Recurrent targeting of anti-ICAM nanocarriers to endothelium in vitro and in vivo. (A) Sequential targeting of 2 doses of anti-ICAM/NCs into TNF-α–activated HUVECs. The number of surface-bound and internalized anti-ICAM/NCs in the first dose is taken as 100% (dashed lines represent the standard deviation of this control value). Binding and internalization of the second dose anti-ICAM/NCs were inhibited at 30 minutes after the internalization of the first dose, yet recovered to 100% by 3 hours. (B) Internalization kinetics for the first dose and second dose at 30 minutes versus 3 hours, determined as percent of internalized nanocarriers. Data are means ± SEM for n > 20 cells from 2 independent experiments. (C) Control mice received a single injection intravenously of either ¹²⁵I-labeled anti-ICAM/NCs (first dose, D1) or IgG/NCs (dashed line) to test targeting to the lungs. In other groups, mice were injected with nonlabeled anti-ICAM/NCs followed by a similar dose of ¹²⁵I-labeled anti-ICAM/NCs (black bars) or ¹²⁵I-IgG/NCs (gray bars) either 15, 30, or 150 minutes later (second dose, D2). In a separate group, mice were injected with a first dose of nonlabeled IgG/NCs and a second dose of ¹²⁵I-labeled anti-ICAM/NCs (white bars). Lung uptake was calculated as percent of injected dose per gram and plotted as percent of the level obtained with a single dose of anti-ICAM/NCs (D1) as M ± SEM, n = 4-5.