Targeting Phagocytes by PS-CNT Figure 4. PS-coated SWCNT effectively bind cyt c, deliver it into RAW264.7 macrophages, and activate apoptotic pathways (caspase 3/7), and cell death. A. AFM images of various SWCNT samples deposited on mica substrates: a) SWCNT with cyt c; b) SWCNT with cyt c and PS/PC. c) The height cross section of the functionalized SWCNT in image b. B. Cyt c delivered into macrophages by PS-coated SWCNT activates caspase 3/7 (a) and increases the number of Trypan Blue positive cells (b). SWCNT were treated with 50 mM cyt c, washed twice and then were coated with PS and PC alone. Cells were incubated with protein/lipid/SWCNT conjugates in the presence of 100 mM chloroquine to trigger endosomal rupture. Data are normalized versus SWCNT/chloroquine. Note that under experimental conditions used chloroquine alone (100 mM, 15 min incubation) did not induce any significant activation of caspase 3/7. The data represent mean6s.d (standard deviation), n = 5, *p,0.05, PS-coated plus cyt c vs PS-coated SWCNT, non-coated SWCNT plus cyt c and SWCNT alone. doi:10.1371/journal.pone.0004398.g004 Furthermore, primary human monocyte-derived macrophages (HMDM) and human monocyte-derived dendritic cells (MDDC) also preferentially recognized and engulfed PS-coated SWCNT compared to PC-coated SWCNT (Fig. 6). The latter data thus confirm that the selective uptake of PS-functionalized SWCNT is a general phenomenon that is seen with phagocytes derived from different species. For the studies of human monocyte-derived phagocytes, flow cytometry was used to quantify the degree of in vitro uptake of NBD-PC- versus NBD-PS-coated nanotubes (Fig. 6A, Fig. 6B), and these data were confirmed using confocal microscopy (Fig. 6C) and TEM evaluation of non-NBD labeled phospholipid-coated SWCNT (data not shown). Interaction of PS-coated SWCNT with HeLa cervical carcinoma cells and SH-SY5Y neuroblastoma cells While ingestion of apoptotic cells is not absolutely specific to phagocytes, the rates of uptake by professional phagocytes - macrophages and microglial cells – are significantly higher compared to non-professional phagocytes (such as endothelial, epithelial cells and fibroblasts) [52]. Typical fluorescence micrographs of cervical carcinoma HeLa cells (Fig. 7Aa) and neuroblastoma SH-SY5Y cells (Fig. 7Ba) demonstrate preferen- tial uptake of NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT by both cell types. Quantitative assessments revealed that PS-coated SWCNT were ingested more actively than NBD- PC-coated SWCNT by HeLa cells, and SH-SY5Y cells, respectively (Fig 7Ab and Fig. 7Bb). However, uptake of NBD- PS-coated SWCNT by these cells was 2.4 times and 2.6 times less effective, respectively, than their ingestion by RAW 264.7 macrophages. Recognition of PS-coated SWCNT by alveolar macrophages in vivo Because PS is an important recognition signal for alveolar macrophages in the lung [15], we reasoned that PS-coated SWCNT could be effectively phagocytozed in vivo. To this end, we used an established mouse model of SWCNT pulmonary exposure through pharyngeal aspiration [36]. Mice were exposed to PS-coated-, PC- coated-, or non-coated SWCNT using procedures described previously [53]. A high magnification TEM image of alveolar macrophages obtained from animals exposed to PS-coated SWCNT clearly demonstrates the presence of ‘‘bamboo shoot’’- like material (Fig. 8A). We also performed quantitative assessments of phagocytosis by counting SWCNT-positive alveolar macrophag- es using TEM images. Macrophages with at least one nanoparticle engulfed were considered as phagocytosis-positive. The data are presented as a plot of percentage of active phagocytes for each condition (Fig. 8B). BAL obtained from PS-coated SWCNT– exposed mice revealed 7268.4% of phagocytosis-positive alveolar macrophages. In contrast, only 40610% and 1868% of macro- phages isolated from mice exposed to PC/SWCNT or non-coated SWCNT contained nanoparticles. Thus, PS-coated SWCNT were ingested at a significantly higher rate in vivo as compared to PC- coated or non-coated SWCNT by alveolar macrophages. Discussion Specific interfacing of SWCNT with phagocytic cells of the immune system – macrophages, microglia, and dendritic cells - is important for several reasons. The first one is that SWCNT can be used for simultaneous targeted delivery of several different PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4398