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.
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 (0.3×106 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 (0.3×106 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 mean±S.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 2.5×105 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 µg/106 cells). At the end of 2 hr incubation, TNF-α was measured in the medium. IL-10 and TGF-β were measured after 4 hr incubation. The cytokines were measured using R&D Quantikine ® immunoassay kit. Data are mean±s.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.
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.
Figure 4.
PS-coated SWCNT effectively bind cyt c, deliver it into RAW264.7 macrophages, and activate apoptotic pathways (caspase 3/7), and cell death.
A. AFM images of various SWCNT samples deposited on mica substrates: a) SWCNT with cyt c; b) SWCNT with cyt c and PS/PC. c) The height cross section of the functionalized SWCNT in image b. B. Cyt c delivered into macrophages by PS-coated SWCNT activates caspase 3/7 (a) and increases the number of Trypan Blue positive cells (b). SWCNT were treated with 50 µM cyt c, washed twice and then were coated with PS and PC alone. Cells were incubated with protein/lipid/SWCNT conjugates in the presence of 100 µM chloroquine to trigger endosomal rupture. Data are normalized versus SWCNT/chloroquine. Note that under experimental conditions used chloroquine alone (100 µM, 15 min incubation) did not induce any significant activation of caspase 3/7. The data represent mean±s.d (standard deviation), n = 5, *p<0.05, PS-coated plus cyt c vs PS-coated SWCNT, non-coated SWCNT plus cyt c and SWCNT alone.
Figure 5.
Primary rat microglia recognized SWCNT functionalized with PS but not with PC.
A. Scanning electron micrographs of microglia treated with PS-coated SWCNT in vitro. Microglia (1.5×105 cells/ml) were incubated for 2 h with PC- or PS-coated SWCNT. At the end of incubation macrophages were washed and fixed for SEM. (Note: The red arrows in all the electron micrographs in figure 5 point SWCNT). B. Transmission electron micrographs of primary microglia exposed to SWCNT in vitro. Microglia (1.5×105 cells/ml) were incubated with PC- or PS-coated SWCNT. At the end of incubation microglia was washed and fixed for TEM. a) Microglia exposed to PS-coated SWCNT; b) Quantitative assessments of SWCNT phagocytosis by RAW 264.7 macrophages. A total of 60 cells from each sample type were analyzed by TEM. Data are mean±S.D., n = 3, *p<0.05, PS-coated SWCNT vs SWCNT, PC coated SWCNT and PS-coated Annexin V–treated SWCNT. C. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by microglia. Microglia (1.5×105 cells/ml) were incubated with NBD-PC- (a), NBD-PS-coated SWCNT (b) or NBD-PS-coated/Annexin V treated SWCNT (c). 1 – bright field image; 2 – blue fluorescence image, Hoechst 33342; 3 – green fluorescence image, NBD-labeled phospholipids; 4 – overlap of blue and green fluorescence images with image under bright field. d) Annexin V treatment of NBD-PS-coated SWCNT prevents their engulfment by microglia. Quantitative evaluation of cell number with engulfed SWCNT. Data are mean±s.d., n = 4. *p<0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT and NBD-PS-coated Annexin V treated SWCNT.
Figure 6.
Primary human monocyte-derived macrophages and dendritic cells recognized SWCNT functionalized with PS but not with PC.
A. M-CSF-activated HMDM (0.5×106 cells/ml) were incubated with NBD-PC- or NBD-PS-coated SWCNT (100 µg/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 (0.5×106 cells/ml) exposed to NBD-PC- or NBD-PS-coated SWCNT (100 µg/ml) for 24 h at 37°C 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 - 63×2. Data are mean±s.d.
Figure 7.
HeLa cervical carcinoma cells and SH-SY5Y neuroblastoma cells interact with PS-coated (but not with PC-coated) SWCNT.
A. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by HeLa cells. a) Overlay of blue and green fluorescence images. b) Quantitative evaluation of cell with engulfed SWCNT. Data are mean±s.d., n = 4. *p<0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT. B. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by SH-SY5Y neuroblastoma cells. a) Overlay of blue and green fluorescence images. b) Quantitative evaluation of cells with engulfed SWCNT. Data are mean±s.d., n = 4. *p<0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT.
Figure 8.
SWCNT functionalized with PS but not with PC engulfed in vivo by murine alveolar macrophages.
A. a)Transmission electron micrographs of bronchoalveolar macrophages isolated from C57BL/6 mice exposed to PS-coated SWCNT; b) high magnification image of the area shown in (a). B. Alveolar macrophages preferentially phagocytoze PS-coated SWCNT in vivo. TEM images were used to calculate the percent of phagocytosis-positive macrophages. At least 150 cells were counted for each treatment. Data are mean±s.d., *p<0.05, PS-coated SWCNT vs non-coated or PC-coated SWCNT. C57BL/6 female mice, 7–8 weeks old, were exposed to SCWNT (40 µg/mouse) by pharyngeal aspiration. Twenty-four hours following exposure, mice were sacrificed using sodium pentobarbital. Mice were lavaged with sterile PBS. The lavage fractions were pooled and centrifuged to obtain the cellular fraction. Cells were then fixed for TEM.