samples. We further used fluorescently-labeled phospholipids to characterize the presence of phospholipid-coated SWCNT in cells. We found that NBD-PS coated SWCNT co-incubated with RAW 264.7 macrophages yielded a higher fluorescence response from cell suspensions than NBD-PC coated SWCNT (Fig. 3Ca). Further, the majority of fluorescence was associated with the cytosolic fraction (containing endo-lysosomal vesicles) and was minimal in the fraction of nuclei and cell debris (Fig. 3Cb). We isolated the endo-lysosomal fraction from RAW 264.7 macro- phages loaded with NBD-PS-coated SWCNT and found that 77618% (n = 3) of the fluorescence response was associated with this fraction. This is in line with the recent demonstration of deposition of SWCNT in endo-lysosomal compartments of macrophages upon prolonged incubation [46]. Additionally, to prove co-localization of SWCNT with phago- lysosomes we used confocal microscopy. To this end, we treated RAW 264.7 macrophages (56105 cells/well) with either NBD-PS- coated SWCNT or NBD-PC-coated SWCNT (150 mg/106 cells). We found that NBD-PS-coated SWCNT were readily taken up by macrophages. As shown in Fig. 3Da,b,c, a robust intracellular fluorescence of NBD-PS (but not of NBD-PC) was co-localized, at least in part, with a lysosomal marker, Lyso-Tracker Red. Moreover, after pre-treatment of macrophages with the cocktail of inhibitors of endocytosis (nystatin (25 mg/ml), genistein (200 mM), chlorpromazine (6 mg/ml) and brefeldin A (10 mg/ ml)), the intensity of intracellular NBD-PS fluorescence was drastically reduced (Fig. 3Dd,e,f). No fluorescence was detected after treatment of macrophages with NBD-PC-coated SWCNT (data not shown). This suggests that NBD-PS-coated SWCNT were effectively taken up by macrophages through an endocytosis- mediated process resulting in their significant accumulation in endo-lysosomes. Effect of SWCNT on cytokine release by RAW264.7 macrophages Apoptotic cells with externalized PS quench the production and release of pro-inflammatory cytokines by macrophages [15,19]. To assess whether PS-coated SWCNT display a similar effect, we employed a standard stimulation of TNF-a formation in RAW264.7 macrophages by zymosan [47,48] and evaluated the effects of SWCNT. PS-coated SWCNT were more potent in inhibiting TNF-a production by stimulated macrophages than PC-coated or non-coated SWCNT (Fig. 2C). Thus, uptake of PS- SWCNT is accompanied by typical PS-dependent suppression of the pro-inflammatory macrophage response. It should be noted that macrophages stimulated with SWCNT alone - in the absence of zymosan - showed a significant increase in the TNF-a production up to 497624 pg/ml vs. 40610 (## P,0.05) in the control. Notably, in PS-coated SWCNT, TNF-a levels dropped to 368633 pg/ml. No significant changes in the content of TNF-a was found after SWCNT coating with PC (445621 pg/ml). In contrast, the production of anti-inflammatory cytokines is known to be stimulated by PS-dependent pathways [16]. Stimulation of RAW264.7 macrophages by zymosan resulted in the accumula- tion of TGF-b and IL-10 at 4 hrs after the challenge. The production of anti-inflammatory IL-10 and TGF-b by zymosan- stimulated macrophages subsequently treated with PS-coated- SWCNT increased 1.5- and 2-fold respectively, compared with the effect of zymosan alone (Fig. 2C). This enhancement of IL-10 and TGF-b production was not observed after exposure of zymosan-stimulated RAW264.7 macrophages to either non- coated SWCNT or PC-coated SWCNT. Targeting Phagocytes by PS-CNT PS-coated SWCNT deliver cyt c into RAW264.7 macrophages We further determined whether PS-coated SWCNT could be employed for the delivery of physiologically active agents to macrophages. Because cyt c released from mitochondria into the cytosol acts as an effective activator of caspases and a death-signal [49] we chose to use it as a cargo. Given that positively charged cyt c readily interacts with negatively charged surfaces we assumed that SWCNT coated with anionic PS would effectively bind to cyt c. Indeed, the binding of cyt c to PS-SWNT was confirmed by direct measurements of specific cyt c absorbance as well as by atomic force microscopy (AFM). Section analysis of bare SWCNT (Fig. 1Cc) demonstrated that the diameter of non-coated SWCNT was 1.3 nm (typically these SWCNTs have diameters in the range of 0.6–1.5 nm [50,51]. Figure 4Aa shows AFM image of cyt c adsorbed on SWCNT sidewalls. AFM image revealed the presence of globular structures of cyt c on the surface and in the contact with SWCNTs (measured height ,1 nm). This image is similar to previously reported [6] picture of SWCNTs functionalized with proteins and in particularly with cyt c. AFM of SWCNT functionalized with PS/PC and cyt c displayed a significantly different image (Fig. 4Ab), where distinct PS/PC molecules were not seen; rather a layer of PS/PC was spread on the SWCNT surface thereby demonstrating lipid adsorption onto SWCNTs. Such an event is likely due to a greater retention of PS/PC aggregation, resulting into multiple PS/PC layers over SWCNTs, while individual globular structures of cyt c are still clearly visible on the nanotube sidewalls. The height cross section of the SWCNT functionalized with PS/PC and cyt c (Fig. 4Ac) shows that the diameter of the structures is 3.5 nm which is significantly larger than bare SWCNT. Importantly, cyt c-loaded PS-SWCNT caused a marked increase of caspase activity (Fig. 4Ba) and PS externalization (Figure S3) in RAW264.7 macrophages upon co-treatment with a disruptor of endosomes, chloroquine. This is in line with previous reports where acid-oxidized SWCNT were utilized for delivery of cyt c into cancer cell lines [6]. PC-coated or non-coated SWCNT pre-incubated with cyt c were ineffective in inducing caspase-3 activation in macrophages. In addition, we found that delivery of cyt c into macrophages resulted in significantly increased numbers of Trypan Blue positive cells (Fig. 4Bb) compared to cells treated with PS-SWCNT with no cyt c attached. Thus, PS-coating makes PS-SWCNT loaded with a protein cargo recognizable, facilitates their uptake by macrophages, and could hence be envisaged as a promising tool for targeted delivery of regulators of macrophage activity/survival. Recognition of PS-coated SWCNT by microglia, human monocyte-derived macrophages (HMDM) and human monocyte-derived dendritic cells (MDDC) To determine whether PS-coated SWCNT can be recognized and taken up by other types of professional phagocytes, we performed experiments using microglia from rat brain. The experiments using SEM (Fig. 5A) and TEM (Fig. 5Ba) demon- strated that, similarly to RAW264.7 macrophages, microglia effectively engulfed PS-coated (Fig. 5Bb), but not PC-coated or non-coated SWCNT (data not shown). Essentially in samples in which PS-coated SWCNT were treated with Annexin V the number of SWCNT positive microglial cells was not different from SWCNT controls or from PC-coated SWCNT (Fig. 5Bb). This was confirmed by quantitative assessments of NBD-PS-SWCNT uptake by fluorescence microscopy (Fig. 5C). PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4398