Semiconducting polymer-based FRET nano-and-microsensors for revealing intracellular disulphide reduction kinetics and mechanism in immune cells
File(s)
Author(s)
Tang, Jiaqing
Type
Thesis
Abstract
An in-depth investigation of the kinetics and mechanisms of intracellular disulphide reduction is of the utmost importance for elucidating the fundamental cellular processes involved in redox homeostasis, as well as aiding the development of efficient and targeted redox-responsive drug delivery systems. However, knowledge of disulphide reduction remains controversial due to the complex redox sources and uncertain intracellular transportation pathways. To address this issue, a reduction-sensitive Förster resonance energy transfer (FRET) nanosensor based on a semiconducting polymer was developed, and this sensor was applied to study spatiotemporal intracellular reduction kinetics and mechanisms in vitro. The nanosensor was first demonstrated to target macrophage (RAW 264.7) and HeLa cells via scavenger receptors. The reduction kinetics were successfully quantified by measuring the FRET ratio of the nanosensor using confocal microscopy and flow cytometry. Advanced imaging techniques such as FRET-correlated light and electron microscopy (FRET-CLEM) provide high-resolution spatiotemporal information about the nanosensor, which enable us to uncover the disulphide reduction mechanisms by combining the inhibition study of endocytosis and disulphide reduction processes together. Though the work in this thesis is mainly focused on characterising disulphide reduction on nanoparticles in the scavenger receptor-mediated pathway, the nanosensor can be utilised as a general sensor tool to study other endocytosis pathways by conjugating alternative targeting moieties on its surface. Finally, the utility of the nanosensor was extended to characterize the phagosomal microenvironment of primary macrophages and dendritic cells by conjugating the above-developed nanosensor to micron-scale beads, and single-phagosome flow cytometry was conducted after cell lysis. In particular, the focus is on the differential impact of activation on redox kinetics and phagosome rupture (phagosomal to cytosol transfer) in various antigen presenting cell (APC) subtypes. By evaluating disulphide reduction within individual phagosomes using this highly sensitive microbead assay, different patterns of phagosomal rupture were observed depending on cell type and activation state with unprecedented intracellular resolution. By combining high-throughput flow cytometry, confocal imaging techniques, and pathway inhibition studies, a detailed description of the dynamic redox potential of antigen-presenting cell (APC) intracellular microenvironments is presented. This description not only explains divergent roles in dendritic cell and macrophage biology but also defines targets for strategic drug delivery in these cells.
Version
Open Access
Date Issued
2023-09-24
Date Awarded
2024-02-01
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Stevens, Molly
Publisher Department
Materials
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)