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 ~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.

Small interfering RNAs (siRNAs), which downregulate gene expression guided by sequence complementarity, can be used therapeutically to block the synthesis of disease-causing proteins. The main obstacle to siRNA drugs — their delivery into the target cell cytosol — has been overcome to allow suppression of liver gene expression. Here, we review the results of recent clinical trials of siRNA therapeutics, which show efficient and durable gene knockdown in the liver, with signs of promising clinical outcomes and little toxicity. We also discuss the barriers to more widespread applications that target tissues besides the liver and the most promising avenues to overcome them.

Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown.Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1.Conclusion: Our findings provide novel insights into cellular uptake of exosomes.Significance: Our data provide potential strategies for how the exosome uptake pathway may be targeted.