18 In vitro dosimetry of multi-walled carbon nanotubes on human bronchial epithelial cells (BEAS-2B)

Abstract Interpreting in vitro toxicology data with nanomaterials requires accurate determination of the dose effectively delivered to cells. For carbon nanotubes (CNTs), this remains particularly challenging due to the difficulty of quantifying inorganic carbon within complex biological matrices. Moreover, existing deposition models typically assume spherical particles, which may not adequately reflect the behaviour of fibrous CNTs. We developed an analytical method to quantify CNTs remaining in suspension, loosely associated with cells, or strongly bound/internalized. The approach combines UV-Vis-NIR spectrophotometry with chemical digestion or lyophilization, depending on the fraction analysed. The method was validated for selectivity, linearity, limits of detection and quantification, lyophilization yield, bias, and precision (within- and between-run). It was applied to ten multi-walled CNTs of various morphologies (long or short, thin or thick) and surface chemistries (hydroxyl- or carboxyl-functionalized), dispersed at four to six exposure concentrations in cell culture medium. Human bronchial epithelial cells (BEAS-2B) were exposed for 96 h prior to quantification. The method successfully estimated both delivered and cellular CNT doses, which did not always match nominal concentrations. CNT distribution among fractions varied with morphology: for short CNTs, the delivered dose closely matched the applied dose, whereas for long CNTs (functionalized or not), up to 20% remained suspended in the medium at the highest concentrations, likely due to a “pool noodle” effect that limits sedimentation. The approach also revealed that cell death contributed to the release of nanotube-containing cells into the culture medium.