Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo Nagarjun V. Konduru1, Yulia Y. Tyurina1, Weihong Feng1, Liana V. Basova1, Natalia A. Belikova1, Hu¨ lya Bayir1, Katherine Clark2, Marc Rubin2, Donna Stolz2, Helen Vallhov5, Annika Scheynius5, Erika Witasp6, Bengt Fadeel6, Padmakar D. Kichambare3, Alexander Star3, Elena R. Kisin4, Ashley R. Murray4, Anna A. Shvedova4, Valerian E. Kagan1,6* 1 Center for Free Radical and Antioxidant Health, Graduate School of Public Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 4 Pathology/Physiology Research Branch, Health Effects Laboratory Division (HELD), National Institute for Occupational Safety and Health (NIOSH), Morgantown, West Virginia, United States of America, 5 Clinical Allergy Research Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden, 6 Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Abstract Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid ‘‘eat-me’’ signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells. Citation: Konduru NV, Tyurina YY, Feng W, Basova LV, Belikova NA, et al. (2009) Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo. PLoS ONE 4(2): e4398. doi:10.1371/journal.pone.0004398 Editor: Arto Urtti, University of Helsinki, Finland Received August 6, 2008; Accepted December 16, 2008; Published February 9, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by NIOSH OH008282, NORA 92700Y, the Swedish Research Council, the Swedish Council for Working Life and Social Research, and the Human Frontier Science Program (HFSP). 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: kagan@pitt.edu Introduction One of the major biomedical applications of carbon nanotubes (CNT) is their use as nanovectors in drug delivery paradigms. Professional phagocytes, particularly macrophages, are very attractive targets for selective drug delivery because these cells: i) host a variety of pathogens with significant public health impact, ii) play a critical role as orchestrators of inflammation as they regulate the production and release of pro- and anti-inflammatory mediators, reactive oxygen (ROS) and nitrogen species (RNS), particularly after exposure to particles [1], and iii) are significant contributors to the distribution of CNT in the body thus determining their potential toxic effects [2,3]. Importantly, non- functionalized nanotubes are poorly recognized by macrophages in vitro and in vivo resulting in the avoidance of CNTs from macrophages-mediated ‘‘surveillance’’ [4,5]. In contrast, functio- nalization of nanotubes induces their recognition by professional and non-professional macrophages and other cells [6–9]. Howev- er, the universal nature of the engulfment of covalently functionalized CNT by different types of cells precludes the possibility of their targeted delivery to specific cells [9]. This stimulated new lines of research on targeted interfacing of single walled carbon nanotubes (SWCNT) with living cells through specific coatings mimicking the cell surface [10]. In particular, glycopolymers - that mimic cell surface mucin glycoproteins and facilitate carbohydrate receptor interactions - have been developed to stimulate targeted engulfment of SWCNT by specific types of cells [10,11]. Macrophage recognition and uptake of apoptotic cells (also termed ‘‘efferocytosis’’) is an important type of cell/cell commu- nications regulating inflammation [12,13]. This interaction triggers not only effective clearance of apoptotic cells but also suppression of the inflammatory response [14,15] or of adaptive immunity [16], thus limiting local tissue responses and normally leading to a quiet cell removal [13]. In contrast, inefficient apoptotic cell clearance is pro-inflammatory and pro-immuno- genic. The recognition of apoptotic cells by macrophages is largely dependent on the appearance on the cell surface of an anionic PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4398