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Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves

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Abstract

Rapidly growing interest in the 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 influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5–20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the 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. Overall, 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 biocargo delivery for future developments in plant nanobiotechnology.

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Fig. 1: DNA-AuNP preparation and characterization.
Fig. 2: Cy3-tagged DNA-AuNP association with plant cells following infiltration into transgenic mGFP5 N. benthamiana leaves.
Fig. 3: TEM of DNA-functionalized AuNS-treated N. benthamiana leaves.
Fig. 4: TEM and μXRF imaging of DNA-AuNP-treated N. benthamiana leaves and mechanism of morphology-dependent AuNP transport.
Fig. 5: Size- and morphology-dependent siRNA-AuNP delivery in mGFP5 N. benthamiana leaves.

Data availability

The key datasets generated and analysed during this study are available in the Zenodo repository with the identifier https://doi.org/10.5281/zenodo.5515736. Additional data related to this study are available from the corresponding author upon reasonable request. Correspondence and requests for materials should be addressed to M.P.L.

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Acknowledgements

We thank the Staskawicz Lab (University of California, Berkeley) for sharing mGFP N. benthamiana seeds, the Falk Lab (University of California, Davis) and the Scholthof Lab (Texas A&M University) for their provision of line 16C N. benthamiana seeds, and M.-J. Cho (Innovative Genomics Institute, Berkeley) for his assistance in N. benthamiana growth. We thank A. Avellan and A. Landolino for helpful discussions regarding sample preparation, and T. Cheng for assistance with performing autocorrelation function calculations. The authors recognize that majority of this work was performed on the territory of Huichin, the unceded land of the Ohlone people. We acknowledge support of a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Stanley Fahn PDF Junior Faculty Grant (award no. PF-JFA-1760), a Bakar Award, a Beckman Foundation Young Investigator Award, a USDA AFRI award, a USDA NIFA award and a Foundation for Food and Agriculture Research (FFAR) New Innovator Award (M.P.L.). This research was supported by the Office of Science (BER), US Department of Energy (DOE; grant no. DE-SC0020366, M.P.L.). M.P.L. is a Chan Zuckerberg Biohub investigator. N.S.G. is supported by a FFAR Fellowship. J.W.W. is a recipient of the National Science Foundation Graduate Research Fellowship. H.Z. acknowledges the start-up funding from Jinan University. S.-J.P. acknowledges support from the LG Yonam Foundation and National Research Foundation of Korea (grant no. NRF-2017R1A5A1015365). We acknowledge the support of UC Berkeley CRL Molecular Imaging Center, the UC Berkeley Electron Microscopy Lab and the Innovative Genomics Institute. We thank the ALS Diffraction and Imaging Program for support. This research used resources of the Advanced Light Source, a US DOE Office of Science User Facility (contract no. DE-AC02-05CH11231). We acknowledge the use of Servier Medical Art elements (http://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported Licence.

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Contributions

H.Z. and N.S.G. conceived the project, designed the study and wrote the manuscript. H.Z. and N.S.G. performed the majority of experiments and data analysis. J.W.W., G.S.D. and E.G.-G contributed key input and advanced project direction. J.W.W. performed confocal microscopy on Cy3-DNA-AuNR3 samples. R.L.P. designed, executed and analysed the dynamic exchange experiments. E.G.-G., A.D.R.F. and R.Z. performed the anion-exchange FPLC experiments. S.C.F. performed the µXRF measurements and processed the data. B.Z. performed the assembly of AuNR3. S.B. performed the initial experiments, verifying project feasibility. All authors edited and commented on the manuscript and gave their approval of the final version.

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Correspondence to Markita P. Landry.

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Supplementary Tables 1–8, Figs. 1–41, Notes 1–6, Discussions 1–3, Methods, Statistics and Data Analysis, and references.

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Zhang, H., Goh, N.S., Wang, J.W. et al. Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves. Nat. Nanotechnol. 17, 197–205 (2022). https://doi.org/10.1038/s41565-021-01018-8

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