Targeting Phagocytes by PS-CNT Figure 7. HeLa cervical carcinoma cells and SH-SY5Y neuroblastoma cells interact with PS-coated (but not with PC-coated) SWCNT. A. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by HeLa cells. a) Overlay of blue and green fluorescence images. b) Quantitative evaluation of cell with engulfed SWCNT. Data are mean6s.d., n = 4. *p,0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT. B. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by SH-SY5Y neuroblastoma cells. a) Overlay of blue and green fluorescence images. b) Quantitative evaluation of cells with engulfed SWCNT. Data are mean6s.d., n = 4. *p,0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT. doi:10.1371/journal.pone.0004398.g007 pathways [67,68]. By contrast, we and others reported that non- functionalized SWCNT are neither effectively recognized nor phagocytozed by macrophages [5,53,69]. The fact that PS-coating leads to recognition and uptake of SWCNT suggests that it is the lack of a recognition signal that is responsible for poor uptake of non-functionalized carbon nanotubes by phagocytes. A variety of specialized receptors on the macrophage surface have been implicated in recognition and tethering of different particles, including ultra-fine particles [64,67,70–72]. Recent studies identified several novel macrophage receptors for which PS is a specific high-affinity ligand [25,26], facilitating uptake of target cells with externalized PS. PC is not specifically recognized by these receptors and its presence on the cell surface does not enhance recognition and uptake of cells by macrophages. Therefore, we chose to use NBD-PC-coated SWCNT as controls in our comparative experiments on assessments of SWCNT uptake by macrophages. Further, we utilized Annexin V – a protein known to selectively bind to PS (but not to PC) and mask PS recognition and uptake by macrophages. The Annexin V mediated suppression of uptake of NBD-PS-coated SWCNT (and lack of fluorescence response from macrophages incubated in the presence of NBD-PS coated SWCNT pre-treated with Annexin V) was employed as an additional specific control of PS-dependent recognition and uptake. Our data indicate that eclipsing of PS with its specific ligand, Annexin V, completely blocks SWCNT recognition by macrophage cell lines and primary phagocytes. Most notably, these PS-dependent recognition patterns are realized in vivo whereby alveolar macrophages display enhanced uptake of PS-coated SWCNT during an inflammatory pulmonary response induced by aspiration exposure. Together, our in vitro and in vivo studies demonstrate that PS-functionalization renders nanotubes appetizing to phagocytes. We believe that analysis of SWCNT uptake by phagocytes based on the employment of fluorescently-labeled phospholipids (NBD-PS and NBD-PC) PLoS ONE | www.plosone.org 13 February 2009 | Volume 4 | Issue 2 | e4398