Uptake and internalization of nanoparticles in Vitis vinifera and phytopathogenic fungi (Botrytis cinerea and Aspergillus spp.)

In recent years, NPs have been applied in several fields of biomedicine. Only recently researchers have begun to explore the potential of nanocarriers in plant biology (1). In the near future, the development of NPs for plant research and agriculture will allow several new applications, includind treatments with pesticides and fertilizers. Poly(lactic-co-glycolic) acid-based nanoparticles (PLGA NPs) are currently considered among the most promising drug carriers (2). For the first time, in this work it has been evaluated the ability of cells and plants of Vitis vinifera to internalize, transport and accumulate PLGA NPs, with an without an outer shell of chitosan. To visualize the cellular uptake, we synthesized PLGA NPs tagged with the high fluorescent probe coumarin 6. The fluorescence-microscopy analysis has shown the ability of PLGA NPs to cross the cell wall and the membrane of in vitro V. vinifera cells. It has been observed a relatively weak and diffuse fluorescence in the cytoplasm and in the nucleus, while a very intense fluorescence signal in large spherical bodies (generally 1 or 2 per cell), whose nature at present remains unknown. Cell viability test has shown that PLGA NPs were not cytotoxic in grapevine cultured cells. Through the use of fluorescent probes and inhibitors of specific endocytic pathways, it has been demonstrated that the internalization involves both the clathrin-dependent and clathrin-independent pathways. TEM analysis on cultured cells showed that PLGA NPs with a diameter ≤ 50 nm were able to enter in grapevine cells, while the larger ones remained adherent to the cell wall. Furthermore, it was demonstrated that PLGA NPs can enter in leaf tissues of V. vinifera through the stomata openings and that they can be absorbed even by the root and transported to aerial organs via the xylem. The cellular uptake of PLGA NPs has been also studied in grapevine pathogenic fungi (Botrytis cinerea, Aspergillus carbonarius, Aspergillus niger), suggesting that PLGA NPs could be used as vectors to deliver antifungal compounds. In contrast to what observed for V. vinifera cells, the PLGA NPs coated with chitosan enter quickly into fungal cells as well as those without chitosan coat. These results suggest a possible role of PLGA NPs surface charge: NPs without chitosan coat, with negative surface charge, are able to enter both in plant and fungal cells; NPs with chitosan coat that made positive the surface charge, selectively enter into fungal cells; NPs with a thin chitosan coat, showing neutral charge, are internalized only in the in vitro grapevine cell suspensions. PLGA NPs could provide targeted delivery systems, using the developed electrostatic reactions between residues of the wall and the surface charge of NPs. These results suggest that PLGA NPs might play a crucial role in the future development of crop management techniques, offering the possibility to deliver chemicals to specific targets in a controlled manner (3). 1) Chronopoulou L., Cutonilli A., Cametti C., Dentini M., Palocci C. (2012). Colloid Surf B 97:117–123 2) De Oliveira J.L., Ramos Campos E. V., Bakshi M., Abhilash P. C., Fraceto L. F. (2014). Biotechnology advances; 32.8:1550-1561. 3) Ghormade V., Deshpande M.V., Paknikar K.M. (2011). Biotechnology Advances; 29(6):792-803.