Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

Abstract

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calciumsensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 M Ca 2؉ , suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 M in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens. magnetic resonance ͉ T2 relaxation ͉ signal transduction ͉ molecular imaging ͉ neuroimaging C alcium ions (Ca 2ϩ ) have been a favorite target in molecular imaging studies because of the important role of calcium as a second messenger in cellular signaling pathways. Fluorescent calcium sensors are used widely in optical imaging, both at the cellular level and at the cell population level. Calcium-sensitive dyes have recently been used in conjunction with laser scanning microscopy to follow neural network activity in small, threedimensional brain areas (1) and to characterize patterns of interaction among cells in developing vertebrate embryos (2). Because of the scattering properties of dense tissue, highresolution optical approaches like these are usually limited to superficial regions of specimens and to restricted fields of view (3). To probe calcium dynamics more globally in living systems, a different imaging modality must be used.Magnetic resonance imaging (MRI) is an increasingly accessible technique for imaging opaque subjects at fairly high spatial resolution, and MRI studies of calcium dynamics could, in principle, complement optical approaches by offering both greatly expanded coverage and depth penetration in vivo (4). Calcium isotopes are unsuitable for direct imaging by magnetic resonance, so attempts to sensitize MRI to calcium have focused around the use of molecular imaging agents. Fluorinated derivatives of the bivalent cation chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) have permitted calcium measurements in vivo by 19 F MRI but only at Ϸ10 Ϫ5 the sensitivity of standard MRI methods (5, 6). Two potentially more powerful proton T1 relaxation-promoting contrast agents were subsequently introduced. The paramagnetic ion manganese (Mn 2ϩ ) mimics calcium by entering cells through calcium channels. Because it accumulates much faster than it is removed, Mn 2ϩ produces an ''integral'' of calcium signaling history that can be d...

In Vivo Characterization of a Smart MRI Agent That Displays an Inverse Response to Calcium Concentration

“…It has already been established that this group behaves in a similar manner to other phosphonates and diphosphonates, forming aggregates in solution that are either hydrogen-bonded or metal-assisted (16). From a practical viewpoint, the use of an aggregation mechanism for the MR imaging of Ca 2þ pubs.acs.org/acschemicalneuroscience Article should be feasible (11). Evidence for the formation of aggregates of GdL in the presence of Ca 2þ was indicated by the measurement of the r 1 in the absence and presence of Ca 2þ as a function of different concentrations of GdL.…”

“…Neurosci. (2010), 1, 819-828 pubs.acs.org/acschemicalneuroscience Article superparamagnetic iron oxide (SPIO) conjugates leads to a change in signal (11). Both approaches have certain advantages and disadvantages determined by the desired application, because many requirements must be fulfiled.…”

Smart MR Imaging Agents Relevant to Potential Neurologic Applications

“…25 The main limitation of this second approach is the relatively long time course of the Ca-dependent aggregation (a few seconds), preventing sensing of fast Ca-concentration changes.…”

Molecular fMRI

“…Functional imaging techniques with improved specificity have been sought by sensitizing MRI acquisition schemes primarily to small blood vessels (Duong et al, 2003), or by trying to tease apart the separate contributions of blood flow, volume, and oxygenation changes to hemodynamic responses using methods, such as "calibrated fMRI" (Hoge, 2012). More radical fMRI approaches have tried to resolve endogenous activity-dependent brain signals arising from nonhemodynamic sources, such as diffusion changes (Le Bihan, 2007), neuronal magnetic fields (Bandettini et al, 2005), and metabolite-dependent spectroscopic signals (Mangia et al, 2009;Hyder and Rothman, 2012). An advantage of such methods is that they can be applied in humans or animals without the need for invasive procedures, but a disadvantage is that most endogenous signals are even smaller than hemodynamic fMRI effects and are therefore difficult to discern unambiguously.…”