Exploring cellular uptake of layered double hydroxide nanosheets for delivery of RNA interference in the diamondback moth, Plutella xylostell

RNA interference (RNAi) is a promising tool for targeted insect pest control due to its ability to silence specific genes. However, RNAi efficiency is inconsistent across insect species and its effectiveness in lepidopteran pests like the diamondback moth (Plutella xylostella) is limited due to factors including degradation and poor cellular uptake of RNA molecules. To help improve RNA stability and delivery, we synthesized layered double hydroxide (LDH) nanosheets as a carrier for interfering RNA molecules. Cellular uptake of RNA typically occurs through endocytosis, which includes pathways such as phagocytosis, pinocytosis, clathrin-mediated endocytosis, and caveolin-dependent endocytosis. This project focuses on understanding the cellular uptake mechanisms of LDH-RNA nanosheets in P. xylostella larvae using pharmacological inhibitors of known endocytosis pathways. To understand which cellular uptake pathway the nanosheets are entering the cell through, larvae were treated with pharmacological inhibitors, then fed LDH-RNA nanosheets targeting genes required for insect survival. RNAi efficiency following treatment with inhibitors of endocytosis pathways was evaluated by recording insect mortality and using qPCR to measure gene expression. These methods will help us improve our understanding of cellular uptake of LDH bound RNA in lepidopteran pests and help design more effective RNAi-based delivery strategies for insect control.