Investigating the theranostic properties of apoA-I-linked porphyrin-lipid nanoparticles in atherosclerosis

Abstract

In an era following advances in lipid-lowering therapies for atherosclerotic cardiovascular disease (CVD), an unacceptably high number of patients still experience cardiovascular events. Atherosclerosis is an inflammatory process involving accumulation of lipid laden plaque in the blood vessel wall. In the coronary arteries, occlusion caused by atherosclerosis can lead to a myocardial infarction. Subsequent percutaneous intervention to revascularise the heart can involve the deployment of a stent. Despite substantial improvements in preventing restenosis in stented regions, concerns remain around complications of an accelerated form of atherosclerosis within the stented region, known as in-stent neoatherosclerosis. Furthermore, current imaging techniques are unable to identify the molecular characteristics of atherosclerosis at a sufficient resolution that impedes accurate prediction of the risk of clinical events. This highlights the need for novel strategies that better identify and prevent atherosclerosis for the improved management of CVD. Nanotechnologies are promising candidates. ApoA-I-linked porphyrin-lipid nanoparticles (apoA-I porphysomes) incorporate theranostic, therapeutic and diagnostic, properties. Porphysomes consist of an outer layer of a fluorescent porphyrin-lipid conjugate with excellent fluorescence imaging capabilities and can be radiolabelled for positron emission tomography (PET) imaging. Also in the outer layer is the apoA-I mimetic peptide R4F that enables diagnostic and therapeutic targeting via binding to the SR-BI receptor, highly expressed in plaque macrophages. Porphysomes have been previously investigated as theranostic nanoparticles for applications in cancer but have not been explored as yet in atherosclerotic models. Accordingly, the objective of this thesis was to investigate the therapeutic and diagnostic capabilities of apoA-I porphysomes in atherosclerotic CVD. Through experimental approaches utilising both in vitro and in vivo atherosclerosis models, this thesis describes the first studies of apoA-I porphysome nanoparticles as a theranostic in atherosclerotic CVD. In vitro studies revealed that porphysomes exhibited anti-atherosclerotic properties in macrophages including inhibiting inflammation and promoting cholesterol efflux. We also demonstrated that apoA-I porphysomes could be internalised by macrophages in vitro, as detected by confocal microscopy of the fluorescent porphyrin-lipid. In vivo studies in Apoe-/- mouse models of stable and unstable plaque found that apoA-I-porphysomes localised to the site of plaque deposition and could be detected with both PET and fluorescence imaging modalities. Flow cytometric and histological analysis suggested that uptake in atherosclerotic plaque in vivo was primarily co-localised with macrophages. Overall, the therapeutic effects of porphysome on plaque development were modest, however, a significant reduction in plaque size occurred in the unstable model. Finally, we made the first foray into the development of a novel mouse model of in-stent neoatherosclerosis with some histopathological changes reminiscent of neoatherosclerosis observed within the stented regions. Further optimisation of the model is required, however, to ensure the patency of the stented vessels are retained consistently. In summary, this thesis explores the highly novel application of apoA-I porphysomes in atherosclerosis to establish as a new potential theranostic for CVD. These studies provide a stepping stone for future development of porphyrin-lipid based nanoparticles into a clinical application for atherosclerotic CVD.