Challenging bio- and eco-coronas in marine nano-ecotoxicology

In meeting the challenges associated with the study of nano-sized objects, a major area of research is represented by the interaction of nanoparticles (NPs) with biological systems. Owing to their characteristics, NPs interact in a fundamentally different fashion with biological entities than molecular species. When in contact with biological fluids, the particle surface is covered by a layer of adsorbed proteins and other biomolecules, in dynamic equilibrium with the surrounding environment, termed the “biomolecular corona”, which provide the as-synthesized particle with a unique biological identity. While within the field of nanotoxicology the importance of the study of the biomolecular corona has been largely recognized as key to untangle the complicate interactions of NPs with cells, environmental toxicology has just begun to consider the study of coronas in facing the upcoming issue of NP pollution of the marine ecosystems. The main aim of this PhD was to investigate the interactions of NPs with biomolecules in the marine environment in order to understand their ecotoxicological implications. As a proxy of the emerging issue of plastic contamination of marine ecosystems, polystyrene nanoparticles, with positive and negative surface charges, were employed throughout the study. Firstly, the nanoparticles interactions with the sea urchin Paracentrotus lividus, as a model invertebrate species, were considered. The biological identity of positive and negative polystyrene nanoparticles entails the potential to promote particlecell association, by assisting their interaction with the highly specialized immune systems of P. lividus. Both positive and negative particles maintained hydrodynamic sizes indicative of a narrow size distributions, mostly encompassing protein coated monomers and dimers, acquiring a net negative surface charge. The observed corona-derived colloidal stability over time suggests that, irrespectively of initial surface charge, in the biological milieu polystyrene nanoparticles can acquire the same physical-chemical characteristics, possibly being presented for bio-nano interactions in a comparable manner. The role of an adsorbed protein in promoting the association of positivelycharged polystyrene nanoparticles was proven to lead to toxic effects sustained by the phagocytic immune cells of the sea urchin, confirming that biomolecular coronas dictate the biological fate of nanoparticles in marine invertebrate species, with a mode of action comparable to what established in “classical” nanotoxicology. The subsequent studies focused on polystyrene NPs in marine waters, as a primary target of their release in the environment. In such abiotic, yet biomolecule rich, environment the interaction with extracellular polymeric substances (EPS) extruded by ubiquitous photoautotrophic planktonic species (e.g. diatoms and cyanobacteria) determine the particle fate. The formation of an “eco-corona” reverses the typical colloidal behavior of positive and negative polystyrene NPs, as solely determined by surface charge, when in high-salinity media. Here, positively-charged particles underwent remarkable aggregation while negative ones were stabilized and aggregates formation was limited upon interaction with EPS, prospecting an opposite environmental destiny in marine ecosystems. Surface charge influenced ecotoxicity, with negative PS NPs being generally non-toxic to microalgae and cyanobacteria. Positively-charge counterparts, however, caused loss of cell viability and oxidative stress, but such effects were significantly reduced when an eco-corona was present, suggesting a buffering action carried out by the adsorbed EPS layer, capable of limiting direct NP-cell interactions and their resulting noxious effects. Overall, a multifaceted scenario for bio-nano interactions in the marine environment emerges from this study, with important ecotoxicological implications. If biological recognition of the biomolecular corona seems the underlying mechanism to ecotoxic effects in complex biological milieus, mere physical effects entailed by the eco-corona may be responsible for modulating the toxicity of NPs dispersed in seawater. This fundamentally different characters of bio-nano interactions forecast an additional dimension of complexity of nano-ecotoxicological research in the marine environment, worthy of being considered in future studies.