Thiol-stimulated crosslinking of gelatin dithiolane to form novel tissue-adhesive hydrogels

Introduction: Corneal injuries make up ~44% of eye-related hospital visits in the United States. Current sealants for corneal injuries promote scar tissue formation, leading to vision loss. There is a need to develop more biocompatible bioadhesives that enhance corneal regeneration. Gelatin dithiolane (GelDT) is a protein based hydrogel modified with dithiolane groups to enable stimuli-responsive crosslinking (under review). Dithiolane rings on the gelatin backbone open into reactive thiols upon exposure to ultraviolet light (UV), forming dynamic covalent bonds that assemble into a viscoelastic 3D polymer network. Unlike other photo-crosslinkable hydrogels, GelDT does not require cytotoxic photoinitiators for crosslinking. In addition to its viscoelastic nature, self-healing ability, and cell-supportive behavior, we have discovered that GelDT robustly adheres to corneal tissue, can act as a stimuli-responsive drug vehicle, and allow the modification of its mechanical properties post-gelation. However, exposing the cornea to UV light carries potential risks, necessitating the development of alternative crosslinking strategies for this application. To address this issue, we investigated whether mixing a thiolated polymer with GelDT can initiate thiol-disulfide exchange, thereby promoting hydrogel formation without the need for UV light stimulation. Indeed, thiol-terminated poly(ethylene glycol) (PEGSH) was found to rapidly form stable polymer networks when mixed with GelDT. In this study, we demonstrate the development of a new thiol-stimulated crosslinking method that enables rapid gelation of GelDT hydrogels, without needing UV light, to form the composite polymer matrix GelDT-PEG-SH, showing significant potential as a biofunctional corneal adhesive.

Materials and

Methods: Gelatin was modified with dithiolane (lipoic acid) using amide coupling. The GelDT prepolymer solution was prepared by dissolving lyophilized GelDT in PBS at 50°C. GelDT hydrogels were subsequently formed under 365 nm ultraviolet (UV) light exposure and confirmed with frequency sweep rheology. Bioadhesive properties of GelDT were tested ex vivo using a tensile rabbit cornea model. In vivo adhesion was tested using a cornea stroma wound model in rabbits, and imaged using Micro-CT. GelDT-PEG-SH gels were formed by mixing GelDT solution with PEG-SH solution: 96 different combinations of PEG-SH and GelDT were studied, varying polymer concentration, PEG molecular weight, PEG arms, and molar ratio of thiol:dithiolane. Qualitative gelation was observed through gelation of 20 μL drops, and stability via incubation in PBS at 37oC overnight. Long term stability of gels was measured through mass retention. Mechanical properties of GelDT and stable GelDT-PEG-SH mixes were characterized using a mechanical tester. GelDT-PEG-SH gelation was quantified with rheology. Biocompatibility was characterized through 3D cell culture using human donor keratocytes and live/dead assay. Ex vivo adhesion of GelDT-PEG-SH was confirmed through tensile adhesion force and ASTM burst pressure test.

Results, Conclusions, and Discussions: The modification of lipoic acid (dithiolane) to gelatin resulted in UV-polymerized hydrogels without requiring cytotoxic photoinitiator (Fig.1a). In vivo adhesion tests in rabbits demonstrated that GelDT adhered to the corneal surface for 24 hours, whereas the commercial control (fibrin glue) did not (Fig.1b). Here, adhesion resulted from UV-stimulated dithiolane ring opening, which generated reactive thiols that bound with native tissue thiols. To eliminate potentially harmful UV exposure, our group discovered that free thiols could open dithiolane rings and initiate polymerization through the thiol-disulfide exchange phenomenon (Fig.1c). 96 different combinations of GelDT-PEG-SH were explored to find the effect of thiolated-PEG parameters on the gelation of GelDT-PEG-SH hydrogels. We discovered a relationship between PEG molecular weight and hydrogel stability. While the relationship is not linear, we found that 10 kDa PEG-SH formed the highest number of stable GelDT-PEG-SH combinations (Fig.1d). This is the first time that a thiol-stimulated crosslinking approach was demonstrated without external stimulation. Mechanical testing of 15% GelDT with 10 kDa PEG-4SH showed a 27% increase in stress relaxation over 15 minutes, from 25% to 52%, compared to UV-cured 15% GelDT (Fig.1e). This indicates a more dynamic polymer network, which is beneficial for cell function. Commercial bio-adhesives like fibrin glue are desirable for their rapid gelation without external stimulation, but they are difficult to modify. We report a novel thiol-stimulated approach to dithiolane crosslinking, enabling instant polymerization into a viscoelastic hydrogel that can be bioactively functionalized without additional chemical modifications. We anticipate that GelDT-PEGSH based novel hydrogels, further modified with signaling molecules and growth factors in future by leveraging the thiol-cysteine interactions, may help to reduce corneal scar tissue and result in improving the corneal vision for corneal patients.

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