Encapsulation of Fludioxonil in lipid-based nanocarriers for the treatment of fungal diseases in agriculture: preliminary results

The aim of this work was to realize lipid nanocarriers (liposomes) loaded with the phytopharmaceutical Fludioxonil (FLUD), for the containment of fungal diseases in agriculture. The application of nanostructured lipid carriers in agriculture finds its motivation in the need to overcome the problems associated with the traditional use of pesticides, such as poor bioavailability, easy degradation, and excessive dispersion in the environment. The research was conducted within the Sos&Bio_for_One Health project of the University of Bari. Two methods were compared for the preparation of liposomes: the Micelle-Vesicle Transition (MVT) method and the extrusion method. Furthermore, three types of vesicles were compared that differed in composition: (I) Plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG coated vesicles, with an additional polyethylene glycol coating; and (III) Cationic vesicles, containing a cationic component capable of modifying the surface charge of liposomes (DDAB). The operating parameters, with particular regard to the total amount of lipids and the lipid/FLUD ratio, have been optimized to obtain stable, nanometric-sized vesicles with good Encapsulation Yields (EE%) and Drug Loading (DL%). In particular, through the MVT method, small unilamellar vesicles (SUVs) having a variable mean diameter in the range 80 nm – 150 nm and EE% higher than 78% were easily obtained. The extruded samples yielded mean diameter sizes between 93 nm and 150 nm, with EE% greater than 90%. With both methods the Zeta potential values measured were, in line with expectations, moderately negative for the Plain vesicles and PEG-coated samples and moderately positive for the Cationic vesicles. Subsequently, in vitro release tests of the systems loaded with FLUD were performed. A fairly slow release was verified in the early stages, to then reach higher values and remain constant in the following hours. In particular, for the Plain vesicles a plateau was reached at 96.6% after about five hours, compared to 73.4% for the PEG-Coated vesicles and 46% for the Cationic vesicles. Finally, preliminary tests of biological activity against the Botrytis cinerea fungus have been carried out. Conidial germination test, inhibition of germ-tube growth and radial growth inhibition test were conducted. In general, FLUD delivered via liposomes showed slightly better performance than free FLUD only at the lowest tested concentrations (0.1-0.3 µg/ml), while it showed similar performance to free drug at high concentrations (1 µg /ml). For intermediate concentration values, free FLUD showed better performance than the liposomal one. Further investigations are underway to evaluate the biological response of FLUD-loaded vesicles by adapting the experimental protocols to the realized systems