Classical plant uptake mechanisms are primarily limited to hydrophilic substances. This study aimed to explore the uptake of hydrophobic nanoparticles (NPs), a less understood area with significant implications for agricultural technologies like targeted fertilizer delivery. The rising global value of vertical farming underscores the practical importance of this research. While previous studies have explored the uptake of various NPs, including metals, metal oxides, and carbon materials, the influence of NP hydrophobicity on uptake efficiency remains largely uninvestigated. Existing methods for efficient plant gene manipulation often rely on pressure-assisted delivery systems, which are not scalable for large-scale agricultural applications. This research hypothesized that hydrophobic surface modifications might enable a more efficient, non-pressure-based delivery method, leveraging the enhanced adhesion properties of hydrophobic materials. This hypothesis was tested by comparing the uptake of hydrophobic and hydrophilic Cu₂ₓSe NPs in tomato plants, a model species chosen for its economic value and well-established physiology. The use of intensely fluorescent, biocompatible Cu₂ₓSe NPs, allowed for the tracking of the NPs in the plant tissue.

Numerous studies have explored plant interactions with nanoparticles (NPs), highlighting their potential to enhance various aspects of plant growth and development. NPs have been shown to improve electron transport rates in photosynthesis, act as ROS scavengers, and improve water and nutrient retention. Furthermore, NPs loaded with active ingredients have been explored for targeted release within plants. Prior work identified passive mechanisms, including diffusion, mass flow, and ion exchange, as well as active, energy-intensive carrier-assisted methods for NP uptake. The role of NP coatings and surface charges on uptake behavior has also been investigated. However, a gap exists in understanding the behavior of hydrophobic NPs in plants, which has motivated this current research.

This study employed two types of copper selenide (Cu₂ₓSe) NPs: hydrophobic oleylamine-coated NPs (CS@OA) and hydrophilic chitosan-coated NPs (CS@CH). The NPs were characterized using various techniques including X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, contact angle measurements, photoluminescence (PL) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Thirty-day-old tomato plants were used for uptake studies. NPs were sprayed onto the roots, and samples were collected at various time points (1.5, 3, 6, 12, and 24 hours) for analysis using inductively coupled plasma mass spectrometry (ICP-MS). To remove NPs merely adhered to the roots, samples were washed with 0.1 M HNO₃ before analysis. Confocal laser scanning microscopy (CLSM) and TEM were used to visualize NP distribution within the plant tissue. AFM was used to measure adhesion forces between the NPs and plant roots. Finally, 2D-polyacrylamide gel electrophoresis (2D-PAGE) was employed to analyze the protein corona associated with each NP type. Toxicity was assessed via ascorbate peroxidase (APOX) and catalase (CAT) activity assays and MTT assay to determine root viability. Detailed procedures for NP synthesis, characterization, plant treatment, sample preparation, and data analysis are provided in the supplementary information.

The hydrophobic CS@OA NPs exhibited significantly higher uptake efficiency (~1.3 times greater) than the hydrophilic CS@CH NPs. The uptake of CS@OA NPs was initially rapid, followed by a gradual decrease in root concentration, likely due to translocation to shoots. The ratio of NP movement from roots to shoots was significantly higher for CS@CH NPs (0.72) compared to CS@OA NPs (0.48). Confocal and TEM imaging confirmed the uptake of intact NPs, with CS@OA NPs predominantly localized in the intercellular regions and aligned within cell membranes, while CS@CH NPs were mainly found in the intracellular region. AFM measurements revealed that the hydrophobic CS@OA NPs exhibited substantially greater adhesion forces (~500 pN more) to the root surface than CS@CH NPs. 2D-PAGE analysis of the protein corona indicated differences in protein binding patterns between the two NP types, potentially contributing to the observed differences in translocation. The toxicity studies showed no significant toxicity of the NPs at the tested concentration, as indicated by no significant differences between APOX and CAT enzyme activity, and MTT assay results showed high root viability (>90%).

The significantly higher uptake efficiency of hydrophobic CS@OA NPs compared to hydrophilic CS@CH NPs demonstrates a non-classical route of plant uptake. The strong adhesion of hydrophobic NPs to the root surface, as shown by AFM, likely facilitates their penetration into the plant tissue. The differences in root-to-shoot translocation and protein corona composition suggest the plant's response to NPs differs depending on their surface properties. The lack of significant toxicity further supports the potential application of this technology in targeted fertilizer application. The observation of CS@OA NPs predominantly localized within cell membranes may allow for future development of spatial targeting strategies.

This study reveals a novel mechanism of plant NP uptake mediated by hydrophobicity, challenging the traditional understanding of plant-nanoparticle interactions. The enhanced uptake of hydrophobic NPs suggests the potential for developing more efficient and targeted delivery systems for fertilizers and other agricultural inputs. Future research should investigate the long-term effects of these NPs on plant growth and development, as well as explore the potential for tailoring NP surface properties to enhance uptake and translocation in different plant species.

This study focused on a specific NP type and plant species under controlled laboratory conditions. The findings may not be directly generalizable to all NP types, plant species, or environmental conditions. The study duration was limited to 24 hours, thus long-term effects of the NPs on plant health remain to be investigated. The concentration of NPs used in the study was relatively high, which might influence toxicity evaluations in the future studies. The impact of the ethanol used as a solvent for CS@OA NPs on uptake was considered through controls but may require further investigation.