Cellular effects of carbon nanotubes

Materials in the nanometer range (nanomaterials, NMs) dramatically change their physico-chemical properties and in general act more toxic than the respective bulk materials. As human exposure to NMs in the future is expected to increase also in medical applications, the toxic mechanism of such NMs has to be studied in more detail. Carbon nanotubes (CNTs) possess features that make them excellent candidates for medical imaging and treatment. CNTs developed for these applications are shorter and more hydrophilic to avoid the adverse effects caused by the CNTs used in technical applications. Epithelial cells and also phagocytes are important cellular targets because they are present at all physiological barriers. Acute exposure may cause cytotoxic and genotoxic effects. As CNTs are not biodegradable also chronic effects and effects of cellular accumulation have to be taken into account. Chronic exposure may impair phagocyte function; induce chronic inflammation and cause lysosomal dysfunction. With the trend towards an extended life expectancy in the population chronic toxic effects become increasingly important. These effects have only rarely been investigated thus far. This project aims to systematically investigate the role of two important parameters in the toxic action of CNTs, functionalization with carboxyl groups and size, with respect to acute and chronic effects in phagocytic and non phagocytic cells. Carboxyl-functionalized and plain single walled CNTs and multi walled CNTs of different sizes in addition to carboxyl-functionalized and plain polystyrene beads as controls will be studied. After physicochemical characterization in physiological fluids, the cytotoxic action is assessed by viability assays and by monitoring of cellular interaction. Necrosis, apoptosis, proliferation and generation of radicals are studied. Similar data are collected after prolonged exposure. In order to highlight pathways important in chronic exposure, dys-regulated pathways are identified by whole genome transcriptome analysis. The effect of accumulation is evaluated by determination of the amount of lysosomes and lysosomal activity according to dye uptake and proteolytic activity of cathepsins B and L. Genotoxicity is assessed by micronucleus formation, detection of the double-strand break marker y-H2AX and hypoxanthine-guanine phosphoribosyltransferase forward mutation assay. Action on macrophage function is analyzed according to phagocytosis, chemotaxis, and production of cytokines, nitric oxide and superoxide. These investigations identify the role of carboxyl functionalization and of the relation of diameter to length in the cellular effects for CNTs upon short- and long-term exposure. These data help to develop less toxic and more biocompatible CNTs.

Carbon nanotubes (CNTs) are used in a variety of industrial applications. For their potential application in medical diagnosis and therapy, it is crucial that they are not toxic. Conventional (long) CNTs have been shown to be cytotoxic, pro-inflammatory and genotoxic. A high ratio of length to diameter (aspect ratio) was identified as decisive parameter for their toxicity. In order to be more biocompatible, CNTs for potential medical applications are shorter. In the project uptake and cellular action of short (<2m) plain and carboxylated CNTs with different diameters were studied and the effect of the suspension medium on size and surface charge was determined. We found that CNTs with nominal length of 2m were much shorter (<500nm) when prepared for the experiments; thin CNTs had the tendency to form bundles of around 1m length. Experiments focused on epithelial cells and phagocytes as they are present at the portals of entry for the most typical exposures to CNTs, inhalation and injection. These cells were exposed to CNTs for up to 4 weeks and cellular reaction studied using different cytotoxicity screening and mode of action assays (proliferation, apoptosis, generation of oxidative stress), and by whole-genome expression analysis. Furthermore, genotoxicity testing was performed. Although cells ingested all particles to a similar extent only the carboxylated thin single-walled CNTs caused adverse cellular effects in concentrations that might be relevant for human exposure. The absence of cell damage suggests that short CNTs could be used for medical applications.