Fabrication and Optimization of Dp44mT-loaded Polymeric Nanoparticles for Treatment of Malignant Cells
Holley, Claire Katherine
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Cancer is the second leading cause of mortality worldwide, resulting in over eight million deaths per year. In the fight against cancer, traditional chemotherapeutics are not very effective due to poor delivery, toxicity to healthy tissues, and ever-increasing cancer resistance. Since neoplastic cells require increased levels of iron (Fe) to proliferate, a promising strategy for cancer treatment is Fe deprivation using metal chelators. One such chelator, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to be extremely toxic towards many types of cancer in its free form (IC50 of 4 - 500 nM), due to its ability to chelate both Fe and copper (Cu), produce reactive oxygen species (ROS) through redox cycling, and overcome multi-drug resistance in malignant cells. Therefore, Dp44mT presents a promising candidate for the treatment of highly aggressive tumors. However, due to the hydrophobicity and toxicity of this compound, encapsulating Dp44mT into a nano-carrier will enhance its therapeutic effectiveness, while also preventing premature drug degradation, improving biodistribution and drug release to the tumor, and mitigating negative side effects to healthy tissues. The objective of this project was the development of a new anti-cancer nano-formulation based on Dp44mT and the in vitro evaluation of this formulation against malignant cells. To this end, we first utilized two distinct techniques, nanoprecipitation and single emulsion, to fabricate nanoparticle of poly(lactic-co-glycolic acid)(PLGA) loaded with Dp44mT (referred to as “Dp44mT-NPs”). During fabrication, Dp44mT encapsulation efficiency and NP size was optimized through the adjustment of the polymer, drug, and surfactant concentrations, as well as injection rate. The resultant Dp44mT-NPs were also characterized for shape, surface potential, colloidal stability and drug release. Next, we assessed, for the first time, the therapeutic effectiveness of both free and encapsulated Dp44mT in glioma (U251, U87) cells, as compared to healthy astrocytes. We further applied our Dp44mT-NPs to other malignant cells, namely breast (MCF7) and colorectal (HT29) cancer cells, to evaluate this nano-formulation as a universal anti-cancer platform. Finally, we modified this nano-formulation, via surface PEGylation and conjugation of a cancer-specific targeting ligand, to improve nanoparticle stability, biodistribution, and delivery for future in vivo applications. We then re-evaluated the efficacy of our PEGylated Dp44mT-NPs against malignant cells, both with and without targeting. Lastly, we assessed the ability of these PEGylated NPs to bypass the endothelial layer of an in vitro Blood-Brain Barrier model. In summary, this dissertation presents the fabrication, optimization, and assessment of a novel nano-formulation, containing anti-cancer chelator Dp44mT, for future application as a chemotherapeutic against malignant cells.