Carbon nanotube biocompatibility with cardiac muscle cells

Purified carbon nanotubes are new carbon allotropes, sharing similarities with graphite, that have recently been proposed for their potential use with biological systems as probes for ill vitro research and for diagnostic and clinical purposes. However the biocompatibility of carbon nanotubes with cells represents an important problem that, so far, remains largely uninvestigated. The objective of this in vitro study is to explore the cytocompatibility properties of purified carbon nanofibres with cardiomyocytes. Cardiac muscle cells from a rat heart cell line H9c2 (2-1) have been used. Highly purified single-walled nanotubes (SWNTs) were suspended at the concentration of 0.2 mg ml(-1) by ultrasound in complete Dulbecco's modified Eagle's medium, and administered to cells to evaluate cell proliferation and shape changes by light microscopy, cell viability by trypan blue exclusion, and apoptosis, determined flow cytometrically by annexin/PI staining. Microscopic observation evidenced that carbon nanotubes bind to the cell membrane, causing a slight modification in cell shape and in cell count only after three days of treatment. Cell viability was not affected by carbon nanotubes in the first three days of culture, while after this time, cell death was slightly higher in nanotube-treated cells (p = ns). Accordingly, nanotube treatment induced little and non-significant change in the apoptotic cell number at day 1 and 3. The effect of nanotubes bound to cells was tested by reseeding treated cardiomyocytes. Cells from a trypsinized nanotube-treated sample showed a limited ability to proliferate, and a definite difference in shape, with a high degree of cell death: compared to reseeded untreated ones, in SWNT-treated samples the annexin-positive/PI-negative cells increased from 2.9% to 9.3% in SWNT (p < 0.05, where p < 0.05 defines a statistically significant difference with a probability above 95%), and the annexin-positive/PI-positive cells increased from 5.2% to 18.7% (p < 0.05). However, overtime cells from a trypsinized nanotube-treated sample continued to grow, and partially recovered the original shape. In conclusion our results demonstrate that highly purified carbon nanotubes possess no evident short-term toxicity and can be considered biocompatible with cardiomyocytes in culture, while the long-term negative effects, that are evidenced after reseeding, are probably due to physical rather than chemical interactions.