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 plastics (nanoplastics) on organisms. Surface characteristics play a key role in controlling interactions between nanoplastics and biological systems, and key amongst these for nanoparticles is the protein complex formed on the surface of nanoplastics when nanoplastics are exposed to biological fluids, the protein corona. However, there are few studies focussing on the nanoparticle-biological interface in the current literature and thus a lack of understanding of the key principles that govern the formation and properties of protein coronae, or how the properties of protein coronae affect the response of biological systems. This work has approached this challenge by first investigating the physical structures that are formed on nanoplastics in the presence of proteins, and then introducing nanoplastic and nanoplastic/protein complexes to in vitro cells and model lipid membranes to investigate their impact. Collectively, the contributory factors were critically assessed – nanoplastic size and charge, and the nature of the protein corona. The initial study involved comparing bare polystyrene (PS) nanoplastics (both large and small, and with both positive and negative surface charge), with the nanoplastics coated with protein coronae formed by exposure to the human serum abundant proteins human serum albumin (HSA), and lysozyme (LYS). The protein coronae were studied using neutron scattering techniques and both hard and soft coronae were found to be produced depending on the conditions (when PS and protein carry same or opposite surface charges, respectively). Soft corona complexes are characterised by a structure where the nanoplastics were surrounded by a loose protein layer (~ 2-3 protein thick, observed for LYS soft corona formed around small PS(+) nanoplastics). In most cases hard-corona coated nanoplastics also formed fractal-like aggregates in solution (except for the HSA hard corona complex with PS(+)large). Nanoplastic size affected the structures of both the protein corona and the intrinsic protein: the selfassociation forces holding the nanoplastic/protein complex together were stronger, and the hard corona proteins underwent significant conformational change, for smaller nanoplastics (20 nm) compared to larger nanoplastics (200 nm). Bare nanoplastics and nanoplastic/protein corona complexes were introduced to cellular environments 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 media caused mild cytotoxicity, although there was no clear correlation between cell death and the physical properties of the nanoplastics (size or surface charge). When the nanoplastics were exposed to in vitro cells they had strong association with cells, and were clearly shown to be adhering to the cellular membrane. On the POPC tethered bilayer damage was observed which was nanoplastic size-dependent and charge-independent — small nanoplastics (20 nm) showed membrane thinning, disruption in headgroup packing, and resistivity decrease, while the large particles (200 nm) did not cause any membrane disruption. Both HSA and LYS protein coronae (soft and hard) altered the way the nanoplastics interacted with in vitro cells and lipid bilayers. In most cases, the presence of the protein corona reduced the bilayer disruption and the extent of cytotoxicity; this reduction was greater for soft corona, independent of the protein type or the nanoplastic size. An exception was found for the LYS hard corona complexes with small PS nanoplastics, where the cytotoxicity effect was not mitigated. The difference may be related to the fractal-like morphology of hard corona nanoplastic/protein complexes, which are known to be harmful to cells. The nanoplastic interaction with cells was not limited to membrane adhesion, however, particle uptake into the cells was indicated in flow cytometry experiments and confirmed with fluorescence microscopy. Three-dimensional reconstructed images of cells showed that some of the uptaken nanoplastics were localised around the cell nuclei, apparently adhering to the nuclear membrane surface, they did not penetrate the nuclei. There was also an indication that chromosomes were found close to the small polystyrene nanoparticles, but not the larger particles. Since these nanoplastics have been associated with reports of delayed reproduction and transgenerational effects, this cellular level observation demonstrates the possibility that small PS nanoplastics (20 nm) could be interacting with DNA. This work therefore determined protein corona formation around PS nanoplastics is mainly dictated by electrostatic interactions and soft and hard protein coronae adopt distinctively different geometries. The presence of protein corona, of different types, can have the impact on cytotoxicity and membrane disruption differently. These findings contribute to the literature surrounding nanoplastic toxicity by establishing the link between molecular level interactions and biological consequences.