Development of a new class of contrast agents for Overhauser Magnetic Resonance Imaging (MRI), based on nitroxyl radicals and responsive to specific enzymatic activity

Publications and Presentations PrologueMolecular imaging is a medical imaging technique that allows visualization of biochemical events at the cellular and molecular level within living cells, tissues, and living organisms.1,2 In recent decades, these approaches have therefore been under intense scrutiny.Compared to conventional in vitro methods, molecular imaging technologies offer numerous advantages, including non-invasive and real-time study of cells in their natural microenvironment, without disturbing the system under investigation.Molecular imaging shows great promise in diagnostics, therapy monitoring, drug discovery and development, and understanding nanoscale reactions such as protein-protein interactions and enzymatic conversion.3 Enzymes play a central role in the cell metabolism and a deregulated enzyme activity is often a relevant marker of a pathological situation.Therefore, mapping enzymatic activity can serve as diagnostic or prognostic markers for many pathologies, such as cancer and inflammatory diseases.Enzymatic imaging has been performed with different imaging techniques, each with its drawbacks.For example, Fluorescence optics 4 are limited to superficial tissue exploration, while Positron Emission Tomography (PET), 5 requires the use of radioactive tracers that show no difference before and after the enzymatic activity and is expensive.Magnetic resonance imaging (MRI) presents limitations in terms of sensitivity and specificity and requires complex ratiometric protocols to quantify the enhancement.A potential new strategy could be the use of MRI, which can explore deep tissues providing sufficiently high-resolution images, but exploiting the Overhauser effect induced by enzymatically activated organic radicals to overcome the lack of specificity.The Overhauser-MRI (OMRI) would require low energy frequency for translation to human or larger animals, necessitating ultra-low or earth field.Therefore, the use of ultra-low field Overhauser-MRI with contrasts agents specific to an enzymatic activity, would allow mapping and quantifying enzymatic activity with a less expensive and more accessible instrumentation.This strategy was proposed in the European project PrimoGaia H2020, that founds this PhD project.as the type of polarizing agent and additional hyperfine interaction that gives only a partial saturation.23,25 In vivo experiments reported in literature have performed Overhauser-enhanced MRI at higher field using RF in the 300 MHz-3.5 GHz range, enabling the detection of free radicals in biological samples and small animals.26 This frequency is not suitable to irradiate organisms as large as humans since it does not penetrate the tissues more deeply than a few centimeters and the energy deposition would be too high.Therefore, the application of the OMRI approach to larger animals and humans can only be achieved by using the OMRI at Ultra-Low Field or earth's magnetic field (25-65 µT).At these fields the electron saturation frequency will be in the range of a 50-200 MHz.These frequencies are routinely applied in clinical human 1.5T and 3T MRI and attainable with a conventional RF coil.Therefore, OMRI at Ultra-Low Field will enable effective penetration of electromagnetic waves in vivo without causing overheating.The significant reduction of the field can also enhance the Overhauser effect.27,28 Indeed, theoretical calculation with a nitroxide radical observed that the DNP enhancement at low field is significantly higher (⁓ 2000) than high field (⁓ 660 for protons).This is largely due to the dominant hyperfine coupling term (Eq.2) between the electron spin and the nitrogen nucleus(2)