Abstract
RNA interference (RNAi) is a post‐transcriptional gene silencing process mediated by short, interfering RNA molecules (siRNAs) targeting complementary mRNA. siRNAs prevent protein expression and create an opportunity to use RNAi for drug target identification, validation, and therapeutics for treating human diseases. Successful application requires efficient delivery approaches, a hurdle which can be overcome through packaging siRNA inside lipid nanoparticles (LNPs). To evaluate biological activities of LNPs, we developed innovative assays and microscopy techniques to monitor tissue biodistribution, cell internalization, endosomal escape, and gene silencing. In combination with fluorescent markers, we visualized Cy5‐labeled siRNA encapsulated in LNPs and quantified kinetics of cell uptake and endosomal escape in various cells. Some LNPs displayed defects in cell uptake whereas other internalized into endosomes and distributed rapidly to the cytosol. Use of endocytosis inhibitors reveal clathrin and non‐clathrin coated pits participate, in part, in internalization in hepatocytes. Multiplexed, image‐based assays were employed to evaluate potential cellular toxicities and bioinformatics tools were developed to visualize multiparameter HCS data and mine phenotypic signatures.
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