Functionalisation and application of gold nanoparticles for enhanced cellular targeting and imaging using four-wave-mixing microscopy

Slesiona, Nicole 2024. Functionalisation and application of gold nanoparticles for enhanced cellular targeting and imaging using four-wave-mixing microscopy. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Previous research has demonstrated that cross-linking of receptors at the cell surface can drive therapeutic payloads to intracellular locations against the flow of endogenous receptors and ligands, thus improving cellular delivery. Gold nanoparticles (GNPs) are particularly promising for this application due to their customisable surface chemistry, which allows for precise control over ligand density essential for receptor cross-linking. GNPs exhibit unique optical properties that allow their use as contrast agents in both linear and non-linear biological imaging, offering advantages over traditional fluorophores that are limited by autofluorescence, photobleaching and therefore time-limited imaging. Despite these benefits, imaging GNPs in cellular environments, especially with linear imaging modalities, is complicated by background signals and the need for live-cell compatible sample preparations. To address these issues, this project utilised Four Wave Mixing (FWM) microscopy, a non-linear imaging method that allows for the background-free visualisation of GNPs. This research has developed and validated a method to quantify ligand density directly on GNPs on a single particle basis using extinction microscopy and single fluorophore bleaching, moving beyond traditional averaged estimates and showing that particle-by-particle quantifications strongly deviate from averaged methodologies. The FWM setup was developed for live cell imaging and demonstrated its utility in tracking GNPs at the single particle level in living cells, marking a substantial step forward in understanding GNP internalisation and receptor cross-linking dynamics. Lastly, previously reported non-colocalisation between GNPs and fluorophores was addressed. We have examined multiple functionalisation procedures using our ligand quantification method and live FWMi, identifying the best functionalisation procedure to be based on utilising pre-functionalised stabilising polymers for functionalisation, resulting in high quality resources for future studies on GNP internalisation. These advancements pave the way for optimising GNP-based cellular targeting, offering new insights into targeted therapeutic interventions at the cellular level.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	6 March 2025
Last Modified:	06 Mar 2025 16:33
URI:	https://orca.cardiff.ac.uk/id/eprint/176672

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