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Title: "Establishing a Method for Modulating Progesterone Using a Nanoparticle-Based System"

Advisory Committee: Catherine M. Klapperich, PhD - BME, MSE, ME (Chair) Joyce Y. Wong, PhD - BME, MSE (Advisor) Joerg G. Werner, PhD - ME, MSE

Abstract: Hormones play a crucial role in promoting and maintaining many important processes. Progesterone, in particular, is involved in reproductive health, pregnancy maintenance, and hormone-dependent cancers. Many hormonal-based therapeutics are delivered systemically, resulting in side effects for the user or the development of resistance to the delivered agent. This project sought to develop a progesterone-specific nanoparticle-based system for localized modulation of progesterone. The aims included development of the particle, with the use of anti-progesterone antibodies, development of a measurement system to determine relevant, physiological levels of progesterone to validate the proof-of-concept studies, and testing of the particle against the developed progesterone measurement system. Development of the particle progressed in stages, beginning with the generation of oleic acid-coated superparamagnetic iron oxide nanoparticles (SPIONs). Much of the particle development efforts focused on the initial thermal decomposition reaction utilized to develop these SPIONs. Optimization focused on improvement of synthesis parameters to improve yield and reduce particle polydispersity, with reaction modifications resulting in improvement of yield more than threefold, a reduction in particle polydispersity, and an increase in the uniformity of particle morphology. The next phase of the particle design consisted of the generation of citric acid-coated SPIONs, followed by addition of polyethylene glycol (PEG) with active sites for the conjugation of anti-progesterone particles. Finally, antibodies were successfully conjugated to the surface of the particles, validated with protein absorbance at 280 nm. Additionally, several standard curves for progesterone, ranging in concentration from 0 to 50 mg/mL, with values of the coefficient of determination for the linear curves greater than 0.9 for all the tested methods, were generated. Specifically, standard curves were generated in ethanol, as well as ethanol diluted in both water and phosphate buffered saline (PBS) to better replicate physiological conditions. All three solutions resulted in linear standard curves for confident determination of the concentration of solutions of progesterone. Finally, the ability of the particles to bind to progesterone was successfully validated using UV absorbance at 241 nm by comparing the progesterone remaining after wash steps for antibody-particles, blank-particles, and the progesterone standard solution. This project resulted in the successful development of anti-progesterone antibody conjugated nanoparticles, validation of the specificity of the particles for progesterone, linear standard curves for progesterone in a variety of solutions, and optimization of the oleic acid SPION synthesis reaction. Future efforts should focus on the detection of progesterone at concentrations below 200 ng/mL, as this was a primary challenge in both the development of the progesterone concentration assay, as well as the testing of the affinity of the particles for progesterone. Additional research should focus on the optimization of both the antibody-conjugation process to maximize coating density while minimizing loss of unconjugated antibody and further development of the testing conditions to determine the duration of treatment and the strength of the affinity of the particles for progesterone.