Figure 1. Binding assay and in vitro and in vivo competition experiments.

(A) Binding assay of cRGD-ZW800-1 on intermediate integrin-expressing HT-29 cells shows an almost linear increase in fluorescence intensity with increasing applied concentrations. In addition, fluorescence signals are increased when cells are incubated at 37°C compared to 4°C with the various concentrations (p < 0.0001). This may be the result of enhanced internalization of the agent. (B) In vitro competition experiment (1:1 ratio) using unlabeled cRGD (competition group) showing decreased NIR fluorescent signals in both the α_vβ₃ positive U-87 MG (*p < 0.05) as well as the α_vβ₃ negative HT-29 cell line compared to the control without unlabeled cRGD. Healthy colon was used as background. (C) In vivo competition experiment using a 200 times higher dose of unlabeled cRGD compared to the dose of cRGD-ZW800-1 (1.24 mg, 2.0 μmol vs. 15.5 μg, 10 nmol) in the orthotopic HT-29 model. Compared to mice in the control group, a significant decrease in TBR was seen at 4 h (*p = 0.02) and 24 h (** p = 0.007). Healthy colon was used as background.