Single‐Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells

Abstract DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single‐particle tracking. We found that the TDNs were rapidly internalized by a caveolin‐dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule‐dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA‐nanostructure‐based drug delivery nanocarriers for targeted therapy.