The Molecular, Physiological, and Morphological Impacts of Carbon Dot Surface Functionalization on Interactions with Plants Microalgae

Abstract

Nanoparticles (NPs) are increasingly utilized in numerous industries, including cosmetics, medicine, and agriculture. However, the diverse nature of nanoparticles makes classifying their interactions with biological systems difficult. This work aims to identify the role of surface functionalization, especially charge, in mediating interactions between nanoparticles, microalgae, and plants. Carbon dots (CDs), which are small, carbon-based nanoparticles with high biocompatibility, were differentially functionalized and used as model particles in this work. First, the microalgae Raphidocelis subcapitata, a model in ecotoxicological studies, was exposed to three CDs with varying surface charges--cationic polyethylenimine (PEI) CDs, anionic carboxylated polyethylenimine (CP) CDs, and near-neutral polyvinylpyrrolidone (PVP) CDs. These experiments were carried out with the aim of understanding how varying nanoparticle surface charge impacts interactions with, and toxicity to, microalgae. Along with traditional toxicology measurements, high-content fluorescence imaging was utilized to screen the treatments for sublethal morphological changes to the algae. The results of this study found that only cationic PEI-CDs were toxic to R. subcapitata, with an EC50 of 42.306 μg/L. Anionic and near-neutral CP and PVP-CDs did not induce toxicity, but did cause sublethal morphological changes to the cell. These included increasing triacylglycerol (TAG) content in the cell and modifying cell area. The findings of this initial project led to the planning of the second chapter of this study. Here, the mechanisms of CP-CD and PEI-CD interactions with microalgae were further studied. To do so, CD exposed cells were morphologically compared to known treatments to identify similarities. The known treatments used were selected to match hypothesized mechanisms of action of the CDs and included exposure to only the polymer used for CD functionalization, growth in low nitrogen conditions, and growth in low light conditions. Hierarchical clustering and random forest classification unveiled that CP-CDs impact cells in a similar manner to nitrogen deprivation. This is likely due to electrostatic interactions between the negative CP-CDs and ammonium present in the media, limiting nitrogen availability to the cell. The PEI-CD treatment was similar to PEI polymer alone. However, it also showed key differences. For example, TAG content was increased in the PEI-CD treatment, but not in the polymer exposure. This suggests that increasing TAG lipid droplet (LD) content may be a nanoparticle specific effect. Finally, the third part of this study aimed to determine if CDs would have a similar effect on terrestrial plants as they did on microalgae. Therefore, tomato (Solanum lycopersicum L.) seeds were vacuum infiltrated with CP-CDs and grown for three weeks. Then, phenotypic traits were measured, and samples collected for RNA sequencing. Measurements of shoot biomass showed no changes despite CP-CD treatment, indicating high biocompatibility consistent with that seen in algal studies. Transcriptomics unveiled an upregulation of genes related to plant biotic stress response, including increased ethylene signaling and expression of parthenogenesis genes. These findings suggest that CP-CDs may also have applications in agriculture, especially in priming plants to be more resilient to biotic stress. The results of this project provide insight into the impacts of nanoparticle functionalization on their interactions with plants. From an ecotoxicological perspective, the findings indicate that cationic NPs pose the greatest risk to microalgae, which are critical primary producers in aquatic ecosystems. Potential applications of CDs to increase lipid content in microalgae are also suggested by this work’s findings. As algae with high TAG content are needed to produce algal biofuels, the evidence that PEI-CDs can increase TAG content in a nanoparticle-specific manner suggests that NPs could play a role in sustainable biofuel production. Finally, CP-CDs were found to be highly biocompatible in both tomato and algae. Their molecular impacts on tomato can inform sustainable design of NPs to increase crop resilience, especially as climate change challenges our global food supply. In summary, this project shows that nanoparticle surface charge impacts their environmental safety and application potential in plants. Future work should investigate additional aspects of nanoparticle surface functionalization, such as charge density, to further enable sustainable nanoparticle design.