This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the asreceived CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma -mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time-and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

This study compares the cytotoxicity to cultured mammalian cells of nine different single-walled carbon nanotube (SWNT) products synthesized by a variety of methods and obtained from a cross section of vendors. A standard procedure involving sonication and centrifugation in buffered bovine serum albumin was developed to disperse all the SWNTs in a biocompatible solution to facilitate comparisons. The effect of the SWNTs on the proliferative ability of a standard cell line was then assessed. Of the nine different SWNT materials tested, only two were significantly toxic, and both were functionalized by carboxylation from different vendors. This was unexpected because carboxylation makes SWNTs more water soluble, which would presumably correlate with better biocompatibility. However, additional purification work demonstrated that the toxic material in the carboxylated SWNT preparations could be separated from the SWNTs by filtration. The filtrate that contained the toxic activity also contained abundant small carbon fragments that had Raman signatures characteristic of amorphous carbon species, suggesting a correlation between toxicity and oxidized carbon fragments. The removal of a toxic contaminant associated with carboxylated SWNTs is important in the development of carboxylated SWNTs for pharmacological applications.

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that approximately 6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.