Tunable Lipid Coatings Enable Cytoplasmic siRNA Delivery by DNA Origami.

DNA origami nanostructures (DONs) offer precise architectural control for small RNA delivery, but their performance is limited by their susceptibility to nuclease degradation, weak cellular uptake, and inefficient endosomal escape. Here, we introduce an electrostatic lipid-coating strategy that introduces membrane-like properties to DONs while maintaining their structural integrity. By coincubating folded DONs with liposomes composed of a defined mixture of zwitterionic and cationic lipids, we identify a formulation window that yields colloidally stable, monodisperse lipid-coated DONs (LCDs). Systematic variation of the zwitterionic-to-cationic lipid ratio revealed that the fraction of cationic lipids strongly regulates the rate and extent of cellular uptake, with the 50 mol % cationic lipid content emerging as the most effective formulation. LCDs exhibited faster and more extensive cellular internalization and reduced early endosomal retention compared with PEG-coated DONs. The 50 mol % cationic formulation enabled efficient siRNA silencing comparable to benchmark lipid nanoparticles, underscoring the importance of rapid uptake and early endosomal escape for functional delivery while maintaining high cell viability. These findings establish an approach for integrating lipid functionalities onto DONs to improve cytoplasmic delivery while preserving full design flexibility.