Development and Screening of Targeted Anticancer Nanomedicines
Gillespie, James William
Type of DegreeDissertation
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Cancer remains a significant healthcare burden worldwide and novel strategies to improve treatment outcomes with improved patient quality of life are needed. Recently, nanomedicines, which encapsulate a therapeutic drug within a protected core, have been added to the repertoire of therapeutic options for a number of diseases. Nanomedicines provide increased circulation time and bioavailability for their encapsulated cargo, and can enhance the toxicity profile of therapeutic molecules in cancer cells. However, there are still significant dose-limiting and off-target toxicities that lead to poor clinical outcomes. Active targeting of nanomedicines to malignant cells may improve the therapeutic index of otherwise non-targeted nanomedicines. Phage display, a tool that uses a bacteriophage or ‘phage’ vector to display randomized peptide fusions within a structural protein of the parent vector, has been used by a number of research groups to identify ligands that interact specifically with the target cancer cells. In this project, two landscape phage display libraries were used to identify a number of novel ligands that interact specifically with overexpressed cell surface receptors on a non-small cell lung cancer (NSCLC) cell line. Two predominate phage clones, ANGRPSMT and VNGRAEAP, were shown to be selective for their target cells and the amino acids surrounding the common NGR motif can modulate the endocytic pathway of uptake and subcellular distribution of intact phage clones. The utility of these two peptides to increase cytotoxicity of a liposomal nanomedicine (Lipodox) by incorporation of the full-length phage major coat protein, pVIII, which displays the cell-specific fusion peptide on the exterior of the liposome membrane after insertion, was demonstrated. The VNGRAEAP-modified Lipodox performed better than ANGRPSMT-modified Lipodox. This showed that internalization of the targeting ligand is required for improved drug delivery. The increase in toxicity was determined to be due to an increased rate of intracellular doxorubicin uptake when compared to untargeted Lipodox. It was hypothesized that this actively-targeted drug delivery system could be translated into a functional screening assay in which selected ligands from either of the landscape phage display libraries could be screened for improved toxicity in the context of a common nanomedicine core. This strategy would prove more advantageous in comparison to a system developed through rational design based on binding data alone. Additionally, a novel pVIII protein isolation procedure, which allows for rapid modification of a common nanomedicine core (Lipodox) with minimal modifications to the liposome integrity and drug retention, was developed. In this context, a novel characterization assay for quantification of protein modification to targeted liposomes using flow cytometry and characterization of protein orientation using a novel dot blot assay was also established. Using this screening assay, seven ligands that displayed increased functional activity when compared to unmodified Lipodox were identified. In a doxorubicin-sensitive cell line (breast cancer; MCF-7), four ligands – DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD – were determined to be functionally active. Similarly, in a doxorubicin-resistant cell line (pancreatic cancer; PANC-1), three ligands with structurally similar YL motifs increased toxicity at least 2-fold in comparison with unmodified Lipodox. Results provide evidence of the utility of this platform for large scale functional screening of targeted nanomedicine preparations in a number of cancer phenotypes.
- Gillespie Dissertation.pdf