Abstract

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since, it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determination of delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery. Single-cell kinetics of siRNA-mediated knockdown unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.