Development of Bioresponsive Small Molecule Probes for Molecular  Magnetic Resonance Imaging

Li, Hao

doi:https://doi.org/10.21985/n2-ww7x-zz36

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Development of Bioresponsive Small Molecule Probes for Molecular Magnetic Resonance Imaging

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In an era of personalized medicine, the clinical community has become increasingly focused on understanding diseases at the cellular and molecular level. Magnetic resonance imaging (MRI) is a powerful imaging modality for acquiring anatomical and functional information. However, it has limited applications in field of molecular imaging due to low sensitivity. To expand the capability of MRI to encompass molecular imaging applications, we introduced bioresponsive Gd(III)-based magnetic resonance contrast agents (GBCAs) in 1997. Since that time, many research groups across the globe have developed new examples of bioresponsive GBCAs. These contrast agents have shown great promise for visualizing several biochemical processes such as gene expression, neuronal signaling and hormone secretion. They are designed to be conditionally retained, or activated, in vivo in response to specific biochemical events of interest. As a result, an observed MR signal change can serve as a read-out for molecular events. A significant challenge for these probes is how to utilize them for noninvasive diagnostic and theranostic applications. This thesis focuses on improving existing and developing new design strategies that underlie bioresponsive probes and describes how these advanced strategies will facilitate the application of bioresponsive GBCAs in vivo. The first strategy is to manipulate the MR signal of the bioresponsive CAs by modulating the hydration number (q) of Gd(III), accomplished through changing the coordination number for Gd(III). Because of the linear relationship between q and T1-w MR signal, an increase in q after bioactivation of a GBCA corresponds to an increased MR signal. Specifically, Chapter 2 describes the design, synthesis and characterization of a new bioresponsive GBCA that has shown field-independent MR turn-on response to β-galactosidase activities, a commonly used reporter gene product. Future work for this project involves in vivo MR imaging using this imaging probe to monitor gene therapy-induced β-gal expression. A fundamental challenge with q-modulated MR probe for molecular imaging is signal validation. Because the local concentration of the probe is unknown, MR signal enhancement cannot be specifically assigned to activated probe versus pooling of the inactive agent. This uncertainty stems from the relatively low sensitivity of MR imaging and the limited dynamic range of most bioresponsive MR probes. Chapter 3 presents a solution to the MR signal validation issue by employing bimodal fluorescence-magnetic resonance (FL-MR) bioresponsive probe design. Because fluorogenic probes have excellent sensitivity and large dynamic range, the FL signal change can be used to substantiate the MR signal enhancement in response to the biological stimulus. Specifically, in Chapter 3 I will describe the design, synthesis, and in vitro evaluations of a new multimodal caspase-activatable imaging probe Caspase Probe-1 (CP1) that exhibits FL-MR turn-on response in both in vitro caspase enzymatic assays and apoptotic HeLa cells. Most importantly, the FL signal of CP1 can be used to quantify the concentrations of the active and inactive probes during caspase-3 assay hence accurately predicting the MR response in vitro. Future experiment includes in vivo MR imaging using CP1 to monitor therapy-induced tumor apoptosis followed by ex vivo fluorescence imaging of apoptotic tumors tissue. In the final Chapter, I used the probe platform detailed in Chapter 3 to develop targeted-activatable GBCAs to imaging prostate-specific membrane antigen (PSMA) in vivo. Specifically I developed the first activatable Gd(III)-based MR contrast agents used to selectively image PSMA+ prostate cancer (PCa) cells. The prostate cancer probe 2 (PCP-2) enabled differentiation between PSMA+ and PSMA- PCa cells in vivo with ease. Importantly, PCP-2 showcased a unique probe design strategy. By simultaneous combining targeting and activation strategies, PCP-2 achieved excellent sensitivity and prolonged signal retention at the target site. Future work of Chapter 4 will focus on applying PCP-2 to PSMA imaging in preclinical and clinical settings.

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