Cassian Nanoparticles—A Novel Method for DNA Delivery into Plant Cells

This research presents for the first time gene expression after DNA delivery into intact plant cells by protein nanoparticles. The successful DNA delivery was carried out by casein nanoparticles (CNPs). The CNPs are self-assembled by the addition of calcium chloride to casein sodium salt solution. A plasmid carrying the red fluorescent protein DsRed sequence was electrostatically bound to CNPs, serving as the model DNA in this study. Tuning the CNPs' zeta potential by adjusting pH facilitated electrostatic interaction with the DsRed plasmid for effective DNA delivery into <i>Nicotiana benthamiana</i> cells. CNPs were covalently modified with the green fluorescent dye 6-Aminofluorescein (6-AF) to track their location in the plant. Infiltration of CNP/DsRed plasmid electrostatic conjugates into <i>N. benthamiana</i> leaves showed successful intracellular and nucleus uptake of CNPs/6-AF/DsRed plasmid conjugates at pH 4.5 (+13.0 mV, 214 nm) and a concentration of 2 mg/mL, with a CNP:plasmid ratio of 1:0.01, confirmed by confocal fluorescence microscopy. RT-PCR analysis confirmed successful gene expression through DsRed protein fluorescence. The initial appearance of the red signal from the DsRed protein was observed 24 hr post-infiltration and persisted for 48 hr.¹<br/><br/>1. Avital Ella Ben-Haima, Reut Amar Feldbauma, Eduard Belausov, Einat Zelinger, Raquel Maria, Einat Nativ-Roth, Karthik Ananth Mani, Omer Barda ,Edward Sionov and Guy Mechrez, DNA Delivery to Intact Plant Cells by Casein Nanoparticles with Confirmed Gene Expression. Advanced Functional Materials 2024, 2314756.