Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves

Abstract

Rapidly growing interest in nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influences transport in plants and use of their cargoes, limiting development and deployment of nanotechnology in plant systems. Here, we employ non-biolistically delivered DNA-modified gold nanoparticles (AuNP) spanning various sizes (5 - 20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana (Nb) leaves. Generally, smaller AuNPs demonstrate more rapid, higher, and longer-lasting levels of association with plant cell walls compared to larger AuNPs. We observe internalization of rod-shaped but not spherical AuNPs into plant cells, yet surprisingly, 10 nm spherical AuNP functionalized with small-interfering RNA (siRNA) are most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery, and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious bio-cargo delivery for future developments in plant nanobiotechnology.