Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Cancer is one of the leading causes of death in the world. Billions of dollars are spent to treat cancer every year. This clearly shows the need for developing improved treatment techniques that are affordable to every person. Early diagnosis and imaging of tumors is equally important for the battle against this disease. This dissertation will discuss new approaches for discovering and developing novel detection and treatment techniques for cancer using organic ligands, and Fe/Fe3O4 core/shell magnetic nanoparticles. A series of o-phenylenediamine derivatives with nitro-, methyl- and chloro- substituents were synthesized and studied their ability to act as anticancer agents by using steady-state, UV/Vis-, and fluorescence spectroscopy. In the absence of zinc(II), intercalation with DNA is the most probable mode of interaction. Upon addition of zinc(II), DNA-surface binding of the supramolecular aggregates was observed. The interaction of the supramolecular (-ligand-Zn2+-)n aggregates with MDA 231 breast cancer cells led to significant cell death in the presence of UVA at λ=313 nm displaying their potential as anticancer agents. Bimagnetic Fe/Fe3O4 core/shell nanoparticles (MNPs) were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected by dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin), a fluorescent dye, was attached to the dopamine-oligoethylene glycol ligands. These modified nanoparticles have the ability to selectively accumulate within the cancerous cells. They are suitable candidates for local hyperthermia treatment. We have observed a temperature increase of 11 ºC in live mice when subcutaneously injecting the MNPs at the cancer site and applying an alternating magnetic field The system is also suitable for Magnetic Resonance Imaging (MRI), which is a diagnostic tool to obtain images of the tumors. Our superparamagnetic iron oxide nanoparticles have the ability to function as T1 weighted imaging agents or positive contrasting agents. We were able to image tumors in mice using MRI. Various proteases are over-expressed by numerous cancer cell lines and, therefore, of diagnostic value. Our diagnostic nanoplatforms, designed for the measurement of protease activities in various body fluids (blood, saliva, and urine), comprise Fe/Fe3O4 core/shell nanoparticles featuring consensus sequences, which are specific for the target protease. Linked to the consensus sequence is a fluorescent organic dye (e.g. TCPP). Cleavage of the sequence by the target protease can be detected as a significant increase in fluorescence occurring from TCPP. We were able to correlate our diagnostic results with cancer prognosis.

The immunophilins are a superfamily of proteins comprising the cyclophilins, the FKBPs and the parvulin sub-families. Members are present ubiquitously in plant and animal cells, acting as both prolyl-isomerases and signalling proteins. Some also have chaperone activity. The prolyl isomerase function of the immunophilins has been identified as being central to progression of a large number of diseases, making them tempting drug targets. Whilst there are several assays which can be used to identify inhibitors of the prolyl isomerase function, they are hampered by one or more problems: multistep mechanisms, poor signal-to-noise ratios, expensive, laborious and unamenable to high throughput screening. Multiple fluorescent systems (fluorescence anisotropy, FRET, 2D-FIDA/FCS) and several technologies (solution and solid phase synthesis, solution and solid phase screening, combinatorial synthesis, and stopped-flow spectrometry) were explored to develop a system suitable for fast, efficient screening of immunophilins. The most promising of these is a prototype assay based on the design, cloning, expression and production of fluorescently labelled mutant of cyclophilin B, which shows an increase in fluorescence emission upon cyclosporin ligand binding.

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein conformation and function as well as active processes involving flippase and floppase proteins. Despite the biological significance, there are limited studies linking the consequences of lipid asymmetry to critical membrane properties and processes involving ion channels. One reason for this is the scarcity of reliable methods to create artificial membrane systems that incorporate both transverse lipid asymmetry and ion channels. Experimental artificial membrane systems incorporate essential cell membrane structures, namely the phospholipid bilayer, in a controllable manner where specific properties and processes can be isolated and examined in an environment much simpler than living systems. It is of particular interest to study asymmetry in transverse lipid composition across the phospholipid bilayer on such a system to probe the effects of the lipid composition and asymmetric arrangement of these lipids on the physicochemical properties of the membrane. By doing so, an understanding of how membrane asymmetry dictates membrane properties and in turn impacts cellular processes will be achieved. The primary goal of this thesis is to develop a platform for fabricating and characterizing compositionally controlled planar, free-standing, asymmetric membranes. This asymmetry was qualitatively demonstrated using a fluorescence quenching assay, and it has been quantified using a combination of anionic and zwitterionic lipids in concert with a patch-clamp amplifier system. Initial measurements of a transmembrane potential on a partially asymmetric bilayer were found to be between 10 and 25 mV. Increasing membrane charge asymmetry increases the offset voltage, as expected, and also modifies the stiffness of the membrane. These initial successes demonstrate a viable pathway to fabricate and quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membrane processes in the future.