erythrocytes derived from female Swiss or C57BL/6 mice were cultured with control and acid-functionalized SWCNTs. To facilitate the interactions between carbon nanotubes and erythro- cytes, the culture tubes were gently rotated (17 RPM) throughout the culture duration. Erythrocyte recoveries at different time points are shown in Figure 1. Treatment with control SWCNTs had no significant effect on the recovery of erythrocytes. A dose and time dependent decline (70 to 90%) in erythrocyte recovery was however seen in cultures treated with AF-SWCNTs. At 50 mg/ml concentration, a significant decline in erythrocyte recovery was seen even at the earliest 4 h time point. At the 24 h time point, erythrocyte recovery in AF-SWCNT treated cultures fell by 80-90%. Uptake of AF-SWCNTs by erythrocytes For examining the interactions between AF-SWCNTs and erythrocytes, AF-SWCNT preparations tagged with fluorescence probe were used. Erythrocytes freshly derived from female C57BL/ 6 mice were incubated with fluorescence tagged AF-SWCNT particles at 50 mg/ml in vitro for 1 h and analysed on flow cytometer before and after washing the erythrocyte preparations. Results in Figure 2 (panel B) show that 69% of erythrocytes incubated with fluorescence tagged AF-SWCNTs were positive for fluorescence indicating an uptake or association with AF-SWCNTs. After washings 18.40% of the erythrocytes (panel C) were still positive for uptake of tagged AF-SWCNTs. These results suggest that AF- SWCNTs could associate with erythrocytes in a loose as well as a relatively stronger manner. Erythrocytes incubated with fluores- cence tagged AF-SWCNTs were also examined by confocal microscopy. Results in Figure 3 show the localization of fluorescence in z-sections of erythrocytes and strongly suggest that some fluorescence tagged AF-SWCNTs could enter erythrocytes. Effect of Carbon Nanotubes on Erythrocytes AF-SWCNT induced membrane effects in erythrocytes Exposure of hydrophobic regions in erythrocyte membrane is associated with cell damage [8]. In order to see if membrane hydrophobicity is induced in erythrocyte membrane exposed to AF-SWCNTs, freshly isolated mature erythrocytes were incubated with AF-SWCNTs at 37uC for 1 h and examined for membrane changes by using ANS (8-anilino naphthalene sulfonic acid), a dye that binds to hydrophobic patches on the cell membrane [13], [14]. Results in Figure 4 show that there was a significant increase in the binding of ANS to erythrocytes treated with AF-SWCNTs. A significant shift of emission maxima (blue shift) from 520 to 480 nm was also noted in AF-SWCNT treated erythrocytes. These results indicate a significant damage of erythrocyte membrane upon exposure to AF-SWCNTs. Phosphatidylserine (PS) externalisation on cell membrane is an early marker of apoptosis and cell death [15]. Results in Figure 5 show a dose and time dependent increase in externalization of PS on erythrocytes incubated with AF-SWNCTs whereas no similar effect was seen upon incubation with control SWCNT prepara- tion. Two hours after incubation with AF-SWCNTs, about 19% of the erythrocytes had externalized PS whereas in control erythrocytes as well as the erythrocytes treated with control SWCNTs, less than 2% cells expressed PS. In vivo effect of control and acid functionalized SWCNTs on blood erythrocytes In order to determine if AF-SWCNTs exerted toxic effect on erythrocytes in vivo too, mice were administered control or acid functionalized SWCNTs through different routes and the erythrocyte count in blood was monitored. Results in Figure 6 (panel A) show that a single dose of AF-SWCNTs (100 mg) given intravenously resulted in a marked fall in erythrocyte blood count. Figure 1. Effect of Control and AF-SWCNT’s on erythrocytes in vitro. Erythrocytes isolated from Swiss mice were cultured in RPMI+10%FBS with different concentrations of control and AF- SWCNT preparations. After different time intervals, residual erythrocyte count was determined. Each point represents Mean6SEM. *p,0.01, **p,0.001 as compared to control untreated groups. doi:10.1371/journal.pone.0022032.g001 PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22032