Design and Sythesis of Conjugages of Amphiphilic Cell-Penetrating Peptides Containing Anticancer Drug and Ligand for Extra Cellular Matrix Biomarker to Provide Efficient Tumor-Targeting

Peptide-drug conjugates (PDCs) have emerging applications in the safer delivery of chemotherapeutics to the site of diseases to avoid the side effects associated with the drug. PDCs also improve the physicochemical property of the anticancer drug. Therefore, we have designed and synthesized various conjugates of anticancer drugs with cancer- targeting peptides using established biomarkers of cancer such as the integrin receptor and extra-domain of fibronectin (EDB-FN) at the extracellular matrix. In the first studies, a series of targeting peptides targeting EDB-FN were synthesized and conjugated to different anticancer drugs. The synthesis, characterization, stability, and hydrolysis were performed. Cell viability studies were performed to evaluate the effect of targeting and efficacy of the drug on different cell lines overexpressing EDB-FN. The confocal microscopy confirmed the targeting of conjugates to the EDB-FN to support our hypothesis to use peptide-drug conjugates for tumor targeting. In the second study, a bifunctional cyclic cell-penetrating peptide was used as a scaffold to the peptide-drug conjugate to increase the water solubility of the hydrophobic drug without decreasing cell-penetration. This approach was used in combination with a targeting peptide (ZD2 or RGDC) to target EDB-FN and integrin biomarkers overexpressed in the extracellular matrix of cancer cells. The synthesis of these peptide-drug conjugates was optimized and successfully performed. A panel of cancer cell lines was selected to evaluate the potency and targeting efficiency of conjugates based on the overexpression of EDB-FN and integrin receptors. The antiproliferative assay was performed using conjugates, drugs, and peptides, and data revealed a decrease in the toxicity of the drug toward non-cancerous kidney epithelial cells (HEK-293) but comparable toxicity toward cancerous cell lines. Peptide-drug conjugates were less toxic as compared to the drug due to slower release kinetics, but it was providing a wider therapeutic window between non-cancerous cells versus cancerous cells. This dissertation includes the data on peptides used for cancer targeting, protocols and methodology of synthesis of the peptide, conjugation of drug to peptide, purification of peptide and peptide-drug conjugates, anti-proliferative assay, hydrolysis and stability assay, cellular uptake study, and confocal microscopy study