Introduction. Most gadolinium-based contrast agents (GBCA) are polar complexes with inadequate cell-permeability, which makes it difficult to use them as molecular sensors for imaging intracellular analytes and processes. We have addressed this challenge by introducing a new class of magnetic resonance imaging (MRI) agents that are paramagnetic complexes of Mn(III) with planar lipophilic ligand systems derived from phenylenediamine (PDA) backbone. Mn(III)-PDA complexes display T₁ relaxivity comparable to that of GBCA and undergo spontaneous cytosolic localization via defined mechanisms. Functionalization of Mn-PDAs with variable molecular moieties provides the opportunity of manipulating the T1-weighted MRI signal as a function of specific process or analyte such as enzymatic activities or fluctuations in concertation of signaling molecules like Ca2+ and nitric oxide (NO). These reagents provide an opportunity to study diverse biological processes in animals and potentially humans. Methods. Manganese based contrast agents were synthesized and characterized by standard analytical methods. MRI data were acquired at 7 T for in vitro experiments and 9.4 T for in vivo studies. All cell experiments were carried out in HEK293 cells unless otherwise specified. Animal experiments were performed in adult rats. Results and discussion. Three classes of non-gadolinium MRI contrast agents are presented here. All agents are derivatives of Mn(III) complexes with phenylenediamido (PDA) backbone. The first class of agents acts as a nitric oxide (NO) sensor in MRI. In the presence of NO, an inner sphere water molecule of this nitric oxide responsive agent (NORA) is irreversibly replaced by a NO radical, which results in lower T₁ relaxivity and darkening of the recorded images. When loaded with this compound, cells ectopically expressing nitric oxide synthase (NOS) isoforms showed MRI signal decreases of over 20% compared to control cells and were also responsive to NOS inhibition or calcium-dependent activation (1). The sensor could also detect endogenous NOS activity in antigen-stimulated macrophages and in a rat model of neuroinflammation in vivo (Figure 1). The second class of probes are Mn-PDAs functionalized with various ester groups cleavable by intracellular enzymes. Addition of ester groups to the backbone of Mn-PDAs provides various intracellular retention times as a function of esterase activity and molecular structure of the substrate (2). Varying the ester residues from the labile acetoxymethyl ester to more stable ethyl ester enable selective labeling of cells expressing appropriate conjugate esterases (Figure 2). The third class of probes are conjugates of the Mn-PDAs with the cell-trappable calcium specific chelator BAPTA-AM. The resulting manganese-based MRI probe, ManICS1-AM, is designed to permeate cells, undergo esterase-mediated cleavage, and allow intracellular calcium levels to be monitored by functional imaging. Cells loaded with ManICS1-AM show changes in MRI contrast when stimulated with pharmacological agents or optogenetic tools (3); responses directly parallel the signals obtained using fluorescent calcium indicators. Introduction of ManICS1-AM into rodent brains furthermore permits MRI-based measurement of neural activation in optically inaccessible brain regions (Figure 3).
Conclusion. We have discussed three new classes of non-gadolinium contrast agents based on the manganese-PDA platform. We also demonstrated the first applications of the contrast agents in living cells and animals for monitoring biological processes at the molecular level. Further development of these contrast agents and their application to functional imaging in living subjects is the focus of our ongoing research.