This thesis is focused on the detailed physicochemical characterisation of zinc oxide (ZnO) nanoparticles (NPs) intended for use in toxicological investigations. A characterisation protocol has been developed in order to determine detailed physicochemical characteristics of 5 ZnO NP samples, selected to investigate in in vitro toxicity assays. The characterisation protocol aims to establish the samples’ distribution of particle sizes, morphology, crystallinity, phase content, purity, surface composition, dispersion and solubility and as such includes the following techniques: inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction (XRD), BET gas adsorption, thermogravimetric analysis with evolved gas analysis by Fourier transform infra-red spectroscopy (TGA-EGA with FTIR), FTIR, X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), plunge freezing transmission electron microscopy (PF-TEM) and Zeta potential measurement. Initially 12 commercial samples were sourced and screened using TEM to obtain information on the average particle size. 3 of the commercial samples were then selected to carry forward to toxicity studies: EN-Z-1, EN-Z-2 and EN-Z-3. Synthesis of ZnO NPs was investigated in house by a flame spray pyrolysis and a polyol route; 2 synthesized samples were carried forward to the toxicity studies: EN-Z-4 and EN-Z-6. A coating was detected on the surface of EN-Z-1 (a colloidal suspension in water), identified as an aliphatic polyether (TGA-EGA, FTIR, XPS and NMR). Physically and chemically adsorbed H2O and CO2 was detected at the surface of the other 4 samples, which were all dry powders. A small amount hydrozincite was present in EN-Z-2 and EN-Z-3, identified by XRD, TGA-EGA, FTIR, XPS and NMR. Additionally, a small amount of diethylene glycol was identified on the surface of EN-Z-6, left over from synthesis.
The dispersion and solubility of the samples in water, Dulbecco’s modified Eagle Medium (DMEM) and bovine serum albumin (BSA) was investigated by DLS, PF-TEM and ICP-MS the results of which are presented in Chapter 6. Most samples showed similar dissolution kinetics and equilibrium solubility with the exception of EN-Z-1 which showed slower dissolution, presumably due to the coating present on
the NPs. Due to the equilibrium solubility of ZnO in DMEM, for concentrations of ZnO below 10 μg/ml, a significant amount is dissolved or re-precipitated as zinc carbonate. The agglomeration and solubility of ZnO NPs was found to increase when suspended in DMEM as compared to in water. The solubility of ZnO was found to increase with decreasing solution temperature (37 and 25 ºC). The addition of BSA was found to have a dispersing effect on ZnO NP suspensions. PF-TEM was investigated as an alternative technique to DLS for measuring size distributions of ZnO NPs in suspensions. It was determined that PF-TEM measurements give an accurate representation of the range of agglomerates in the suspension, however the sampling size is very low and data processing is time consuming and therefore the technique should be used as a complementary technique to DLS.
The cytotoxicity and genotoxicity of ZnO NPs was assessed using the MTT (thiazolyl blue tetrazolium bromide) and comet assay respectively. A lower toxicity was measured for ZnO NP powder samples as compared to the coated colloidal dispersion. The polymer coating enhances NP interaction with lipid membranes which may increase toxicity through increased cellular uptake followed by intracellular dissolution. Coated and uncoated ZnO NPs are taken up by A549 cells and were located in both the cytoplasm and the nucleus. In cells exposed to 100 μg/ml EN-Z-4 and EN-Z-6, smaller than primary particle size NPs were located in the cells, suspected to be zinc carbonate particles formed from dissolved zinc precipitating from solution in the extracellular or intracellular environment.
Altmetric
Altmetric