In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or (125)I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5≫EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various (125)I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of (125)I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.