Development of novel formulations for dsRNA based on cationic interpolyelectrolyte complexes

Growing concerns over negative effects of pesticides on the environment and on human health are the cause of an increased demand for environmentally sustainable plant protection agents. A promising substitute for synthetic pesticides is double stranded RNA (dsRNA). Its application triggers RNA-induced gene silencing, also known as RNA interference (RNAi), a cellular mechanism of eucaryots to protect themselves against pathogens.

The aim of our investigation is to develop a spray-induced gene silencing approach with dsRNA artificially produced in a bacterial host system and formulated with biologically degradable components to stabilize and protect the dsRNA and increase its uptake in planta.

To overcome plant barriers such as size-exclusion limits and charge and to provide protection, we developed a cationic dsRNA-biopolymer carrier formulation based on the electrostatic interaction of selected cationic and anionic biopolymers. To moderate the plant uptake, biopolymers with appropriate characteristics, e.g. molecular weight and charge, were chosen and formulated with the optimal parameters, such as charge ratio, polymer concentration, and energy input, to achieve a formulation with a positive zeta potential, indicating charge masking of negatively charged dsRNA.

The hydrodynamic diameter of our formulations was determined by dynamic light scattering and was 93.7 +- 2.9 nm with a PDI of 0.214 ± 0.024 at the optimal charge ratio of 1.25 (+/-) and at a total polymer concentration of 0.005 %. Scanning electron microscopy analysis confirmed the diameter and revealed spherical particles. Furthermore, energy-dispersive X-ray spectroscopy verified dsRNA integration into the particles by the presence of a phosphorous peak. Moreover, electrophoretic light scattering measurements indicated a positive zeta-potential of +20 to +50 mV, which confirms that the dsRNA was successfully masked.

By optimizing loading ratio and polymer concentration during the process, dsRNA was formulated in sherical particles with a hydrodynamic diameter of < 100 nm and a positive zeta potential, which should improve the uptake of dsRNA in plants.