Anti-PECAM/NCs targeting and effects on endothelial cells in vitro and in vivo. (A) VE-cadherin staining in HUVECs incubated in the absence or presence of anti-PECAM/NCs at 37°C for 15 or 60 minutes. VE-cadherin (top panels) was continuously distributed around the entire periphery of control cells and cells exposed to NCs (lower panels). Magnification bar represents 20 μm. (B) HUVECs grown on 0.4-μm-pore transwell filters were incubated with ¹²⁵I-labeled BSA either in the absence or presence of anti-PECAM/NCs, control IgG/NCs, or 100 nM thrombin as a positive control for barrier disruption. After incubation for 1 hour at 37°C, media in the lower chamber was collected and radioactivity measured to calculate the percentage of ¹²⁵I-BSA transported across the endothelial monolayer with respect to total ¹²⁵I-BSA added. Data were normalized to ¹²⁵I-BSA transport in control cells. Data are mean plus or minus SEM (n = 4 wells; *P < .001). (C) Transmission electron microscopy of mouse lungs 3 hours after injection with anti-PECAM/NCs. Arrows indicate anti-PECAM/NCs bound to the surface of endothelial cells and being incorporated into vesicular invaginations. Arrowheads indicate anti-PECAM/NCs internalized by pulmonary endothelial cell (EC). Magnification bar represents 200 nm. (D) Lung-to-blood ratio of ¹²⁵I-labeled BSA 3 hours after injection in mice either in the absence of presence of anti-PECAM/NCs, control IgG/NCs, or TNFα. Data are mean plus or minus SD (n ≥ 4 mice; *P < .05).