Novel optical contrast agents containing both DFO and multi-RGD peptides

A series of novel near-infrared fluorescent compounds containing both desferoxamine (DFO) and multi-RGD peptides, i.e. DFO-Cypate-(RGD) _n-NH₂ (1), were designed and synthesized based on a dicarboxylic acid-containing near-infrared fluorescent carbocyanine (Cypate) scaffold. The trimeric 1 (n=3) showed the strongest cellular internalization into A549 cells in vitro among the four analogs of 1 (n=1, 2, 3, 4), suggesting that such a linear array of three RGD peptide motifs might be optimal for synergistic effects on cellular internalization. The four analogs showed higher internalization than an integrin α_vβ₃-targeting cyclic RGD peptide analog DFO-cypate-[RGDfK(∼)] (2) after 1h of incubation, indicating that the linear arrays of multi-RGD peptides might be different from the cyclic RGD peptide analog in the internalization kinetics and mechanism of receptor targeting. Confocal microscopy showed that 1 (n=4) could localize at least in part to the mitochondria. Noteworthy, the two compounds 1 (n=2, 3) resulted in a 1.5 to 2 fold increase in fluorescence of the calcium indicator fluo4 after 30 min of incubation. These results suggest the possible effects of these compounds on the cellular function by internalization. Such a type of near-infrared fluorescent cypate analogs containing both DFO and multi-RGD peptides could provide a platform for discovering and developing novel multifunctional optical contrast agents for integrin receptor targeting as well as related tumor imaging and therapy.