Dissociation of ICAM-1 from internalized anti-ICAM nanocarriers. (A) Fluorescence microscopy of green FITC-labeled anti-ICAM/NCs in HUVECs at indicated time at 37°C. In 4 columns, red labeling depicts markers of early endosomes (anti–EEA-1), lysosomes (anti–LAMP-1), Texas Red dextran, or ICAM-1 cytosolic domain. Yellow color, vesicles in which anti-ICAM/NC particles colocalize with indicated markers (arrowhead); green color, anti-ICAM/NC–containing vesicles negative for indicated markers (arrow); red color, anti-ICAM/NC–free vesicles positive for indicated markers; blue color, noninternalized anti-ICAM/NCs (asterisk); white color, sites of ICAM-1 clustering by anti-ICAM/NC at the cell surface. Bar = 1 μm. (B) Percentage of localization of the markers (EEA-1, LAMP-1, Texas Red [TR] dextran, or cytosolic ICAM-1) to anti-ICAM/NC–containing vesicles, quantified by image analysis and plotted as a function of the time after anti-ICAM/NC internalization. (C) Schema of ICAM-1 recycling. ICAM-1 enters ECs via nascent vesicles along with anti-ICAM/NCs. Anti-ICAM/NCs traffic to lysosomes via early endosomes, whereas ICAM-1 partially escapes lysosomal pathway and recycles to the plasma membrane. The fluid phase TR dextran labels both the lysosomal and the recycling route. (D) Single-labeled dextran-containing vesicles, diverging from anti-ICAM/NC–containing vesicles, can be detected 1 hour after internalization and disappear by 3 hours, likely due to release from recycling compartments. Data in panels B and D are shown as M ± SD for n > 10 cells from 2 independent experiments.