at various visible light exposure times. This ex vivo test was performed to measure the burst pressure of rabbit corneas with 2 mm full thickness incisions (FIGS. 16A and 16B). The burst pressure of the engineered GelMA was greater than 200 mmHg, approximately 10 times the pressure of a healthy eye, and significantly greater than the burst pressure of the commercial control, ReSure® (FIG. 16B).

[0122] Ex vivo evaluation of smoothness, permeability and retention. Ex vivo studies were performed to evaluate the GelMA bioadhesive using explanted rabbit corneas. The bioadhesive was applied ex vivo to 3 mm >50% deep corneal defects in New Zealand rabbits. For this, a 20% w / v GelMA solution containing 0.05 mM eosin Y, 0.4% w / v TEOA and 0.4% w / v VC was used. The solution was exposed to visible light for 120 seconds, forming a hydrogel layer over the corneal defect, indicating the firm adhesion of the bioadhesive to the corneal stroma. In addition, the bioadhesive was permeable and had a smooth surface, as shown in FIG. 17.

[0123] After this procedure, the eyes were maintained in PBS at 4°C. Changes in the bioadhesive over time were evaluated using slit lamp biomicroscopy and serial evaluation by anterior segment optical coherence tomography (AS-OCT). During the 30-day evaluation period, the bioadhesive was found to remain intact (fully retained thickness and diffusivity) and fully attached to the cornea in all tested eyes. Slit lamp biomicroscopy showed that the bioadhesive remained clear with a smooth surface during this period, without any biomicroscopic changes in shape or contour (Figure 18). Additionally, AS-OCT confirmed no changes in the thickness or shape of the bioadhesive.

[0124] In vivo evaluation of biocompatibility and biointegrability. A corneal injury model in New Zealand white rabbits was used by creating a 50%-depth corneal defect. After general anesthesia with intramuscular injection of ketamine and xylazine, a central 50%-depth corneal incision was created in the right eye, followed by application of the bioadhesive. Immediately after photocrosslinking, firm adhesion of the bioadhesive to the corneal defect was observed. One day after surgery (Figures 19A-19C), the bioadhesive was transparent and had a smooth surface, and the surrounding cornea was clear and non-inflamed. AS-OCT also showed complete adhesion to the stromal bed. One week after surgery (Figures 19D-19F), the bioadhesive was still transparent, and constant migration of the corneal epithelium on the bioadhesive was observed.

[0125] Histological evaluation of rabbit corneas harvested 2 weeks after surgery demonstrated epithelial cell migration onto the bioadhesive and keratocyte migration into the bioadhesive (FIG. 20C). In addition, preliminary IHC studies demonstrated that corneas filled with GelMA bioadhesive showed higher expression of CD45 + It showed that cell invasion was reduced by 20.9%.

[0126] Example 8: Crosslinking of GelMA hydrogels 1 HNMR analysis. 1 HNMR analysis was performed to obtain the crosslinking degree of gelatin methacryloyl (GelMA) hydrogels fabricated by using various visible light exposure times including 1, 2 and 4 min (FIG. 21). 1 To perform HNMR studies, uncrosslinked GelMA prepolymers and GelMA hydrogels made with various visible light exposure times were dissolved in deuterated DMSO. To quantify the degree of crosslinking, all spectra were normalized to the phenylalanine signal (δ = 6.9-7.3 ppm). Signals related to the protons of the methacrylate groups in GelMA have been reported to appear as peaks located at δ = 5.30 and 5.64 ppm. 47,48 The degree of crosslinking was calculated by the following formula, which represents the ratio of C=C remaining in the methacrylated groups after the crosslinking process: TIFF0007626793000002.tif10128

[0127] 1 HNMR results. 1 Based on HNMR analysis, the degree of crosslinking was calculated from the disappearance of C=C bonds associated with methacrylated groups at δ=5.30 and 5.64 ppm. The degree of crosslinking of 20% (w / v) GelMA hydrogels increased from 63.4±2.7 at 1 min crosslinking time to 88.9±7.8 at 4 min, respectively (FIG. 22 and Table 2). Furthermore, at 10% (w / v) GelMA concentration, 86.8±1.3 of the original methacrylated groups were consumed after 1 min reaction time (FIG. 22). The degree of crosslinking was 90.7±0.2 and 92.9±2.2% after 2 and 4 min, respectively.

[0128] (Table 2) 1 Quantification of the cross-linking degree of GelMA hydrogels fabricated by using 10% and 20% (w / v) prepolymer concentrations at various visible light exposure times (1, 2, and 4 min) based on HNMR spectra TIFF0007626793000003.tif34162

[0129] References TIFF0007626793000004.tif67160TIFF0007626793000005.tif242160TIFF00076267930 00006.tif242160TIFF0007626793000007.tif242160TIFF0007626793000008.tif82160

[0130] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications cited throughout this application, are expressly incorporated herein by reference for purposes of explanation and disclosure, for example, to explain and disclose the methodologies described in such publications that may be used in combination with the technology described herein. These publications are provided solely because of their disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an admission that the inventors are not entitled to claim a date prior to such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute an admission as to the accuracy of the dates or contents of these documents.

Claims

1. A prepolymer solution for corneal reconstruction comprising methacryloyl-substituted gelatin, a visible light activated photoinitiator, and a pharma- ceutically acceptable carrier, the methacryloyl-substituted gelatin has a degree of methacryloyl substitution of 30% to 85%; the methacryloyl-substituted gelatin is present in the prepolymer solution at a concentration of 5% to 15% (w / v); The prepolymer solution further comprising at least one corneal cell.

2. 2. The prepolymer solution of claim 1, wherein the methacryloyl-substituted gelatin comprises methacrylamide and methacrylate substitutions, the ratio of methacrylamide to methacrylate substitution being from 80:20 to 99:

1.

3. 3. The prepolymer solution of claim 1 or 2, wherein the methacryloyl-substituted gelatin is present in a concentration of 5% (w / v), 10% (w / v) or 15% (w / v), optionally the methacryloyl-substituted gelatin is present in a concentration of 5% (w / v).

4. Visible light activated photoinitiators include Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 4. The prepolymer solution of claim 1, wherein the prepolymer is selected from (trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, or any combination thereof.

5. 4. The prepolymer solution of claim 1, wherein the visible light activated photoinitiator is a mixture comprising Eosin Y, triethanolamine, and vinylcaprolactam.

6. 6. The prepolymer solution of claim 5, wherein the concentration of Eosin Y is 0.0125-0.5 mM, the concentration of triethanolamine is 0.1-2% w / v, and the concentration of vinylcaprolactam is 0.05-1.5% w / v.

7. The prepolymer solution according to any one of claims 1 to 6, wherein the corneal cells are epithelial cells.

8. A method for producing a crosslinked methacryloyl-substituted gelatin hydrogel comprising exposing the prepolymer solution of any one of claims 1 to 7 to visible light.

9. The method of claim 8, wherein the prepolymer solution is exposed to visible light having a wavelength in the range of 450 to 550 nm.

10. The method of claim 8 or 9, wherein the prepolymer solution is exposed to visible light for a period of from 10 to 300 seconds.

11. A crosslinked methacryloyl-substituted gelatin hydrogel produced from the prepolymer solution of any one of claims 1 to 7.

12. 12. The crosslinked methacryloyl-substituted gelatin hydrogel of claim 11, wherein the at least one corneal cell is contained within the construct of the crosslinked hydrogel.

13. 13. The crosslinked methacryloyl-substituted gelatin hydrogel according to claim 11 or 12, wherein the hydrogel has a Young's modulus of 250 to 350 kPa.

14. The crosslinked methacryloyl-substituted gelatin hydrogel according to any one of claims 11 to 13, wherein the hydrogel has a degree of crosslinking between 85% and 95%.

15. 15. The crosslinked methacryloyl-substituted gelatin hydrogel according to any one of claims 11 to 14, wherein the hydrogel has a swelling ratio of less than 20%.

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