(16c) Influence of Dextran and Surface Charge on Nanoparticle-Mediated siRNA Delivery

Understanding the relationship between delivery vehicle characteristics and the endocytosis and intracellular trafficking of short interfering RNA (siRNA) remains a critical bottle neck in the development of siRNA therapeutics. As a result, it is difficult to design a delivery vehicle for a novel target or specific cell type or to pinpoint the characteristics contributing to the inefficiency, toxicity, or immunogenicity of a vehicle.

Our aim is to explore the mechanism by which the chemical and physical characteristics of a nanoparticle influence siRNA delivery. As a model, we have chosen to use silica nanoparticles (sNPs) due to their straightforward and flexible synthesis. Our functional readouts are: silencing efficacy, accumulation of siRNA in the cells, intracellular location and degree of siRNA-sNP complex dissociation, and intracellular location and degree of siRNA strand separation. We are investigating how vehicle size, shape, charge density, and surface functionalization can be varied to synthesize optimal vehicles for delivery to a variety of cell types.

The presentation will discuss our current finding on the role of sNPs characteristics (dextran functionalization and zeta potential) on siRNA delivery. Our work explores the preferential mechanism of endocytosis (clathrin, caveolin, ARF6, GRAF1, flotillin, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). Additionally, we will discuss findings from our confocal microscopy-based intracellular trafficking assay, which independently tracks the vehicle and siRNA strands across endosomal vesicles (early, late, slow/fast recycling, and lysosomal), the Endoplasmic Reticulum, and the Golgi apparatus. Our results to date demonstrate that multiple vehicle characteristics affect siRNA delivery and a critical role of intracellular trafficking in maximizing siRNA potency.