Physical-chemical characterization of tungsten carbide nanoparticles as a basis for toxicological investigations.

One task in risk assessment of engineered nanoparticles is toxicological studies. A suitable interpretation of these investigations demands a comprehensive physical-chemical characterization. Here, we present an approach to gain well-dispersed nanoparticles in physiological media. Therefore, a step-by-step procedure is demonstrated on two different tungsten carbide nanopowders which can be transferred to other powders. The procedure includes a comprehensive powder characterization, followed by a preparation of a non-physiologic, electrostatically stable nanoparticle suspension and finally closes with investigations of the particles' behavior in different physiological media. Our study showed that the particles agglomerate in protein-free media. In this context, dependencies of mass- and surface-based nanoparticle concentrations as well as of different physiological media were analyzed. In the presence of bovine serum albumin (BSA) or serum, the agglomeration process is decelerated or, at the appropriate protein amount, prevented.

Physico-chemical characterization in the light of toxicological effects.

Toxicological investigations on nanoparticles require a comprehensive physico-chemical characterization to get useful information about the powder as well as the behavior of the suspended nanoparticles in water and physiological media. Therefore, we characterized the often used TiO(2) P25 and developed procedures to get stable, homogeneous, and well-defined nanoparticle suspensions. A titration of the zeta potential as a function of the pH yielded the conclusion that the TiO(2) suspension is stable at a pH of 4 or lower. In this region the zeta potential is higher than 30 mV, which guarantees a high stability of the suspended particles. Hence, a stable TiO(2) initial suspension was prepared in 0.1 mM HCl having a mean particle size of 170 +/- 5 nm, which was determined by dynamic light scattering. Furthermore, the initial suspension was added to different physiological media (0.9% NaCl solution, phosphate-buffered saline [PBS], Hanks balanced salt solution [HBSS], Dulbecco's modified Eagle's medium [DMEM]) for studying the agglomeration behavior. As a result, the agglomeration kinetics at the same TiO(2) concentration is independent of the used media. Investigations with PBS containing bovine serum albumin (BSA) and DMEM supplemented with 10% FBS revealed that these protein additions inhibit the agglomeration of the particles. Thus, the physiological media contains particles that are stabilized through the steric or electrosteric effect of BSA and of the proteins in FBS, respectively. Consequently, the particles keep their size from the initial suspension. Finally, our procedure demonstrated on TiO(2) P25 results in homogeneously suspended particles in physiological media. This definite status of the particles means an improvement for toxicological testing and understanding.

Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line.

Due to their increased production and use, engineered nanoparticles are expected to be released into the aquatic environment where particles may agglomerate. The aim of this study was to explore the role of agglomeration of nanoparticles in the uptake and expression of toxicity in the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1. This cell line was chosen as model because it is known to be amenable to culture in complete as well as greatly simplified exposure media. Nano-sized tungsten carbide (WC) with or without cobalt doping (WC-Co), two materials relevant in the heavy metal industry, were applied as model particles. These particles were suspended in culture media with decreasing complexity from L15 with 10% fetal bovine serum (FBS) to L15 to L15/ex, containing only salts, galactose and pyruvate of the complete medium L15. Whereas the serum supplement in L15 retained primary nanoparticle suspensions, agglomerates were formed quickly in L15 and L15/ex. Nevertheless, scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) elemental analysis revealed an uptake of both WC and WC-Co nanoparticles into RTgill-W1 cells irrespective of the state of agglomeration of nanoparticles. The localisation seemed to be restricted to the cytoplasm, as no particles were observed in the nucleus of cells. Moreover, reduction in cell viability between 10 and 50% compared to controls were observed upon particle exposure in all media although the pattern of impact varied depending on the medium and exposure time. Short-term exposure of cells led to significant cytotoxicity at the highest nominal particle concentrations, irrespective of the particle type or exposure medium. In contrast, long-term exposures led to preferential toxicity in the simplest medium, L15/ex, and an enhanced toxicity by the cobalt-containing WC nanoparticles in all exposure media. The composition of the exposure media also influenced the toxicity of the cobalt ions, which may dissolve from the WC-Co nanoparticles, with cells reacting much more sensitively toward cobalt ions in the absence of FBS. However, the toxicity observed by ionic cobalt alone did not explain the toxicity of the WC-Co nanoparticles, suggesting that the combination of metallic Co and WC is the cause of the increased particle toxicity of WC-Co. Taken together, our findings indicate that minimal exposure media can lead to rapid agglomeration of nanoparticles but that agglomeration does not prevent uptake into cells and the expression of toxicity.

Toxicity of tungsten carbide and cobalt-doped tungsten carbide nanoparticles in mammalian cells in vitro.

BACKGROUND: Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. OBJECTIVE: We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobalt-doped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. METHODS: We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendrocyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). RESULTS: Chemical-physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. CONCLUSIONS: Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.