Selective Delivery of Doxorubicin to EGFR+ Cancer Cells by Cetuximab–DNA Conjugates

Doxorubicin is a hydrophobic anticancer drug that has poor selectivity, due to the lack of active targeting capability. Here, learning lessons from the success of antibody–drug conjugates, we have designed a new doxorubicin delivery system without conjugating doxorubicin to antibody directly. In this setup, cetuximab, an antibody that targets the epidermal growth factor receptor (EGFR) in cancer cells, was conjugated to a single-stranded DNA with a carefully designed sequence in a site-selective manner by using the DNA-templated protein conjugation (DTPC) method. The DNA duplex in the conjugates serves as a carrier of doxorubicin through noncovalent intercalation, and cetuximab functions as the targeting agent; this could drastically decrease systemic toxicity and potentially avoid under- or overdosing. The size of conjugates loaded with doxorubicin was about 8.77 or 16.61 nm when characterized by dynamic light scattering and atomic force microscopy, respectively. In vitro cytotoxicity and selective cancer cell killing was investigated against two EGFR⁺ cell lines (KB and MDA-MB-231) and one EGFR^? cell line (NIH-3T3). Cytotoxicity and flow cytometry data showed that doxorubicin loaded in cetuximab–DNA conjugates was more potent in terms of cell cytotoxicity than free doxorubicin in EGFR-overexpressed cell lines, thus suggesting that the conjugates were more selectively and easily taken up into cells, followed by rapid release of doxorubicin from the system into the cytoplasm from endosomes.