Mansoor M. Amiji
Robert Campbell, Rebecca L. Carrier
Date of Award
Master of Science
Department or Academic Unit
Bouvé College of Health Sciences. Department of Pharmaceutical Sciences.
Pharmaceutical science, Oncology, Heat shock proteins, HSP70, Nanoemulsion, Temperature-sensitive
Pharmacy and Pharmaceutical Sciences
As the second leading cause of death worldwide, cancer is one of the most debilitating diseases that continue to mystify researchers and clinicians. Despite advances in new drug discoveries and treatment combinations, the mortality rate due to cancer has not changed significantly since the 1950's. The inability to detect cancer in its preliminary stage accounts for lethality, which is then associated with poor prognosis as a result of dissemination to other organs. Besides physical limitations of conventional chemotherapy such as targeted drug delivery and drug accumulation, it also suffers from chemical limitation of resistance development by the cancer cells. Therefore, there is a strong need for a paradigm shift in the approach to cancer therapy. One of the differences between normal cell and tumor cell, at molecular level, is the differences in the ability of express Heat Shock Proteins (Hsp) over an extended period of time, when subjected to heat. Cancer cells are not capable of producing Hsp, a chaperone molecule, which are required to refold the denatured proteins, which otherwise would result in proteo-toxicity resulting in cell death. Besides exploiting the inherent tumor characteristic of being susceptible to heat, local hyperthermia also aids in higher drug delivery and increased intratumoral drug perfusion. The objective of this study was to develop a delivery system that could respond to temperature stimuli, resulting in higher payload at elevated temperatures and to evaluate the potential of heat therapy when co-administered with a derivative of doxorubicin, a pro-apoptotic chemotherapeutic agent. The delivery system selected was oil-in-water nanoemulsion prepared using polyunsaturated fatty acids (PUFA) rich oil as the internal phase and water as the external phase. The optimized formulations were characterized for oil droplet size, surface charge, morphology and stability. Pine-nut oil nanoemulsions were successfully formulated with an approximate hydrodynamic diameter of 110 nm and a -40 mV surface charge. A hydrophobic doxorubicin derivative (i.e., doxorubicin stearate) was efficiently encapsulated in the oil phase of the nanoemulsion. TEM analysis showed that the oil droplets of the nanoemulsion had a spherical shape and smooth surface morphology. Temperature responsiveness of the optimized formulation was evaluated in-vitro using a lipophilic fluorophor which does not degrade at elevated temperatures. SKOV3 human ovarian adenocarcinoma cells and SKOV3TR resistant cells were cultured and intracellular delivery of nanoemulsion formulation was examined qualitatively and quantitatively by fluorescence microscopy and fluorescence spectroscopy respectively. The cell viability and IC50 values were determined upon co-administration of heat and drug treatment using MTT assay in both types of cell lines. Western blot analysis showed upregulation of Hsp70 in response to any noxious stimuli and the downregulation in response to hyperthermia for sufficiently long duration, which explains the mechanistic pathway of heat induced cancer cell death. Qualitative evaluation of apoptosis was done by TUNEL assay while quantitative evaluation was done by caspase-3/7 and FACS analysis. All the above result allowed us to understand the synergistic therapeutic effect of combinational chemo- and thermal therapies.
Bakshi, Chinmay, "Combination chemo- and thermal therapies with temperature-sensitive nanoemulsions in drug resistant tumor cells" (2009). Pharmaceutical Science Master's Theses. Paper 2. http://hdl.handle.net/2047/d10019418
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