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Defining endocytic pathways to characterise the cellular uptake of extracellular vesicles

Roberts-Dalton, Hope 2017. Defining endocytic pathways to characterise the cellular uptake of extracellular vesicles. PhD Thesis, Cardiff University.
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Abstract

There is a need for vectors that, with high efficiency, can deliver small and macromolecular therapeutics into cells and defined intracellular locations. Extracellular vesicles (EVs), including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration, but are believed to involve endocytosis. For endocytic characterisation a significant challenge is having the ability to label exosomes directly or indirectly with fluorescent probes to qualitatively and quantitatively monitor exosome-cell interaction and uptake without compromising functionality. In this thesis, techniques to inhibit different pathways of endocytosis were investigated and further developed order to establish high content in vitro platforms to study the uptake mechanisms of potential drug delivery vectors focusing here exosomes as a model system. These techniques involved siRNA depletion of prospective endocytic proteins and chemical inhibitors of endocytosis. A simple and rapid method for fluorescent labelling purified Du145 exosomes covalently was for the first time utilised to allow comprehensive analysis of the 2 cellular uptake of differentiation competent prostate cancer derived EVs in live cells using confocal microscopy. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that a fluidphase endocytosis and/or macropinocytosis-like mechanism dependent on dynamin were involved in exosome internalisation. Over a period of six hours exosomes were observed to increasingly co-localise with lysosomes, indicating a possible intracellular terminus with significance with respects to utilisation of exosomes as drug delivery vectors. Overall this labelling method, when used in conjunction with established models of endocytic inhibition, provides new opportunities for analysing the cellular dynamics of exosomes and other extracellular vesicles as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors. The work also further enhances our ability to study multiple endocytic pathways as potential routes for entry of other drug delivery vectors to gain important information towards the design of more efficient formulations targeting a number of diseases.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Pharmacy
Subjects: Q Science > Q Science (General)
Uncontrolled Keywords: endocytosis, extracellular vesicle, fluorescent-labelling, intracellular trafficking
Date of First Compliant Deposit: 9 March 2018
Last Modified: 19 Oct 2019 03:29
URI: http://orca-mwe.cf.ac.uk/id/eprint/109685

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