Cell entry of anionic nano-objects has been observed in various types of viruses and self-assembled DNA nanostructures. Nevertheless, the physical mechanism underlying the internalization of these anionic particles across the negatively charged cell membrane remains poorly understood. Here, we report the use of virus-mimicking designer DNA nanostructures with near-atomic resolution to program “like-charge attraction” at the interface of cytoplasmic membranes. Single-particle tracking shows that cellular internalization of tetrahedral DNA nanostructures (TDNs) depends primarily on the lipid-raft-mediated pathway, where caveolin plays a key role in providing the short-range attraction at the membrane interface. Both simulation and experimental data establish that TDNs approach the membrane primarily with their corners to minimize electrostatic repulsion, and that they induce uneven charge redistribution in the membrane under the short-distance confinement by caveolin. We expect that the nanoscale like-charge attraction mechanism provides new clues for viral entry and general rules for rational design of anionic carriers for therapeutics.

Mechanistic understanding of the endocytosis and intracellular trafficking of nanoparticles is essential for designing smart theranostic carriers. Physico-chemical properties, including size, clustering and surface chemistry of nanoparticles regulate their cellular uptake and transport. Significantly, even single nanoparticles could cluster intracellularly, yet their clustering state and subsequent trafficking are not well understood. Here, we used DNA-decorated gold (fPlas-gold) nanoparticles as a dually emissive fluorescent and plasmonic probe to examine their clustering states and intracellular transport. Evidence from correlative fluorescence and plasmonic imaging shows that endocytosis of fPlas-gold follows multiple pathways. In the early stages of endocytosis, fPlas-gold nanoparticles appear mostly as single particles and they cluster during the vesicular transport and maturation. The speed of encapsulated fPlas-gold transport was critically dependent on the size of clusters but not on the types of organelle such as endosomes and lysosomes. Our results provide key strategies for engineering theranostic nanocarriers for efficient health management.