Date of Award

January 2014

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Mark Saltzman

Second Advisor

Jiangbing Zhou

Abstract

Cancer continues to be a highly prevalent and lethal disease, despite significant advances in understanding tumor biology and developing new chemotherapies. Major obstacles in cancer chemotherapy are drug resistance and systemic toxicities. Potential strategies for addressing these problems include delivering combination therapies to overcome drug resistance, and utilizing synergistic agents to minimize dosing and subsequently drug toxicity. In turn, delivery can also be optimized to target the tumor site and consequently minimize systemic side effects. Polymer nanocarriers are gaining interest as vehicles for cancer therapeutics for their abilities to not only deliver multiple agents, but also target the tumor itself. Our goal is to design multi-layered polymer nanoparticles (MLNPs) for efficient delivery of small molecules and genetic material towards synergistically inhibiting tumor growth. The MLNPs were first optimized for transfection in vitro through delivery of plasmids encoding for luciferase (pLuc) and green fluorescent protein (pGFP). The particles were then evaluated for effective delivery of both a candidate small molecule, camptothecin (CPT), and a plasmid encoding for TNF related apoptosis inducing ligand (pTRAIL) (CT MLNPs). Co-delivery of CPT and pTRAIL via CT MLNPs were then evaluated for growth inhibition of brain, colorectal, and breast cancer cells in vitro. MLNPs were approximately 116 nm in diameter. They were able to delivery approximately 575 ng of plasmid per mg of particle, and between 0.1 mg to 0.01 µg of CPT per mg of particle. MLNPs were non-toxic, and human embryonic kidney cells (293T) transfected with pLuc loaded MLNPs expressed comparable amounts of luciferase as cells transfected with the gold standard lipid formulation, Lipofectamine 2000. Thirty-seven percent of transfected 293T cells expressed GFP 72 h after transfection. Studies on tumor death kinetics related to CPT exposure and pTRAIL transfection suggested that simultaneous transfection and drug exposure provided the greatest inhibition of cell growth. MLNPs were able to provide the optimal timing for delivery of both agents. Synergy analysis of co-delivering CPT and pTRAIL via CT MLNPs, using the Chou-Talalay method, provided a combination index at 50% inhibition ranging between 0.31 and 0.53 for all cell lines. These CI values indicate a synergistic interaction between the two agents. For obtaining a 50% effect level, co-delivery with MLNPs resulted in providing 3.14-7.38 fold reduction in CPT and 4.66 to 6.09 fold reduction in pTRAIL. These initial results support our hypothesis that MLNPs can deliver both small molecule and genetic agents towards synergistically inhibiting tumor growth.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/28/2019

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