Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting

Tumor vasculature can be targeted by peptides containing an RGD (Arg–Gly–Asp) sequence, which bind to α vβ 3 and α vβ 5 integrins on angiogenic endothelial cells. By covalently attaching cyclic RGD-peptides (cRGDfK) to a protein backbone, we prepared a multivalent peptide–protein conjugate with increased affinity for α vβ 3/α vβ 5 integrins. We demonstrated that RGDpep–protein conjugate bound to HUVEC, whereas the conjugate prepared with the control RAD peptide was devoid of any binding. RGDpep–protein conjugate was furthermore functional in inhibiting the adhesion of HUVEC to α vβ 3/α vβ 5 ligand vitronectin, and direct binding of the radiolabeled conjugate to HUVEC was inhibited by α vβ 3/α vβ 5-specific RGD peptides. Finally, RGDpep–protein conjugate was shown to be internalized and degraded by HUVEC, a process that could be inhibited by lysosomal degradation inhibitors chloroquine and ammonium chloride. This cellular handling was significantly influenced by the presence of cations, which strongly inhibited internalization. This is the first study that shows direct evidence that primary endothelial cells are capable of internalizing RGD-containing macromolecular proteins. This feature makes them attractive carriers for the intracellular delivery of potent anti-angiogenic drugs into endothelial cells for the treatment of cancer and chronic inflammatory diseases.