Targeting Phagocytes by PS-CNT Figure 2. SWCNT functionalized with PS but not with PC engulfed by murine RAW264.7 macrophages. A. Scanning electron micrographs of RAW264.7 macrophages treated with SWCNT in vitro. RAW264.7 macrophages (0.36106 cells/ml) were incubated for 2 h with non- coated, PC- or PS-coated SWCNT. At the end of incubation macrophages were washed and fixed for SEM. a) Recognition of PS-coated SWCNT by RAW264.7 macrophages; (Note: The red arrows in all the electron micrographs in figure 2 point SWCNT. Sub-panel A-a represents scanning electron micrograph of SWCNT alone.) b) PC-coated SWCNT, c) non-coated, d) PS-coated Annexin V treated SWCNT are poorly recognized by macrophages. A total of 150 cells from each sample type were analyzed by SEM. B. Transmission electron micrographs of RAW264.7 macrophages treated with SWCNT in vitro. RAW264.7 macrophages (0.36106 cells/ml) were incubated for 2 h with non-coated, PC- or PS-coated SWCNT. At the end of incubation macrophages were washed and fixed for TEM. a) A high-power image of the macrophage with engulfed PS-coated SWCNT. b) A typical TEM image of PS-coated SWCNT. Samples of SWCNT for TEM imaging were processed in a fashion similar to that of cells for electron microscopy. Arrows indicate SWCNT. Representative TEM images are presented. c) Quantitative assessments of SWCNT phagocytosis by RAW 264.7 macrophages. A total of 150 cells from each sample type were analyzed by TEM. Data are mean6S.D., n = 3, *p,0.05, PS-coated SWCNT vs SWCNT, PC-coated SWCNT and PS-coated Annexin V-treated. C. Effect of SWCNT on cytokine production by zymosan-stimulated RAW 264.7 macrophages. Macrophages were seeded at 2.56105 cells/well in 48 well plates and co-incubated with zymosan (0.25 mg/ml) in the presence of non- coated, PC-coated and PS coated SWCNT (150 mg/106 cells). At the end of 2 hr incubation, TNF-a was measured in the medium. IL-10 and TGF-b were measured after 4 hr incubation. The cytokines were measured using R&D Quantikine H immunoassay kit. Data are mean6s.d., n = 3. ##p,0.05, SWCNT vs control. *p,0.05, PS-coated plus zymosan vs SWCNT plus zymozan and PC-coated plus zymosan. doi:10.1371/journal.pone.0004398.g002 agreement with independent assessments performed by confocal microscopy of NBD-labeled phospholipid-coated SWCNT (see below). Uptake of SWCNT functionalized with fluorescently labeled phospholipids To more quantitatively characterize uptake of phospholipid- coated SWCNT by macrophages we utilized fluorescently labeled PS, NBD-PS, for coating SWCNT; NBD-PC was utilized in control experiments. After extensive washings, SWCNT incubated with NBD-PS or NBD-PC displayed characteristic fluorescence spectra confirming that the coating with phospholipids was successful (Fig. 3A). We studied the time-course of uptake of NBD-PC-coated SWCNT, NBD-PS-coated-SWCNT and NBD- PS-coated and Annexin V treated SWCNT by RAW264.7 macrophages. To this end, we co-incubated RAW 264.7 macrophages with either NBD-PC- or NBD-PS coated nanopar- ticles for up to 4 h (Fig. 3Ba). At this time point, viability test using Trypan Blue exclusion revealed only 5.560.9% (n = 3) dead cells. Uptake of NBD-PS-coated nanoparticles was time-dependent. NBD-PS fluorescence was already detectable from cells after PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4398