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In Vivo Near-Infrared Fluorescence Imaging of Integrin α2β1 in Prostate Cancer with Cell-Penetrating-Peptide–Conjugated DGEA Probe

The overexpression of integrin α2β1 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 α2β1 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 α2β1 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 α2β1–positive). Receptor specificity was confirmed with blocking experiments and evaluation in a CWR-22 control tumor model with low α2β1 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.