Uptake of fluorescent polymeric nanoparticles in plant pathogenic fungi

In recent decades, there has been considerable research interest in the field of drug delivery for small and large molecules. A special interest is being focused on biopolymeric nanoparticles (NPs) as they are non-toxic, ecofriendly, effective at lower doses, and more importantly, can be used for controlled release formulations. Over the years, a variety of natural and synthetic polymers have been explored for the preparation of NPs. Among them, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymer (PLGA) have been extensively investigated due to its proven biocompatibility and biodegradability. Nevertheless, most research on PLGA-NP has been carried out on mammals and human systems and consequently few information is available about the uptake in fungal cells, although numerous pathogenic fungi cause immeasurable economic damage to agriculture every year. Among the most common pathogens there are Aspergillus niger, cause of black mold disease, and mycotoxin production, and Botrytis cinerea, cause of grey mould disease. Due to the huge damage that these plant pathogenic fungi cause in terms of productivity, there is increasing interest to counteract their infections. The present research has been designed to study PLGA-NPs uptake in A. niger and B. cinerea. The PLGA-NPs loaded or not with the high fluorescent probe coumarin-6 have been synthethized by microfluidic technology. A. niger and B. cinerea reference strains were growth as planktonic cells and biofilm. The Cu6-PLGA NPs were administered in the various stages of development. The uptake capacity and the localization of Cu6-PLGA NPs into fungal cells were observed with a fluorescence microscope (Apotome Zeiss). The observations revealed that 50 nm NPs have the capacity to penetrate into fungi cells. Furthermore, we observed that NPs penetrates inside the conidia depending on the stage of development. In the first stage of conidia development the protective envelope didn’t allow the uptake of NPs. In later stage of conidia development, when envelope breaks, fluorescence inside the conidia was observed (Fig. 1). NPs were clearly visible in the hyphae (Fig. 2). The fluorescence was found up to 1μm below the fungal wall. By administering the NPs to the biofilm of A. niger and B. cinerea, the coumarin-6 florescent signal in the entire thickness of the biofilm, also after washing with saline solution biofilm, was observed (Fig. 3). This work lays the foundations to understand NPs uptake in the fungal cells for a future application in agriculture. In fact, although there are numerous fungicides available on the market, their efficacy to fight these pathogens is poor, many treatments have to be done and their application can cause negative effects on the environment and also on the plants and human health. Nanotechnology can play a very important role in solving these problems.