Linking Protein Corona Composition to Ecotoxicological Outcomes: A comparative Study of different Engineered Nanoparticles in Mediterranean Sea Urchin Paracentrotus lividus

Abstract

Once released into the environment, nanoparticles (NPs) rapidly interact with biomolecules present in biological fluids, leading to the formation of a protein corona that strongly influences their biological fate and interaction with living beings. In the field of ecotoxicology, investigating this bio-nano interface is crucial to understand how NPs are recognized and processed by marine organisms. In our study, we analyzed the formation of the protein corona around four NPs, two metal-based, AgcitLcys and TiO₂, and two polymer-based, PS-COOH and PS-NH₂, after incubation in the coelomic fluid (CF) of the sea urchin Paracentrotus lividus. NPs were characterized before and after incubation in CF using DLS and TEM, while proteomic analysis enabled the identification of adsorbed proteins and the estimation of their relative abundance. The composition of the coronas differed depending on the chemical nature and surface functionalization of the NPs, suggesting selective interactions with components of the CF biological matrix. In vitro bioassays on sea urchin’s coelomocytes revealed distinct ecotoxicological outcomes among NPs: AgcitLcys induced the strongest effects, significantly reducing cell viability and lysosomal membrane stability at environmentally relevant concentrations (25 µg/L) while PS and TiO₂ elicited milder responses. A combined analysis of proteomic and in vitro data suggests that the protein corona is essential for mediating interactions between NPs and coelomocytes. The different affinity of NP surfaces for specific extracellular proteins may influence their recognition, internalization, and impact on cellular physiology. For instance, the corona formed around AgcitLcys contained proteins that bind to and transport other molecules, which could facilitate cellular interactions and activate redox and inflammatory pathways at the extracellular level, thereby modulating particle toxicity. Overall, our findings highlight the importance of accounting for NP-biological corona in the ecotoxicological assessment of NPs and confirm the protein corona as a key determinant in mediating nano-bio interactions in marine organisms.