(555e) Internalization and Endocytic Trafficking of 3WJ RNA Nanoparticles for siRNA Delivery

RNA nanotechnology is becoming increasingly successful as a therapeutic agent for the treatment of cancer and genetic diseases. The intrinsic features of RNA allow the bottom-up assembly of complex structures and nano-scale devices in numerous geometries. Though the RNA structure is conceptually similar to DNA, it offers elasticity, flexibility, and the ability to form non-canonical base pairing. Guo and co-workers have constructed RNA nanoparticles with controllable shape, size, and stoichiometry using the stable phi29 packaging RNA three-way junction (3WJ) motif as a scaffold, which displays high chemical and thermodynamic stability. A benefit of these particles is the ability to functionalize them with targeting agents, therapeutic siRNA/miRNA, and fluorescent imaging molecules. These particles have shown success in preliminary studies in vivo but the mechanisms of cellular entry and intracellular trafficking have not been explored. This project aims to elucidate these mechanisms in order to inform rational design to improve particle targeting specificity and survive endosomal processing.

RNA cellular internalization through endocytosis occurs primarily through clathrin-dependent, caveolin-dependent, and macropinocytosis pathways. These pathways can be inhibited through pharmacological small-molecule inhibitors, which can give insight into the impact of each pathway on particle internalization and subsequent endosomal processing. A variety of endosomal assays can be used to track the processing of particles. However, this report is interested in whether or not the particle uptake pathway leads to an acidified endosome. Bafilomycin A1 (BafA1) inhibits endosomal proton ATPases, which prevents the acidification of the endosome. Thus, transfections in the presence of BafA1 can elucidate the reliance of particle survival on the endosomal environment, and fluorescent co-localization reveals visual proof of particle sequestration to pathway-specific endosomes.

3WJ RNA nanoparticles comprise a siRNA targeting the luciferase reporter gene, an Alexa647 fluorescent probe, and an epidermal growth factor receptor-targeting aptamer (EGFR). Particles are transfected into MDA-MD-231 cell line stably expressing the luciferase reporter gene (MDA-luc). Protein knockdown is measured by a decrease in bioluminescence when lysate is exposed to the luciferase substrate. The dose providing highest knockdown is used in transfections preceded by endocytic pathway inhibition by small molecule inhibitors to elucidate pathway impact on particle efficiency. Similarly, flow cytometry is used to determine particle uptake after pathway inhibition. BafA1 is then used to determine the impact of acidic environments on the uptake and knockdown efficiency of particles entering through various pathways.

The EGFR receptor is known to internalize through clathrin-dependent endocytosis. Therefore, inhibition of the clathrin-dependent endocytic pathway is expected to result in a compensatory uptake through alternative pathways. Previous reports show that, when clathrin is inhibited, delivery is enhanced. This study is expected to follow this trend. Inhibition through BafA1 is expected to have a positive effect on knockdown when particles are processed by acidified endosomes (clathrin-dependent endocytosis and macropinocytosis) and no effect when not (caveolin-dependent endocytosis). These implications suggest that increased delivery and knockdown efficiency can be achieved through the targeting of alternative, non-acidified pathways or appending endosomal buffering moieties to the particles that inhibit acidification.