(467a) Gold Nanoparticle-Enhanced siRNA Silencing in Plants Can Occur Independent of NP Internalization

Efficient delivery across a plant’s cell wall and cell membrane remains a key challenge to the transport of biomolecular cargo into plant cells. Nanoparticle (NP)-mediated delivery of DNA and RNA to plants is an increasingly studied alternative to current delivery strategies^1–3, however, little is known about how the size and shape of nanoparticles influences their transport in plants and the resulting efficiency of cargo delivery.

This 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. 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 using both confocal microscopy and transmission electron microscopy. Generally, smaller AuNPs demonstrate more rapid and higher 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 AuNPs functionalized with small-interfering RNA (siRNA) are most efficient at siRNA delivery and inducing GFP gene silencing in mature transgenic Nb plant leaves.

Based on our results, we propose a ‘reservoir’ mechanism in which NPs embed in the cell wall, protecting the siRNA from degradation, whilst slowly releasing cargo into plant cells to achieve gene silencing. Contrary to initial expectations, our results demonstrate that NP-mediated cargo delivery can be achieved without NP internalization into plant cells. 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.

References:

(1) Demirer, G. S.; Zhang, H.; Matos, J. L.; Goh, N. S.; Cunningham, F. J.; Sung, Y.; Chang, R.; Aditham, A. J.; Chio, L.; Cho, M. J.; Staskawicz, B.; Landry, M. P. High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material without DNA Integration in Mature Plants. Nat. Nanotechnol. 2019, 14 (5), 456–464. https://doi.org/10.1038/s41565-019-0382-5.

(2) Mitter, N.; Worrall, E. A.; Robinson, K. E.; Li, P.; Jain, R. G.; Taochy, C.; Fletcher, S. J.; Carroll, B. J.; Lu, G. Q.; Xu, Z. P. Clay Nanosheets for Topical Delivery of RNAi for Sustained Protection against Plant Viruses. Nat. Plants 2017, 3 (2), 1–10. https://doi.org/10.1038/nplants.2016.207.

(3) Torney, F.; Trewyn, B. G.; Lin, V. S. Y.; Wang, K. Mesoporous Silica Nanoparticles Deliver DNA and Chemicals into Plants. Nat. Nanotechnol. 2007, 2 (5), 295–300. https://doi.org/10.1038/nnano.2007.108.