Biomimetic modeling of the nitrogen-centered radical postulated to occur during the inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N·) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N·-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H 2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

An Investigation on Biocompatibility of Bio-Absorbable Polymer Coated Magnesium Alloys

Advances in biomaterials have enabled medical practitioners to replace diseased body parts or to assist in the healing process. In situations where a permanent biomaterial implant is used for a temporary application, additional surgeries are required to remove these implants once the healing process is complete, which increases medical costs and patient morbidity. Bio-absorbable materials dissolve and are metabolized by the body after the healing process is complete thereby negating additional surgeries for removal of implants. Magnesium alloys as novel bio-absorbable biomaterials, have attracted great attention recently because of their good mechanical properties, biocompatibility and corrosion rate in physiological environments. However, usage of Mg as biodegradable implant has been limited by its poor corrosion resistance in the physiological solutions. An optimal biodegradable implant must initially have slow degradation to ensure total mechanical integrity then degrade over time as the tissue heals. The current research focuses on surface modification of Mg alloy (MZC) by surface treatment and polymer coating in an effort to enhance the corrosion rate and biocompatibility. It is envisaged that the results obtained from this investigation would provide the academic community with insights for the utilization of bio-absorbable implants particularly for patients suffering from atherosclerosis. The alloying elements used in this study are zinc and calcium both of which are essential minerals in the human metabolic and healing processes. A hydrophobic biodegradable co-polymer, polyglycolic-co-caprolactone (PGCL), was used to coat the surface treated MZC to retard the initial degradation rate. Two surface treatments were selected: (a) acid etching and (b) anodization to produce different surface morphologies, roughness, surface energy, chemistry and hydrophobicity that are pivotal for PGCL adhesion onto the MZC. Additionally, analyses of biodegradation, biocompatibility, and mechanical integrity were performed in order to investigate the optimum surface modification process, suitable for biomaterial implants. The study concluded that anodization created better adhesion between the MZC and PGCL coating. Furthermore, PGCL coated anodized MZC exhibited lower corrosion rate, good mechanical integrity, and better biocompatibility as compared with acid etched.