In vitro toxicity testing of two multi-walled carbon nanotubes determined for the purpose of targeted drug delivery

Carbon Nanotubes (CNTs) are novel, carbon based nanomaterials that gain more and more interest in fields like mechanical engineering or pharmacy and medicine. This study was an initial determination of the cytotoxic potential of two multi-walled Carbon Nanotubes that were determined for the development of a multifunctional drug delivery carrier. A549 lung epithelial cells and HepG2 cells were employed for the experiments. The cells were exposed with a dose of 1, 10 and 25µg/cm² growth area (24 and 48h) of the two CNT types. The two CNT types were produced using the identical chemical vapour deposition (CVD) synthesis for both types (IFW; Dresden, Germany). The first type was used as produced, still containing an iron catalyst that was added during synthesis (Fe-CNT). The second type was heat treated after synthesis for 1h at 2600°C to eliminate all remaining iron from the CNT structure (nonFe-CNT). Both CNT types showed an identical morphology (16.5µm (± 8) length, 48nm (±12) diameter) and appeared curved/flexible in REM investigation. The heat treated CNT type (nonFe) showed a significantly reduced number of surface defects, indicated by a reduction in d-peak intensity during Raman spectroscopy. The WST-8 assay was applied to evaluate cell viability and the LDH cytotoxicity assay was used to detect potential membrane damage. To investigate the production of reactive oxygen species (ROS) the DCFH-DA assay was applied. Additionally a cell cycle analysis was performed to assess cell proliferation. The results showed a time and dose dependent decrease in cell viability for both cell lines. In A549 cells the severe leakage of lactate dehydrogenases (LDH) after 48h of exposure resulted in a total cytotoxicity of 25% (Fe-CNT) and 45% (nonFe-CNT). The DCFH-DA assay showed an increase in ROS production, primarily under the influence of the nonFe-CNTs. The cell cycle analysis demonstrated in heat treated CNTs an increased number of cells in the G2/M-phase while the number in S-phase was reduced, indicating a G2-phase arrest. Summarizing, most cytotoxic effects were more prominent for the heat treated (nonFe) CNTs. This indicates that the number of surface defects plays an important role in CNT-related cytotoxicity. Additionally we conclude that the CNTs can't be used as drug delivery carriers without achieving biocompatibility via further modification.