DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYMERIC MICELLES FOR GENE-DIRECTED ENZYME PRODRUG THERAPY

Gene-directed enzyme prodrug therapy is a form of cancer therapy in which delivery of a gene that encodes an enzyme is able to convert a prodrug, a pharmacologically inactive molecule, into a potent cytotoxin. Currently delivery of gene and prodrug is a two-step process. Here, we propose a one-step method using polymer nanocarriers to deliver prodrug, gene and cytotoxic drug simultaneously to malignant cells. Prodrugs acyclovir, ganciclovir and 5-doxifluridine were used to directly to initiate ring-opening polymerization of epsilon-caprolactone, forming a hydrophobic prodrug-tagged poly(epsilon-caprolactone) which was further grafted with hydrophilic polymers (methoxy poly(ethylene glycol), chitosan or polyethylenemine) to form amphiphilic copolymers for micelle formation. Successful synthesis of copolymers and micelle formation was confirmed by standard analytical means. Conversion of prodrugs to their cytotoxic forms was analyzed by both two-step and one-step means i.e. by first delivering gene plasmid into cell line HT29 and then challenging the cells with the prodrug-tagged micelle carriers and secondly by complexing gene plasmid onto micelle nanocarriers and delivery gene and prodrug simultaneously to parental HT29 cells. Anticancer effectiveness of prodrug-tagged micelles was further enhanced by encapsulating chemotherapy drugs doxorubicin or SN-38. Viability of colon cancer cell line HT29 was significantly reduced. Furthermore, in an effort to develop a stealth and targeted carrier, CD47-streptavidin fusion protein was attached onto the micelle surface utilizing biotin-streptavidin affinity. CD47, a marker of self on the red blood cell surface, was used for its antiphagocytic efficacy, results showed that micelles bound with CD47 showed antiphagocytic efficacy when exposed to J774A.1 macrophages. Since CD47 is not only an antiphagocytic ligand but also an integrin associated protein, it was used to target integrin alpha(v)beta(3), which is overexpressed on tumor-activated neovascular endothelial cells. Results showed that CD47-tagged micelles had enhanced uptake when treated to PC3 cells which have high expression of alpha(v)beta(3). The synthesized multifunctional polymeric micelle carriers developed could offer a new platform for an innovative cancer therapy regime.

NEW MICROFLUIDIC SYSTEM TO INCREASE ROBUSTNESS OF ELECTRODE PERFORMANCE AND DEVELOP POINT-OF-CARE HEMATOCRIT DEVICE

The present dissertation aimed to develop a new microfluidic system for a point-of-care hematocrit device. Stabilization of microfluidic systems via surfactant additives and integration of semipermeable SnakeSkin® membranes was investigated. Both methods stabilized the microfluidic systems by controlling electrolysis bubbles. Surfactant additives, Triton X-100 and SDS stabilized promoted faster bubble detachment at electrode surfaces by lowering surface tension and decreased gas bubble formation by increasing gas solubility. The SnakeSkin® membranes blocked bubbles from entering the microchannel and thus less disturbance to the electric field by bubbles occurred in the microchannel. Platinum electrode performance was improved by carbonizing electrode surface using red blood cells. Irreversibly adsorbed RBCs lysed on platinum electrode surfaces and formed porous carbon layers while current response measurements. The formed carbon layers increase the platinum electrode surface area and thus electrode performance was improved by 140 %. The microfluidic system was simplified by employing DC field to use as a platform for a point-of-care hematocrit device. Feasibility of the microfluidic system for hematocrit determination was shown via current response measurements of red blood cell suspensions in phosphate buffered saline and plasma media. The linear trendline of current responses over red blood cell concentration was obtained in both phosphate buffered saline and plasma media. This research suggested that a new and simple microfluidic system could be a promising solution to develop an inexpensive and reliable point-of-care hematocrit device.