Lymph node imaging with magnetic resonance, positron emission tomography and fluorescence techniques

Abstract

The knowledge that unbound gadolinium was responsible for NSF highlighted the need for alternative and safer imaging agents for MRI applications. Nanoparticles (used in conjunction with MRI) were identified as offering the potential and promise, to be safer across a multitude of applications; some dedicated for the investigation of specific disease processes. In particular, iron oxide nanoparticles have been considered as offering the greatest possibility as an MRI contrast (imaging) agent in both the research and clinical arenas. Advantages offered by T10 dextran coated iron oxide nanoparticles include human biocompatibility with a safe and known excretion pathway. The dextran coating can be functionalised, thus providing opportunities for creative compounds to be created. For example, experimental work contributing to this thesis has resulted in T10 dextran coated iron oxide nanoparticles being radiolabelled with 68Ga – and this is being presented for consideration as a potential imaging agent for PET/MRI; that is, 68Ga providing an imaging agent effect on PET imaging while the iron oxide component provides simultaneous imaging contrast with MRI. 68Ga itself provides an advantage over 99mTc (the most commonly used PET imaging agent); it has an improved imaging sensitivity over 99mTc and is less costly to generate, requiring 68Ge (a radio-isotope of germainium) and a gallium generator. The clinical benefit to developing iron oxide nanoparticles radio-labelled with 68Ga is also to improve the imaging of lymph nodes in oncology patients (as well as a PET/MRI contrast agent). Also presented here are the PET imaging findings, identifying the prostate draining lymph nodes, from four prostate cancer patients having had these nanoparticle preparations directly injected into their prostate glands. Iron oxide nanoparticles can also be loaded into immune cells, in vitro; for this thesis, murine dendritic cells (bone marrow derived) and human dendritic cells (monocyte derived) were used, as dendritic cells are known to migrate to lymph nodes. The combination of iron oxide nanoparticles radiolabelled with 68Ga, and in vitro cell loading, offer the potential to re-visit cellular MRI to determine if imaging advances can be made in this area. To support these achievements and claims, this thesis includes in vitro murine dendritic cell and in vitro human dendritic cell studies and also in vivo murine and in vivo human imaging studies. Chapter 1 provides a review of the literature, identifying; the advantages of iron oxide nanoparticles over the limitations of gadolinium based contrast agents; their relevance to MRI and also their capabilities of being internalised by certain cells for targeting imaging applications. The overall aims of the thesis are presented. Chapter 2 explains in detail the materials used, the methods employed and the physical and chemical processes that underpin all of the in vitro and in vivo experiments. The processes used draw upon knowledge from a range of disciplines, including cell biology, immunology, chemistry, physics, medical imaging, nuclear medicine and radiopharmaceuticals. This chapter describes how the iron oxide nanoparticles were prepared and the radiolabelling process used. The functionalised method used for the T10 dextran coating is explained, thus providing attachment sites for either 68Ga or fluorescent markers such as FITC or R-PE for in vitro (murine and human) and in vivo (murine) experiments. Also described are the in vitro cellular (murine and human) experimental process with dendritic cells and iron oxide nanoparticles, investigating dose dependent and time dependent uptake. Chapter 3 provides an analysis of the physical characteristics of the laboratory produced T10 dextran coated iron oxide nanoparticles (using TEM and SEM) and an important assessment of how these nanoparticles behaved in a magnetic environment, namely at 1.5T and 3.0T clinical MRI environment; calculating and graphing the R2 and T2 relaxivity rate values of nanoparticle concentration at a specific TE value and magnetic field strength. The methodology of radiolabelling these nanoparticles with 68Ga is described and the results of TLC are provided, demonstrating the levels of bound and unbound 68Ga. Any effects T10 dextran coated iron oxide nanoparticles could cause on cell proliferation was assessed with PBMC using the MTS assay technique and compared with the effect that Dotarem® (safest gadolinium based contrast agent) may have on these same cells. Chapter 4 investigates the characterisation of nanoparticle uptake by murine bone marrow derived dendritic cells (GM-CSF and Flt3) in vitro, with dose (concentration) dependent and time dependent uptake studies. These same BM-DCs were observed for apoptotic effects, in vitro, using various nanoparticle concentrations over a 24 hour incubation period. A murine CBA kit was used to also ascertain any inflammatory response in these BM-DCs to the presence of nanoparticles; assessed using the supernatant from in vitro experiments Chapter 5 reports on the characterisation of nanoparticle uptake by human monocyte derived DCs, in vitro. Dose (concentration) dependent and time dependent uptake in CD14+ and CD11+ cells were identified. In vitro supernatant assessment for an inflammatory response using a human CBA kit was used. Dose dependent uptake, in vitro, was also quantified in other cells that play varying roles in an immune response; namely PBMC, lymphocytes, granulocytes/monocytes. Chapter 6 examines in vivo applications of nanoparticle preparations; murine and human. Lymphatic drainage to the popliteal and inguinal lymph nodes in C57/BL6 female mice were assessed using flow cytometry; following local subcutaneous injection of T10 dextran coated iron oxide nanoparticles tagged with FITC. To observe any systemic effects of these nanoparticles in these mice, biochemical analysis of blood serum was conducted and histopathological assessment performed, using H&E and Perls Prussian Blue, of the heart, lungs, liver, spleen and kidneys and PAS staining of the kidneys (to observe the basement membrane). Following footpad injections of T10 dextran coated IONPs tagged with FITC and radiolabelled with 68Ga, separate PET and MRI imaging of mice was conducted and on both imaging modalities, image contrast effects due to 68Ga (PET) and iron oxide nanoparticles (MRI) were identified. Most importantly, PET imaging results from patients (positive for prostate cancer) undergoing insertion of gold seeds (for later radiotherapy treatment) having T10 dextran coated IONPs radiolabelled with 68Ga demonstrated observed drainage to lymph nodes with the PET component of a PET/CT scanner