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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize
Journal of Nanobiotechnology Open Access 07 May 2022
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Liu, Y. et al. A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice. Nat. Biotechnol. 33, 301–305 (2015).
Li, T., Liu, B., Spalding, M. H., Weeks, D. P. & Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 30, 390–392 (2012).
Karny, A., Zinger, A., Kajal, A., Shainsky-Roitman, J. & Schroeder, A. Therapeutic nanoparticles penetrate leaves and deliver nutrients to agricultural crops. Sci. Rep. 8, 7589 (2018).
Torney, F., Trewyn, B. G., Lin, V. S. Y. & Wang, K. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotechnol. 2, 295–300 (2007).
Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).
Kwak, S.-Y. et al. Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat. Nanotechnol. 14, 447–455 (2019).
Mitter, N. et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat. Plants 3, 16207 (2017).
Demirer, G. S. et al. Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Sci. Adv. 6, eaaz0495 (2020).
Zhang, H. et al. DNA nanostructures coordinate gene silencing in mature plants. Proc. Natl Acad. Sci. USA 116, 7543 (2019).
Lei, W.-X. et al. Construction of gold-siRNANPR1 nanoparticles for effective and quick silencing of NPR1 in Arabidopsis thaliana. RSC Adv. 10, 19300–19308 (2020).
Zhang, H. et al. Gold-nanocluster-mediated delivery of siRNA to intact plant cells for efficient gene knockdown. Nano Lett. https://doi.org/10.1021/acs.nanolett.1c01792 (2021).
Martin-Ortigosa, S. et al. Mesoporous silica nanoparticle-mediated intracellular Cre protein delivery for maize genome editing via loxP site excision. Plant Physiol. 164, 537–547 (2014).
Liu, Q. et al. Carbon nanotubes as molecular transporters for walled plant cells. Nano Lett. 9, 1007–1010 (2009).
Bao, W., Wang, J., Wang, Q., O’Hare, D. & Wan, Y. Layered double hydroxide nanotransporter for molecule delivery to intact plant cells. Sci. Rep. 6, 26738 (2016).
Avellan, A. et al. Nanoparticle size and coating chemistry control foliar uptake pathways, translocation, and leaf-to-rhizosphere transport in wheat. ACS Nano 13, 5291–5305 (2019).
Spielman-Sun, E. et al. Protein coating composition targets nanoparticles to leaf stomata and trichomes. Nanoscale 12, 3630–3636 (2020).
Zhang, S., Gao, H. & Bao, G. Physical principles of nanoparticle cellular endocytosis. ACS Nano 9, 8655–8671 (2015).
Herd, H. et al. Nanoparticle geometry and surface orientation influence mode of cellular uptake. ACS Nano 7, 1961–1973 (2013).
Xie, X., Liao, J., Shao, X., Li, Q. & Lin, Y. The effect of shape on cellular uptake of gold nanoparticles in the forms of stars, rods, and triangles. Sci. Rep. 7, 3827 (2017).
Chithrani, B. D., Ghazani, A. A. & Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662–668 (2006).
Yi, X., Shi, X. & Gao, H. A universal law for cell uptake of one-dimensional nanomaterials. Nano Lett. 14, 1049–1055 (2014).
Huang, C., Zhang, Y., Yuan, H., Gao, H. & Zhang, S. Role of nanoparticle geometry in endocytosis: laying down to stand up. Nano Lett. 13, 4546–4550 (2013).
Shi, X., von dem Bussche, A., Hurt, R. H., Kane, A. B. & Gao, H. Cell entry of one-dimensional nanomaterials occurs by tip recognition and rotation. Nat. Nanotechnol. 6, 714–719 (2011).
Vácha, R., Martinez-Veracoechea, F. J. & Frenkel, D. Receptor-mediated endocytosis of nanoparticles of various shapes. Nano Lett. 11, 5391–5395 (2011).
Hui, Y. et al. Role of nanoparticle mechanical properties in cancer drug delivery. ACS Nano 13, 7410–7424 (2019).
Houston, K., Tucker, M. R., Chowdhury, J., Shirley, N. & Little, A. The plant cell wall: a complex and dynamic structure as revealed by the responses of genes under stress conditions. Front Plant Sci. 7, 984 (2016).
Cunningham, F. J., Goh, N. S., Demirer, G. S., Matos, J. L. & Landry, M. P. Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2018.03.009 (2018).
Schwab, F. et al. Barriers, pathways and processes for uptake, translocation and accumulation of nanomaterials in plants – critical review. Nanotoxicology 10, 257–278 (2016).
Wang, P., Lombi, E., Zhao, F.-J. & Kopittke, P. M. Nanotechnology: a new opportunity in plant sciences. Trends Plant Sci. 21, 699–712 (2016).
Hubbard, J. D., Lui, A. & Landry, M. P. Multiscale and multidisciplinary approach to understanding nanoparticle transport in plants. Curr. Opin. Chem. Eng. 30, 135–143 (2020).
Corredor, E. et al. Nanoparticle penetration and transport in living pumpkin plants: in situ subcellular identification. BMC Plant Biol. 9, 45 (2009).
Bao, D. P., Oh, Z. G. & Chen, Z. Characterization of silver nanoparticles internalized by Arabidopsis plants using single particle ICP-MS analysis. Front. Plant Sci. https://doi.org/10.3389/fpls.2016.00032 (2016).
Zhang, P. et al. Shape-dependent transformation and translocation of ceria nanoparticles in cucumber plants. Environ. Sci. Technol. Lett. 4, 380–385 (2017).
Giraldo, J. P. et al. Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nat. Mater. 13, 400–408 (2014).
Santana, I., Wu, H., Hu, P. & Giraldo, J. P. Targeted delivery of nanomaterials with chemical cargoes in plants enabled by a biorecognition motif. Nat. Commun. 11, 2045 (2020).
Zhang, X., Servos, M. R. & Liu, J. Instantaneous and quantitative functionalization of gold nanoparticles with thiolated DNA using a pH-assisted and surfactant-free route. J. Am. Chem. Soc. 134, 7266–7269 (2012).
Yang, G. et al. Implications of quenching-to-dequenching switch in quantitative cell uptake and biodistribution of dye-labeled nanoparticles. Angew. Chem. Int. Ed. 60, 15426–15435 (2021).
Sattelmacher, B. The apoplast and its significance for plant mineral nutrition. New Phytol. 149, 167–192 (2001).
Yu, M. et al. Rotation-facilitated rapid transport of nanorods in mucosal tissues. Nano Lett. 16, 7176–7182 (2016).
Matsuoka, K., Bassham, D. C., Raikhel, N. V. & Nakamura, K. Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells. J. Cell Biol. 130, 1307–1318 (1995).
Elkin, S. R. et al. Ikarugamycin: a natural product inhibitor of clathrin-mediated endocytosis. Traffic 17, 1139–1149 (2016).
Aniento, F. & Robinson, D. G. Testing for endocytosis in plants. Protoplasma 226, 3–11 (2005).
Reynolds, G. D., Wang, C., Pan, J. & Bednarek, S. Y. Inroads into internalization: five years of endocytic exploration. Plant Physiol. 176, 208–218 (2018).
Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).
Tiwari, M., Sharma, D. & Trivedi, P. K. Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Mol. Biol. 86, 1–18 (2014).
Bennett, M., Deikman, J., Hendrix, B. & Iandolino, A. Barriers to efficient foliar uptake of dsRNA and molecular barriers to dsRNA activity in plant cells. Front. Plant Sci. https://doi.org/10.3389/fpls.2020.00816 (2020).
Pinals, R. L., Yang, D., Lui, A., Cao, W. & Landry, M. P. Corona exchange dynamics on carbon nanotubes by multiplexed fluorescence monitoring. J. Am. Chem. Soc. 142, 1254–1264 (2020).
Geilfus, C.-M. The pH of the apoplast: dynamic factor with functional impact under stress. Mol. Plant 10, 1371–1386 (2017).
Chehab, E. W., Eich, E. & Braam, J. Thigmomorphogenesis: a complex plant response to mechano-stimulation. J. Exp. Bot. 60, 43–56 (2009).
Mori, I. C. & Schroeder, J. I. Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol. 135, 702–708 (2004).
Baldock, B. L. & Hutchison, J. E. UV–visible spectroscopy-based quantification of unlabeled DNA bound to gold nanoparticles. Anal. Chem. 88, 12072–12080 (2016).
Marcus, M. A. et al. Beamline 10.3.2 at ALS: a hard X-ray microprobe for environmental and materials sciences. J. Synchrotron Radiat. 11, 239–247 (2004).
Mitov, M. I., Greaser, M. L. & Campbell, K. S. GelBandFitter – a computer program for analysis of closely spaced electrophoretic and immunoblotted bands. Electrophoresis 30, 848–851 (2009).
Toni, L. S. et al. Optimization of phenol-chloroform RNA extraction. MethodsX 5, 599–608 (2018).
O’Leary, B. M., Rico, A., McCraw, S., Fones, H. N. & Preston, G. M. The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example. J. Vis. Exp. https://doi.org/10.3791/52113 (2014).
Nicot, N., Hausman, J.-F., Hoffmann, L. & Evers, D. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J. Exp. Bot. 56, 2907–2914 (2005).
Selvakesavan, R. K. & Franklin, G. Nanoparticles affect the expression stability of housekeeping genes in plant cells. Nanotechnol., Sci. Appl. 13, 77–88 (2020).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).
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.
The authors declare no competing interests.
Peer review information Nature Nanotechnology thanks Mohamed El-Shetehy, Shadi Rahimi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
This article is cited by
Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize
Journal of Nanobiotechnology (2022)
Nature Reviews Materials (2021)