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 (0.3×10⁶ 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 mean±s.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 mean±S.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 37°C. 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 37°C (a,b,c). In the experiments with inhibitors of endocytosis, macrophages were pretreated with a mixture containing nystatin (25 µg/ml), genistein (200 µM), chlorpromazine (6 µg/ml) and brefeldin A (10 µg/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.