Oxidative Stress on Human Cells in the Presence of Nano-Sized Titanium Dioxide

Nanosized TiO2 (nanoTiO2) is one of the most widely used nanomaterials, with applications ranging from paints, self-cleaning coatings, pharmaceuticals, to food and cosmetics. In spite of this massive use of nanoTiO2, its biological activity and toxicity remains subject of the intense debate. In particular, there is still considerable uncertainty in the current understanding of the relationship between physico-chemical parameters of nanoTiO2, such as crystalline phase, size, aspect ratio, surface properties, surface defects and surface chemistry and its potential toxicological effects. Motivated by this general problem, this thesis provides a multidisciplinary experimental insight into to the toxicity and photo-toxicity of various forms of nanoTiO2. Firstly, since all of the widely accepted models of nanoTiO2 toxicity involve reactive oxygen species (ROS), electron spin resonance (ESR) in combination with spintrapping was used to measure ROS formation efficiency for the two most industrially important polymorphs of TiO2, anatase and rutile. The study was performed in an unprecedentedly broad range of particle sizes: 3.8 nm to 150 nm and 5 nm to 215 nm, for anatase and rutile nanoTiO2, respectively. Moreover, the photocatalytic and toxic properties of custom-made anatase-based TiO2 nanowires (with a diameter of 35 nm and a length of 0.5-1 µm) were characterized for the first time. For pure anatase nanoTiO2, the maximum ROS generation efficacy was found for nanoparticle sizes in the range of 25 – 30 nm. The ROS generation efficacy of the custom-made TiO2 nanowires was ca. 30% lower, being close to that of the commercial anatase nanoTiO2 with primary grain sizes of 5.3 nm. Secondly, this thesis addressed challenging, complex and still poorly understood processes occurring when nanoTiO2 particles are brought into contact with living cells. In particular, the comparative nanotoxicity study towards human melanoma Lu1205 and WM793 cell lines was performed for three selected nanomaterials: the custom-made anatase-based TiO2 nanowires, the commercial anatase nanoTiO2 with a primary particle size of 5.3 nm and similar in vitro ROS formation efficacy, and the industrial photocatalytic standard, P25 Degussa (a formulation consisting of 80% anatase and 20% rutile, with primary grain sizes of 25 nm). This comparative nanotoxicity study was performed using very low concentrations of nanoTiO2 (2 – 2.5 µg/mL), considerably lower than that applied in the majority of previous ex vivo cell culture studies. A wide palette of spectroscopic and microscopic methods was applied to verify the cellular response to the presence of selected nanoTiO2-based particles, both in the dark and under illumination with UV-A light of low intensity (λ = 365 nm, 1 mW/cm2). In particular, to detect intracellular ROS in cells incubated with the selected nanoTiO2, ESR spin-trapping with an intracellular spin-trap (ACP) and histochemical detection with a ROS-sensitive dye (NBT) were used. These studies were completed by optical microscopy visualization of ROS-induced changes in cell morphology and actin filament organization (FITC-phalloidin staining). Moreover, atomic force microscopy (AFM) was used to follow the changes in the cell topology and elastic properties (AFM force spectroscopy) of cells incubated with nanoTiO2 and exposed to photo-oxidative stress under UV-A illumination. All of these techniques revealed, either directly or indirectly, deleterious effects induced by incubation of living cells with nanoTiO2 and by photo-oxidative stress under illumination with UV-A. Primarily, optical microscopy visualization of selectively stained actin stress fibers and AFM topography images pointed to a rapid disorganization of the actin cortex for cells exposed to photo-oxidative stress in the presence of nanoTiO2. These results corroborated the changes in the cell shape and elasticity measured by AFM. The cell elasticity measurements, quantitatively described in terms of local Young's modulus values, pointed to an internal remodeling of the cytoskeleton in cells exposed to the presence of nanoTiO2 and photo-oxidative stress. The custom made anatase-based TiO2 nanowires revealed generally stronger cytotoxic effects than the anatase nanoTiO2 with a particle size of 5.3 nm, despite having similar photocatalytic properties in vitro. These observations were confirmed by AFM measurements of the cellular elasticity, histochemical detection with a ROS-sensitive dye (NBT) and by intracellular detection of ROS with ESR spin-trapping. The latter technique provided also a new important observation pointing to the enhanced ROS formation in cells incubated with nanoTiO2 in the dark. Overall, the findings stemming from cell toxicity studies were much more complex than those obtained from measurements of ROS generation efficacy in the presence of nanoTiO2 in vitro. In contrast to a marked evolution of ROS generation efficacy as a function of the primary grain size in vitro, the cellular work pointed to the lack of direct and simple correlations between the physical parameters of nanoTiO2, such as particles size, their aspect ratio, and aggregation state, and cell toxicity.

Process monitoring of alumina nanoparticle synthesis by inductively coupled RF thermal plasma

Jong-Won Shin

Since the first inductively coupled plasma (ICP) torch was developed in the 60's, RF thermal plasmas have found a variety of applications in material processing such as crystal growth, thermal spray coatings, spheroidization and vaporization of refractory materials. In recent decades, inductively coupled thermal plasmas have been used for the synthesis of high purity nanoparticles, thank to their remarkable advantages such as high energy density, variable operating pressures and low product contamination. The formation of nano-sized particles in thermal plasmas particularly using solid refractory precursor is a complex process. Hereby the injected precursor powders are heated by the high temperature of the thermal plasma. The heated powders are vaporized and decomposed into vapor species. During the following cooling, the vapor species are condensed to nano-sized particles. In this synthesis process, characterized by evaporation-condensation, the properties of the synthesized particles such as particle size, size distribution, phases and morphology are affected by various process parameters. Gas pressure, flow rates of the different torch gases, size of the precursor powders and powder loading in the plasma are considered as the influencing process parameters. In order to optimize and control the process for tailor-made nanoparticle synthesis, it is necessary to understand the effects of the process parameters on plasma properties and on the particle-plasma interactions resulting in final properties of the synthesized powders. In the present work, firstly, the enthalpy probe technique, a well-established plasma diagnostic tool for high pressure thermal plasma, has been applied to characterize the inductively coupled Ar-H2 thermal RF plasma used for alumina nanoparticle synthesis at different operating conditions. The plasma enthalpy has been determined, and the plasma temperature under a given gas pressure could subsequently be calculated assuming local thermodynamic equilibrium (LTE), once the gas composition has been determined by mass spectrometry. The gas velocity of the plasma jet could also be obtained by using the Bernoulli equation and stagnation pressure. Secondly, the influence of flow rates of central gas and precursor feed rates on the Al2O3 precursor evaporation has been investigated by process monitoring. Optical emission spectroscopy (OES) and laser light extinction (LE) measurements have been carried out to monitor in-situ the injection of the alumina precursors and to obtain information on the ongoing precursor evaporation. The emission line intensities of the aluminum vapor were used to determine the dependence of process parameters on precursor evaporation. Furthermore, the laser light extinction was used to calculate the number density of non plasma-treated powders, from which the number fraction of evaporated powders could be deduced. The size and morphology of the synthesized particles have been characterized ex-situ. Finally, the influence of quenching conditions on the condensation and formation of the nano-sized alumina particle has been studied by process analysis of the gas phase reactions of alumina. Optical emission spectroscopic was performed at different axial positions to monitor the gas phase reactions of the vaporized particles. Axial profiles of the emission line intensities of the main vapor species of alumina, Al(g) and AlO(g), were compared with the results of the enthalpy probe measurements and the thermal decomposition reactions of alumina found in literature. The results allow explanation of the alumina reactions in the thermal plasma as well as gives indications for the optimal axial position of the quenching gas injection. In addition to the determination of the optimal quenching position, the influence of the flow rates of quenching gas on the nanoparticle formation has been investigated. The synthesis of the submicron alumina using vapor-condensation principle predominantly leads to various thermodynamically metastable crystal structures called transition alumina. Therefore, the occurrence of different transition aluminas is discussed with regards to the flow rates of the quenching gas. The results presented in this thesis show that the synthesis process of the nanoparticle can be described on the basis of experimental results and basic information on the powder materials. The explanation of the influence of process parameters on powder evaporation and nanoparticle formation shows the usefulness of the in-situ plasma process monitoring, and the large potential for optimizing and controlling the nanopowder synthesis process in an inductively coupled RF thermal plasma.

EPFL2006

Photocatalytic and phototoxic properties of TiO2-based nanofilaments: ESR and AFM assays

László Forró, Andrzej Kulik, Arnaud Magrez, Katarzyna Pierzchala, Andrzej Sienkiewicz

There is uncertainty in understanding of the relationship between physico-chemical parameters of nanosized titanium dioxide (nano-TiO2) and its toxicity when brought into contact with living cells. This study provides a multidisciplinary experimental insight into the toxicity and phototoxicity of the custom-made TiO2-based nanowires (TiO2-NWs). We employed electron spin resonance (ESR) to detect reactive oxygen species (ROS) generated in aqueous suspensions of TiO2-NWs and combined these results with atomic force microscopy (AFM) to trace the onset of toxic effects towards human melanoma cells. The cells were treated with low concentrations (similar to 2.5 mu g/ml) of TiO2-NWs and Degussa P25. High-resolution AFM surface topography and cell elasticity measurements revealed toxic effects both in cells incubated with TiO2-NWs in the dark and exposed to the photo-oxidative stress under UVA radiation. In contrast to ROS generation efficacy in the absence of cells in vitro, no direct correlation was found between the physical parameters of nano-TiO2 and cell toxicity.

Informa Healthcare2012

Innovative photo-Fenton catalysis by PE-FeOx films leading to methylene blue (MB) degradation: Kinetics, surface properties and mechanism

Huiyu Dong, Juan Kiwi, César Pulgarin, Rosendo Sanjines, Laura Victoria Suárez Murillo

This study reports an innovative preparation polyethylene-FeOx (PE-FeOx films) leading to the degradation of the methylene blue (MB) dye under low intensity sunlight/visible light (>400 nm). The intermediate highly oxidative radicals generated by PE-FeOx leading to MB discoloration/degradation in the presence of H2O2 were identified. The relative contribution to the MB discoloration by the Fe ions leached from the PE-FeOx films into solution was determined quantitatively. This allows estimating the contribution of the heterogeneous and of the homogeneous leading to MB-degradation. The shift in the peak binding energy (BE) for Fe was detected by photoelectron spectroscopy (XPS) after the MB degradation providing the evidence for redox catalysis occurring during MB-degradation at the PE-FeOx surface. The particle size and roughness of the PE-FeOx surfaces were determined by atomic force spectroscopy (AFM). The spectral properties of PE-FeOx films are reported and insight is provided for the mechanism of MB-degradation. (C) 2016 Elsevier B.V. All rights reserved.

Elsevier2016

Process monitoring of alumina nanoparticle synthesis by inductively coupled RF thermal plasma

Jong-Won Shin

EPFL2006