The ongoing challenge of determining carbon nanotube toxicity

(Nanowerk Spotlight) It seems that with every new study on the toxicity of nanomaterials there remain more questions afterwards than before. Environmental, occupational and public exposure to engineered nanoparticles will increase dramatically in the near future as a result of the widespread use of nanoparticles for consumer and industrial products. The extent of future exposure to nanoparticles associated with these new products is still unknown.
So far only limited data is available regarding carbon nanotube (CNT) toxicity. As a result still not much is known about their impact on biological systems including humans. Discussions regarding the potential risks of their widespread use, as well as their possible positive impact are just beginning to take place.
In order to provide a basis for comparison to existing epidemiological data, a group of researchers in Switzerland and Germany have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human cancer cells. The cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference.
While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibers at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.
CNTs are generally contaminated, among others, by amorphous or graphitic carbon, fullerenes and metal catalyst residues. For most technical applications, however, CNTs of high purity are required and conventional purification methods are based on acidic and optional oxidative treatment. Furthermore, because of their geometry and hydrophobic surface, CNTs have a tendency to form agglomerates with a bundle-like form. Various agents, which by themselves are cytotoxic, are being used to disperse CNTs.
The existence of contaminants as well as dispersant agents complicates the interpretation of results of toxicological studies on CNTs (and other engineered nanoparticles in general).
A recent study ("Oxide Nanoparticle Uptake in Human Lung Fibroblasts: Effects of Particle Size, Agglomeration, and Diffusion at Low Concentrations") on the in vitro uptake of oxide nanoparticles in human lung cells has underlined the need for thorough nanomaterials characterization prior to in vitro biological evaluation in order to make at all statements on cytotoxicity.
Another study revealed evidence that the choice of the time point at which possible effects are assessed as well as the kind of parameter used to quantify latter effects are critical for the toxicity evaluation ("In vitro evaluation of possible adverse effects of nanosized materials").
The paper by the Swiss-German team appears to be the first study ("The degree and kind of agglomeration affect carbon nanotube cytotoxicity") were out of the same CNT raw material different qualities of suspensions were produced, carefully characterized and with the same in vitro test assessed. In contrast to nanoparticles, the researchers observed that agglomerated CNT showed a more adverse effect then well-dispersed CNT.
Morphology changes of MSTO-211H cells after 3 days of exposure to 15 µg/ml of different fractions of CNTs and asbestos. (a) Untreated MSTO-211H cell culture and (b) cell culture exposed to asbestos. Arrows indicate needles of asbestos. (c) Cells treated with CNT-agglomerates were round-shaped and lost their adherence on the cell culture plate. Arrow point to CNT agglomeration. (d) Cells exposed to CNT-bundles showed no visible morphological changes compared to the control cells. (e) Effect of CNT-pellet fraction. Non-tubes material agglomerated during the incubation period to micro-sized structures. (f) Cells incubated with CNT raw material. Arrow indicates agglomerated CNT material: scale bar 20 µm. (Reprinted with permission from Elsevier)
"In our recent study we tried to answer basically two questions: 1) in how far the degree of dispersion and agglomeration affects CNT cytotoxicity and 2) in how far CNTs cytotoxicity is comparable to that of crocidolite asbestos" Dr. Arie Bruinink explained to Nanowerk. "Asbestos was included in our study as references not only because of the similarities in shape and possibly the persistency of the material, but also since numerous reports have been published on the effects of this material on animals and cells."
Bruinink is a researcher in the Materials-Biology Interactions Lab at the Empa research institute in St. Gallen/Switzerland. Other researchers involved in the study are from ETH Zurich and the Max Planck Institute for Solid State Research in Stuttgart/Germany.
The researchers used a form of human cancer cells, the mesothelioma cell line MSTO-211H, because this provided the best possible comparison to existing toxicological data of asbestos. Further a biocompatible method was developed to disperse CNT which is based on PS80, a widely used food additive and supplement for certain cell culture medium.
Light microscopic analysis of CNT treated cultures revealed CNT induced morphological changes of the cells and that these changes depended on the type of CNT preparation.
"We found that dispersed CNT-bundles were less cytotoxic than micron-size agglomerates of CNTs" says Bruinink. "CNT-agglomerates evoked similar effects on cell morphology and cell performance as the asbestos reference. Additionally, the study underlines the need for in-depth materials characterization used for toxicological studies."
The researchers point out that the direct comparison of CNT-bundles and CNT-agglomerates both with comparable amount of impurities clearly showed that the quality of the dispersion of CNTs is of extreme importance and is needed to know to make an accurate interpretation of the cytotoxic data.
"What our study does is underline the need for thorough materials characterization prior to toxicological studies" says Bruinink. "Critical features that seem to determine CNT toxicity are the presence of carboneous material and the degree of CNT dispersion but not the content of entrapped contaminants such as yttrium and nickel."
Michael Berger By – Michael is author of three books by the Royal Society of Chemistry:
Nano-Society: Pushing the Boundaries of Technology,
Nanotechnology: The Future is Tiny, and
Nanoengineering: The Skills and Tools Making Technology Invisible
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