Bryce Feltis

Relating Cytotoxicity, Zinc Ions, and Reactive Oxygen in ZnO Nanoparticle-Exposed Human Immune Cells

Although zinc oxide (ZnO) nanoparticles (NPs) have been widely formulated in sunscreens, the relationship between reactive oxygen species (ROS) generation induced by these particles, zinc ions, and cytotoxicity is not clearly understood. This study explores whether these factors can be accurately quantified and related. The study demonstrates a strong correlation between ZnO NP-induced cytotoxicity and free intracellular zinc concentration (R (2) = .945) in human immune cells, indicating a requirement for NP dissolution to precede cytotoxicity. In addition, although direct exposure to ZnO NPs was found to induce cytotoxicity at relatively high concentrations, indirect exposure (via dialysis) was not cytotoxic, even at extremely high concentrations, highlighting a requirement for NP-to-cell contact. Elevated levels of ROS present in NP-exposed cells also correlated to both cytotoxicity and intracellular free zinc. Although the addition of antioxidant was able to reduce ROS, cytotoxicity to ZnO NPs was unaffected, suggesting ROS may be, in part, a result of cytotoxicity rather than a causal factor. This study highlights both the requirement and role of intracellular dissolution of zinc nanomaterials to elicit a cytotoxic response. This response is only partially ROS dependent, and therefore, modification of NP uptake and their intracellular solubility are key components in modulating the bioactivity of ZnO NPs.

Quantification of ZnO Nanoparticle Uptake, Distribution, and Dissolution within Individual Human Macrophages

The usefulness of zinc oxide (ZnO) nanoparticles has led to their wide distribution in consumer products, despite only a limited understanding of how this nanomaterial behaves within biological systems. From a nanotoxicological viewpoint the interaction(s) of ZnO nanoparticles with cells of the immune system is of specific interest, as these nanostructures are readily phagocytosed. In this study, rapid scanning X-ray fluorescence microscopy was used to assay the number ZnO nanoparticles associated with ∼1000 individual THP-1 monocyte-derived human macrophages. These data showed that nanoparticle-treated cells endured a 400% elevation in total Zn levels, 13-fold greater than the increase observed when incubated in the presence of an equitoxic concentration of ZnCl2. Even after excluding the contribution of internalized nanoparticles, Zn levels in nanoparticle treated cells were raised ∼200% above basal levels. As dissolution of ZnO nanoparticles is critical to their cytotoxic response, we utilized a strategy combining ion beam milling, X-ray fluorescence and scanning electron microscopy to directly probe the distribution and composition of ZnO nanoparticles throughout the cellular interior. This study demonstrated that correlative photon and ion beam imaging techniques can provide both high-resolution and statistically powerful information on the biology of metal oxide nanoparticles at the single-cell level. Our approach promises ready application to broader studies of phenomena at the interface of nanotechnology and biology.

Formation of zinc-containing nanoparticles from Zn2+ ions in cell culture media: Implications for the nanotoxicology of ZnO

Zinc ions generate a range of poorly soluble Zn-containing nanoparticles when added to commonly used mammalian cell culture media. The formation of these nanoparticles confounds the use of soluble Zn salts as positive controls during cytotoxicity testing of other Zn-containing nanoparticles, such as ZnO. These nanoprecipitates can either be crystalline or amorphous and vary in composition depending upon the concentration of Zn(II) within the medium. The cytotoxicity and immune system response of these nanoparticles in situ are similar to those of 30 nm ZnO nanoparticles. The low residual level of truly soluble Zn species (taken as species passing through a 2 kDa membrane) in cell culture media with serum is insufficient to elicit any appreciable cytotoxicity. These observations highlight the importance of employing appropriate controls when studying ZnO nanoparticle toxicity and suggest a re-evaluation of the conclusions drawn in some previous cytotoxicity studies.

Independent cytotoxic and inflammatory responses to zinc oxide nanoparticles in human monocytes and macrophages

Abstract Significant public and scientific concerns remain for the use of nanoparticles (NPs) in commercial products, particularly those applied topically for skin care. There are currently a range of metal oxides formulated into many sunscreens that are present at the nanoscale. In this study, we sought to determine the effect of the size and dispersion of one type of these NPs (zinc oxide) on immune cell function and cytotoxicity for human macrophages and monocytes, which are key cells for particle and debris clearance in the skin. We have found that particle size and coating, but surprisingly, not agglomeration, are key determinates of nanoparticle cytotoxicity in an in vitro culture system of human immune cells. Most importantly, we found that this nanoparticle-induced cellular immune signalling, can be decoupled from cytotoxicity and surface coating, so that at an equivalent cytotoxic load, smaller particles induce a greater cellular response.

Porous 45S5 Bioglass®-based scaffolds using stereolithography: Effect of partial pre-sintering on structural and mechanical properties of scaffolds

Scaffolds made from 45S5 Bioglass® ceramic (BG) show clinical potential in bone regeneration due to their excellent bioactivity and ability to bond to natural bone tissue. However, porous BG scaffolds are limited by their mechanical integrity and by the substantial volume contractions occurring upon sintering. This study examines stereolithographic (SLA) methods to fabricate mechanically robust and porous Bioglass®-based ceramic scaffolds, with regular and interconnected pore networks and using various computer-aided design architectures. It was found that a diamond-like (DM) architecture gave scaffolds the most controllable results without any observable closed porosity in the fired scaffolds. When the pore dimensions of the DM scaffolds of the same porosity (~60vol%) were decreased from 700 to 400μm, the compressive strength values increased from 3.5 to 6.7MPa. In addition, smaller dimensional shrinkage could be obtained by employing partially pre-sintered bioglass, compared to standard 45S5 Bioglass®. Scaffolds derived from pre-sintered bioglass also showed marginally improved compressive strength.

ZnO nanoparticles and organic chemical UV-filters are equally well tolerated by human immune cells

Abstract An important part of assessing the toxic potential of nanoparticles for specific applications should be the direct comparison of biological activities with those of alternative materials for the same application. Nanoparticulate inorganic ultraviolet (UV) filters, such as zinc oxide (ZnO), are commonly incorporated into transparent sunscreen and cosmetic formulations. However, concerns have been raised about potential unwanted effects, despite their negligible skin penetration and inherent advantages over organic chemical UV-filters. To provide useful application-relevant assessments of their potential hazard with/without UVA co-exposure, we directly compared cytotoxic and immune response profiles of human THP-1 monocytic cells to ZnO nanoparticles (30 nm) with bulk ZnO particulates (200 nm) and five conventional organic chemical UV-filters – butylmethoxydibenzoylmethane (avobenzone), octylmethoxycinnamate, octylsalicylate, homosalate and 4-methylbenzylidene camphor. High exposure concentrations of both organic and particulate UV-filters were required to cause cytotoxicity in monocyte and macrophage cultures after 24 h. Co-exposure with UVA (6.7 J/cm2) did not alter cytotoxicity profiles. Particle surface area-based dose responses showed that ZnO NPs were better tolerated than bulk ZnO. Organic and particulate UV-filters increased apoptosis at similar doses. Only particulates increased the generation of reactive oxygen species. Interleukin-8 (IL-8) release was increased by all particulates, avobenzone, homosalate and octylsalicylate. IL-1β release was only increased in macrophages by exposure to avobenzone and homosalate. In conclusion, direct effects were caused in monocytes and macrophages at similar concentrations of both organic UV-filters and ZnO nanoparticulates – indicating that their intrinsic cytotoxicity is similar. With their lower skin penetration, ZnO nanoparticles are expected to have lower bioactivity when used in sunscreens.

Titanium dioxide nanoparticles as radiosensitisers: An in vitro and phantom-based study

Objective: Radiosensitisation caused by titanium dioxide nanoparticles (TiO2-NPs) is investigated using phantoms (PRESAGE® dosimeters) and in vitro using two types of cell lines, cultured human keratinocyte (HaCaT) and prostate cancer (DU145) cells. Methods: Anatase TiO2-NPs were synthesised, characterised and functionalised to allow dispersion in culture-medium for in vitro studies and halocarbons (PRESAGE® chemical compositions). PRESAGE® dosimeters were scanned with spectrophotometer to determine the radiation dose enhancement. Clonogenic and cell viability assays were employed to determine cells survival curves from which the dose enhancement levels “radiosensitisation” are deduced. Results: Comparable levels of radiosensitisation were observed in both phantoms and cells at kilovoltage ranges of x-ray energies (slightly higher in vitro). Significant radiosensitisation (~67 %) of control was also noted in cells at megavoltage energies (commonly used in radiotherapy), compared to negligible levels detected by phantoms. This difference is attributed to biochemical effects, specifically the generation of reactive oxygen species (ROS) such as hydroxyl radicals (•OH), which are only manifested in aqueous environments of cells and are non-existent in case of phantoms. Conclusions: This research shows that TiO2-NPs improve the efficiency of dose delivery, which has implications for future radiotherapy treatments. Literature shows that Ti2O3-NPs can be used as imaging agents hence with these findings renders these NPs as theranostic agents.

Bismuth Sulfide Nanoparticles as a Complement to Traditional Iodinated Contrast Agents at Various X-Ray Computed Tomography Tube Potentials

Background: Investigation of the contrast enhancement effects on the CT images of the conventional iodinated contrast media (CM) or bismuth sulfide nanoparticles (Bi2S3 NPs) with increasing x-ray tube potentials. Materials and methods: A phantom was filled with iodinated CM or Bi2S3 NP solutions at concentrations ranging from 0 to 65 mM and the phantom was scanned with a CT scanner using tube potentials of 80, 100, 120 and 140 kVp at a fixed current of 200 mA. The CT contrast enhancement and contrast-to-noise ratios (CNR) of all scanned images were calculated. Results: This study demonstrated that greater image contrast was observed with Bi2S3 NPs compared to the iodinated CM at all concentrations and energies (80-140 kVp) tested. For example, at a concentration of 65 mM and tube potential of 140 kVp, a CNR enhancement of three to four times was observed for Bi2S3 NPs compared to iodinated CM. Even at a conventional tube potential of 80 KVp, one to two-fold increase in CNR was noted for Bi2S3 NPs. Results are also demonstrated that CNR increased with increasing concentration of Bi2S3 NPs at a fixed tube potential. Conclusion: These results highlight the effects of the different physical densities and atomic numbers of the two elements (I versus Bi), and their role in enhancing the probability of Compton scattering (CS) and photoelectric effects (PEs). As tube potential was increased, the CT numbers for both Bi2S3 NPs and iodinated CM decreased, consistent with a decrease in the probability of CS and PEs with increasing beam energy. However, the rate of decrease for iodinated CM was larger than Bi2S3 NPs. A good correlation was observed between the experimental results and the theoretical spectra based on linear attenuation coefficients .