Morphology-Dependent Biomolecule Delivery in Plants with Gold Nanoparticles

Plant genetic engineering has the potential to ameliorate the effects of climate change and rising populations via the creation of high-yield crop varieties and climate-resilient plants. To this end, we require a plant genetic engineering toolset that is (i) plant-species independent, and (ii) able to bypass physical barriers present in mature plant tissues. A principal challenge remains the efficient delivery of biological cargo across a plant’s cell wall and cell membrane. Traditional plant genetic engineering tools such as Agrobacterium and biolistic delivery suffer limitations in species range, cargo diversity, tissue damage, efficiency, and transgene integration resulting in regulatory oversight. Recently, nanoparticles (NPs) have emerged as promising materials for use as cargo carriers into plant cells showing high transgene expression without transgene integration.

A lack of heuristics in NP design for unassisted (non-biolistic) DNA, RNA, and protein delivery in plants limits a rational design-based approach for future developments in plant biomolecule delivery. To this end, we utilize DNA-coated gold nanoparticles (AuNPs) as model materials to elucidate the effect of NP morphology on internalization timescale, efficiency, and mode of uptake. We investigate the impact of NP morphology for fluorescent NP internalization into transgenic GFP Nicotiana benthamiana (GFP Nb) plant cells, demonstrating that size and shape are both internalization-determining parameters. By comparing NP fluorescence against cytosolic GFP fluorescence, we show nanorods experience peak internalization faster than nanospheres of a similar diameter. We then investigate endocytosis as a mechanism of NP uptake through the use of an endocytosis inhibitor, demonstrating morphology-dependent effects. Finally, we build upon morphology studies to deliver siRNA-loaded AuNPs to GFP Nb and demonstrate unassisted NP-based gene silencing in mature plants. Our study confirms that NP morphology impacts both time required for and extent of internalization, and confirms NP utility for biomolecule delivery in plants.