Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications

Summary With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell–MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to examine in depth the effects induced by MWCNTs-COOH and, in particular, the timing of the MWCNT-COOH–cell interaction. Lay description Due to their unique properties, carbon nanotubes (CNTs) are exploited in different industrial and biomedical sectors. However, the fibrous morphology, the high surface energy and the structural similarities with asbestos fibers of CNTs raises several health concerns. Moreover, CNT modification, made to enhance their hydrophilicity and dispersion in water and in biological media, may affect CNT effects. Numerous studies have been conducted to evaluate how chemical functionalization may affect multiwalled CNT (MWCNT) effects; however, controversial data have been reported. The aim of this work was the evaluation of modification induced by carboxylated-MWCNTs (MWCNTs-COOH) on epithelial cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells were incubated with MWCNTs-COOH for several exposure times and concentrations (10 μg/ml for 1,2,4h; 5, 10, 20 μg/ml for 24h). MWCNTs-COOH were observed associated with plasma membrane and in contact with microvilli just after short incubation time. After 24 h exposure, MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells. Analysis revealed that nanotube agglomerates entered into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions.