Chemical exchange saturation transfer (CEST) offers many advantages as a method of generating contrast in magnetic resonance images. However, many of the exogenous agents currently under investigation suffer from detection limits that are still somewhat short of what can be achieved with more traditional Gd3+ agents. To remedy this limitation we have undertaken an investigation of Ln3+ DOTA-tetraamide chelates (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that have unusually rigid ligand structures: the nitrobenzyl derivatives of DOTA-tetraamides with (2-phenylethyl)amide substituents. In this report we examine the effect of incorporating hydrophobic amide substituents on water exchange and CEST. The ligand systems chosen afforded a total of three CEST-active isomeric square antiprismatic chelates; each of these chelates was found to have different water exchange and CEST characteristics. The position of a nitrobenzyl substituent on the macrocyclic ring strongly influenced the way in which the chelate and Ln3+ coordination cage distorted. These differential distortions were found to affect the rate of water proton exchange in the chelates. But, by far the greatest effect arose from altering the position of the hydrophobic amide substituent, which, when forced upwards around the water binding site, caused a substantial reduction in the rate of water proton exchange. Such slow water proton exchange afforded a chelate that was 4.5 times more effective as a CEST agent than its isomeric counterparts in dry acetonitrile and at low temperatures and very low presaturation powers.

Although paraCEST is a method with immense scope for generating image contrast in MRI, it suffers from the serious drawback of high detection limits. For a typical discrete paraCEST agent the detection limit is roughly an order of magnitude higher than that of a clinically used relaxation agent. One solution to this problem may be the incorporation of a large payload of paraCEST agents into a single macromolecular agent. Here we report a new synthetic method for accomplishing this goal: incorporating a large payload of the paraCEST agent DyDOTAM3+ into a Reverse Assembled nano-Capsule. An aggregate can be generated between this chelate and polyacrylic acid (PAA) after the addition of ethylene diamine. Subsequent addition of polyallylamine hydrochloride (PAH) followed by silica nanoparticles generated a robust encapsulating shell and afforded capsule with a mean hydrodynamic diameter of 650 ± 250 nm. Unfortunately this encapsulation did not have the effect of amplifying the CEST effect per agent, but quenched the CEST altogether. The quenching effect of encapsulation could be attributed to the effect of slowing molecular tumbling, which is inevitable when the chelate is incorporated into a nano-scale material. This increases the transverse relaxation rate of chelate protons and a theoretical examination using Solomon Bloembergen Morgan theory and the Bloch equations shows that the increase in the transverse relaxation rate constant for the amide protons, in even modestly sized nano-materials, is sufficient to significantly quench CEST.

Generating MR image contrast from exogenous contrast media though chemical exchange saturation transfer (CEST) offers several exciting new possibilities, such as multicolored imaging, the interleaving of pre-and post-contrast images, and the potential to perform ratiometric metabolic imaging. The major limitation of the deployment of CEST imaging is the comparatively high detection limits of exogenous agents and particularly at the low B1 power levels required to meet SAR requirements. The large chemical shifts afforded by paramagnetic (paraCEST) agents permit more rapid exchange kinetics and therefore potentially more effective contrast agents. Despite comparatively large chemical shifts, many Ln 3+ DOTA-tetraamide (DOTAM) chelates traditionally investigated as CEST agents are predicted to have exchange kinetics that are considerably faster than optimal at very low B1 powers. This work explores two methodologies for slowing water exchange kinetics in Ln 3+ DOTAM chelates and improving CEST imaging: structural manipulation and encapsulation. In the first method, rigid Ln 3+ NB-DOTAM chelates with hydrophobic amide substituents was thoroughly studied using NMR spectroscopy techniques in order to assess their ability to produce CEST contrast at low B1 power levels. NMR techniques utilized included 1 H NMR, variable temperature, COSY, and CEST experiments. The phenyl amide substituent in the pseudo-axial position afforded chelates with considerably slow water proton exchange rates and appreciably more CEST contrast than isomeric chelates with the amide substituent in the pseudo-equatorial position. The second method involved characterizing a vesicle system to be used for encapsulating a Ln 3+ DOTAM chelate. The vesicles prepared were analyzed using the following NMR ii techniques: 1 H NMR, T1, shift reagent, and CEST experiments. The vesicle system chosen for study did not afford slow water exchange kinetics to enhance CEST contrast. A second vesicle system was attempted but the vesicle synthesis was difficult, parameters studied were not optimized, and the second system did not exhibit slow water exchange with the limited amount of experiments run and data collected.iii