Poster image credits: Jaquesta Adams
Upon entering graduate school, I was fascinated by the prospect of applying genetic engineering to plants towards generating desirable traits in plants. Traditional approaches in plant genetic engineering of whole plants (i.e. the use of Agrobacterium or the gene gun) suffer various limitations on the type of cargo delivered, plant species that could be targeted, and physical tissue damage. The Landry Lab strives to add to plant biologists’ toolkits towards enabling genetic engineering of diverse plant species. To this end, we use nanoparticles as vehicles for transporting biomolecules into plant cells for genetic engineering (DNA, RNA, and proteins). Nanoparticles are materials that have their smallest dimension in the 1 – 100 nm range. Their small size, tunable physicochemical and unique optical properties, and high surface-to-volume ratio render them versatile scaffolds to be functionalized as carriers or probes for therapeutic and diagnostic purposes1–3. In plant genetic engineering, nanomaterials have been used to deliver plasmid DNA4–6, small-interfering RNA7–11, and proteins12 to mature plants.
Within the lab, Dr. Gozde Demirer created single-walled carbon nanotube-based delivery constructs capable of delivering plasmid DNA and small-interfering RNA (siRNA) to plants and inducing gene expression and gene silencing respectively. Similarly, Dr. Huan Zhang demonstrated that both DNA nanostructures and gold nanoclusters are capable of delivering siRNA, with her former work elucidating the dependence on gene silencing efficiency on DNA nanostructure size, shape, and stiffness.
The landscape of nanoparticles used in plant genetic engineering is extremely diverse, spanning various sizes, shapes, materials, and loading strategies. I was astounded by this diversity, which made me all the more curious – how might we design a nanoparticle that enables efficient delivery of biomolecular cargo to plants? Given its small size relative to a plant cell, does a nanoparticle’s shape and size impact its transport within a plant leaf, and accordingly, how might this affect its delivery efficiency? In partnership with Dr. Huan Zhang, I set off to seek answers to these questions.
The plant cell wall is a dominant barrier in restricting biomolecule delivery to plants. It has a size-exclusion limit (SEL) of 5 – 20 nm – in order to deliver a construct across the cell wall without physical or chemical disruption, we designed constructs that did not exceed the SEL in their smallest dimension. From the outset, our hypothesis could be encapsulated by “the smaller, the better”. We anticipated smaller NP constructs being able to bypass the cell wall and cell membrane more efficiently compared to larger NPs. Central to this hypothesis was the assumption that having bypassed the cell wall, smaller NPs would be more likely to internalize into plant cells.
We created a library of five gold nanoparticles (AuNPs) for our experiments – gold nanospheres (AuNS) 5, 10, 15, and 20-nm in diameter and a gold nanorod (AuNR) 13 nm by 67 nm. To directly visualize the interaction of AuNPs with plant cells, we performed transmission electron microscopy (TEM) on leaf sections that had been treated with AuNPs. What we witnessed was surprising: contrary to expectations that smaller NPs would bypass cell walls or at least travel further past the extracellular-cell wall boundary, none of our AuNSs showed significant travel into the cell wall. Conversely, we noted several instances of AuNRs that were internalized into cells, suggesting they had gone through both the cell wall and cell membrane.
Based on these results, we hypothesized that siRNA-loaded gold nanorods would be the most efficient at delivering siRNA into the cell. We studied siRNA delivery efficiency by quantifying the abundance of mRNA transcripts for our target gene via quantitative PCR. In an unexpected turn, samples treated with siRNA-functionalized 10 nm AuNSs experienced far greater silencing than siRNA-AuNR treated leaves (99 % silencing versus 39 %). Taken together with our TEM observations, we found that 10 nm AuNSs resulted in the greatest amount of gene silencing despite not having bypassed the plant cell wall. The fact that silencing occurred suggests that siRNA delivery into the cell was successful, demonstrating that nanoparticle internalization is not needed to attain siRNA delivery in plants.
What implications do these findings have? They help shed light on what nanoparticle parameters might be important for siRNA delivery and other forms of nanoparticle-mediated delivery. For instance, we could leverage the respective properties of AuNSs not internalizing and AuNRs internalizing into cells to design functional nanoparticles with a desired fate. On a mechanistic level, they raise more questions – how does the presence of nanoparticles enhance silencing compared to free siRNA? What causes the desorption of siRNA from nanoparticles? What is the mechanism of entry for the desorbed siRNA? Why do 10 nm AuNSs specifically induce high levels of silencing compared to smaller and larger AuNSs?
We attempt to answer some of these mechanistic questions in our study, though this phenomenon demands further investigation in the context of nanoparticle-mediated delivery in plants. For one, the introduction of nanoparticles into a complex biological environment like plant tissue results in the adsorption of biomolecular constituents (proteins, lipids, and metabolites) onto the nanoparticle, forming a coating known as a ‘corona’. This corona impacts nanoparticle-cell interactions, intracellular localization, and toxicity to the organism13. Thus, a robust understanding of how nanoparticle properties influence the corona composition and in turn nano-bio interactions can inform nanoparticle design strategies14. To further investigate nanoparticle interactions with plants on a cellular or molecular level, however, more tools are also needed to provide greater resolution on nanoparticle and cargo transport. Currently, a common technique for visualizing nanoparticles in relation to plant cells is the use of fluorescence microscopy. Not only could fluorescent labeling of nanoparticles alter their resulting signal and biological distribution15, fluorescence microscopy is also diffraction-limited and thus unable to resolve intracellular components. Tools that can non-invasively track the travel of nanoparticles and cargoes at scales below visible wavelengths are sorely needed to advance our fundamental understanding of nano-bio interactions.
Working on this study has been an edifying experience, providing the opportunity to coordinate and collaborate with scientists across institutions and even across international borders. In this work, we uncovered fascinating and unexpected dependencies of nanoparticle size and shape on internalization characteristics in leaf tissues. While our initial design heuristics for nanoparticle-mediated delivery were intended allow for nanoparticles to bypass the cell wall, our findings show that we do not necessarily need nanoparticle internalization into plant cells to efficiently deliver siRNA to plants. This significantly opens up the design space for nanoparticles and allows us to prioritize other design elements like subcellular-targeting peptides or endosomal disruptors.
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