Effect of Carbon Nanotubes on Erythrocytes Figure 8. Effect of single intravenous dose of AF-SWCNTs on the proportions of erythrocyte cohorts of different age groups in mouse blood. Mice were prepared by DIB technique as described in materials and methods. AF-SWCNTs (100 mg) was administered i.v. and proportions of erythrocyte cohorts of different ages in blood circulation were monitored. Erythrocyte cohorts were identified by biotin levels on erythrocytes as per the DIB technique [9]. Results of a representative experiment in panel A show the distribution of the three erythrocytes cohorts [Old erythrocytes, (biotinhigh, age.40 days, top boxes); intermediate age erythrocytes(biotinlow age10–40 days, middle boxes); young erythrocytes (biotinnegative age,10 days, lower boxes)] before and 3h after intravenous administration of AF-SWCNT (100 mg) in 6–8 weeks old Swiss mice. Blood samples were also taken from these mice at different intervals of time and effect of AF-SWCNT administration on the distribution of the three erythrocyte cohorts was assessed at each time points (panel B). Proportion of young, intermediate and old erythrocytes at zero time point were taken as hundred and changes induced by AF-SWCNTs have been depicted as percent of control. Each data point in panel B represents mean 6 SEM of data from swiss mice. *p,0.05 as compared to control groups by ANOVA. doi:10.1371/journal.pone.0022032.g008 process to generate AF-SWCNTs involved extensive dialysis for several days and it is therefore unlikely that some material leaching from AF-SWCNTs was responsible for the toxic effect on erythrocytes. To further test this possibility, we separated AF- SWCNTs from erythrocytes by a dialysis membrane (10,000 mol wt cut off) during incubation and found that no loss of erythrocytes occurred (results not shown). Interestingly control SWCNTs had no significant effect on survival of erythrocytes. The reason for this lack of effect of control SWCNTs could be that the control SWCNTs preparations are highly agglomerated and do not interact effectively with cells. These results corroborate our previous findings on the effects of control and acid functionalized SWCNTs on LA4 lung epithelial cells lines where too the AF- SWCNTs induced a marked cellular damage but control SWCNTs lacks a significant cytopathic effect [5]. Using fluorescence tagged AF-SWCNTs, we could demonstrate a significant binding of AF-SWCNTs with erythrocytes. Flow cytometric analysis indicated that almost 70% of erythrocytes incubated with fluorescence tagged AF-SWCNTs bound nano- tubes. Further confocal microscopic studies suggested that some AF-SWCNTs could enter the erythrocyte also. Since an active uptake of AF-SWCNTs by erythrocytes appear unlikely, the observed association of AF-SWCNTs could reflect loose binding of AF-SWCNTs with erythrocyte membrane or a stronger binding perhaps reflecting embedding of nanotubes in erythrocyte membrane or even some passive entry of the nanotubes into the cells. Our results showing that 18% of the erythrocytes retained the association with fluorescence tagged AF-SWCNTs even after three rigorous washings suggest that AF-SWCNTs may interact effectively with erythrocytes. Membrane damage as well as PS externalization seen in response to treatment with AF-SWCNTs could be a direct consequence of interactions of the AF-SWCNTs with erythrocytes. Erythrocytes treated with AF-SWCNTs were found to have a marked increase in ANS binding within one hour of treatment indicating that the membrane damage in erythrocytes may be early consequence of interaction with AF-SWCNTs. Erythrocytes treated with AF-SWCNTs also displayed increasing externaliza- tion of PS in the membrane that was related to the dose and time period of treatment. While these results clearly show a response similar to apoptotic response in nucleated cells, it is not clear from our results if PS externalization alone lead to the death/removal of erythrocytes. We also examined the AF-SWCNT induced erythrolysis by time lapse micrography (Video S1) and found that the AF-SWCNT exposed erythrocyte gradually disintegrated and disappeared from the field in a random manner and this process started within 30 min of treatment with 75 mg/ml of AF- SWCNTs. Results of in vitro studies established the toxic effect of AF- SWCNTs on erythrocytes, which was further confirmed by in vivo studies. Intratracheal and intravenous administration of AF- SWCNTs induced transient anemia in mice. As observed in vitro, control SWCNT preparations did not result in decrease in erythrocyte count. Interestingly, intra-peritoneal and intra-gastric administration of AF-SWCNTs didn’t reduce the RBC count too. The drop in erythrocyte count in blood seen in case of intravenous administration of AF-SWCNTs could be due to an efficient interaction of the nanotubes with erythrocytes in circulation. In all other forms of in vivo treatments, nanotubes were not deposited into blood circulation directly. As a result, the immediate physical PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22032