Multifrequency EPR studies of zero field splitting of Gd(III) based MRI contrast agents in solids and liquids

Magnetic Resonance Imaging (MRI) offers an amazing number of ways to probe the human body. It is a wonderful symbiosis of engineering, science and medicine. The power of MRI is its ability to probe a variety of physical mechanisms that can then not only reveal delicate anatomical structures but also discover important information about the physiology of tissue function in normal and diseased states. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent MRI contrast agents. They decrease the T1 and T2 relaxation times of nearby water protons. The mechanism of T1 relaxation is generally a through space dipole-dipole interaction. This interaction is between the unpaired electrons of a paramagnetic ion, such as Gd(III) and the water molecules that are in fast exchange within the ion's inner-coordination sphere. The lanthanide ion Gd(III) is by far the most frequently chosen metal centre in MRI contrast agent, because it has a very high magnetic moment (S = 7/2) and a symmetric electronic ground state, 8S7/2. As it is generally highly toxic, acyclic and macrocyclic polyaminocarboxylates ligands have been synthesized forming thermodynamically and kinetically stable complexes with Gd(III). The coupling of the seven unpaired electrons of the Gd(III) ion with the surrounding water proton spins observed in MRI is crucial for the contrast agent effectiveness, expressed as relaxivity. Therefore, understanding the behavior of the electron spin system can provide valuable information for the development of new compounds. Since this ion has S >1, its spin Hamiltonian includes a significant Zero Field Splitting (ZFS) contribution. This work was devoted to the determination by Electron Paramagnetic Resonance (EPR) spectroscopy of parameters governing the strength of ZFS of Gd(III)-based MRI contrast agents in aqueous solution, frozen solution, and powders. The analysis of multiple frequency and temperature EPR data has been conducted on selected commercial and potential MRI contrast agents. The studied complexes have been designed in the aim to optimise other important parameters which influence the relaxivity, like the rotational diffusion of the complexes and the water residence time in the first coordination shell. This work provides a survey of electron spin relaxation through the determination of the ZFS parameters for Gd(III)-based contrast agents (1) in solution where a rigorous derivation of the relaxation rates was applied to the analysis of the full EPR line shape at multiple frequencies, and (2) in glasses and powders where a direct and straightforward determination of ZFS parameters allowed, for the first time, to establish a correlation between the sign of the axial ZFS parameter and the nature of the chelating ligand in Gd(III) complexes.

Highly efficient MRI contrast agents

Loïck Moriggi

Since contrasts agents are used in magnetic resonance imaging (MRI), numerous efforts have been done to increase their relaxivity (parameter allowing to quantify contrast agent efficiency). Few years ago, the Merbach group developed the chelating agent DTTA (H4DTTA = diethylenetriaminetetraacetic acid = N,N'-[iminobis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]), which contains when complexed with gadolinium(III) in aqueous solution two inner-sphere coordination sites for water molecules. This chelate unit complexed with gadolinium(III) presents also a relatively fast inner-sphere water exchange rate which is favorable for the relaxivity. Three types of compounds have been studied in this thesis, all of them containing the DTTA chelate unit. The first is considered to be the monomer and allows studying the stability of the chelate unit. The second is a close derivative to the metallostar compound {Fe[Gd2bpy-DTTA2(H2O)4]3}4-, when the iron(II) central ion is replaced by a ruthenium(II) ion. The third is represented by the DTTA thiol derivative coated to gold nanoparticles. The first part of this thesis, chapter II, is thus the study of the physicochemical properties of the DTTA chelating moiety. For this purpose, the methylated derivative H4DTTA-Me (N,N'-[(methylimino)bis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]) was synthesized. Protonation and deuteration constants of the ligand were determined in an aqueous solution by potentimetry and 1H NMR pH titration and compared to various DTTA derivatives. Stability constants were measured for the chelates formed with Gd3+ (log KGdL = 18.60 ± 0.10) and Zn2+ (log ΚZnL = 17.69 ± 0.10). A novel approach of determining in a direct way the relative conditional stability constant of two paramagnetic complexes by 1H NMR relaxometry is presented and was used for the Gd3+ complexes [Gd(DTTA-Me)(H2O)2]- (L1) and [Gd(DTPA-BMA)(H2O)] (L2) [Κ*L1/ L2 (at pH 8.3, 25°C) = 6.4 ± 0.3]. The transmetalation reaction of the Gd3+ complex with Zn2+ in phosphate buffer solution (pH 7.0) was measured to be twice as fast for [Gd(DTTA-Me)(H2O)2]- in comparison to that for [Gd(DTPA-BMA)( H2O)]. This can be rationalized by the higher affinity of Zn2+ toward DTTA-Me4- if compared to DTPA-BMA3-. The formation of a ternary complex with L-lactate, which is common for DO3A-based heptadentate complexes, has not been observed for [Gd(DTTA-Me)( H2O)2]- as monitored by 1H NMR relaxometric titrations. From the results, it was concluded that the heptadentate DTTA-Me4- behaves similarly to the commercial octadentate DTPA-BMA3- with respect to stability. Considering recent suspicions against [Gd(DTPA-BMA)( H2O)] for being involved in Nephrogenic Systemic Fibrosis (NSF) disease, DTTA-type chelates will not be admitted as contrast agents in clinical MRI. However, their use in vitro and in animal studies is absolutely conceivable, mainly at high magnetic fields, where the increase of inner-sphere-coordination water actually seams to be the only promising way to increase the relaxivity markedly. The second part of this thesis, chapter III, presents the synthesis in aqueous solution and the magnetic and optical properties of the Ru(II)-based metallostars Na4{Ru[Ln2bpy-DTTA2(H2O)4]3} (Ln = Y, Gd, and Eu). The synthesis and the purification of the new, highly stable heptametallic entities have been optimized for the diamagnetic Y3+ complex and followed by 1H NMR. The europium(III) ruthenium-based metallostar {Ru[Eu2bpy-DTTA2(H2O)4]3}4- displays sensitized 5D0→7FJ luminescence upon excitation of the tris-(2,2'-bipyridyl)ruthenium(II) unit in both the ultraviolet around 293 nm, as well as in the visible around 450 nm (1MLCT state). NMRD profiles at two temperatures (25°C and 37°C) were measured on the {Ru[Gd2bpy-DTTA2(H2O)4]3}4-. NMRD profiles of the ruthenium-based {Ru[Gd2bpy-DTTA2(H2O)4]3}4- and iron-based {Fe[Gd2bpy-DTTA2(H2O)4]3}4- metallostars were fitted with SBM theory coupled to the model-free Lipary-Szabo method for internal motion as well as with the modified Florence approach. Comparison of both fitting methods shows that the Florence approach is able to fit NMRD profiles up to 100 MHz, fails however at higher frequencies because it does not account for internal motion. Overall, the results detailed point to the heptametallic self-assembled edifices being potential relaxivity and luminescence bimodal bioprobes. The third part of this thesis, chapter IV, is devoted to nano objects. We developed small, water-dispersible, stable nanoparticles covalently linked to Ln-DTTA derivative complexes (with Ln=Gd and Y) on the surface. Characterizations of these DTTA thiol capped gold nanoparticles (Gd-DtNP) using TEM images, dynamic light scattering technique and STEM with EDX analysis indicate 1.5-15.5 nm particle diameters, coated by Ln-Dt complexes on the surface. Molecular modeling shows a rough distance in the range of 1.3 nm between the lanthanide(III) ion and gold core surface and the structure that possess Ln-DtNP offers to lanthanide(III) a tightly bonding on the surface of particles. Accurate Au, Gd and Y concentrations have been determined by ICP-MS technique. Bulk magnetic susceptibility of samples with different gadolinium concentration has been established following the Evans method and no significant magnetic contribution can be attributed to the gold core. NMRD profiles of Gd-DtNP at 25°C show very high relaxivities and present broad relaxivity humps indicating very large systems with slow rotational motion. The modified Florence approach allows fitting well the experimental NMRD profil of Gd-DtNP, and the fitted electronic relaxation parameters are in good coherence with what was expected from the Ru-based metallostar values. Our Gd-DtNP systems are very rigid nano-object.

EPFL2008

Luminescent lanthanides bioprobes emitting in the visible and/or near-infrared ranges

Steve Comby

This Ph.D. work deals with the development of lanthanide-based bioprobes for biomedical analyses and imaging. For this purpose, two different approaches have been followed, one focusing on visible-emitting complexes, while the second addresses the sensitization of NIR-emitting LnIII ions. The homoditopic ligands H2LC2 and H2LC3 have been tailored to self-assemble with lanthanide ions (LnIII) into neutral triple-stranded bimetallic helicates of overall composition [Ln2(L)3] and also to provide a versatile platform for further derivatization. The grafting of polyoxyethylene groups onto the pyridine (H2LC2) or the benzimidazole (H2LC3) units ensures water solubility, while maintaining large thermodynamic stability and adequate antenna effect for the excitation of LnIII ions. The conditional stability constants are large at physiological pH, with logβ23 reaching values in the range 22-25 for helicates with H2LC2 and H2LC3. The ligands triplet states feature adequate energy to efficiently sensitize the EuIII luminescence (QEu L = 21 and 11 % for L = H2LC2 and H2LC3, respectively) in aerated water at pH 7.4. Conversely, energy back transfer occurring in [Tb2(LC3)3] precludes a good sensitization of the TbIII luminescence, and only the TbIII helicate with H2LC2 displays sizeable quantum yield (QTbL = 11 %). Furthermore, it has been demonstrated that ligand H2LC2 sensitizes the emission of other visible- and NIR-emitting LnIII ions, such as SmIII or YbIII, for which in cellulo luminescence is evidenced, a rare observation. The EuIII emission spectra of both helicates arise from a main species with pseudo-D3 symmetry and without coordinated water. Cytotoxicity experiments reveal no significant effect of the helicates on the viability of several cancerous cell lines and cell imaging properties of the EuIII helicates are demonstrated for the HeLa cell line by luminescence microscopy. Bright EuIII emission is seen after relatively short time (15-30 min) and low helicate incubation concentrations (10 µM). Similar images were obtained for [Sm2(LC2)3] and [Tb2(LC2)3] using slightly higher loading concentration. The helicates stain the cytoplasm and the permeation mechanism is likely to be endocytosis. The described luminescent helical stains are robust chemical species which remain mostly undissociated in the cell medium and which resist to the presence of other complexing agents, as demonstrated in the case of H2LC2. All these properties make these helicates potential bioprobes for interacting with biological material and for cell imaging applications. As near-infrared (NIR) light, in addition to be less energetic than visible light, displays better penetrability into the human body, the development of bioprobes emitting in the near-infrared range is of prime importance for deeper tissue imaging, especially for early diagnosis of tumors. We therefore investigate the ability of new polydentate podands to sensitize the NIR luminescence of NdIII and YbIII ions. These ligands have been designed in a way to take advantage of the chelating effect of bidentate (Tsox, TsoxMe) or tridentate 8-hydroxyquinolinate (8-HQ) subunits (T2soxMe). The interaction between the ligands and LnIII ions in aqueous solution leads to the formation of stable 1:1 chelates (pLn in the range of 12-16), all the chromophoric 8-HQ units being coordinated to the metal center, exploiting the entropic effect generated by the anchor. At physiological pH, one major species is present for all the ligands regardless of the LnIII ion used. The low energy triplet states of the ligands are particularly well-suited for an efficient sensitization of the NdIII- or YbIII-centered luminescence (QNdL = 0.02-0.04 % and QYbL = 0.13-0.37 %) in buffered aqueous solution. ErIII NIR luminescence is also detected in water for the chelates with Tsox and TsoxMe. Lifetime determinations point in all cases to the absence of coordinated water molecules in the inner coordination sphere of the LnIII ion. It has also been demonstrated that methylation of the amide functions of Tsox removes the quenching mechanism induced by the proximate N-H groups and increases both the lifetimes and quantum yields of the TsoxMe chelates, with the YbIII complex being one of the most NIR luminescent lanthanide-based edifices described so far. Ligand TsoxMe thus appears to be the best candidate of the series regarding to the development of NIR probes for bioanalyses. Furthermore, other applications, for instance in telecommunications, are still conceivable since the incorporation of the Yb-TsoxMe chelate into sol-gel silica films has proven to be successful, the chelate withstanding the sol-gel process, while providing a slight enhancement of photophysical properties compared to the same chelate in aqueous solution.

EPFL2008

Luminescent lanthanides bioprobes emitting in the visible and/or near-infrared ranges

Steve Comby

EPFL2008

Highly efficient MRI contrast agents

Loïck Moriggi

EPFL2008