Abstract:
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.