Phd in ENVIRONMENTAL AND EVOLUTIONARY BIOLOGY

Thesis title: Internalization mechanisms of biopolymeric nanoparticles in plants and fungi and application in integrated pest management

PhD in Environmental and Evolutionary Biology (XXXIV cycle) PhD Student: Giulia De Angelis Tutor: Prof. Gabriella Pasqua. The application of biodegradable poly lactic-co-glycolic acid nanoparticles (PLGA-NPs) to develop controlled release systems of bioactive molecules has shown enormous potential in agriculture. PLGA is a non toxic harmless biocompatible polymer that degrades into nontoxic byproducts. The hydrolysis of lactide and glycolide monomers leads to lactic and glycolic acids, respectively, which are metabolized by the Krebs cycle releasing carbon dioxide and water. The main advantage of nanoformulations consists in protecting the load from volatilization, infiltration, outflow, leaching and photo-, chemio- or bio-degradation. The expected benefit could be a decrease in the amount of active chemicals incorporated into plants and soils, and a low environmental impact. However, the use of PLGA-NPs in agriculture needs an accurate evaluation of nanoparticle-plant interactions, including the comprehension of the mechanisms of their uptake, translocation and accumulation. In this PhD project, PLGA-NPs were used for a dual purpose. The first one was to clarify the mechanisms of internalization of PLGA-NPs, sorting and targeting into plant and fungal cells according to morphology and physico-chemical properties of NPs. The second one was to use NPs as vectors to deliver antifungal molecules against plant pathogenic fungi cause of immeasurable economic damage in agriculture. It is supposed that the major route for the internalization of PLGA-NPs in plant cells is the clathrin-mediated endocytosis, which starts through the formation of clathrin-coated membrane invaginations, also termed clathrin-coated pit. In this context, dynamin is a GTPase protein essential for vesicle detachment. However, in addition to the clathrin-dependent endocytosis pathway, emerging researches have revealed clathrin-independent pathways are not all dependent on dynamin in plant cells. In this study, PLGA-NPs (30 nm) loaded with the high fluorescent probe coumarin-6 (Cu6-PLGA NPs) have been synthetized by microfluidic technology. The localization of Cu6-PLGA NPs has been studied in suspended cells and roots of A. thaliana seedling by confocal microscope apparatus and by fluorescence microscopy at different times (0min, 10min, 30min, 1h, 2h and 24h in suspended cells; 0min, 10min, 30min, 80min and 5 h in roots). To investigate the mechanisms of NPs uptake and internalization, the Dynasore, a specific inhibitor of dynamin, was added to cultured cells and roots. Dynasore, whose activity is well known in animal cells, is able to block the GTPasic activity of the dynamin preventing the detachment of the vesicles from the membrane. The obtained confocal images showed that Cu6-PLGA NPs (30 nm) at a concentration of 15 mg/L, were rapidly internalized by A. thaliana cells, as revealed by the small highly fluorescent round bodies (endosomes) and a low diffuse background observed after 10 min from the treatment with Cu6-PLGA NPs. After 30 min from treatment, the endosomes were coalescing by appearing to be less numerous and greatest. Cu6- PLGA NPs were accumulated in the cytoplasm and seemed to be stable over time: at 24 h from Cu6-PLGA NPs treatment, the fluorescence distribution pattern was similar to the one observed at 60 min. The treatment with Dynasore, at 80 µM or 160 µM for 10 minutes before the addition of 15 mg/L NPs did not prevent the NPs uptake in A. thaliana suspended cells, as revealed by fluorescence visible in the cytoplasm and in spherical vesicles. The confocal analysis of in vitro roots of A. thaliana seedlings showed that 15 mg/L Cu6-PLGA NPs enter the root in a few minutes. In particular, Cu6-PLGA NPs penetrated through the root hairs 10 min after treatment, and the fluorescence was visible not only in the root hairs, but in almost all cells of the epidermis at longer times (30 and 80 min). Cu6-PLGA NPs stayed at the epidermis level also after 5 hours. The treatment with Dynasore at 80 or 160 µM for 30 and 60 minutes before the addition of 15 mg/L Cu6-PLGA NPs for 10 minutes did not prevent the uptake of NPs observed in the epidermis. Even treating the roots with a higher concentration of Dynasore (320 µM) for 30 and 60 minutes before Cu6-PLGA NPs treatment, intense fluorescence continued to be visible in the root epidermis. The cytotoxicity test with propidium iodide showed that the root cells were not viable after 120-minute treatment with Dynasore at the highest concentration (320 µM). Our results suggested that the internalization of NPs could occur by a dynamin-independent pathway and consequently, the pathway of clathrin-independent endocytosis could be involved, in agreement with previous results obtained by using clathrin-dependent endocytosis inhibitors on grapevine cells. In addition, the PLGA-NPs were translocated from the root to the hypocotyl in the A. thaliana seedling. The results of this research add new information to the understanding of PLGA-NPs internalization pathways in plants with potential applications in the agronomic field, where NPs could be applied for purposes of sustainable agriculture. In another study, Botrytis cinerea, and Aspergillus brasiliensis, were selected in order to investigate the uptake of nanoparticles in fungi. Botrytis cinerea is known to be the cause of grey mould disease, Aspergillus brasiliensis is a species belonging to the Nigri section. Aspergillus species are responsible for a variety of disorders in various plants and plant products, mainly as opportunistic storage molds They produce several mycotoxins cause of pulmonary aspergillosis, otomycosis and eye infections in humans. In order to study the uptake of nanoparticles in fungi, B. cinerea conidia and mycelium and A. brasiliensis conidia, mycelium and biofilm were treated with 50 nm PLGA-NPs uploaded with high fluorescent probe coumarin-6 (Cu6-PLGA NPs) and analyzed by ApoTome fluorescence microscopy. The fluorescence microscopy observations revealed that, after 10 minutes from the treatment, 50 nm Cu6-PLGA NPs penetrated rapidly B. cinerea conidia and hyphae as well as A. brasiliensis hyphae and biofilm. In an effort to investigate the antimicrobial activity of antifungals delivered by nanoparticles, pterostilbene, a naturally-derived stilbenoid or fluopyram, a synthetic antifungal, used as control, were encapsulated in PLGA-NPs and supplied at different stages of B. cinerea development. Pterostilbene and fluopyram loaded in PLGA NPs showed higher antifungal activity against B. cinerea than the free compounds. In addition, pterostilbene encapsulated in PLGA-NPs was tested for its antifungal ability against A. brasiliensis biofilm. Pterostilbene delivered by PLGA-NPs reduced A. brasililiensis biofilm formation and mature biofilm demonstrating that the PLGA-NP system can enhance the bioavailability of natural compounds in A. brasiliensis biofilm crossing the biofilm matrix barrier and delivering the molecule to fungal cells. Moreover, pterostilbene loaded in PLGA NPs was tested in an in vivo model by selecting the larvae of Galleria mellonella as a fungal in vivo model. G. mellonella was infected with A. brasiliensis conidia and treated with pterostilbene delivered by PLGA-NPs or pterostilbene supplied in free form. In vivo experiments showed an infected larvae reduction in mortality after the injection of pterostilbene loaded in PLGA-NPs compared to pterostilbene supplied in free form at the same concentration. Based on the obtained results, PLGA NPs could represent a promising method to deliver bioactive compounds for sustainable purposes in agriculture and to enhance the effectiveness of natural and synthetic antifungals through controlled and targeted drug delivery with the aim to reduce environmental toxicity and agricultural costs.