Targeting Phagocytes by PS-CNT Figure 1. Physico-chemical characterization of SWCNT. A. Evaluation of phospholipid content of PC-coated or PS-coated SWCNT. a) Typical one-dimensional HPTLC of phospholipids extracted from PC-coated or PS-coated SWCNT. b) Phospholipid content of phospholipid-coated SWCNT. B. Typical fluorescence spectrum of Annexin V bound to PS-coated SWCNT. Note that a robust fluorescence response (Annexin V-FITC binding) was recorded from PS-coated but not from non-coated or PC-coated SWCNT. C. Physico-chemical characterization of bare SWCNT. a) Typical histograms of size distribution assessed by dynamic light scattering; b) AFM images of bare SWCNT deposited on mica substrates; c) The height cross section analysis of bare SWCNT. doi:10.1371/journal.pone.0004398.g001 engulfment of PC-coated and non-coated SWCNT was docu- mented (Fig. 2Bc). In addition, macrophages co-incubated with PS-coated SWCNT had more endocytotic vesicles with entrapped nanoparticles compared to macrophages incubated in the presence of PC-coated or non-coated SWCNT (data not shown). At higher magnifications (Fig. 2Ba), PS-coated SWCNT fibers within phagosomes could be seen. To distinguish SWCNT from autophagic bodies inside of phago-lysosomes we used a high magnification TEM imaging (100,0006). Further, in order to better identify nanoparticles in cells we performed TEM imaging of nanotubes alone which were processed in a fashion similar to that of cells for electron microscopy. SWCNT alone appear as ‘‘bamboo shoots’’ and resemble SWCNT that have been engulfed by RAW264.7 macrophages (Fig. 2Bab). Markedly less of ‘‘bamboo shoot’’-like material was detected in RAW264.7 macrophages exposed to PC-coated or non–coated SWCNT (Fig. 2Bc). Quantitative assessments of the number of nanotubes present in an endosome within a 60 nm ultrafine section of cells could be complicated by the fact that the same SWCNT could be cut 1, 2 or more times, especially SWCNT bundles or ‘‘bird’s- nest’’-like aggregates. Therefore, we presented the data as a plot of percentage of active phagocytes in analyzed samples. We considered as active macrophages those that engulfed at least one nanoparticle. We found that amongst the various treatment groups, 70% macrophages treated with PS-coated SWCNT showed active phagocytosis with nanoparticles inside, which was approximately twice the phagocytosis-positive population of cells co-incubated with PC-coated SWCNT. Annexin V that masks PS [44] and affects its recognition [45], effectively blocked the recognition by 50% as indicated by a lower cell population with active phagocytes (Fig. 2Bc). Notably, these TEM-based evalua- tions of phagocytotic activity towards SWCNT were in good PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4398