Nanoplastics and Protein Corona - Investigating the Corona Structure and their Biological Impacts

This thesis explores the chemical and biological bases behind the reported effects of nano-scale \nplastics (nanoplastics) on organisms. Surface characteristics play a key role in controlling \ninteractions between nanoplastics and biological systems, and key amongst these for \nnanoparticles is the protein complex formed on the surface of nanoplastics when nanoplastics \nare exposed to biological fluids, the protein corona. However, there are few studies focussing \non the nanoparticle-biological interface in the current literature and thus a lack of understanding \nof the key principles that govern the formation and properties of protein coronae, or how the \nproperties of protein coronae affect the response of biological systems. \nThis work has approached this challenge by first investigating the physical structures that are \nformed on nanoplastics in the presence of proteins, and then introducing nanoplastic and \nnanoplastic/protein complexes to in vitro cells and model lipid membranes to investigate their \nimpact. Collectively, the contributory factors were critically assessed – nanoplastic size and \ncharge, and the nature of the protein corona. \nThe initial study involved comparing bare polystyrene (PS) nanoplastics (both large and small, \nand with both positive and negative surface charge), with the nanoplastics coated with protein \ncoronae formed by exposure to the human serum abundant proteins human serum albumin \n(HSA), and lysozyme (LYS). The protein coronae were studied using neutron scattering \ntechniques and both hard and soft coronae were found to be produced depending on the \nconditions (when PS and protein carry same or opposite surface charges, respectively). Soft \ncorona complexes are characterised by a structure where the nanoplastics were surrounded by \na loose protein layer (~ 2-3 protein thick, observed for LYS soft corona formed around small \nPS(+) nanoplastics). In most cases hard-corona coated nanoplastics also formed fractal-like \naggregates in solution (except for the HSA hard corona complex with PS(+)large). Nanoplastic \nsize affected the structures of both the protein corona and the intrinsic protein: the selfassociation \nforces holding the nanoplastic/protein complex together were stronger, and the hard \ncorona proteins underwent significant conformational change, for smaller nanoplastics (20 nm) \ncompared to larger nanoplastics (200 nm). \nBare nanoplastics and nanoplastic/protein corona complexes were introduced to cellular \nenvironments of human alveolar epithelial (A549) cells and tethered POPC lipid bilayers. For bare nanoplastics the introduction of bare PS nanoplastics to the A549 cells in serum-free \nmedia caused mild cytotoxicity, although there was no clear correlation between cell death and \nthe physical properties of the nanoplastics (size or surface charge). When the nanoplastics were \nexposed to in vitro cells they had strong association with cells, and were clearly shown to be \nadhering to the cellular membrane. On the POPC tethered bilayer damage was observed which \nwas nanoplastic size-dependent and charge-independent — small nanoplastics (20 nm) showed \nmembrane thinning, disruption in headgroup packing, and resistivity decrease, while the large \nparticles (200 nm) did not cause any membrane disruption. \nBoth HSA and LYS protein coronae (soft and hard) altered the way the nanoplastics interacted \nwith in vitro cells and lipid bilayers. In most cases, the presence of the protein corona reduced \nthe bilayer disruption and the extent of cytotoxicity; this reduction was greater for soft corona, \nindependent of the protein type or the nanoplastic size. An exception was found for the LYS \nhard corona complexes with small PS nanoplastics, where the cytotoxicity effect was not \nmitigated. The difference may be related to the fractal-like morphology of hard corona \nnanoplastic/protein complexes, which are known to be harmful to cells. \nThe nanoplastic interaction with cells was not limited to membrane adhesion, however, particle \nuptake into the cells was indicated in flow cytometry experiments and confirmed with \nfluorescence microscopy. Three-dimensional reconstructed images of cells showed that some \nof the uptaken nanoplastics were localised around the cell nuclei, apparently adhering to the \nnuclear membrane surface, they did not penetrate the nuclei. There was also an indication that \nchromosomes were found close to the small polystyrene nanoparticles, but not the larger \nparticles. Since these nanoplastics have been associated with reports of delayed reproduction \nand transgenerational effects, this cellular level observation demonstrates the possibility that \nsmall PS nanoplastics (20 nm) could be interacting with DNA. \nThis work therefore determined protein corona formation around PS nanoplastics is mainly \ndictated by electrostatic interactions and soft and hard protein coronae adopt distinctively \ndifferent geometries. The presence of protein corona, of different types, can have the impact \non cytotoxicity and membrane disruption differently. These findings contribute to the literature \nsurrounding nanoplastic toxicity by establishing the link between molecular level interactions \nand biological consequences.