A molecular toolkit for immunological imaging

Chronic inflammation is prevalent in society with a wide range of inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease, as well as association with cancer, strokes, and neurodegenerative conditions. Due to the complexity in the inflammatory pathways and a lack of understanding of those processes, there are limited therapeutic options available that aim to resolve the causes of inflammation, with most treatments instead aiming to manage symptoms. More bespoke imaging agents are required to help advance our understanding of the fundamental processes governing these disease states.
To overcome current imaging limitations such as quantification, correlation, depth penetration and resolution quality, this work takes a multimodal molecular imaging approach to study one type of immune cells, macrophages, that are of particular importance in rheumatoid arthritis. Such methods aim to aid the non-invasive characterisation of disease phenotypes and therapeutic response in inflammatory diseases such as IBD and rheumatoid arthritis.
The general structure of selective imaging probes is an imaging moiety, a targeting group to localise at a specific site and a linker group to attach the two. The work within this thesis focusses on designing a toolkit of imaging probes that target macrophages by using folate and tuftsin derivatives to target receptors with macrophage selective expression. A variety of imaging modalities are explored in this work such as lanthanide luminescence, magnetic resonance and photoacoustic, with the aim to permit the choice of the most appropriate modality required for a given spatial resolution, scale, and depth penetration. In addition, different bioconjugation strategies have been investigated, such as amide linkages and azide-alkyne cycloadditions, to enable access to a wider variety of macrophage imaging agents. By using a toolkit of molecular components that can be easily combined, this work demonstrates preliminary work for luminescent microscopy, magnetic resonance, and photoacoustic imaging to enable the study of macrophage populations in tissue and model systems. Chapter 1 outlines the processes governing inflammation and the current methods of imaging inflammation. It provides a general overview of the different imaging modalities, with a specific focus on lanthanide chemistry and its photophysics, magnetism and stability in relation to its in vivo imaging. Moreover, the suitability of the targeting moieties – folate and tuftsin – are discussed and the prior work in these areas.
Chapter 2 outlines the synthesis, paramagnetic NMR spectroscopy and photophysical characterisation of alkyne appended DO3A derivatives. The purpose of these alkynes is for use in bioconjugation to the targeting moieties with an azide functionality for use in CuAAC and SPAAC.
Chapter 3 describes the synthesis of multiple terbium-folate conjugates using solid-phase synthesis, as well as the generation of folate-azides for conjugation to the alkynes discussed in chapter 2. The NMR spectroscopy and photophysical properties of these terbium-folate conjugates are discussed.
Chapter 4 details the synthesis of lanthanide-tuftsin conjugates for use in optical and magnetic resonance imaging. The relaxivity and cell diffusion NMR spectroscopy of the gadolinium complexes were studied in blood plasma and whole blood and was compared to a commercially available non-specific gadolinium complex, DOTAREM.
Chapter 5 discusses the preparation of macrophage-targeted photoacoustic probes and their optical properties. The probes are investigated for their photoacoustic properties and stability.
Chapter 6 provides a summary of work and conclusions that can be drawn, with opportunities for future work proposed.
Finally, chapter 7 contains the experimental procedures.

Keywords: INDIANA
optoacoustic
click chemistry
lanthanide
folate
inflammation
imaging agent
tuftsin
diffCEST
magnetic resonance
lanthanide fluorescence
photoacoustic
Advisor: Faulkner, S