Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion

Abstract

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Keywords: Drug delivery; Pesticide nanocarriers; Plant virus; Precision agriculture; Tobacco mild green mosaic virus.

Figure 1

Schematic representation of the “breathing” phase transition diagram. AI = active ingredient.

Figure 2

TEM and image analysis of the non-covalently conjugated viral nanoparticles. ****p-value is < .00001, n = 30. CTD = clothianidin, FLP = fluopyram, IVM = ivermectin, RIF = rifampicin.

Figure 3

Circular dichroism spectra for TMGMV samples loaded with AI as indicated (control = native TMGMV). CTD = clothianidin, FLP = fluopyram, IVM = ivermectin, RIF = rifampicin.

Figure 4

Comparison of the length of virions after AI infusion. Image analysis from transmission electron microscopy: (A) pH breathing methodology; (B) DMSO-induced breathing methodology. CTD = clothianidin, FLP = fluopyram, IVM = ivermectin, RIF = rifampicin.

Figure 5

Surface charge distribution of AI molecules. A ball and stick model of the Ais with a space-filling mesh surface surrounding them. Atoms are colored so grey is carbon, white is hydrogen, blue is nitrogen, red is oxygen, yellow is sulfur, chlorine is green, and chartreuse is fluorine. The surface mesh is colorless if there is no electrostatic potential, blue if positive, and red if negative. The conformations of the molecules are energy minimized.

Figure 6

Docking of AI to TMGMV. TMGMV coat protein (PDB: 1VTM) where red arrows indicate the location of nucleotides (A). A space filling model of a putative binding site for RIF (B), IVM (C), FLP (D), and CTD (E). Red and blue spheres indicate negative and positive electrostatic contributions, respectively. The three-letter amino acid abbreviation are followed by the residue number.