A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is
the inefficiency of their delivery across the plasma and endosomal membranes to the
cytosol, where they interact with the RNA interference machinery. With the aim of
improving endosomal release, a poorly understood and inefficient process, we studied
the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles,
by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA
release occurred invariably from maturing endosomes within similar to 5-15 min of
endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted
it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release.
Gene knockdown occurred within a few hours of release and required <2,000 copies of
cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand
how it is regulated will facilitate the development of rational strategies for improving
the cytosolic delivery of candidate drugs.
