Comparison of ⁶⁸Ga-labeled RGD mono- and multimers based on a clickable siderophore-based scaffold

Cyclic pentapeptides containing the amino acid sequence arginine-glycine-aspartic (RGD) have been widely applied to target α_vβ₃ integrin, which is upregulated in various tumors during tumor-induced angiogenesis. Multimeric cyclic RGD peptides have been reported to be advantageous over monomeric counterparts for angiogenesis imaging. Here, we prepared mono-, di-, and trimeric cyclic arginine-glycine-aspartic-D-phenylalanine-lysine (c (RGDfK)) derivatives by conjugation with the natural chelator fusarinine C (FSC) using click chemistry based on copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC). The α_vβ₃ binding properties of ⁶⁸Ga-labeled mono-, di-, and trimeric c(RGDfK) peptides were evaluated in vitro as well as in vivo and compared with the references monomeric [⁶⁸Ga]GaNODAGA-c(RGDfK) and trimeric [⁶⁸Ga]GaFSC(suc-c(RGDfK))₃. All ⁶⁸Ga-labeled c(RGDfK) peptides displayed hydrophilicity (logD = -2.96 to -3.80), low protein binding and were stable in phosphate buffered-saline (PBS) and serum up to 2 h. In vitro internalization assays with human melanoma M21 (α_vβ₃-positive) and M21-L (α_vβ₃-negative) cell lines showed specific uptake of all derivatives and increased in the series: mono- < di- < trimeric peptide. The highest tumor uptake, tumor-to-background ratios, and image contrast were found for the dimeric [⁶⁸Ga]GaMAFC(c(RGDfK)aza)₂. In conclusion, we developed a novel strategy for direct, straight forward preparation of mono-, di-, and trimeric c(RGDfK) conjugates based on the FSC scaffold. Interestingly, the best α_vβ₃ imaging properties were found for the dimeric [⁶⁸Ga]GaMAFC(c(RGDfK)aza)₂.