PhoCoil—An Injectable and Photodegradable Single-Component Recombinant Protein Hydrogel for Localized Therapeutic Delivery

Hydrogels have found widespread utility in many research areas including controlled therapeutic delivery and tissue engineering. Many gel systems have been developed that mimic properties of the extracellular matrix (ECM), providing biocompatibility and enabling 3D cell culture. Traditionally, hydrogels have been formed from polymeric precursors that are either naturally occurring or synthetic. Natural gels are derived from naturally occurring biomolecules, often extracted from tissues. As such, many of these hydrogels mimic the ECM at a chemical and/or structural level, making them a top choice for biomedical applications, but lack in their tunability, providing researchers with few options to control these systems in a defined manner. They also frequently suffer from batch-to-batch variation, creating technical challenges in their implementation. In contrast, hydrogels formed from synthetic polymers retain batch-to-batch similarity and provide significantly more tunability through incorporation of diverse chemistries during synthesis or crosslinking. However, many of these materials are dissimilar to the chemical and/or structural makeup of the ECM. On top of this, both synthetic and natural hydrogels are intrinsically polydisperse. This results in networks with an ill-defined inter-crosslink distance, impacting material elasticity and contributing to overall heterogeneity in these ideally homogenous systems. To unite the advantages of synthetic and natural hydrogels, there has been growing interest in engineering materials from recombinant proteins. These efforts remain nascent but promise perfectly monodisperse, customizable gel networks, with molecular tunability achieved through engineering at the amino acid and fusion protein levels, while also mimicking the proteinaceous nature of the ECM and conferring high biocompatibility. Our work is focused on further enabling external tunability of recombinant hydrogels by installing stimuli-responsivity, which has often been limited to synthetic polymers.<br/><br/>We have developed and characterized a novel, stimuli-responsive recombinant gel platform named PhoCoil. This material is injectable, photodegradable, and formed from a single protein. PhoCoil is crosslinked through homopentameric, physical coiled-coil interactions, resulting in a reversible gel-sol transition in response to applied shear force. Coil domain pairs are connected by an intrinsically disordered protein, originally engineered for high expression and low immunogenicity, providing intra-protein mobility and enabling intermolecular interactions without steric restriction. At the center of the protein is PhoCl, a photolabile green-fluorescent protein which undergoes irreversible cleavage of the polypeptide backbone in response to visible light. The resultant PhoCoil protein hydrogel is shear thinning and self-healing due to its physical crosslinking, enabling easy injection, as well as degradable on-demand in response to cytocompatible light. PhoCoil degradation can be spatiotemporally triggered through conventional mask-based and laser-scanning photolithography. Controlled hydrogel softening can also be achieved by modulating the light dosage, or through co-formulation with a non-light-responsive coiled-coil protein network. PhoCoil is the first fully recombinant protein hydrogel to utilize PhoCl as a cleavable linker and is differentiated from other previously developed photoresponsive recombinant hydrogels by its stability in ambient light and its simple single-component formulation, which does not require any small molecule or metal additives. PhoCoil hydrogels have applications in <i>in vitro</i> cell culture, as they show high cytocompatibility and enable bioorthogonal post-encapsulation release for single-cell analysis. Of particular interest is the use of PhoCoil for controlled, localized drug or cell delivery via post-injection photodegradation, which has been demonstrated in proof-of-concept <i>ex</i> <i>vivo</i> studies.