MRI With Non-Gadolinium Metals
Ali Barandov1
1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States

Synopsis

A new class of non-gadolinium cell-permeable MRI contrast agents have been developed for monitoring intracellular analytes and processes at the molecular level. In this talk, we discuss the design, synthesis and applications of such probes for acquiring spatially resolved functional images of fluctuations in concentrations of specific analytes in the brains of living subjects. By improving the technology with more sensitive contrast agents and better brain delivery strategies, it will be possible to measure and map an expanding array of neurophysiological processes in animals and ultimately in humans.

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.


Acknowledgements

Acknowledgements

Funding came from the MIT Simons Center and NIH grants R21-MH102470 and U01-NS090451 to A.J

References

References

1. A. Barandov, S. Ghosh, N. Li, B. B. Bartelle, J. I. Daher, M. L. Pegis, H. Collins, A. Jasanoff, Molecular Magnetic Resonance Imaging of Nitric Oxide in Biological Systems. ACS Sensors (2020), doi:10.1021/acssensors.0c00322.

2. A. Barandov, B. B. Bartelle, B. A. Gonzalez, W. L. White, S. J. Lippard, A. Jasanoff, Membrane-Permeable Mn(III) Complexes for Molecular Magnetic Resonance Imaging of Intracellular Targets. J. Am. Chem. Soc. 138, 5483–5486 (2016).

3. A. Barandov, B. B. Bartelle, C. G. Williamson, E. S. Loucks, S. J. Lippard, A. Jasanoff, Sensing intracellular calcium ions using a manganese-based MRI contrast agent. Nat. Commun. 10, 897 (2019).

Figures

Figure 1. Nitric oxide responsive manganese-based MRI contrast agent. (A) Schematic presentation of NO-sensing mechanism and corresponding MRI signal changes. (B) Experimental timeline (top) and representative T1-weighted MR images of rats treated with LPS and infused with NORA in the absence (left) or presence (right) of the iNOS inhibitor 1400 W. (C) R1 maps associating with the experiment presented in B.

Figure 2. Cell-trappable MRI contrast agent responsive to intracellular enzymatic activity. (A) Molecular structure of cell-permeable manganese-based contrast agents with various ester residues. (B) Manganese content of cells compartments, cytosol (C), nucleus (N) and membrane (M), after incubated with contrast agents. Contrast agents with ethyl esters show various cleavage rate in cells expressing porcine liver esterase (PLE) compared to control cells.

Figure 3. Design of cell permeable sensors for calcium-dependent molecular fMRI. (A) Presentation of the manganese-based intracellular calcium sensor (ManICS1-AM) . MRI response to intracellular calcium fluctuations upon optical (B) and chemical (C) stimulations. (D) ManICS1-AM enables detection of neural activation in rat brain. Region of interest analysis shows the time course of signal changes observed during K+ or Na+ in the presence of ManICS1-AM (red and cyan, respectively), and during K+ stimulation in the presence of calcium-insensitive MnL1F (blue).

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)