Targeting Phagocytes by PS-CNT 30 min and increased in the course of incubation. Ingestion of NBD-PS-coated nanoparticles was approximately 3-fold higher than that of PC-coated SWCNT. We observed a robust and time- dependent increase in the fluorescence response from RAW 264.7 macrophages exposed to PS-coated SWCNT which was not saturable over 4 hrs of incubation. In contrast, a much weaker and saturable (after 1 hr) fluorescence was detectable from RAW 264.7 macrophages treated with PC-coated SWCNT or PS-coated SWCNT in the presence of Annexin V. Quantitatively, these results are summarized in Fig. 3Bb where fluorescence from NBD- phospholipid containing macrophages is shown. Clearly, PS coated SWCNT were specifically and effectively recognized and taken up by RAW264.7 macrophages. In addition, we performed experiments with primary human monocyte-derived phagocytes at two time-points, 2 h and 24 h, and the preferential ingestion of PS-coated SWCNT versus PC-coated SWCNT was evident also at the later time-point, thus arguing against a non-selective mode of uptake of the functionalized SWCNT (see below). Intracellular localization of PS-coated SWCNT Visually, we observed accumulation of ‘‘black’’ (SWCNT- containing) pellets after sedimentation of cells. To quantitatively characterize this effect, we performed measurements of optical density of cell suspensions incubated with PS-coated SWCNT and PC-coated SWCNT. A broad absorbance of SWCNT with characteristic maxima at 1050–1060 and 1260–1270 nm was detected. The intensity of absorbance at 1050–1060 nm was 1.3- fold higher in PS-coated SWCNT than in PC-coated SWCNT Figure 3. In vitro assessment of uptake of NBD-PS-coated or NBD-PC-coated SWCNT by RAW264.7 macrophages. A. Typical fluorescence spectra obtained from NBD-PC- and NBD-PS-coated SWCNT. B. Time-dependent uptake of NBD-PS-coated but not NBD-PC-coated SWCNT. a) RAW264.7 macrophages (0.36106 cells/ml) were incubated for up to 4 hrs with NBD-PC- or NBD-PS-coated SWCNT. Annexin V prevents engulfment of NBD-PS-coated SWCNT by RAW264.7 macrophages. Overlapped blue and green fluorescence images are presented. b) Quantitative evaluation of cell number with engulfed SWCNT. Data are mean6s.d., n = 3. *p,0.05, NBD-PS-coated vs NBD-PC-coated SWCNT and NBD-PS-coated Annexin-V treated SWCNT. C. Assessment of NBD-phospholipid-coated SWCNT in whole cells and subcellular fractions isolated from RAW264.7 macrophages. a) Uptake of PS-coated and PC-coated SWCNT by RAW264.7 macrophages. Data are mean6S.D., n = 3, *p,0.05, NBD-PS-coated SWCNT vs NBD-PC coated SWCNT. Inset: typical fluorescence spectra obtained from endosomal/lysosomal fraction isolated from RAW264.7 macrophages. b) Intracellular localization of PS-coated SWCNT in RAW264.7 macrophages. Macrophages were incubated with PC-coated or PS- coated SWCNT for 15 min at 37uC. At the end of incubation, subcellular fractions were isolated and examined for the presence of NBD fluorescence. D. Typical confocal microscopy images of RAW 264.7 macrophages with NBD-PS-coated SWCNT. RAW macrophages were treated with NBD-PS- coated SWCNT in the presence of Lyso-Tracker Red for 5 min at 37uC (a,b,c). In the experiments with inhibitors of endocytosis, macrophages were pretreated with a mixture containing nystatin (25 mg/ml), genistein (200 mM), chlorpromazine (6 mg/ml) and brefeldin A (10 mg/ml) for 30 min prior to incubation with NBD-PS-coated SWCNT (d, e, f). a and d - green fluorescence is from NBD-phospholipid coated SWCNT; b and e - red fluorescence is from Lyso-Tracker Red, c and f - overlay of green and red fluorescence. doi:10.1371/journal.pone.0004398.g003 PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4398