Design, Synthesis, and Evaluation of a Novel Cytotoxic Peptide-Doxorubicin Conjugate Targeting Triple-Negative Breast Cancer

The aim of this thesis was to design, synthesize, and characterize a novel peptide-doxorubicin conjugate, evaluate in vitro stability and cytotoxicity properties of the conjugate and study its mechanism for uptake into the triple-negative breast cancer (TNBC) cells. For the synthesis of the conjugate, first, doxorubicin (Dox) was conjugated to a highly efficient established cross-linker, MCC, to give an intermediate product, MCC-Dox. MCC-Dox was characterized using NMR spectroscopy, mass spectrometry, and RP-HPLC and purified for subsequent reaction. Second, the 11-mer peptide 18-4 (NH2-CWxEAAYQrFL-CONH2) with high proteolytic stability and specificity for breast cancer cells was reacted with MCC-Dox to synthesize the final product peptide-Dox conjugate which was also purified and characterized. The evaluation of the stability in water, media, and human serum showed that conjugate possesses decent stability. The results from the evaluation of stability of conjugate in human serum up to 48 h showed that half-life for the conjugate was 18 h. Furthermore, when the conjugate was incubated in aqueous conditions with pH 7, more than 80% of it was still intact after 48 h. However, the half-life of the conjugate in the aqueous conditions with pH 5, or cell culture medium was 24 h and ~ 48 h, respectively. The cytotoxicity studies showed that the conjugate was as toxic as free Dox toward the TNBC cells, MDA-MB-231 and MDA-MB-468, and was 30 times less toxic toward the non-cancerous breast cells, MCF-10A, compared to the free doxorubicin. Furthermore, results from evaluating surface expression levels of keratin 1 (K1) in different breast cancer cell lines and a normal breast cell line using immunohistochemistry staining suggested that cell-surface expression of K1 in normal breast MCF-12A cell line is much less than that of TNBC MDA-MB-231 and MDA-MB-468 cell lines. The results presented here highlight the potential of a targeted peptide-Dox conjugate as a new modality for TNBC treatment