Cell type-specific intracellular protein delivery with inactivated botulinum toxin

The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms but existing approaches are limited by immunogenicity, lack of cell type specificity, or their multicomponent nature. Here we explore inactivated botulinum toxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain could be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3-55 kD, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a heterogenous mixture. These explorations suggest that BoNT can be a versatile platform for targeted protein and peptide delivery into mammalian cells.