Philip S. Casey

Effects of surface chemistry on cytotoxicity, genotoxicity, and the generation of reactive oxygen species induced by ZnO nanoparticles.

The relationship between the toxicity of zinc oxide (ZnO) nanoparticles (NPs) and their surface chemistry was investigated. Cytotoxicity, genotoxicity, and the ability to generate reactive oxygen species (ROS) were assessed for well-characterized ZnO NPs whose surface chemistry was varied from its pristine state by coating with oleic acid (OA), poly(methacrylic acid) (PMAA), or components adsorbed from cell culture medium (medium-soaked). It was found that uncoated NPs showed ROS accumulation and diminished cell viability whereas all tested surface coatings assisted in reducing ROS production and cytotoxicity. The ability of coatings to reduce the cytotoxicity of ZnO NPs was ranked in the following order: medium-soaked ≈ PMAA > OA. However, PMAA-coated ZnO had significant genotoxicity compared to uncoated ZnO and the other coated NPs, highlighting the need to investigate thoroughly the effects of NP surface modification on both cytotoxicity and genotoxicity assays. The lowest toxicity was achieved with a surface coating of components from a cell culture medium.

Electrical conductivity and space charge in LDPE containing TiO/sub 2/ nanoparticles

Electrical conductivity (DC) and space charge accumulation were studied in samples of low density polyethylene to which nano-sized and micro-sized TiO/sub 2/ (anatase) particles and a dispersant had been added. Sample thicknesses were in the range 150-200 /spl mu/m. At applied field strengths of 10 and 20 kV/mm, the conductivity at 30 /spl deg/C, measured in vacuum in samples containing 10 % w/w nano-sized TiO/sub 2/, decreased by 1-2 orders of magnitude relative to samples with dispersant but without TiO/sub 2/, and by three orders of magnitude at 70 /spl deg/C. In air at 30 /spl deg/C the corresponding decrease was an order of magnitude at 10 kV/mm, and a factor of four at 20 kV/mm. In samples containing 10 % w/w micro-sized TiO/sub 2/ the conductivity increased in air and in vacuum, but only by factors in the range 2-10 depending on temperature and field. Space charge profiles were obtained using the laser-intensity-modulation-method (LIIMM), irradiating both surfaces of the sample. The micro-sized TiO/sub 2/ particles are associated with increased charge injection from the electrodes and increased charge trapping in the sample bulk, increasing the conductivity overall. The nano-sized particles generate very little charge in the sample bulk, but render the electrodes partially-blocking and so lower the conductivity.

Conductivity and space charge in LDPE containing nano- and micro-sized ZnO particles

DC conductivity and ac impedance measurements were made in air and in vacuum on samples of low density polyethylene to which nano-sized and micro-sized ZnO particles and a dispersant had been added. The samples were 150-200 mum thick. The temperature range was 30-70degC. The temperature dependence of the vacuum dc conductivity in samples containing the dispersant and 10% w/w nanosized ZnO followed an Arrhenius relationship closely, the conductivity being 1-2 orders of magnitude lower than that of a sample containing dispersant only. The addition of 10% w/w microsized ZnO had very little effect on the dc conductivity. The ac measurements were made in the frequency range 10 mHz-1 MHz. Addition of nanoparticles increased the ac conductivity at higher frequencies but decreased it at lower frequencies, the cross-over frequency increasing with increasing temperature. The real part of the relative permittivity of samples with nanoparticles was increased relative to that of samples containing dispersant only, at all temperatures, but the corresponding values in samples with microparticles were unchanged, within experimental error. Space charge profiles were obtained using the laser-intensity-modulation-method (LIMM). Space charge densities of order 300 Cm-3 were measured in the bulk near the electrodes, several hours after poling at field strengths around 30 kV/mm.

Comparison of dermal absorption of zinc from different sunscreen formulations and differing UV exposure based on stable isotope tracing.

In a pilot study to determine if zinc (Zn) from zinc oxide nanoparticles in sunscreen can penetrate human skin in vivo, nanoparticles (~30nm) of a stable isotope (52% (68)Zn enrichment) were incorporated into an essentially phytochemical-based formulation and applied to the backs of 3 human subjects twice daily for 5 days during the Southern Hemisphere winter. Blood and urine were collected prior to application and at regular intervals and up to 50 days. As observed in a larger outdoor trial following this pilot study but with a different formulation and with UV exposure: values of (68)Zn in blood continued to increase beyond the 5 day application phase with the highest measurement at 14 days after the first application; variable amounts of the (68)Zn tracer were observed in urine; and the amounts of extra Zn added to blood were small and indicate very low levels of absorption (minimal estimate <0.01% of the applied dose) through the skin. Reasons for differences in absorption detected in the stable isotope trials and previous investigations include: the sensitivity of the stable isotope method; the duration of the investigations; the number of applications of sunscreen formulation; in vitro methods with excised skin; lack of measurement of blood and urine; no skin flexing; and lack of UV exposure.

Effects of iron or manganese doping of ZnO nanoparticles on their dissolution, ROS generation and cytotoxicity

ZnO nanoparticles (NPs) have found wide applications due to their unique optoelectronic and photocatalytic characteristics. However, their safety aspects remain a concern especially considering that they are common constituents in sunscreen formulations. Production of reactive oxygen species (ROS) and dissolution of particles to ionic zinc are identified as two key determinants for the toxicity of ZnO. Doping transitional metals into the ZnO lattice has been demonstrated to be effective in suppressing NP dissolution and lowering cytotoxicity. However, the possibility of triggering excessive ROS by these transition metals has not been discussed. In this study, the behaviour of particle dissolution and the ability for ROS generation of iron and manganese doped ZnO NPs were studied in detail and further correlated with their cytotoxicity. Although Fe doping significantly reduced the level of released Zn ions, the cytotoxicity did not decrease as expected compared with undoped ZnO because more ROS were activated and damaged cells through oxidative stress. For Mn-doped ZnO NPs, their dramatically elevated intracellular ROS level was associated with high cytotoxicity compared with undoped ZnO even though both of them released similar amounts of free Zn ions.

An Experimental and Computational Approach to the Development of ZnO Nanoparticles that are Safe by Design.

Zinc oxide nanoparticles have found wide application due to their unique optoelectronic and photocatalytic characteristics. However, their safety aspects remain of critical concern, prompting the use of physicochemical modifications of pristine ZnO to reduce any potential toxicity. However, the relationships between these modifications and their effects on biology are complex and still relatively unexplored. To address this knowledge gap, a library of 45 types of ZnO nanoparticles with varying particle size, aspect ratio, doping type, doping concentration, and surface coating is synthesized, and their biological effects measured. Three biological assays measuring cell damage or stress are used to study the responses of human umbilical vein endothelial cells (HUVECs) or human hepatocellular liver carcinoma cells (HepG2) to the nanoparticles. These experimental data are used to develop quantitative and predictive computational models linking nanoparticle properties to cell viability, membrane integrity, and oxidative stress. It is found that the concentration of nanoparticles the cells are exposed to, the type of surface coating, the nature and extent of doping, and the aspect ratio of the particles make significant contributions to the cell toxicity of the nanoparticles tested. Our study shows that it is feasible to generate models that could be used to design or optimize nanoparticles with commercially useful properties that are also safe to humans and the environment.

Reducing the cytotoxicity of ZnO nanoparticles by a pre-formed protein corona in a supplemented cell culture medium

The safety of zinc oxide (ZnO) nanoparticles (NPs) remains a critical concern considering that they are a common constituent in cosmetics and sunscreen formulation. In our study, the cytotoxicity of pristine ZnO NPs in a human hepatocellular carcinoma (HepG2) cell line was found to be significantly reduced when the NPs were pre-incubated in a supplemented cell culture medium for 24 h prior to actual cell exposure. These pre-coated particles developed a stable protein layer on their surfaces, which facilitated further protein adsorption during the cell culture process. The amount of proteins adsorbed on pre-coated NPs was significantly larger and the affinity between the NPs and proteins was stronger, which inhibited both ROS generation and ZnO dissolution and resulted in lower cytotoxicity compared to pristine NPs. Our studies on the continued evolution of a hard protein corona on ZnO NPs in a supplemented cell culture medium and its effects on cytotoxicity demonstrate an effective and convenient way to achieve safe biomedical and environmental applications of ZnO NPs and may be extrapolated to other classes of engineered nanomaterials.

Effects of aspect ratio (AR) and specific surface area (SSA) on cytotoxicity and phototoxicity of ZnO nanomaterials

With growing interests in the applications of high aspect ratio (AR) ZnO nanomaterials (NMs), their potential toxicity to human health and nature environment remains a critical concern because these NMs have extremely large specific surface area (SSA) that could dramatically enhance interactions between NMs and surrounding molecules. In addition, originated to their intrinsic photocatalytical activity, ZnO NMs may induce phototoxicity under environmentally UV exposure. In this paper, ZnO spheres, grains, rods and needles with increasing AR (from 1.1 to 17.8) and various SSA (from 2.0 m(2) g(-1) to 27.4 m(2) g(-1)) were used to study the influence of AR and SSA on viabilities of WIL2-NS human lymphoblastoid cells in both dark and UV conditions. In dark, SSA was the main influencing factor for toxicity of NMs with low AR (spheres, grains and rods) and smaller SSA leads to higher cell viability. However, ZnO needles with the smallest SSA did not further enhance cell viability which may be possibly attributed to its high AR. Cell viability measured under UV irradiation did not show evident relationship with SSA or AR, but indicated that lower photocatalytic activity could mitigate phototoxicity.

Size-dependent cytotoxicity and genotoxicity of ZnO particles to human lymphoblastoid (WIL2-NS) cells

The relationship between particle size and cytogenotoxicity of ZnO particles was systematically studied in vitro using WIL2‐NS human lymphoblastoid cells. Before toxicity measurements, the ZnO particles of three different sizes (26 nm, 78 nm, and 147 nm) were well characterized for their physical and chemical properties to ensure that variations in other properties including surface chemistry and particle shape, which also may influence particle toxicity, were minimal. Cell viability testing showed that increasing cytotoxicity was associated with decreasing particle size. Both the dissolution kinetics of ZnO particles in supplemented cell culture medium and the apparent numbers of ZnO particles internalized by cells were size dependent and showed strong correlation with cytotoxicity. Genotoxicity, as measured by micronucleus formation, was significantly enhanced in the presence of the medium‐sized and large‐sized particles. The observation that necrosis increased with smaller‐ sized particles but micronuclei were present to a greater extent with larger‐ sized particles suggests that different mechanisms of cell damage induction or susceptibilities are operating depending on particle size. Environ. Mol. Mutagen. 56:767–776, 2015.

A comparative interlaboratory study on photocatalytic activity of commercial ZnO and CeO2 nanoparticles

AbstractPhotocatalytic activity (PCA) was one of a number of physicochemical end points identified by the Organisation for Economic Cooperation and Development (OECD) as relevant to environmental safety and human health as part of their Sponsorship Programme for the Testing of Manufactured Nanomaterial. Photoactive surfaces can produce reactive oxygen species including free radicals which have the potential to cause oxidative stress in tissue or even oxidative damage to DNA. Here we report a study that involved three laboratories in Australia that independently characterised the PCA of commercially available ZnO and CeO2 NPs provided by the OECD programme. This inter-laboratory comparison found that PCA is a stable characteristic of NPs which was insensitive to variations in interlaboratory protocols and with ZnO NPs being more photoactive (by an order of magnitude) than CeO2 NPs. Comparisons were made between NPs of different sizes and the effect of the presence or absence of a surface coating on PCA. Because the competition between surface and volume effects determined PCA, a critical particle size was found for CeO2 NPs to achieve maximum PCA. The presence of a surface coating appeared to significantly mitigate, but not eliminate PCA.