In vivo characterization of a smart MRI agent that displays an inverse response to calcium concentration

Abstract

Contrast agents for magnetic resonance imaging (MRI) that exhibit sensitivity toward specific ions or molecules represent a challenging but attractive direction of research. Here a Gd(3+) complex linked to an aminobis(methylenephosphonate) group for chelating Ca(2+) was synthesized and investigated. The longitudinal relaxivity (r(1)) of this complex decreases during the relaxometric titration with Ca(2+) from 5.76 to 3.57 mM(-1) s(-1) upon saturation. The r(1) is modulated by changes in the hydration number, which was confirmed by determination of the luminescence emission lifetimes of the analogous Eu(3+) complex. The initial in vivo characterization of this responsive contrast agent was performed by means of electrophysiology and MRI experiments. The investigated complex is fully biocompatible, having no observable effect on neuronal function after administration into the brain ventricles or parenchyma. Distribution studies demonstrated that the diffusivity of this agent is significantly lower compared with that of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).

Keywords: Smart contrast agents; calcium signaling; functional magnetic resonance imaging (fMRI); in vivo brain imaging.

Figure 1

Structure of GdL.

Scheme 1. Synthesis of L

Conditions and yields: (a) Boc₂O, H₂O/CH₂Cl₂, 62%; (b) tris-t-Bu-DO3A, K₂CO₃, CH₃CN, 80 °C, 82%; (c) TFA, CH₂Cl₂, 68%; (d) H₃PO₃, formaldehyde, H₂O, 100 °C, 58%.

Figure 2

Dependence of r₁ on the concentration of GdL (bottom) in the absence of Ca²⁺ (◼) and the presence of 3 equiv of Ca²⁺ (●) and relaxometric Ca²⁺ titration curve (top) with GdL concentration = 2.5 mM at 9.4 T, 25 °C, and pH 7.3 (HEPES buffer).

Figure 3

Injection of the compound GdL into the cerebroventricles: (a) overlay of the anatomical image and the rat brain atlas; (b) pre-injection; (c) 15 min after injection; (d) 2 h after injection; (e) time courses of signal increase due to GdL with the respective trendlines.

Figure 4

A comparison of the diffusivity of Gd−DTPA and GdL following their simultaneous injection in brain tissue: (a) the injection sites of Gd−DTPA (left) and GdL (right) with the corresponding sections (green lines) used to produce the diffusion diagrams; diffusion diagrams for (b) Gd−DTPA and (c) GdL.

Figure 5

Electrophysiological recording. Multiunit activities (MUA) before and after GdL injection. Red and blue traces represent MUAs before and after the injection, respectively. Shaded area (blue) represents the standard error of mean for MUAs after the injection (2−3 h). MUA responses during the stimulated period (shaded as green) remained in a similar way as before and no significant effect on MUA by GdL was observed.

Figure 6

An fMRI map demonstrating functional activity at the injection site. Percentage signal change (−3 to 3; teal to yellow) of gradient echo EPI with microstimulation after GdL injection overlaid upon T₁-weighted MDEFT (modified driven equilibrium Fourier transformed, with 0.2 mm square voxels) anatomy demonstrating functional activity at the injection side. Parameter: activated voxels were selected with p < 0.1, gradient echo EPI and microstimulation as described in the Methods section.