The Formation of Self-Assembled Fluorinated Nanostructures for 19F Magnetic Resonance Imaging Cell Tracking Agents

The major aim of this thesis is to investigate methods into the preparation of bimodal imaging agents (dual 19F MRI and fluorescence imaging agents), how imaging performance (i.e. 19F signal intensity and acquisition time) is related to the agent’s structure, and how successful these imaging agents are in labelling various cell lines both in vitro and in vivo for cell trafficking applications. A bimodal imaging agent (PFPE-FITC) comprising of a linear, fluorinated perfluoropolyether (PFPE) oligomer was covalently conjugated to a common fluorescent dye, fluorescein isothiocyanate (FITC) and subsequently self-assembled with charged ammonium carboxylate PFPE oligomers (PFPE-NH4) to produce self-assembled fluorinated nanoparticles. To gain some fundamental understanding of these nanoparticles, TEM, cryo-TEM, fluorescence and 19F NMR measurements were made to provide information on particle size, chain mobility, 19F imaging parameters and the fluorescent moiety. These investigations found that the ammonium carboxylate PFPE oligomers formed spherical micelles in solution with a diameter of 8 nm while the PFPE oligomers incorporated with PFPE-FITC were shown to be spherical in shape, dispersed in solution with diameters of 16 nm. Fluorescence measurements of the fluorescent (FITC) moiety between the unconjugated FITC and the conjugated PFPE-FITC linker show a minimal Stokes shift of ~15nm which is common with conjugating fluorescent dyes to other molecules. Examination with 19F NMR showed that the PFPE-FITC–PFPE-NH4 nanoparticles had a T2 (spin-spin) relaxation time eight times greater than the PFPE-NH4 nanoparticles which allowed for 19F images of these nanoparticles to be obtained in a short time period of 15 seconds. In order to compare the 19F PFPE-FITC–PFPE-NH4 nanoparticles with traditional 1H MRI contrast agents; solutions comprising of superparamagnetic iron oxide nanoparticles (SPIONs) were made and compared to the 19F PFPE-FITC–PFPE-NH4 nanoparticles using NMR relaxation and imaging experiments. These investigations found that the fluorine based-nanoparticles had significantly longer T2 relaxation times which allowed them to be imaged using smaller concentrations than the SPIONs. The advantage of the 19F imaging technique over the 1H imaging technique was also demonstrated with the 19F imaging producing a single image directly from the 19F compound while the 1H MR imaging produced two signals, one from the SPION compound and one from the background 1H signal in the aqueous solution. To determine their potential use in cell trafficking studies the PFPE-FITC–PFPE-NH4 nanoparticles were used to label a phagocytic mouse dendritic cell line. The nanoparticles were taken up easily without the need for any transfection agents. Characterisation techniques showed that the intracellular uptake of the PFPE-FITC label was evenly distributed in the cytoplasm of the dendritic cell, fluorescence intensity increased linearly with concentration and molecular mobility was unhindered once it entered the cell. Overlaid 1H/19F MRI in vitro images of labelled cells were highly sensitive and allowed for the accurate localisation of the labelled dendritic cells. In vivo trafficking studies of PFPE-FITC labelled dendritic cells in male, C57/BL mice (injected through the footpad and tail vein) were monitored by 1H/19F MRI. In these studies, mice models and their respective lymph nodes and organs that showed positive 19F signal were surgically isolated, processed and analyzed. Fluorescence based techniques (i.e. FACS, fluorescence microscopy) detected high levels of fluorescence (confirming the presence of PFPE-FITC) in the harvested lymph nodes and organs. Different cancer cell lines were labelled with fluorinated nanoparticles comprising of rhodamine isothiocyanate (RITC)-folate (folate-conjugated) and PFPE-FITC (non-folate conjugated), in order to show the potential in attaching targeting ligands to imaging agents. Characterisation techniques determined there was no toxicity from the labelling procedures of both types of nanoparticles and that the folate-conjugated nanoparticles (RITC-Folate) were engulfed by the cancer cells while the non-folate conjugated nanoparticles (PFPE-FITC) were not up taken up. In vivo experiments comprising of a mouse model with a breast cancer (T47D) tumour were conducted using both folate-conjugate and non-folate conjugate nanoparticle solutions. MRI studies of the folate-conjugated (RITC-Folate) nanoparticles post-injection found the presence of nanoparticles at the site of the tumour, kidneys and liver (while the non-folate conjugated nanoparticles passed relatively quickly through the animal). As these organs and the cancer tumour contained folate-receptors it was expected that the RITC-Folate nanoparticles would be taken up in significant numbers allowing for easy detection. In summary, this work provides a study into cellular labelling and tracking using different imaging modalities with fluorescent and 19F-nanoparticles. This work will provide a basis to future studies of more complex systems involving molecular imaging, for example, in vivo studies of targeted molecular events in disease