Title: Studies of the impact of core-shell polystyrene nanoparticles on cell membranes and biomimetic models.
Author: Jacqueline MAXIMILIEN
National thesis number: 2015COMP2180
- This project s aim was to study polymeric nanoparticle-membrane interactions using both live cells and biomimetic models with the idea to validate such nanoparticles for use in bio-applications. Core-shell polymeric nanoparticles below 100 nm, as this small size is capable of penetrating plasma membranes, were synthesised. Nanoparticles (NPs) with the same chemical composition but with hydrodynamic diameters of ~250 nm, were also prepared in an effort to highlight any effect of NP size on cell internalisation. In this thesis, an innovative method is presented for the synthesis of water-compatible, iniferter-bound polystyrene core shell NPs (~30 nm) using a one-pot synthetic method. A plethora of functionalities could be added to the nanoparticles via shell grafting from the surface of the polystyrene core in the presence of additional monomers via controlled living radical polymerisation. Shell thickness could be tuned as a function of monomer s concentration and polymerisation time. The nanoparticles were fully characterised by dynamic light scattering, Fourier transform infra-red spectroscopy, microelemental analysis and transmission electron microscopy. Further, the interactions of polystyrene core NPs possessing neutral and anionic shells were investigated using neonatal human epidermal keratinocytes (NHEK), human primary fibroblasts and HaCaT cells. Cytotoxicity studies performed using propidium iodide and lactate dehydrogenase indicated no evidence of cytotoxicity in either cell line. However, cell proliferation monitored by electric cell substrate impedance sensing (ECIS) protocols indicated that anionic nanoparticles induced a dramatic decrease in cell proliferation in keratinocytes. The cellular internalisation of NPs was confirmed by confocal microscopy and no co-localisation was found with early endosomes, lysosomes or actin. Additionally, fluorescence activated cell sorting (FACS) data support the theory that an energy-dependent mechanism is employed for neutral NP internalisation but less so for negatively charged NPs. Biomimetic membrane models were used to investigate specific nanoparticle-lipid interactions under controlled conditions. Employing giant vesicles coupled with fluorescent spectroscopy techniques revealed that core-shell nanoparticles interact deep in the hydrophobic region of bilayers only when the membrane is in the fluid phase. Their mode of entering artificial cells (i.e giant vesicles) appears to cause the formation of pores. Anionic nanoparticles interact with the choline moiety of phosphatidylcholine and confer a rigidifying effect on phosphocholine containing bilayers. Therefore we conclude that the polymeric nanoparticles that we synthesized are versatile tools for cell interaction and imaging studies. These nanomaterials could eventually be applied to drug delivery studies by incorporation of the drug in for instance a thermoresponsive polymeric shell. Furthermore, it is clear that NPs coated with anionic and neutral polymeric shells present a lower toxicity profile than previously reported cationic nanoparticles. Both nanoparticles increase the order lipid bilayer vesicles composed of POPC (the most common glycerophospholipid) in animal and plants. Anionic nanoparticles in particular exhibit a rigidifying effect on POPC lipid bilayers and their mode of entry into cells may be due to the formation of pores which was determined to not induce cell death.