Resisting Resistance: Development of Nanoparticle-mediated Delivery Systems to Explore RNA Interference as a Solution to Biological Resistance

Abstract

Biological resistance to conventional pesticides and antifungals presents a significant challenge in agriculture and healthcare, necessitating the development of alternative control strategies. RNA interference (RNAi) offers a highly specific and environmentally sustainable approach to managing resistance, but its widespread application is hindered by the lack of efficient RNA delivery methods. This dissertation explores nanoparticle-mediated delivery systems for double-stranded RNA (dsRNA) to enhance RNAi efficiency in both fungal and insect models.

While RNAi is a promising tool for gene regulation, the clear structural and physiological differences between yeast and insects necessitate distinct delivery approaches. For delivery in yeast, we developed the first nanoparticle-mediated photoporation system focused on delivery into fungal cells. This required the meticulous optimization of laser power, wavelength, irradiation time, and several nanoparticle characteristics to maximize RNA uptake while maintaining cell viability. All optimizations were done in Saccharomyces cerevisiae before moving on to gene silencing in the clinically-relevant pathogen, Candida albicans. In contrast, we investigated branched amphiphilic peptide nanocapsules (BAPCs), a biologically-derived peptide-based nanoparticle, for RNAi delivery in the fall armyworm (Spodoptera frugiperda), a major agricultural pest, where nanoparticle concentration and silencing efficiency were studied. Additionally, cellular uptake and trafficking mechanisms were studied using Sf9 cells. Gene silencing efficacy was evaluated in both models using RT-qPCR.

Our findings demonstrate that photoporation successfully facilitates the intracellular delivery of various molecules in yeast, marking the first application of this technique in fungal systems. Additionally, fall armyworms readily consume BAPCs complexed with dsRNA, resulting in effective gene silencing. Evidence suggests that transcytosis plays a role in nanoparticle transport from the gut to systemic tissues, highlighting a potential mechanism for RNAi-based pest control.

This work enhances the field of drug delivery through the development and optimization of of new delivery methodologies and providing insights into mechanisms surrounding nanoparticle-mediated RNA delivery in fungal and insect systems. By bridging the gap between laboratory research and field applications, these nanoparticle-mediated RNAi delivery strategies provide a foundation for novel RNA-based fungicides and insecticides as sustainable alternatives to current chemical treatments.