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theses

Since no treatment exists to cure advanced solid tumors, there is a growing need for innovative strategies to treat these patients. Antivascular therapy works by delivering therapeutics to tumor neovasculature to cut off blood supply and nutrients to the tumor, resulting in apoptotic or necrotic death. This study aims to selectively deliver to tumor vasculature adequate concentrations of the chemotherapeutic doxorubicin encapsulated in pH-tunable liposomes with doubly fusogenic properties for enhanced bioavailability. A PSMA (prostate specific membrane antigen)-targeting ligand is conjugated on the surface of liposomes to achieve high specificity to tumor endothelium, which uniquely expresses PSMA since normal endothelium is consistently PSMA-negative. A pH-responsive fusogenic peptide (GALA) is also attached on the liposome surface and remains masked during circulation of liposomes in the blood stream. Upon PSMA-mediated endocytosis of liposomes by tumor endothelial cells, pH acidification induces lipid phase-separation and domain formation on liposome membranes activating two fusion mechanisms — (1) the fusion peptides change their conformation from random coils to alpha-helices, become unmasked and available to interact with the endosomal membrane, and (2) the liposomal domain boundaries serve as sites to nucleate fusion with the endosomal membrane. As a result liposomes are expected to release the encapsulated cargo directly into the cytoplasm of tumor endothelial cells, avoiding entrapment in the endosomal pathway. The first part of this study uses a parallel plate flow chamber to mimic blood flow conditions and measures binding and internalization of PSMA-targeting liposomal doxorubicin by prostate adenocarcinoma cells expressing varying levels of PSMA and human endothelial cells induced to express PSMA. The second part of this study aims to evaluate the membrane activity of GALA–labeled liposomes by varying membrane parameters. To monitor fusogenic activity, the self-quenching relief of a fluorescent dye encapsulated in endosomal-analogue vesicles incubated with GALA-labeled liposomes is measured. Since self-quenching is inversely proportional to content release, we observe that GALA-labeled liposomes exhibit pH-triggered membrane activity and induce content release of the endosomal analogues at late endosomal pH values.

ETD_5152

M.S.

Includes bibliographical references

by Ana Maria Gomez

doi:10.7282/T3416V3N

ETD graduate

The author owns the copyright to this work.