RNAi in Spodoptera frugiperda Sf9 Cells via Nanomaterial Mediated Delivery of dsRNA: A Comparison of Poly-L-arginine Polyplexes and Poly-L-arginine-Functionalized Au Nanoparticles

摘要

This work focused on the delivery of dsRNA either complexed with poly-L-arginine (PLR-polyplex) or loaded onto poly-L-arginine functionalized 20 nm Au nanoparticles (PLR/Au NPs). RNA interference (RNAi) of a luciferase gene expressed in Spodopteria frugiperda Sf9 stable cell line (Sf 9_LUC) was used as a model system. The binding affinity of dsRNA with two modes of functionalization of Au NPs was investigated: the electrostatic binding of PLR on citrate stabilized Au NPs (e-PLR/Au NPs) via the layer-by-layer method or the covalent-linking reaction of the polymer with NHS groups on the Au NPs surface (c-PLR/Au NPs) with excess groups quenched with either hydroxylamine (cPLR/Au/Hyd NPs) or bovine serum albumin (c-PLR/Au/BSA NPs). The formation of PLR-polyplex particles resulting from the complexation of PLR and dsRNA were revealed by transmission electron microscope (TEM), scanning transmission electron microscope (STEM), and elemental mapping by energy dispersive X-ray spectroscopy (EDS). Luciferase gene knockdown was evaluated after exposure of Sf9 cells for 72 h to 600 ng of dsRNA. Gene knockdown (GK) was found to be more efficient by exposing Sf9 cells to nanoscaled (size <100 nm) PLR-polyplex (58% GK), in contrast to microscaled (size >1 mu m) PLR-polyplex (20% GK) or covalent PLR/Au/Hyd (7% GK) particles. The replacement of hydroxylamine by bovine serum albumin as ligand has significantly enhanced the efficiency of GK (31% GK). Investigation of endosomal escape, a key physiological barrier for dsRNA delivery, with CypHerSE labeled dsRNA showed the good ability for the dsRNA conjugated to the different nanosystems to enter the cells compared to the unconjugated one. This study provides valuable information concerning the required properties of materials used for dsRNA delivery in lepidopteran cells such as nanoparticle size, stability in the cell culture media, the polymer molecular weight and binding strength to the nanoparticle, and the nature of ligands on the surface.