In vivo near-infrared fluorescence imaging of integrin α2β1 in prostate cancer with cell-penetrating-peptide-conjugated DGEA probe.

UNLABELLED The overexpression of integrin α(2)β(1) has been demonstrated to correlate with prostate tumor aggressiveness and metastatic potential. Recently, we reported that the DGEA peptide is a promising targeting ligand for near-infrared fluorescence and microPET imaging of integrin α(2)β(1) expression in prostate cancers. Here, we aimed to further improve the targeting efficacy of this peptide by incorporating a series of cell-penetrating peptides (CPPs) into the DGEA sequence. METHODS After the conjugation with appropriate fluorescent dyes, the CPP-DGEA peptides were evaluated in human prostate cell lines (PC-3, CWR-22, and LNCaP) that contain different integrin α(2)β(1) expression levels. In addition, to reduce excess kidney uptake, a carboxypeptidase-specific sequence Gly-Lys was incorporated into the probe design, allowing for cleavage by the kidney brush border enzymes of the CPP before uptake by proximal tubule cells. RESULTS Although the CPP motif greatly facilitated the translocation of CPP-DGEA without affecting binding specificity in vitro, fluorescent dye-labeled CPP-DGEA demonstrated extremely high kidney uptake in vivo. Kidney uptake was dramatically decreased after a carboxypeptidase-specific peptide linker (Gly-Lys) had been incorporated into the probe design. The optimized probe demonstrated a prominent accumulation of activity in PC-3 tumor (integrin α(2)β(1)-positive). Receptor specificity was confirmed with blocking experiments and evaluation in a CWR-22 control tumor model with low α(2)β(1) expression. CONCLUSION This study demonstrated that the introduction of a CPP sequence can facilitate the internalization of an integrin-targeted peptide probe in vitro. Moreover, a cleavable peptide linker successfully reduced kidney uptake while preserving good tumor uptake in vivo.