Effect of Carbon Nanotubes on Erythrocytes Figure 2. Uptake of fluorescenated AF-SWCNTs by erythrocytes in vitro. Erythrocytes were incubated with fluorescenated AF-SWCNTs (50 mg/ml) for 1 h and examined flow cytometrically either before (panel B) or after (panel C) three washings with fresh medium. Panel A shows the flow histogram for control unstained erythrocytes. Panel D shows the dose dependent uptake of AF-SWCNTs by erythrocytes with or without three washings. Results of a representative experiment have been shown. In panel D all data points represent mean 6 SEM of three observations. doi:10.1371/journal.pone.0022032.g002 Maximum decline in blood count was about 23% and occurred 12 and 24 h after the infusion of AF-SWCNTs. The blood erythrocyte count returned to normal levels 72 h after the administration of AF-SWCNTs. A lesser though significant fall in blood erythrocyte count (about 10%) was also observed when AF-SWCNTs were administered intratracheally (Figure 6A). Administration of AF-SWCNTs through oral gavage and intraperitoneal route did not result in a significant reduction in the blood erythrocyte count (Figure 6A). A concomitant transient drop in blood haemoglobin levels was also seen in mice administered a single dose of 100 mg of AF-SWCNTs through intravenous or intra tracheal route (Figure 6B). Results above show that treatment with AF-SWCNTs resulted in a transient anemia in mice. A second dose of AF-SWCNTs given 24 h after the first dose resulted in a more sustained anemia that was not alleviated even at 72 h time point (Figure 7). Administration of two doses of control SWCNTs however still had no significant effect on the blood erythrocyte count. Age dependence of susceptibility of blood erythrocytes to AF-SWCNTs Results so far indicate that the intravenous administration of AF-SWCNTs caused a marked and transient anemia in mice. It was of interest to determine if the toxic effect of AF-SWCNTs Figure 3. Localization of AF-SWCNTs in side erythrocytes by confocal microscopy. Fluorescenated AF-SWCNTs were prepared as described in Materials and Methods. Erythrocytes were incubated in RPMI +1%FBS with 50 mg/ml fluorescenated AF-SWCNTs for 1 h and unbound/loosely attached particles were removed by three extensive washings with PBS. After that erythrocyte suspension was taken for confocal microscopy. Above figure shows fluorescence images of erythrocytes incubated with alexa fluor 633 hydrazide tagged AF-SWCNTs as z-sections. (Magnification 100X). doi:10.1371/journal.pone.0022032.g003 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22032