There is evidence that the risk of MWCNT nanotoxicity increases with their length. Our study showed that highly purified, well-dispersed MWCNTs exhibit different degree of toxicity depending on their length as well as cell types tested. In addition, the experimental design and methods used in the present study provide valuable information for interpretation of cell toxicity analysis and for risk evaluation in biomedical applications of MWCNTs.
In the evaluation of the MWCNT toxicity in vitro, choice of appropriate methods for cell viability is crucial in view of conflicting data in some of the reported studies, which can be explained by the various in vitro cell viability tests. In many of these studies, MTT assay has been used. Subsequently, a problem with the MTT assay was identified as the MTT-formazan crystals bind to the CNTs, accounting for the false positive results by this assay . Other authors have reported interactions of CNTs with various indicator dyes: Commasie Blue, Alamar Blue (same as CellTiter-Blue), neutral red, MTT, and WST-1 (same as MTS) [19, 20]. One report evaluating the interference of MWCNTs with resazurin confirmed a significant decrease in the signal (approximately 15 % to 20 %) during fluorescence detection in the presence of MWCNTs . The present study also revealed similar interference between the CellTiter-Blue reagents and MWCNTs (data not shown). To minimize this effect in the CellTiter-Blue assays used in the present study, excessive MWCNTs in media were thoroughly washed and removed before adding the reagents. However, this failed to remove most MWCNTs attached to cell membranes when checked with microscopy. Therefore, it seems likely that during CellTiter-Blue assay the interference between MWCNTs (both intracellular and membrane bound) and assay reagents persists and could contribute to the false high cell viability results when validated by manual trypan blue counting. More complex mechanisms such as cell membrane-MWCNT interaction may also be involved in determining cellular responses to MWCNTs , all of which merit detailed further investigation.
As the immune system plays a key role in the protection against foreign materials, studies of MWCNT toxicity to immune cells are essential for in vivo translation of therapies based on MWCNTs. In this respect, tissue and circulating macrophages constitute an important component of the effector immune system responsible for the ingestion and clearance of particulate foreign bodies and, thus, deserve attention in nano-toxicity studies. Our data suggest that precautions are needed even when using short MWCNTs.
To study the possible mechanisms underlying the difference in their biocompatibility, S- and L-MWCNTs cellular uptake was investigated. RAW264.7 cells internalised more MWCNTs than the MCF-7 cells, partially explaining the difference in cytotoxicity by MWCNTs in two cell types, as the larger load of internalised MWCNTs may be responsible for increased disruption of the integrity of cell membrane and intracellular organelles. Cellular uptake cannot, however, be the only factor determining cell viability. As MWCNT-cell membrane interaction could affect cellular response and, therefore, cytotoxicity, the higher toxicity of L-MWCNTs may also be explained by such mechanism, which is yet to be clarified. It has been shown that macrophages experience difficulty in engulfing long CNTs, and incomplete internalisation or simply binding of MWCNTs by macrophages could impair the plasma membrane . Additionally, membrane damage caused by carbon-based nanomaterials can be also ascribed to lipid peroxidation following contact between the nanomaterial and cell membrane .
Pro-inflammatory cytokine secretion is an important parameter in inducing apoptosis of immune cells as well as an indicator of the inflammatory response. Our studies have shown that in response to MWCNT treatment, RAW264.7 cells secreted considerable amount of TNF-α, indicative of the inflammatory response by these cells. However, in contrast to the cytotoxicity, S-MWCNTs induced more TNF-α production than L-MWCNTs. This finding is in agreement with the previous report by Brown et al.  that straighter and shorter nanotubes induced higher TNF-α production compared with other CNT samples. Since our viability data showed that L-MWCNTs were more cytotoxic to RAW264.7 cells, other mechanisms are probably involved, e.g. membrane-nanotube contact causing more cell membrane and intracellular organelle damage, lipid peroxidation, incomplete uptake of L-MWCNT, etc. IL-12 production by RAW264.7 in response to both types of MWCNTs was, however, minimal during 24-h incubation. Nevertheless, we cannot exclude IL-12 release during longer incubation periods. In addition to pro-inflammatory cytokine secretion by RAW264.7 cells, ROS generation by both cell lines was employed in the present study to compare MWCNT toxicity. Although previously reported, ROS production induced by CNT exposure was considered to be caused by the metal impurities [8, 24]; our data demonstrate that both S- and L-MWCNTs, even when highly purified, induced excessive of ROS production within 24 h in dose- and length-dependent manner, suggesting the nanoscale nature of the MWCNTs itself influences the oxidative stress response of cells.
The present study has produced evidence that the current panel of cell viability assays for nanomaterials is not sensitive enough for providing an accurate toxicity profile of nanomaterial. In the short term, it is recommended that in vitro assays of nanomaterial toxicity should be confirmed by trypan blue exclusion, but there is an urgent need for the development of improved testing-based label-free approaches for measuring nanotoxicity in living cells. These approaches are being explored in our laboratory.