Internalization of RGD peptide conjugates of near-infrared fluorescent probes in different cell lines occurs via different integrin receptor subtypes

Expression of integrin α _vβ ₃ is upregulated in a number of cancers including colon, pancreas, lung and breast. Previous studies demonstrated that near infrared (NIR) fluorescent probes designed to target αvβ3 accumulated both in vitro and in vivo in α _vβ ₃-positive tumor cells. To evaluate the selectivity of some NIR-labeled RGD peptides for α _vβ ₃, the molecular probes were incubated in different cells, including the α _vβ ₃-positive U87 and A549 cells, and α _vβ ₃-negative HT29 cells. Whereas the RGD compounds tested internalized in the A549 cells, their uptake by the HT29 cell line, which is positive for α _vβ ₅ and α _vβ ₆, was low. The uptake of these probes in U87 depended on the structural features of the compounds. Further studies with functional blocking antibodies showed that the internalization in the α _vβ ₃-positive cells may be mediated by different integrin receptor subtypes. The preliminary results suggest that the internalization of linear RGD peptides is mediated by the α _vβ ₃ heterodimer but rearrangement of the peptide sequence could alter the selectivity of the molecular probes for different integrin subunits in the dimeric α and β proteins. Thus, a careful choice of RGD peptides can be used to monitor the functional status of different integrins in cells and tissues.