Structural and mechanistic insight into the biomolecular corona formation of engineered nanomaterials and nanoparticles to unravel their impact on marine biodiversity

Engineered nanoparticles (NPs) are increasingly released into marine environments as a consequence of their widespread application in industrial, medical, and consumer products. Once dispersed in seawater, NPs undergo complex physicochemical transformations that strongly influence their environmental fate and biological interactions. This PhD thesis aims to investigate the mechanisms governing nano-bio interactions in marine organisms, with particular emphasis on the formation and biological role of the protein corona in the sea urchin Paracentrotus lividus. The introductory section provides a general overview of nano-bio interactions, describing how they have been studied in ecotoxicology and outlining the main processes that affect NP behavior in seawater, including aggregation, dissolution, and surface modification. Special attention is dedicated to the adsorption of marine biomolecules onto NP surfaces, leading to the formation of an interfacial layer known as the bio-corona. This acquired biological identity plays a crucial role in modulating NP recognition, cellular uptake, and downstream biological effects. Within this framework, the immune system of P. lividus, particularly its coelomic fluid and immune cells (coelomocytes), is presented as a powerful model to unravel molecular and cellular responses to NPs. The thesis is organized into four interconnected chapters, each addressing a specific aspect of NP interactions with marine biological systems. Chapter 1 focuses on the in vitro formation of the protein corona in P. lividus coelomic fluid and its correlation with coelomocyte responses. By integrating physicochemical characterization, shotgun proteomics, and cellular bioassays, this chapter demonstrates that NP surface properties drive selective protein adsorption, which in turn governs immune cell recognition, internalization, and toxicity. Chapter 2 expands the investigation to the in vivo level, exploring how NPs transform in natural seawater and how these transformations influence organismal responses under environmentally realistic exposure conditions. The combined use of cellular and enzymatic biomarkers reveals that eco-corona formation is a key determinant of physiological and immune alterations in exposed sea urchins. Chapter 3 addresses the molecular mechanisms underlying corona-mediated effects by examining whether protein adsorption induces structural modifications in bound biomolecules. Using circular dichroism spectroscopy, this chapter investigates conformational changes in toposome, a major immune-relevant protein of P. lividus, and evaluates how such alterations may affect its biological function. Finally, Chapter 4 tackles the methodological challenge of tracking coronated nanoparticles. A novel fluorescent labeling strategy is developed and validated to enable visualization of NP corona complexes at the single-particle level without disrupting their interfacial organization. This approach provides a valuable tool for studying nano-bio interactions with improved accuracy and reliability. Overall, the thesis reconstructs the progressive acquisition of biological identity by nanoparticles in the marine environment and demonstrates that the protein corona represents a central element in mediating nano-bio interactions across multiple organizational levels. By combining molecular, cellular, and organismal perspectives, this work contributes to a deeper understanding of NP ecotoxicology and highlights the importance of considering biological interfaces in the environmental risk assessment of engineered nanomaterials.