Efficient targeting and internalization of anti-ICAM/α-Gal nanocarriers in micro- and macro-vascular endothelial cells. (a) Fluorescence microscopy images and quantification of TNFα-activated HBMECs and HUVECs incubated with FITC-labeled anti-ICAM/α-Gal NCs at 37°C for 30 min, washed and incubated in cell medium for 30 min. Cells were fixed and surface-bound NCs and nuclei were stained with Texas-Red-labeled anti-mouse IgG and DAPI, respectively. Internalization was compared to that of anti-ICAM NCs, shown in Figure S2. (b) Uptake of anti-ICAM/α-Gal NCs was also tested in the presence of amiloride or MDC, which inhibit CAM- vs clathrin-mediated endocytosis, respectively. In both (a) and (b), single-labeled green NCs are internalized (arrow) vs double-labeled (green+red) yellow NCs, which are located in the cell surface (arrowhead). Dashed lines mark the cell border, determined by phase-contrast. Scale bar, 10 μm. Data are mean±SEM (n≥55 cells, duplicated). *** is p≤0.001, by Student’s t-test.