Uptake and Bioactivity of Chitosan/Double-Stranded RNA Polyplex Nanoparticles in Caenorhabditis elegans

In this study, we investigated chitosan/dsRNA polyplex nanoparticles as RNAi agents in the nematode Caenorhabditis elegans. By measurement of an easily observed phenotype and uptake of fluorescently labeled dsRNA, we demonstrate that chitosan/dsRNA polyplex nanoparticles are considerably more effective at gene knockdown on a whole body concentration basis than naked dsRNA. Further, we show that chitosan/dsRNA polyplex nanoparticles introduce dsRNA into cells via a different mechanism than the canonical sid-1 and sid-2 pathway. Clathrin-mediated endocytosis is likely the main uptake mechanism. Finally, although largely reported as nontoxic, we have found that chitosan, as either polyplex nanoparticles or alone, is capable of downregulating the expression of myosin. Myosin is a critical component of growth and development in eukaryotes, and we have observed reductions in both growth rate and reproduction chitosan exposed C. elegans. Given the increased potency, noncanonical uptake, and off-target effects that we identified, these findings highlight the need for a rigorous safety assessment of nano-RNAi products prior to deployment. Specifically, the potential adverse effects of the nanocarrier and its components need to be considered.