Franco Fedeli

Paramagnetic lanthanide(III) complexes as pH-sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications

The recently introduced new class of contrast agents (CAs) based on chemical exchange saturation transfer (CEST) may have a huge potential for the development of novel applications in the field of MRI. In this work we explored the CEST properties of a series of Lanthanide(III) complexes (Ln = Eu, Dy, Ho, Er, Tm, Yb) with the macrocyclic DOTAM‐Gly ligand, which is the tetraglycineamide derivative of DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). These complexes possess two pools of exchangeable protons represented by the coordinated water and the amide protons. Yb‐DOTAM‐Gly displays the most interesting CEST properties when its amide N‐H resonance (16 ppm upfield H2O signal) is irradiated. Up to 70% suppression of the water signal is obtained at pH 8. As the exchange rate of amide protons is base‐catalyzed, Yb‐DOTAM‐Gly results to be an efficient pH‐responsive probe in the 5.5–8.1 pH range. Moreover, a ratiometric method has been set up in order to remove the dependence of the observed pH responsiveness from the absolute concentration of the paramagnetic agent. In fact, the use of a mixture of Eu‐DOTAM‐Gly and Yb‐DOTAM‐Gly, whose exchangeable proton pools are represented by the coordinated water (ca. 40 ppm downfield H2O signal at 312K) and amide protons, respectively, produces a pH‐dependent CEST effect which is the function of the concentration ratio of the two complexes. Magn Reson Med 47:639–648, 2002.

Relaxometric evaluation of novel manganese(II) complexes for application as contrast agents in magnetic resonance imaging

Abstract. Three novel Mn(II) complexes bearing benzyloxymethyl functionalities are reported and their ability to enhance water (1H and 17O) relaxation times is investigated in detail. Two of them contain one coordinated water molecule and display relaxivity values only slightly smaller than those shown by the most clinically used contrast agents (e.g. [Gd(DTPA)(H2O)]2–). Moreover, in these Mn(II) chelates the exchange rate of the coordinated water is ca. one order of magnitude higher if compared to the exchange rates previously reported for Gd(III) complexes with octadentate ligands. The occurrence of such fast exchange rates of the coordinated water is exploited in the formation of macromolecular adducts with human serum albumin to attain systems displaying relaxivity values in the upper range of those so far reported for analogous Gd(III) systems. These results strongly support the view that Mn(II) complexes, in spite of the lower effective magnetic moment, can be considered as viable alternatives to the currently used Gd(III) complexes as contrast agents for MRI applications.

High-Relaxivity Contrast Agents for Magnetic Resonance Imaging Based on Multisite Interactions between a β-Cyclodextrin Oligomer and Suitably Functionalized GdIII Chelates

The results reported in this work show that tightly assembled adducts formed by trisubstituted GdIII complexes and a beta-CD multimer (Poly-beta-CD, d.p. ca. 12) may represent very interesting candidates for novel MRI applications wherein a high number of paramagnetic ions endowed with high relaxivity (per GdIII ion) are necessary. The relaxivities found for the paramagnetic adducts represent a remarkable step forward on the relaxivity scale. However, a detailed investigation of the determinants of the relaxation enhancement in these systems shows that their relaxivities are still limited by a nonoptimal tauR and a relatively long exchange lifetime of the coordinated water(s). Moreover, the exchange rate of the water molecule(s) coordinated to the GdIII ion further decreases upon binding to the Poly-beta-CD. It is suggested that this finding is related to the structural properties of the supramolecule, which brings a high density of hydroxyl groups into the proximity of the guest complexes, and this yields an overall reinforcement of the hydrogen-bonding network involving the coordinated water(s). On the other hand, such a tight arrangement appears responsible for an enhanced contribution to the observed relaxivity arising from water molecules in the second coordination sphere of the metal center.

Improved paramagnetic liposomes for MRI visualization of pH triggered release

This work aims at assessing the in vitro potential of paramagnetic pH sensitive liposomes as imaging tools for visualizing drug-delivery and release processes by Magnetic Resonance Imaging (MRI). pH sensitive liposomes (pSLs) were formulated using the fusogenic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the membrane stabilizer D-α-tocopherol-hemisuccinate (THS), and were loaded with several paramagnetic complexes including the clinically approved Gadoteridol (marketed as ProHance™). The proposed formulation allows the fast and full release of Gadoteridol at pH 5.5. The leakage of the imaging reporter from the vesicles was associated with a relaxivity enhancement that allowed its visualization by MRI. It was observed that the release mechanism implies the protonation of the THS basic sites that leads to vesicle aggregation, thus enabling the expression of the fusogenic property of POPE. Attempts for improving the MRI properties of pSLs were pursued through the encapsulation of imaging agents with higher relaxivity than Gadoteridol, but it was observed that the release kinetic can be significantly affected by the probe size. Aiming at preparing stealth pSLs, PEG chains were conjugated to the external surface of the vesicles via cleavable disulphide bridges. Such nanomedicines do not release their content at acidic pH as long as the coating polymer is not removed from the surface. The results obtained suggest that the liposomal formulation investigated in this work has the potential for visualizing drug-delivery and release processes by in vivo MRI preclinical studies.

Poly-β-cyclodextrin based platform for pH mapping via a ratiometric 19F/1H MRI method

The in vitro validation of a new pH mapping MRI method based on a supramolecular poly-beta-cyclodextrin-19F-Gd adduct is reported.

Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic Resonance Imaging Cell Labeling

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

An MRI Method To Map Tumor Hypoxia Using Red Blood Cells Loaded with a pO2-Responsive Gd-Agent

Hypoxia is a typical hallmark of many solid tumors and often leads to therapy resistance and the development of a more aggressive cancer phenotype. Oxygen content in tissues has been evaluated using numerous different methods for several imaging modalities, but none has yet reached the required standard of spatial and temporal resolution. Magnetic Resonance Imaging (MRI) appears to be the technique of choice and several pO2-responsive probes have been designed for it over the years. In vivo translation is often hampered in Gd-relaxation agents as it is not possible to separate effects that arise from changes in local concentration from those associated with responsive properties. A novel procedure for the MRI based assessment of hypoxia is reported herein. The method relies on the combined use of Gd-DOTP- and Gd-HPDO3A-labeled red blood cells (RBCs) where the first probe acts as a vascular oxygenation-responsive agent, while the second reports the local labeled RBC concentration in a transplanted breast tumor mouse model. The MRI assessment of oxygenation state has been validated by photoacoustic imaging and ex vivo immunofluorescence. The method refines tumor staging in preclinical models and makes possible an accurate monitoring of the relationship between oxygenation and tumor growth.

Targeting exofacial protein thiols with GdIII complexes. An efficient procedure for MRI cell labelling

Cells display on the outer surface of the plasma membrane a large number of protein thiols that can be reversibly labelled with suitably designed Gd(III)-based contrast agents for cell tracking by MRI.

In vivo imaging of inhibitory, GABAergic neurons by MRI

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.

Macrocyclic chelants, their chelates and uses thereof in the diagnostic field

This invention relates to novel compounds able to chelate paramagnetic bi- or trivalent metal ions, their chelates with said metal ions and their use as contrast agents in magnetic resonance imaging (MRI).