Opening the Proteome to Analysis by Magnetic Resonance ImagingPublic Deposited
The development of metal-based probes has provided major benefits to understanding basic biological process and clinical outcomes. Metals offer access to geometries that carbon alone cannot attain, along with valuable magnetic, optical, and binding properties. Metals have proven to be particularly useful in advancing molecular imaging, a field that seeks to identify the location of events of biochemical interest in intact specimens. Such imaging is commonly achieved with a clinical modality such as positron emission tomography, single-photon emission computed tomography, or X-Ray computed tomography. However, the modality with the best combination of spatial resolution and imaging depth is magnetic resonance imaging (MRI). Metals with interesting magnetic properties have played a major role in expanding MRI from a strictly anatomical imaging technique to a modality that can be used for molecular imaging. Three different probes were designed with the goal of expanding the functionality of MRI in three unique ways. First, a nanoparticle based probe was developed to create an MRI reporter gene that could integrate into the existing HaloTag platform. An analysis was performed that sought to couple well-understood properties of relaxation theory to quantification of protein expression levels. This analysis was then applied to the design of an MRI probe consisting of a gold nanoparticle core functionalized with Gd(III)-bearing DNA strands. Finally, those strands were coupled to a HaloTag targeting group. This nanoparticle displayed the ability to bind to HaloTag expressed on the cell surface, cause differential uptake in HaloTag-expressing cells, and HaloTag-dependent contrast in cell pellet images. Second, a series of probes were created with the goal of imaging amyloid plaques in the brain. These plaques are indicative of Alzheimer’s disease, however there are no suitable MR probes to detect them in live samples. These Stilbene-based probes were tested for their ability to bind to amyloid fibrils and bypass the blood-brain barrier. In addition, they were evaluated for the capacity to serve as cell labeling agents by measuring their toxicity, ability to accumulate in cells, serve as bimodal agents through stilbene fluorescence, and imaging them in cell pellets. These data conclude that, contrary to published reports, Stilbene-chelates do not cross the blood-brain barrier. Third, iron oxide nanoparticles were applied to the development of a method for quantifying experimental metastases in the brain. Using a high-relaxivity probe developed in the Meade lab, breast cancer cells were labeled and injected in the heart of live mice. These mice were then imaged over seven days and showed clear hypointense voxels indicative of the presence of labeled micrometastases. This work outlines a procedure count metastases via MRI, identify successful injections through the use of bioluminescence, validated these results histologically fluorescence. Finally, the basic lessons learned in probe design from HaloTag-targeting were expanded to endogenously expressed surface receptors. A second generation of targeted nanoparticles with improved particle stability and Gd(III) payload. Folate receptor and PSMA were selected using the parameters outlined in the HaloTag study. Probes targeted to each, along with HaloTag, were synthesized and characterized. Preliminary in vitro data showed that the second generation nanoparticles are capable of outperforming the first generation in regard to particle stability, loading, synthetic ease, and protein binding in the case of Folate Receptor-targeted nanoparticles. These agents show promise for successful translation into in vivo experiments.