Novel contrast agents and strategies for MR molecular imaging

Molecular imaging is a rapidly emerging field of research, which can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to conventional clinical diagnostic imaging, this novel field focuses on the in vivo visualization of molecular and cellular processes that underlie disease, thereby allowing its early detection and characterization. For this purpose, conventional imaging modalities are combined with the use of so-called targeted contrast agents that are intended to specifically bind to receptors, which are abundantly expressed at diseased sites. The objective of the present thesis was to develop novel methods for magnetic resonance (MR) molecular imaging of apoptosis (programmed cell death) and angiogenesis (the formation of new blood vessels from pre-existing vessels), as these processes play a key role in the etiology as well as treatment of various disorders with a high prevalence, such as cancer and cardiovascular disease. Magnetic resonance imaging (MRI) was used as this imaging modality provides excellent soft tissue contrast at a high spatial resolution throughout the whole body without using ionizing radiation. MRI however suffers from relatively low sensitivity, and therefore potent contrast agents were designed that contained a high quantity of contrast-generating material such as iron oxide crystals or gadolinium ions. Furthermore, novel molecular imaging methods were introduced and applied to examine important aspects of biomarker-specific MRI. Chapter 1 gives a general introduction to contrast agents for MRI, in which basics such as T1 and T2 (*) relaxation, relaxivities (r1 and r2 (*)) and contrast-generating mechanisms, as well as contrast agent applications are discussed. Moreover, the main molecular pathways of apoptosis and angiogenesis are described. The following three chapters concentrate on molecular imaging of the apoptotic process. An important hallmark of apoptosis is the appearance of the phospholipid phosphatidylserine (PS) in the outer leaflet of the cell membrane, which can be exploited as a diagnostic marker for apoptosis by using annexin A5 conjugates. Chapter 2 and 3 contain in vitro studies on annexin A5-functionalized contrast agents, whereas an in vivo application is described in Chapter 4. In Chapter 2 three different types of lipid-based annexin A5-functionalized MR contrast agents were designed with either a micellular or liposomal morphology. MR contrast was generated by incorporation of T1-reducing paramagnetic gadolinium lipids in the lipid (bi)layer or by encapsulation of T2 (*)-reducing superparamagnetic iron oxide particles in the micellular core. Bimodality was obtained by additional inclusion of fluorescent labels, i.e. fluorescent lipids or quantum dots. The resulting annexin A5-functionalized paramagnetic micelles (~ 10 nm), superparamagnetic micelles (~ 10 nm), and paramagnetic liposomes (~ 100 nm) all demonstrated high specificity for apoptotic Jurkat cells, and could be easily detected with MRI and fluorescence microscopy. Their differences in size, magnetic and fluorescent properties provide the possibility to choose the optimal contrast agent for future in vivo applications. Chapter 3 showed specific association of commercially available annexin A5-functionalized polysaccharide-coated iron oxide particles to apoptotic Jurkat cells. Importantly, it was shown that cell membrane association led to internalization of these particles when coincubated with the apoptotic stimulus, which is also expected to occur in vivo. In the present study, marginal differences were observed between the r2 */r2 ratios of membraneassociated and internalized iron oxide particles, which suggested that both T2- and especially T2 (*)-weighted imaging sequences are suitable for their detection. In Chapter 4 annexin A5-functionalized paramagnetic micellular contrast agents were applied for the non-invasive MRI-based detection of PS-exposing cells in atherosclerotic lesions. Both in vivo MRI as well as ex vivo near-infrared fluorescence imaging (NIRF) showed higher signal enhancement in aortas of apoE-/- mice at 24 hours post-injection of the annexin A5-micelles compared with untargeted control micelles. Confocal fluorescence microscopy confirmed association of the target-specific contrast agent with macrophages and apoptotic cells. These results may be of great value for future diagnostics of atherosclerotic lesion type, as the presence of PS-exposing cells is believed to strongly correlate with plaque vulnerability. The last part of this thesis deals with the pharmacokinetic behavior of liposomal contrast agents as presented in Chapter 2. These nanoparticles were coated with poly(ethylene glycol) (PEG), which is known to increase their circulation half-life and should allow for extensive accumulation at the targeted site. However, the resulting target-to-background ratio may be reduced due to circulating agents that are also detected in the MR images. Therefore, in Chapter 5 we incorporated biotin in the bilayer of the paramagnetic liposomes and successfully introduced a novel strategy to rapidly clear these liposomes from the blood circulation in C57BL/6 mice through a so-called avidin chase. Avidininduced clearance was confirmed both with dynamic in vivo MRI and by determination of the Gd content in blood samples ex vivo. In Chapter 6 the avidin chase was applied to remove non-bound RGD-biotin-liposomes from the blood pool in B16F10 tumor-bearing C57BL/6 mice. These liposomes target the avß3 integrin, which is strongly expressed in angiogenic blood vessels. The avidin chase demonstrated that besides target-associated contrast agent, the circulating contrast agent contributed significantly to the MR contrast enhancement in the tumor as well. These results were confirmed by ex vivo fluorescence microscopy. Consequently, this clearance methodology may be used to increase the specificity of molecular MRI of tumor angiogenesis. Finally, Chapter 7 concludes with a general discussion on the preceding chapters, followed by some thoughts on future perspectives of the work presented