Development of plug-and-deliverable intracellular protein delivery platforms based on botulinum neurotoxin

Intracellular protein delivery systems have great potential in the fields of therapeutics development and biomedical research. However, targeted delivery, passing through the cell membrane without damaging the cells, and escaping from endosomal entrapment of endocytosed molecular cargos are major challenges of the system. Here, we present a novel intracellular protein delivery system based on modularly engineered botulinum neurotoxin type A (BoNT/A). LH_NA domain, consisting of light chain and endosomal escape machinery of BoNT/A, was genetically fused with SpyCatcher (SC) and EGFR targeting affibody (EGFRAfb) to create SC-LH_NA-EGFRAfb, a target-specific and protein cargo-switchable BoNT/A-based intracellular protein delivery platform. SC-LH_NA-EGFRAfb was purely purified in large quantities, efficiently ligated with multiple ST-fused protein cargos individually, generating a variety of protein cargo-containing intracellular delivery complexes, and successfully delivered ligated protein cargos into the cytosol of target cells via receptor-mediated endocytosis, followed by endosomal escape and subsequent cytosolic delivery. SC-LH_NA-EGFRAfb enhanced intracellular delivery efficiency of protein toxin, gelonin, by approximately 100-fold, highlighting the crucial roles of EGFRAfb and LH_NA domain as a targeting ligand and an endosomal escape machinery, respectively, in the delivery process. The BoNT-based plug-and-deliverable intracellular protein delivery system has the potential to expand its applications in protein therapeutics and manipulating cellular processes.