Thiomethyltetrazine Based Reversible Covalent Chemistry Hydrogels to Support 3D Cell Culture

• Shelby Nelson, Biomedical Engineering, University of Delaware

• Joseph Fox, Chemistry and Biochemistry, University of Delaware

For many biomedical applications, 3D cell cultures are used through creating microenvironments for in vitro tissue studies. To create an environment that is biomimetic to extracellular matrices, a dynamic system that imitates the viscoelasticity and stiffness of human tissue is needed. The lamina propria layer of the vocal fold tissue is responsible for the vibration that causes the human voice to be produced. Different pathologies can cause the natural, vibrating nature of the vocal fold to be harmed, leading to various vocal disorders. Creating a 3D tissue culture model of the vocal fold could aid in discovering treatments for vocal fold disorders and further studies of tissue function. The Fox Group at the University of Delaware has recently discovered a new type of reversible covalent chemistry, utilizing thiomethyltetrazines, that can be made irreversible via tetrazine- trans-cyclooctene (TZ-TCO) ligation. The TZ-TCO ligation is the fastest known bioorthogonal reaction, which can occur in biological systems without affecting normal biological processes, and has been utilized in a variety of applications including interfacial polymerizations and hydrogel formation. The goal of this work is to use the  thiomethyltetrazine reversible covalent chemistry as a way to make dynamic hydrogels capable of supporting 3D tissue culture. This dynamic hydrogel system consists of a hyaluronic acid-thiol backbone functionalized with thiomethyltetrazines through a reversible nucleophilic aromatic substitution (SNAr) reaction. The hydrogel gel can be temporally controlled by introducing TCO at different time points to induce the bioorthogonal reaction locking the thiomethyltetrazine into place. This leads to an irreversible system, potentially increasing the stiffness of the hydrogel. Spatiotemporal control over the TZ-TCO ligation can be used to alter the mechanical properties of hydrogels being used for 3D tissue culture to progress treatment for vocal fold disorders.