Engineered nano-bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery-a core concept in fundamental and translational biomedical research-holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA-SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB-SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin-1 at the cell-NT interface, suggesting the presence of endocytic pits. Small-molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT-mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F-actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT-enhanced molecular delivery platform has strong potential to support gene editing technologies.
Keywords: Cas9 RNP; gene editing; intracellular delivery; siRNA knockdown; silicon nanotubes.
© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.