Absolute quantification and single-cell dose-response of cytosolic siRNA delivery

Abstract

Endosomal escape and subsequent cytosolic delivery of small inhibitory 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 detect cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP 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.