Date of Award

8-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Committee Member

Dr. Tzuen-Rong J. Tzeng, Committee Chair

Committee Member

Dr. Olin Thompson Mefford

Committee Member

Dr. Yanzhang Wei

Committee Member

Dr Xiuping Jiang

Abstract

Magnetic nanoparticles (MNPs) are currently being extensively in multitude of biomedical applications on account of their exceptional biocompatibility. By attaching different targeting ligands/molecules, MNPs have been broadly used in magnetic hyperthermia, cancer therapy, targeted drug delivery, MRI imaging, pathogen detection, and biological cell-separation. In this dissertation, MNPs coated with polyethylene oxide (PEO) based polymer (PEO-MNPs) and functionalized with bacterial adhesin-specific glycoconjugate molecule Neu5Ac(α2-3)Gal(β1-4)-Glcβ-sp (GM3-MNPs), are investigated for their interactions with enterotoxigenic Escherichia coli (E. coli). It also describes the feasibility of using alternating magnetic fields (AMF) for targeted killing of E. coli K99 (EC K99) strain using MNPs. Lastly, the interactions of MNPs with normal human colon cells CCD-18Co are explored to assess their in vitro biocompatibility. To begin with, GM3-MNPs were synthesized by based on 'click chemistry' platform. Bacteria specific aggregation of EC K99 was seen due to interactions occurring between GM3-MNPs and adhesin molecules of EC K99. These interactions were observed by means of fluorescence microscopy, transmission electron microscopy (TEM), and colony forming units (CFU) assays. The preliminary cytotoxicity assay performed on normal colon cells CCD-18Co indicated excellent biocompatibility of GM3-MNPs. Thus, such glycoconjugate functionalized MNPs can be effectively utilized as anti-adhesion and anti-bacterial agents for reducing gastro-intestinal (GI) tract infections. Next, GM3-MNPs were used along with AMF for targeted killing of EC K99 cells. CFU/ml assays indicated that killing rate of EC K99 was mainly dependent on concentration of GM3-MNPs and AMF exposure time. Clinically relevant reduction in CFU/ml of EC K99 was achieved after 120 minutes of AMF exposure in presence of GM3-MNPs in both pure and mixed bacterial culture environment. Enormous cell-membrane damage was observed via fluorescence microscopy and TEM imaging of EC K99 cells after AMF exposure in presence of GM3-MNPs. AMF exposure in presence of GM3-MNPs also caused significant decrease in intracellular ATP levels of EC K99. These results suggest that bacterial specific glycoconjugate MNPs along with AMF can be efficiently employed as novel non-antibiotic platform to inactivate targeted bacterial pathogens. Finally, the overall biocompatibility of GM3-MNPs was examined in CCD-18Co cells and compared to PEO-MNPs. GM3-MNPs were found to have relatively stable hydrodynamic diameter in cell-culture media DMEM whereas PEO-MNPs drastically increased their size on account of protein-corona formation. Both cytotoxicity and ATP assays revealed that GM3-MNPs exhibited great biocompatibility in the cells. CCD-18Co cells also maintained their overall cell-membrane integrity in presence of GM3-MNPs. Interestingly, GM3-MNPs were able to substantially decrease the glutathione (GSH) levels in the cells leading to increased oxidative stress. Thus, by properly controlling surface properties of glycoconjugate functionalized MNPs and attaching different drugs, they can be potentially used as colon specific drug-delivery carriers for therapeutic applications.

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