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Title: Endocytosis of nanomaterials : the development and application of imaging tools
Author: King, Philippa J.
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
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Endocytosis is the active process by which useful biomolecules cross the plasma membrane into the cell. There are several mechanisms, carried out by an extensive machinery of lipids and proteins, the most well-characterised of which is clathrin-mediated endocytosis (CME). The mechanism of endocytosis influences the trafficking pathway and terminal destination of a cargo once it has entered the cell. When attempting to deliver nanoparticles carrying drugs or RNA to cells, or when assessing the toxic effect of unintentional exposure to industrial nanoparticles, it is crucial to have knowledge of these pathways, specific to each cargo, to enhance the design and understanding of nanoparticle efficacy. In this thesis, two novel techniques for studying the uptake of nanoparticles, both quantitative and qualitative, were developed. These were used to study and characterise a model system of 50 nm silica particles endocytosed by retinal pigment epithelial cells. The methods were: • A MATLAB® programme to analyse uptake data obtained using confocal microscopy; • A particle tracking technique, based on optical tweezers, used on a single cell level. The uptake mechanism of 50 nm silica particles, analysed using confocal imaging of cells exposed to the particles under inhibition conditions, and quantified using the MATLAB® programme, was found to be mainly CME. Intracellular trafficking, analysed using confocal and electron microscopy, took place via the early and late endosomes, and most of the particles ended up in the lysosomes. The optical-tweezers based tracking technique was developed with the aim of following the endocytosis and trafficking pathway of a particle in a live cell, and although this was not achieved for 50 nm silica, single particle measurements were made of 500 nm latex on the surface live cells, to show proof-of-principle of the use of the technique. In summary, this thesis presents a holistic approach to studying the uptake and trafficking of nanoparticles, and demonstrates the design and implementation of widely-applicable techniques for this purpose
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available