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Title: Tracing uptake, biodistribution and efficacy of NanoDrugs in aquatic organisms using advanced imaging
Author: Windell, D.
ISNI:       0000 0004 7962 9249
Awarding Body: University of Exeter
Current Institution: University of Exeter
Date of Award: 2019
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Gold nanoparticles (AuNPs) are increasingly being used in biomedicine for enhancing drug delivery and facilitating imaging, as well as directly in applications such as cancer photothermal therapy. Despite this, very little is known about their potential impacts on the environment. Importantly, the effectiveness of AuNPs in such applications depends on a range of particle specifications that have been optimised during development to serve their specific purpose. The manipulations include their size and the addition of coatings, usually to enhance biocompatibility. How these different particle characteristics affect their bioavailability and biological effects on wildlife once released into the environment, has received very little research attention. Consequently, the main aims of this thesis were to investigate, principally via imaging methods, how the characteristics of size and surface coatings of gold nanoparticle (AuNP) affected bioavailability, tissue distribution and potential for toxicity using zebrafish (Danio rerio) as an experimental model. To undertake this work, a second major objective of the thesis work was to construct an imaging platform to trace the uptake and biodistribution of the gold nanoparticles. For this, I constructed an open source selective-plane illumination microscopy system (OpenSPIM) which allowed me to quantitatively trace fluorescently tagged AuNPs in exposed zebrafish embryos with relatively high-throughput. Applying SPIM through multiple angles, the fluorescence tag on the AuNPs was detectable throughout the zebrafish embryo body enabling concentration-response uptake analyses. Having built the OpenSPIM system from the ground up, careful method developments were undertaken to optimise the SPIM which included the imaging of multiple transgenic models of zebrafish such as the MPO:GFP model with fluorescent neutrophils, the elavl3:GCaMP6s utilised in chapter 6 to measure neural activity, and the casper model which was utilised in all uptake studies. Studies into the toxicity of the AuNPs were carried out with models for detecting assess oxidative stress responses in the 3EpRE:hsp70:mCherry model (using the Zeiss inverted microscope) and the pod::NTR-mCherry/l-fabp::VDBP-GFP model to assess for damage to the pronephric glomerular filtration barrier (using the EVOS fluorescent microscope). The OpenSPIM system was applied to image uptake and distribution of quantum dots (CdTe) in the casper model which indicated minimal uptake in the entire zebrafish embryo. Extensive oxidative stress was detected for exposures to aqueous levels of CdTe QDs down to 10 µg/L in the lateral line neuromasts, fionocytes and pronephros (undeveloped kidney) but there were no effects to the glomerular filtration barrier. Aqueous exposure to AuNPs illustrated a size selective uptake with the highest uptake for 40-80 nm AuNPs across a size range between 10 to 100 nm. This was principally found accumulating in the pronephros and gut with some fluorescence visualised in the heart. The effect of AuNP functionalisation on uptake in the casper model demonstrated varied levels of uptake depending on surface modification. Modifications of the AuNPs resulted in enhanced uptake levels for coatings with PEG and TNFα but not for NHS/PAMAM where there was decreased uptake. Depuration studies with all AuNP studies indicated that the AuNPs were cleared (but not completely) from the zebrafish embryos over a period of 4 days. No toxic effects were seen in any study with AuNP exposure in concentrations that exceed environmentally relevant doses (2 mg/L). This body of work illustrates the utility of SPIM for detecting the uptake and fate of selected NPs in exposed fish embryos. When combined with transgenic models that allow for functional analyses across specific tissue targets and effect mechanisms, thus providing a powerful tool for nanoparticle screening. Finer level details relating to the biological effects of NPs, however, require imaging methods that allow for sub-cellular resolution to analyse cell mechanics and how NPs influence cellular functions.
Supervisor: Tyler, C. ; Owen, S. ; Moger, J. Sponsor: AstraZeneca UK Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Gold Nanoparticles ; Light sheet ; Nanodrugs ; Selective Plane Illumination Microscopy ; Zebrafish ; Nanotoxicology ; Ecotoxicology ; Microscopy