Publication Date

12-2018

Date of Final Oral Examination (Defense)

11-29-2018

Type of Culminating Activity

Dissertation

Degree Title

Doctor of Philosophy in Biomolecular Sciences

Department

Interdisciplinary Studies

Major Advisor

Denise G. Wingett, Ph.D.

Advisor

Daniel Fologea, Ph.D.

Advisor

Dmitri Tenne, Ph.D.

Advisor

Juliette Tinker, Ph.D.

Abstract

The production of materials at the nanoscale leads to novel properties and has made the field of nanotechnology a part of everyday life. Numerous applications of nanomaterials have led to their use in electronics, optics, and medicine. However, creating materials at such a small size brings them on the same scale as many biomolecules and cellular components, altering their interactions with biological systems. This can lead to unintended biological impacts as many nanomaterials are considerably more toxic than their bulk counterpart material. ZnO nanoparticles (nZnO) are particularly interesting in this context. The FDA classifies ZnO as a generally recognized as safe substance, but numerous reports have demonstrated that they are inherently toxic when produced in the nanoscale. Much research has been conducted on understanding what makes them toxic in order to modify their properties for specific applications. While this would seem to impede their commercial use, many of the properties that make nZnO toxic have been exploited for the treatment of various diseases.

A wealth of knowledge has been generated about the physicochemical properties of nZnO that contribute to their toxicity, yet controversy remains about the toxicity mechanism. Numerous factors contribute to the toxicity of nZnO and the mechanism can vary greatly between cell types and the properties of the nanoparticles (NPs) under study. Even with all the data available, more work is needed to understand the complex interplay of nZnO with cells to fully exploit their commercial use and development as a potential therapeutic. In this regard, studies described in this dissertation were conducted to help further the understanding of what aspects contribute to the cytotoxicity of nZnO and how different properties of the material can be utilized for biological applications.

The first chapter demonstrates that biological buffers can significantly impact the dissolution property of nZnO and may influence the conclusions that have been drawn from previously reported studies. All the biological buffers tested, including HEPES and MOPS that are routinely utilized in cellular media and biological imaging solutions, induced the rapid dissolution of nZnO. This observation extended to other experiments that demonstrate the inclusion of biological buffers in commonly used RPMI media, impacted the conversion of nZnO to other chemical species and altered its structural morphology. As dissolution has been implicated as one of the primary sources of nZnO toxicity, cellular viability experiments were conducted and the inclusion of HEPES in the media was found to significantly increase the toxicity of nZnO towards leukemic Jurkat T cells. These results highlight the fact that environmental factors need to be carefully considered when assessing the toxicity of nZnO.

Ion channels are critical to a cell’s ability to maintain homeostasis and are imperative for the correct functionality of many cells. Therefore, the second chapter focused on what effects nZnO may have on transmembrane transport. For these assessments, lysenin, a pore forming toxin that mimics ions channels with respect to ionic transport and regulation, was utilized to investigate potential interactions of protein channels with nZnO. The conductance of lysenin was greatly diminished in the presence of nZnO and is believed to depend on electrostatic interactions. Lysenin’s conductance can be inhibited from zinc ions, but the dissolution of nZnO was ruled out as being responsible for the modulation of the transport capabilities of the protein. We concluded that the positively charged nZnO interacts with negative residues within lysenin to alter the conductance and these observations may translate to a potential contributor to the cytotoxicity of nZnO.

Many of the assessments on nZnO toxicity have relied on end-point observations. This is due, in part, to the fact that their small size makes it extremely difficult to track their interplay with cells in real-time and fluorescent labeling of the NPs may alter how unmodified nZnO interacts with cells. Direct fluorescence imaging of the NPs could substantially help in tracking the complex interactions of nZnO with cells. However, the band gap of nZnO is 3.37 eV which correlates to 368 nm and therefore requires the use of UV excitation sources to generate photo emissions. Unfortunately, UV excitation sources and detectors are generally absent in most conventional fluorescence microscopes making this approach difficult. The third study of this project sought to modulate certain physical properties of nZnO to create a way to fluorescently track the NPs in living cells without changing the composition of the material. To achieve this goal, a systematic control of defects in the crystal of nZnO was carried out to alter its properties. By producing a relatively high number of defects in nZnO, the band gap of the material was lowered to ~3.1 eV (400 nm) and produced a narrow emission in the visible spectra, with a peak at 425 nm. These changes allowed for the use of a 405 nm laser, generally available on fluorescent microscopes, to image the nZnO with confocal microscopy. Initial live-cell imaging experiments were conducted to demonstrate the feasibility of utilizing these new nZnO to track their interactions with cells.

The final piece of this dissertation sought to utilize the ability of nZnO to generate ROS when photo irradiated for a new drug delivery platform. Towards this end, the nZnO were encapsulated within a lipid coating and in vitro studies demonstrated that the encapsulation of the NPs essentially removed the toxicity at concentrations up to 10 times the IC50 of bare nZnO. This feature could be extremely helpful in preventing off-target effects when treating patients with nZnO but requires reestablishing the toxicity when the NPs reach the cancerous environments. To this end, the cancer cells that were treated with the encapsulated nZnO were exposed to irradiation and the cytotoxicity of the NPs was restored. To further expand upon this strategy, a fluorescent dye was co-encapsulated with the nZnO to simulate hydrophilic drug loading and allow for evaluations on the ability to trigger the release of the dye. Studies on the release kinetics demonstrated a rapid release of the dye upon irradiation and gave insights into optimizing the encapsulation of the NPs. To demonstrate that this strategy is not just a novelty of fluorescent dyes, the hydrophobic chemotherapy drug Paclitaxel was co-encapsulated with nZnO. Both Jurkat T cell leukemia and T47D breast cancer cells were treated with the co-encapsulated nZnO and Paclitaxel. In both cases, the triggered release groups showed improved toxicity towards the cells with the most pronounced difference noted for the breast cancer cells. Taken together, the cumulation of this dissertation helps further the progress in understanding the cytotoxicity of nZnO, offers a new way to study the interactions of essentially pure nZnO with cells and provides a novel strategy for the use of nZnO as a therapeutic and potential diagnostic tool.

DOI

10.18122/td/1472/boisestate

Available for download on Saturday, December 19, 2020

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