Carbon-based nanomaterials are becoming more prominent in a variety of nanotechnology applications; for example, as contrast agent transporters for imaging. The growing use of these materials will result in greater exposure by both professionals and the public — and in mounting debate over the toxicity of such materials. A team of researchers at Ecole Polytechnique Fédérale de Lausanne, the Universities of Fribourg and Lausanne, and Laboratoire Cytopath, all in Switzerland, has found that exposure to carbon-based nanomaterials has a lethal effect on cell proliferation. Cells of the human lung-tumor line H596 were exposed in vitro to three types of carbon-based nanomaterials: nanotubes, with an average diameter of 20 nm and aspect ratios ranging from 80 to 90; nanofibers, measuring an average of 150 nm across and with aspect ratios ranging from 30 to 40; and submicron particles, with an aspect ratio of 1. Cells were exposed to each of the materials with concentrations of 0.002, 0.02 and 0.2 μg/ml for four days. The effect of the materials was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which is based on the accumulation of dark blue formazan crystals inside the living cells. The researchers quantified the concentration of the crystals with a spectrophotometer from Dynex Technologies of Chantilly, Va., to measure the absorbance at 570 nm. A linear relationship was established between the number of living cells and optical density, which enabled the quantification of changes in the rate of cell proliferation. In all cases, carbon nanoparticles proved to be the most toxic, followed by nanofibers and nanotubes. Although dose-dependent toxicity was predictable, the size-dependent toxicity was contrary to the researchers’ expectations, given that nanotubes have thus far been of primary concern within the science community. It is possible that the toxicity — apparent after only 24 hours — correlates to the presence of dangling bonds, which appear in high density in carbon nanoparticles as compared with nanotubes. Researchers used three types of carbon-based nanomaterials to determine their toxic effect on cells: (from left to right) multiwalled carbon nanotubes, carbon nanofibers and carbon black nanoparticles. Scale bars correspond to 2 μm. Images reprinted with permission from Nano Letters. Although the experiment demonstrates inhibition of cell proliferation, other studies find carbon-based nanomaterials to be less toxic than previously used materials such as metals and silicon. Researcher Arnaud Magrez speculates that the findings may differ because of the specific processing method of the experiments or because the dose limit under which the nanomaterials are not toxic is much lower than the concentrations used by the research team. For safe use in biological applications, the scientists believe that the amount of carbon-based nanomaterials must be extremely low. However, researcher László Forró warned, “Our observations point out that carbon-based nanomaterials can be toxic. This means, before using them as contrast agent transporters, we have to make sure that they do not act as toxic.” After a one-day treatment with 0.02 μg/ml of multiwalled carbon nanotubes, cells have lost their mutual attachments and retracted their cytoplasm. The nuclei appear smaller and more condensed. It is possible for the toxicity to be controlled with specific functionalization of the materials’ surfaces. Another important aspect is determining their stability inside the body and how they would be eliminated. Much higher concentrations are used in composites, and their use in construction or textiles would bring people in contact with them on a daily basis. Thus, it is not enough to know that these nanomaterials are toxic, which is why this group hopes to determine the origin of the cell death and to understand the cell death pathways. Forró and his team may extend their research to carbon-based nanomaterials with modified surfaces to find out which surfaces are the most toxic and what can be done to avoid them. Nano Letters, ASAP, May 20, 2006.