Cytotoxic Effect of Poly-Dispersed Single Walled Carbon Nanotubes on Erythrocytes In Vitro and In Vivo Sumedha Sachar, Rajiv K. Saxena* School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Abstract Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF- SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF- SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age ,10 days) and old (age.40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed. Citation: Sachar S, Saxena RK (2011) Cytotoxic Effect of Poly-Dispersed Single Walled Carbon Nanotubes on Erythrocytes In Vitro and In Vivo. PLoS ONE 6(7): e22032. doi:10.1371/journal.pone.0022032 Editor: David L. McCormick, IIT Research Institute, United States of America Received March 11, 2011; Accepted June 13, 2011; Published July 19, 2011 Copyright: 2011 Sachar, Saxena. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Department of Science and Technology (DST) grant (SR/SO/HS-133/2007) to RKS, and a fellowship from DBT to SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rajivksaxena@hotmail.com Introduction Nano-sized particles are finding increasing practical applica- tions and commercial use. Consequently, the possibility of occupational exposure to nano-particles has increased. Further- more, as the use of nanoparticles increases, they may diffuse and accumulate in environment increasing the risk of environmental exposure. Health effects of exposure to nanoparticles are now under investigation by many researchers [1], [2], [3]. Nanopar- ticles have a tendency to agglomerate into particles of larger size that may not have the same biological effects as non-agglomerated nanoparticles. For studying the biological effects therefore it is necessary to test non-agglomerated forms of nanoparticles. For Single walled Carbon Nanotubes (SWCNTs) disaggregation can be achieved by acid functionalization that introduces negative charge, without changing the essential structural features of SWCNTs [4], [5]. We have previously reported cytostatic and toxic effects of acid functionalized SWCNTs (AF-SWCNTs) on LA4 lung epithelial cell line [5]. We also found that the administration of AF-SWCNTs intratracheally resulted in cardiac damage in mice [6]. Since AF-SWCNTs were toxic to some types of nucleated cells that are capable of self repair, we hypothesized that the AF- SWCNTs may have a more aggravated toxic effect on erythrocytes that have no nucleus and lack substantial cellular repair mechanisms. This proposition was examined in the present study. Our results indicate that unlike pristine SWCNTs, AF- SWCNTs are significantly toxic to mouse blood derived erythrocytes in vitro as well as in vivo. AF-SWCNTs were found to bind with erythrocytes and cause significant membrane damage as well as externalization of phosphatidylserine. Administration of AF-SWCNTs intravenously induced a significant though transient anemia in mice. Older as compared to the younger subpopulation of erythrocyte in blood circulation, was more susceptible to the toxic effect of AF-SWCNTs. Taken together our results show that non-agglomerated form of SWCNTs may exert significant and rapid toxic effects on blood erythrocytes. Materials and Methods Experimental model Inbred Swiss and C57BL/6 female mice (6–12 weeks old, 20–25 g body weight) were used throughout this study. Animals were bred and maintained in the animal house facility at JNU, New Delhi or obtained from the National Institute of Nutrition, Hyderabad. The animals were housed in positive-pressure air PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22032