Enhancing the delivery and stability of lipid nanoparticle–dsRNA formulations in the RNAi-recalcitrant fall armyworm (Spodoptera frugiperda)

The fall armyworm (FAW, Spodoptera frugiperda ) is an invasive lepidopteran pest of staple crops. Its broad host range, ability to spread rapidly, and increasing resistance to pesticides pose a major threat to global food security. RNA interference (RNAi) offers a sustainable and targeted alternative to broad-spectrum chemical pesticides, but its efficacy is limited in lepidopterans primarily by the rapid degradation of double-stranded RNA (dsRNA) in the midgut and poor epithelial uptake. Here, we investigated lipid nanoparticles (LNPs) as a delivery strategy to enhance dsRNA stability and uptake in FAW larvae. LNP–dsRNA complexes (40–50 nm, +39 to +56 mV) were generated by the microfluidic mixing of a ternary lipid blend. Encapsulation protected dsRNA from degradation by gut enzyme extracts for up to 1 h, even under highly alkaline conditions (pH 11.5). The analysis of larvae exposed to Cy3-labeled dsRNA by fluorescence microscopy demonstrated that LNPs improved internal distribution beyond the gut lumen, whereas unformulated dsRNA mainly accumulated at the peritrophic membrane. These results indicate that LNPs resist the gut environment and overcome limited systemic uptake, the two major physiological barriers to RNAi in lepidopterans, enabling the more efficient delivery of dsRNA. This study establishes a lipid nanoparticle-based dsRNA delivery platform that overcomes key physiological barriers in FAW, providing a prerequisite for future in vivo gene knockdown and efficacy studies.