The structural and electronic properties of SWNTs lend themselves to a variety of biomedical applications involving the detection and treatment of diseases, most notably cancer [1
]. For example, the structural change in DNA upon shifting from the B to Z conformation sufficiently perturbs the electronic structure of SWNTs such that the change can be detected optically from living cells that have taken up DNA-SWNT complexes [7
]. This and other works demonstrate how CNTs can be used as sensors within living cells [8
]. In another example, exposing cells containing SWNTs to near infrared radiation kills the cells due to the efficient optical-to-thermal energy conversion of SWNTs, demonstrating that they can potentially be used in targeted cancer therapies to eliminate cancer cells [10
]. Finally, there are a number of reports that CNTs facilitate the transport of bound oligonucleotides, peptides, and proteins across the plasma membrane [1
]. However, despite these and other intracellular applications not listed here, there remain technical challenges towards realizing the potential benefits of CNTs in biomedicine. Namely, CNTs are extremely hydrophobic, bundle together, and are insoluble in water.
Two approaches have been used to modify the hydrophobic surface of CNTs to make them water soluble and biocompatible. The first has been to debundle and disperse CNTs in aqueous solution by covalently attaching water soluble substances to the CNT surface, and the second has involved the noncovalent association of material to the CNT surface [20
]. In both approaches, a wide variety of organic adducts and biological materials have been used successfully including oligonucleotides [7
], peptides [14
], proteins [8
] (most notably, bovine serum albumin (BSA) [60
]), an assortment of polymers [64
], and various cell culture media formulations [19
]. While covalently attaching material to CNTs is advantageous for many applications, one serious drawback is that the covalent attachment introduces defects in the surface of the CNTs that often interfere with the electronic and optical properties that make CNTs so useful.
Beyond CNT dispersal, another challenge in the field is assessing whether CNTs are inherently cytotoxic [73
]. At present, there are roughly as many publications reporting no apparent cytotoxicity [10
], as there are reporting varying degrees of significant cytotoxicity [68
]. Two major considerations in this area are how the CNTs are presented to the organism and the purity and concentration of the CNTs. For example, pulmonary toxicity of SWNTs has been established when large doses of dry, unpurified SWNTs have been blown into the lungs of rats [89
]. This method of presentation is not relevant to the small measured doses of CNTs that would be used in chemotherapy and drug delivery. In fact, the biodistribution of chemically modified SWNTs injected into mice or rabbits was studied recently, and the CNTs were reported to be cleared rapidly with no evidence of toxicity [85
]. CNT purity is also absolutely crucial. Many CNT syntheses use metal catalysts that are known to be toxic. Such impurities, and other carbonaceous impurities, must be removed from the samples in order to reach conclusions about inherent CNT toxicity, and it is not always clear from the published reports that they have been removed. Finally, many accounts of CNT toxicity have used MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as a reporter of cell viability, and it was recently shown by Worle-Knirsch et al. that MTT itself binds to CNTs (quenching its fluorescence) and thereby introducing uncertainty in this assessment of toxicity [65
]. In summary, while the question of whether CNTs have long-term toxicity in biomedical applications requires further research, early reports raising the alarm of toxicity in model cell culture systems have not been adequately verified.
Recently, our group reported that HiPco SWNTs, dispersed in a peptide solution or in media supplemented with serum, were taken up by HeLa cells in a time- and temperature-dependent fashion and did not affect the HeLa cell growth rate, evidence that the SWNTs inside cells were not toxic under these conditions [19
]. This work also demonstrated that our dispersion preparation protocol (involving probe sonication and multiple centrifugations) was effective in removing metals from the raw, as-received SWNT-containing powder. Herein, we present the characterizations of an as-received CoMoCAT SWNT-containing powder using thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM), and of SWNTs dispersed in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS) using atomic force microscopy (AFM), inductively coupled plasma – mass spectrometry (ICP-MS), and absorption and Raman spectroscopies. The resulting purified dispersions, termed DM-SWNTs, are next shown to have no effect upon the morphologies and growth rates of HeLa cells – a thoroughly characterized human epithelial-like cell line. Using confocal microRaman spectroscopy, it is shown that DM-SWNTs were taken up by cells in a time- and temperature-dependent fashion. Evaluation of the distribution of intracellular DM-SWNTs was performed using transmission electron microscopy (TEM) which revealed SWNT-like material in vacuoles. Finally, intracellular superoxide dynamics of cells exposed to DM-SWNTs were evaluated using fluorescence-based flow cytometry and MitoSOX™ Red – a selective indicator of superoxide in mitochondria. The MitoSOX™ Red fluorescence detected from control cells was statistically similar to that observed for cells incubated in DM-SWNT dispersions. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNTs are not inherently cytotoxic to HeLa cells.