Magnetic resonance imaging has developed into a powerful diagnostic technique characterized by a very high spatial resolution and an inherently relatively low sensitivity. In order to improve the contrast of MRI images, contrast agents are commonly injected into the patients before an examination. These substances are paramagnetic, superparamagnetic, or ferromagnetic compounds that shorten the relaxations times of the water hydrogen atoms. At present, most of the contrast enhanced clinical exams are performed with gadolinium complexes. They are particularly useful as their ability to change the relaxation rate (or relaxivity) can be very high. Several factors have a strong influence on the relaxivity of MRI contrast agents but the water exchange time τm and the rotational correlation time τr are particularly important for obtaining an increased relaxivity. These parameters can be adjusted by suitable chemical modifications of the Gd(III) complexes. For instance, decreasing the tumbling rate by linking a Gd(III) complex to a macromolecule leads to an increased relaxivity. This goal can be achieved through a covalent or a noncovalent linkage with synthetic polymers, particles or biomacromolecules. However, the covalent bonding has a detrimental effect on the clearance of the metal complexes thus exposing the patient to the toxicity of released Gd(III) ions and metabolites. This problem could be circumvented by using covalent links that are cleaved by endogenous biomolecules or after administration of exogenous compounds following the exam. In this context, our approach was to bind Gd(III) chelates to macromolecules through disulfide links as the latter are known to be reduced in vivo by thiols present in the body. Towards this aim, we have developed two bifunctional chelator agents bearing a methanethiosulfonate group (MTS) which reacts specifically with thiols, thus spontaneously establishing a disulfide bond between the Gd(III) chelate and the thiolated macromolecule. The first ligand that we have prepared (MTS-ADO3A) is a monoamide derivative of DOTA with an ethyl-MTS substituent.
This compound is relatively easily synthesized but amide arms such as the one it features are known to have a detrimental effect on relaxivity through the lengthening of water exchange times. The conjugate obtained by binding Gd(III) chelates of this ligand to albumin or to polythiolated silica nanoparticles has been studied by nuclear magnetic relaxation dispersion (NMRD),17O NMR and luminescence analyses. These measurements confirm that the method is suitable to increase the relaxivity (20 mM-1s 1, 20 MHz, 25°C) but that this relaxivity increase (of 300%) is limited by a slow water exchange (660 ns). To overcome this limitation, a second ligand called MTS-CyDOTA has been synthesized. This ligand is a DOTA ligand grafted with a cyclohexyl ring featuring a MTS function.
The synthesis is more demanding but faster water exchange times are expected because of a more sterically crowded coordination sphere. Moreover, this second ligand has a more rigid structure that could limit the independent rotation of the chelate from the macromolecule. As expected, the water exchange time of the Gd(III) chelate of this ligand (120 ns) is clearly lower than the one determined for Gd MTS-ADO3A. After binding to albumin or to silica nanoparticles a notable relaxivity increase was expected. Unfortunately, if the obtained relaxivity is higher (30 mM-1s 1, 20 MHz, 25°C), it’s not as high as it could have been expected in view of the size of the conjugate and of the water exchange time of the free chelate. Results obtained in this work suggest that fixation on silica nanoparticles or on albumin drastically decreases the water exchange rate which remains the limiting parameter. This effect has already been reported for Gd(III) chelates linked to albumin by non-covalent bonds and has been assigned to stable layers of water molecules on the macromolecule surface. Thanks to the high loading of the silica nanoparticles (10000 Gd(III) per particle), we have reached very high molecular relaxivities (>200000 mM-1s-1). Stability tests carried out on the disulfide links formed suggest that the small amount of free thiols in the circulation is not sufficient to cause a significant degradation of the disulfide bond in the conjugate within a reasonable length of time. An injection of glutathione would be necessary to achieve a complete degradation.
To avoid the problem of water exchange lenghtening, we propose to increase the distance between Gd(III) chelates and macromolecules without loss of rigidity by developing double anchor chelates with substituents grafted on the side of the ring. Considerable synthetic efforts have devoted to the synthesis of such a system and are discussed in chapter VI. At present, this work is still in progress in the laboratory and recent results suggest that it should be possible to evaluate this double “arms” system in a near future. On the fringe of this synthesis, we present a relaxometric study on the interaction between HSA and a hydrophobic Gd(III) chelate obtained during the preparation of our double anchor chelate.
Finally, a chapter of this work is devoted to the study of two compounds, phenEDTA and phenDTPA, which are ditopic chelates featuring a dihydro-1,10-phenanthroline unit that spontaneously self-assemble in the presence of a transition metal ion.
The tris-complex generated by this process rotates more slowly in solution and thus presents an increased relaxivity (+130%). As part of this work, we have determined by potentiometric titration the acidity constants of phenEDTA and its stability constant with Gd(III). Moreover, the protonation scheme of this ligand has been studied by NMR titration. The particular behavior of Gd phenDTPA and Fe(Gd phenDTPA)3 in the presence of Zn(II) has also been studied by relaxometry, luminescence and EXAFS.