Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

Abstract

Superoxide overproduction is known to occur in multiple disease states requiring critical care; yet, noninvasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by "click conjugating" paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced in vivo to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species-in particular, superoxide-and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for T contrast at low field (<3.0 T) and T contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for a "quenchless fluorescent" bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.

Supramolecular and biomacromolecular enhancement of metal-free magnetic resonance imaging contrast agents

“…Most importantly, the ORCA can survive in the presence of ascorbate for periods of time that exceed-to the best of our knowledge (Table S1 †)-currently known aminoxylbased ORCAs by 20-fold. 24,[40][41][42][43][44][45] Our results indicate that with the combined approach of (i) utilizing macrocycles for steric shielding to inhibit reduction in vivo along with (ii) enhancing T 1 relaxivity by conjugating these supramolecular agents to biomacromolecular scaffolds, ORCAs are moving closer to clinically viable contrast agents.…”

“…24,25 However, two major issues prevent aminoxyl-based ORCAs from replacing traditional contrast agents based on Gd: (i) their single unpaired electron provides weaker contrast compared to the seven unpaired electrons of Gd and (ii) they are reduced rapidly to MRI-silent hydroxylamines in physiological conditions by compounds including ascorbate, saccharides, and cysteine-rich proteins. 24 Contrast issues in ORCAs have improved greatly in recent years by attaching them to polymeric or biomacromolecular nanoparticle systems [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] that both create high local concentrations of aminoxyl moieties and decrease the diffusional and rotational motion of the attached ORCAs. Because rotational correlation time is inversely proportional to relaxivity, attaching contrast agents onto large macromolecules will-in generalimprove their performance.…”

Viral nanoparticles for drug delivery, imaging, immunotherapy, and theranostic applications

“…Previous studies show that this system can be detected at concentrations as low as 230 fM, which is near the forefront of current standards. In another study, TMV loaded with a metal-free paramagnetic nitroxide organic radical contrast agent (ORCA) for MRI and electron paramagnetic resonance probes for superoxide detection showed enhanced in vitro r1 and r2 relaxivities [204]. The probes functioned as both T1 and T2 contrast agents making this platform suitable for pre-clinical and clinical MRI scanners.…”

Assessment of the Relaxation-Enhancing Properties of a Nitroxide-Based Contrast Agent TEEPO-Glc with In Vivo Magnetic Resonance Imaging

“…However, nitroxides with bulky side groups have shown remarkable stability in conditions mimicking biological matrix [23,24]. e research of nitroxide-based contrast agents comprises both macromolecular [25][26][27][28][29][30][31] and small molecule [32][33][34][35][36] systems. It is often considered that the advantage of using polymers or nanomaterials as a backbone for nitroxides is the possibility to attach several radical centers to the contrast agent molecule.…”