Biological Evaluation of a Novel Doxorubicin−Peptide Conjugate for Targeted Delivery to EGF Receptor-Overexpressing Tumor Cells

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial malignancies and thus can be used for EGFR-targeted therapy to improve antitumor efficacy. Therefore we synthesized a novel conjugate of doxorubicin (DOX) with an EGFR-binding peptide (NH₂-CMYIEALDKYAC-COOH; EBP) via an ester bond at position 14 of DOX through a glutarate spacer. To confirm that the DOX-EBP conjugate is capable of targeting tumor cells overexpressing EGFR, we compared the cellular accumulation, intracellular distribution and in vitro cytotoxicity of DOX-EBP and free DOX. After treating with equimolar concentration of DOX-EBP or free DOX, the conjugate accumulated at significantly higher levels in EGFR-overexpressing cells than in non-EGFR-overexpressing cells, while the intracellular accumulation of free DOX was almost the same in all the cells. However, the intracellular accumulation of DOX-EBP was significantly reduced in EGFR-overexpressing cells preincubated with inhibitory anti-EGFR monoclonal antibody, demonstrating the involvement of EGFR pathway in the transport of the conjugate. Confocal fluorescence microscopy reveals that the conjugate was distributed in cytoplasmic and perinuclear areas during the first 30 min, whereas the free DOX was accumulated in both cytoplasm and nuclei. After 24 h, however, the DOX signal in the cells treated with DOX-EBP was also distributed in the nuclei, suggesting the release of DOX from the conjugate and entry into the nuclei. Biodistribution and in vivo antitumor experiments, together with in vitro cytotoxicity, indicate that the therapeutic competence of DOX-EBP was due to its increased accumulation in EGFR-expressing tumor cells. Furthermore, the survival of tumor-bearing mice treated with DOX-EBP was significantly higher than that with free DOX. These data demonstrate the enhanced anticancer efficacy and reduced systemic toxicity of DOX-EBP conjugate with targeting ability to EGFR-overexpressing tumor cells.