Targeting Phagocytes by PS-CNT Figure 6. Primary human monocyte-derived macrophages and dendritic cells recognized SWCNT functionalized with PS but not with PC. A. M-CSF-activated HMDM (0.56106 cells/ml) were incubated with NBD-PC- or NBD-PS-coated SWCNT (100 mg/ml) for 2 h and then subjected to flow cytometric evaluation of uptake of nanotubes. Macrophages incubated without SWCNT were included as an autofluorescence background control, and values are reported as percentage of background control. Representative histograms are shown below the bar graph. PS- coated SWCNT were observed to be taken up by HMDM to a higher degree than PC-coated SWCNT (p,0.002) (n = 4). B. MDDC (0.56106 cells/ml) exposed to NBD-PC- or NBD-PS-coated SWCNT (100 mg/ml) for 24 h at 37uC were assessed by flow cytometry. Data are reported as above. A tendency toward higher degree of uptake of PS-coated SWCNT was observed compared to PC-coated SWCNT in these cells (p,0.06) (n = 3), was seen for these cells. Similar results were obtained when uptake was monitored at 2 h (data not shown). C. Confocal microscopic imaging of MDDC incubated in the absence of SWCNT (a), or in the presence of NBD-PC-coated SWCNT for 2 h (b) or 24 h (d), or NBD-PS-coated SWCNT for 2 h (c) or 24 h (e), respectively. Counterstaining with antibodies to HLA-DR (red) was performed to visualize the plasma membrane of dendritic cells and the yellowish green color represents NBD-PS-coated SWCNT inside the cells. Original magnification - 6362. Data are mean6s.d. doi:10.1371/journal.pone.0004398.g006 Understanding of major principles of particle recognition by macrophages has long been a controversial issue. Because non- functionalized nanoparticles are prone to aggregation, sonication of non-coated SWCNT was performed before adding them to cells. Under these conditions, no significant uptake of non-coated SWCNT by RAW 264.7 macrophages occurred during the 2 h incubation period. In contrast, Dumortier et al. [65] have recently reported that SWCNTs tend to re-aggregate and form large clusters that are eventually (after 24 h of co-incubation) phagocy- tozed by professional macrophages. It is likely that macrophage uptake of big clusters of SWCNT formed during prolonged incubation times is related to the reduced solubility of these non- coated nanotubes. It has been also reported that geometry and shape act as determinants of particle recognition [66]. Several studies have demonstrated that functionalized SWCNT are recognizable by cells and taken up through endocytosis-dependent PLoS ONE | www.plosone.org 12 February 2009 | Volume 4 | Issue 2 | e4398