Developing Protein-based injectable hydrogels using PCNA and SpyCatcher for controlled cargo load and release

Abstract

Three-dimensionally crosslinked hydrogels have been widely developed for various biomedical applications such as scaffolds, drug delivery, and biomaterial implants. However, many hydrogels use chemical crosslinkers that limit their applications due to their potential toxicity and non-biodegradabiliy. To overcome these limitations, protein-based hydrogels have emerged as promising alternatives. Herein, we designed two different non-fibrous recombinant protein building blocks; a core trimeric proliferating cell nuclear antigen (PCNA) and a bridging SpyCatcher protein (SC) dimer. We genetically introduced SpyTag peptide (ST) to the (PCNA) to form ST-PCNA to serve as the core building blocks, and fused two SCs to form SC-SC as the connecting building blocks. Simple mixing of these two building block proteins (ST-PCNA and SC-SC) spontaneously led to the PCNA-based 3D networks, facilitating rapid gelation and resulting in stable protein-based hydrogel formations both in vitro and in vivo. The newly formed protein-based hydrogels possessed a 3D porous network structure that allows for control of cargo loading and release, making them well-suited for use as sustained release systems. To take advantage of the attractive properties of protein-based hydrogels, we evaluated injectability of hydrogels and the controlled loading and sustained release of various cargos, including fluorescent dyes, therapeutic drugs, and proteins, both in vitro and in vivo. These non-fibrous protein building block-based hydrogels may offer new opportunities to develop biocompatible and versatile platforms for sustained cargo delivery, scaffold formation, and implantable biomaterials.