
doi: 10.25560/89499
handle: 10044/1/89499
Unregulated inflammation is implicated in a variety of disease states such as stroke, cancer and neurodegenerative conditions and often the extent of inflammatory response can determine the prognosis for a particular disease state. Human neutrophils express formyl peptide receptors one (FPR1) and two (FPR2/ALX, also known as the lipoxin A4 receptor) and are known to both drive vascular inflammation and at the same time be involved in its resolution. Therefore, these receptors represent an attractive target for non-invasive visualisation of inflammation in disease states. The work within this thesis focusses on designing luminescent imaging probes that selectively tag neutrophils in an inflammatory context. To achieve this, a small-molecule FPR2/ALX ligand was chosen based on ease of synthesis, ability for further functionalisation and a dose-dependent neutrophil response observed in vitro. The targeting system was initially combined with a rhodamine-based dye and binding via the neutrophil FPR family was confirmed via pharmacological blocking. In vitro, the probe did not affect neutrophil responses and in vivo preferential uptake by neutrophils in a model of acute inflammation compared to control groups was demonstrated. Via minor synthetic alterations, a pH-responsive version based on the rhodamine spirocyclisation equilibrium was prepared (pKcycl = 3.97) and the compound was shown to be fluorescent in stimulated neutrophils in vitro but remained non-fluorescent under normal physiological conditions. By appending the same targeting group to metal chelates, compounds with long-lived luminescence signals that can be separated from cellular autofluorescence via lifetimes, were prepared. Time-resolved microscopy on activated and unactivated human neutrophils incubated with FPR2/ALX-targeted lanthanide-based compounds was performed, but high concentrations were required. A second iteration of probe design, using a Re(I) tricarbonyl motif, resulted in time-resolved images of neutrophils at lower probe concentration. Finally, a construct containing a lanthanide unit, a pH-responsive fluorophore and the targeting group was formed and the energy transfer processes in the Tb(III) analogue were investigated. The observed energy transfer provides a unique opportunity to enhance emission properties of the compound for time-resolved imaging via donor-sensitised, long-lived emission, providing an alternative radiative pathway that circumvents the formally forbidden, characteristically weak lanthanide emission.
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