Highly efficient MRI contrast agents:from monomers to nanoparticles

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

Physicochemical and MRI characterization of Gd3+-loaded polyamidoamine and hyperbranched dendrimers

Generation 4 polyamidoamine (PAMAM) and, for the first time, hyperbranched poly(ethylene imine) or polyglycerol dendrimers have been loaded with Gd3+ chelates, and the macromolecular adducts have been studied in vitro and in vivo with regard to MRI contrast agent applications. The Gd3+ chelator was either a tetraazatetracarboxylate DOTA-pBn4− or a tetraazatricarboxylate monoamide DO3A-MA3− unit. The water exchange rate was determined from a 17O NMR and 1H Nuclear Magnetic Relaxation Dispersion study for the corresponding monomer analogues [Gd(DO3A-AEM)(H2O)] and [Gd(DOTA-pBn-NH2)(H2O)]− (k ex 298 =3.4 and 6.6×106s−1, respectively), where H3DO3A-AEM is {4-[(2-acetylaminoethylcarbamoyl)methyl]-7,10-bis(carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl)}-acetic acid and H4DOTA-pBn-NH2 is 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. For the macromolecular complexes, variable-field proton relaxivities have been measured and analyzed in terms of local and global motional dynamics by using the Lipari-Szabo approach. At frequencies below 100MHz, the proton relaxivities are twice as high for the dendrimers loaded with the negatively charged Gd(DOTA-pBn)− in comparison with the analogous molecule bearing the neutral Gd(DO3A-MA). We explained this difference by the different rotational dynamics: the much slower motion of Gd(DOTA-pBn)−-loaded dendrimers is likely related to the negative charge of the chelate which creates more rigidity and increases the overall size of the macromolecule compared with dendrimers loaded with the neutral Gd(DO3A-MA). Attachment of poly(ethylene glycol) chains to the dendrimers does not influence relaxivity. Both hyperbranched structures were found to be as good scaffolds as regular PAMAM dendrimers in terms of the proton relaxivity of the Gd3+ complexes. The in vivo MRI studies on tumor-bearing mice at 4.7T proved that all dendrimeric complexes are suitable for angiography and for the study of vasculature parameters like blood volume and permeability of tumor vessel

Physicochemical Properties of the High-Field MRI-Relevant [Gd(DTTA-Me)(H2O)2]− Complex

To study the physicochemical properties of the DTTA chelating moiety (H4DTTA = diethylenetriaminetetraacetic acid = N,N′-[iminobis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]), used in several compounds proposed as magnetic resonance imaging (MRI) contrast agents, the methylated derivative H4DTTA-Me (N,N′-[(methylimino)bis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]) was synthesized. Protonation 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 KZnL = 17.69 0.10). A novel approach of determining the relative conditional stability constant of two paramagnetic complexes in a direct way 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) [KL1/L2*(at pH 8.3, 25 °C) = 6.4 0.3]. The transmetalation reaction of the Gd3+ complex with Zn2+ in a 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. The use of [Gd(DTTA-Me)(H2O)2]− as an MRI contrast agent in vitro and in animal studies is conceivable, mainly at high magnetic fields, where an increase of the inner-sphere-coordination water actually seems to be the most certain way to increase the relaxivity

A ruthenium-based metallostar: synthesis, sensitized luminescence and 1H relaxivity

Ru-based metallostars Na4{Ru[Ln2bpy-DTTA2(H2O)4]3} (Ln = Y, Gd, and Eu) have been self-assembled in aqueous solution and their relaxivity and optical properties unravelled. The ynthesis 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 etallostar {Ru[Eu2bpy-DTTA2(H2O)4]3}4- displays sensitized 5D0 →7FJ luminescence upon excitation of the tris(2,2¢-bipyridyl)ruthenium(II) unit in the ultraviolet around 293 nm, as well as in the visible round 450 nm (1MLCT state). NMRD profiles at two temperatures (25 ◦C and 37 ◦C) were performed n {Ru[Gd2bpy-DTTA2(H2O)4]3}4-. NMRD profiles of the ruthenium-based {Ru[Gd2bpy- TTA2(H2O)4]3}4- and the iron-based {Fe[Gd2bpy-DTTA2(H2O)4]3}4- metallostars were fitted with BM theory coupled to the model-free Lipary-Szabo method for internal motion as well as with the mdified Florence approach. Comparison of both fitting methods shows that the Florence approach is ble to fit NMRD profiles up to 100 MHz, fails however at higher frequencies because it does not ccount for internal motion. Overall, the results detailed point to the heptametallic self-assembled difices being potential relaxivity and luminescence bimodal bioprobes for use in animal odels

Towards the Rational Design of MRI Contrast Agents: Electron Spin Relaxation Is Largely Unaffected by the Coordination Geometry of Gadolinium(III)–DOTA-Type Complexes

Electron‐spin relaxation is one of the determining factors in the efficacy of MRI contrast agents. Of all the parameters involved in determining relaxivity it remains the least well understood, particularly as it relates to the structure of the complex. One of the reasons for the poor understanding of electron‐spin relaxation is that it is closely related to the ligand‐field parameters of the Gd3+ ion that forms the basis of MRI contrast agents and these complexes generally exhibit a structural isomerism that inherently complicates the study of electron spin relaxation. We have recently shown that two DOTA‐type ligands could be synthesised that, when coordinated to Gd3+, would adopt well defined coordination geometries and are not subject to the problems of intramolecular motion of other complexes. The EPR properties of these two chelates were studied and the results examined with theory to probe their electron‐spin relaxation propertie