Non-covalent conjugates between cationic polyamino acids and GdIII chelates: a route for seeking accumulation of MRI-contrast agents at tumor targeting sites

Three novel Gd chelates containing on their external surface pendant phosphonate and carboxylate groups, which promote the interaction with the positively charged groups of polyornithine and polyarginine, have been synthesized. Their solution structures have been assessed on the basis of 1H- and 31P-NMR spectra of the Eu and Yb analogues. A thorough investigation of the relaxometric (1H and 17O) properties of the Gd chelates has been carried out and the observed relaxivities have been accounted for the sum of three contributions arising from water molecules in the first, second, and outer coordination layers, respectively. It has been found that the occurrence of a tight second coordination coating renders the dissociation of the water molecule directly coordinated to the Gd ion more difficult. The binding interactions between the negatively charged Gd chelates and the positively charged groups of polyornithine (ca. 140 residues) and polyarginine (ca. 204 residues) have been evaluated by means of the proton relaxation enhancement (PRE) method. Although the binding interaction decreases markedly in the presence of competitive anions in the solution medium, the affinity is strong enough that in blood serum it is possible to meet the conditions where most of the chelate is bound to the polyamino acid substrate. On this basis one may envisage a novel route for a MRI location of tumors as it is known that positively charged polyamino acids selectively bind to tumors having a greater negative charge than non-tumor cells.     

Molecular dynamics simulation of [Gd(egta)(H(2)O)](-) in aqueous solution: internal motions of the poly(amino carboxylate) and water ligands, and rotational correlation times

Molecular dynamics simulations of [Gd(egta)(H(2)O)](-) (egta(4-)=3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioate(4-)) have been performed without any artificial constraint on the first coordination sphere, such as covalent bonds between the Gd(3+) and the coordination sites. Two new crystallographic structures were determined for this gadolinium chelate and used to start two molecular dynamics simulations. [Gd(egta)(H(2)O)](-) and [Gd(egta)](-) were both observed during the simulations, with a mean volume for the reaction of dissociation [Gd(egta)(H(2)O)](-)-->[Gd(egta)](-)+H(2)O of +7.2 cm(3)mol(-1), which corroborates the previously published experimental value of +10.5 cm(3)mol(-1). Changes in the conformation of the complex with the inversion of several dihedral angles are observed in the simulations independently from the water dissociation. Very fast changes of the third-order rotation axis direction of the Gd(3+) coordination polyhedron (of symmetry D(3h)) are observed during the simulations and are related to the mechanism of electronic relaxation of the complex. Different rotational correlation times (tau(R)) were calculated from the simulations on various observables of the complex. Protons of the inner sphere have different tau(R). The mean tau(R) of the two Gd-HW(HW=hydrogen of water molecule) vectors is 72% lower than tau(R) of the complex, and 75% lower than tau(R) of the vector Gd-OW (OW=oxygen of water molecule). This discrimination of the tumbling rates should be taken into account in future global (17)O NMR, EPR and NMRD (nuclear magnetic relaxation dispersion) data analysis.     

High-Relaxivity contrast agents for magnetic resonance imaging based on multisite interactions between a beta-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.     

A highly stable gadolinium complex with a fast, associative mechanism of water exchange

The stability and water exchange dynamics of gadolinium (GdIII) complexes are critical characteristics that determine their effectiveness as contrast agents for magnetic resonance imaging (MRI). A new heteropodal GdIII chelate, [Gd-TREN-bis(6-Me-HOPO)-(TAM-TRI)(H2O)2] (Gd-2), is presented which is based on a hydroxypyridinate (HOPO)-terephthalamide (TAM) ligand design. Thermodynamic equilibrium constants for the acid-base properties and the GdIII complexation strength of TREN-bis(6-Me-HOPO)-(TAM-TRI) (2) were measured by potentiometric and spectrophotometric titration techniques, respectively. The pGd of 2 is 20.6 (pH 7.4, 25 degrees C, I = 0.1 M), indicating that Gd-2 is of more than sufficient thermodynamic stability for in vivo MRI applications. The water exchange rate of Gd-2 (kex = 5.3(+/-0.6) x 107 s-1) was determined by variable temperature 17O NMR and is in the fast exchange regime - ideal for MRI. Variable pressure 17O NMR was used to determine the volume of activation (DeltaV) of Gd-2. DeltaV for Gd-2 is -5 cm3 mol-1, indicative of an interchange associative (Ia) water exchange mechanism. The results reported herein are important as they provide insight into the factors influencing high stability and fast water exchange in the HOPO series of complexes, potentially future clinical contrast agents.     

High-relaxivity magnetic resonance imaging (MRI) contrast agent based on supramolecular assembly between a gadolinium chelate, a modified dextran, and poly-beta-cyclodextrin

Nanosized contrast agents have great potential in magnetic resonance molecular imaging applications for clinical diagnosis. This study proposes new nanoparticles spontaneously formed under mild conditions and composed of a noncovalent adduct between a gadolinium complex, a polymer of beta-cyclodextrin (pbetaCD: MW 1.5 x 10(6) g mol(-1)) and a dextran grafted with alkyl chains (MD). The formation of this supramolecular nanoassembly is based upon a "lock-and-key" recognition process in which the hydrophobic alkyl chains of MD and the adamantyl moieties of macrocyclic Gd(III) chelates are included in the cavities of pbetaCD. The large number of betaCDs contained in the pbetaCD resulted in the formation of 200 nm diameter nanoparticles, each entrapping 1.8 x 10(5) molecules of a low-molecular-weight Gd complex. This system, which exhibits a great relaxivity enhancement (48.4 mM(-1) s(-1), at 20 MHz and 37 degrees C) compared to the Gd(III) chelate itself (5.2 mM(-1) s(-1)), appears to be a promising strategy for the in vivo targeted delivery of Gd(III) complexes. The mechanisms of particle formation, conjugation strategies, and relaxometric characterizations in the field of contrast-enhanced magnetic resonance imaging are discussed.     

New hyperpolarized contrast agents for 13C-MRI from para-hydrogenation of oligooxyethylenic alkynes

Two alkyne derivatives, which contain one and two oligooxyethylenic chains respectively, showed to be good substrates for para-hydrogenation reactions, yielding the corresponding hyperpolarized alkenes in good yields. A suitable theory has been developed to account for the observed results, fully explaining the different para-H 2 induced effects observed upon the para-hydrogenation of symmetrically and asymmetrically substituted alkynes in ALTADENA and PASADENA modes. The oligooxyethylenic substituent provides good water solubility to the para-hydrogenated symmetrical derivative. (13)C-MR in vitro images of the latter derivative were obtained both in acetone and in water solutions (130 mM), using the ALTADENA procedure and after application of the field cycling procedure which allows acquisition of an in-phase (13)C carbonyl resonance. The finding that the hydrogenated product is water-soluble in contrast to the parent alkyne which is not allows for the pursuit of a fast phase-transfer separation from the organic solvent, the unreacted substrate, and the catalyst to obtain a "ready-to-use" water solution suitable for further in vivo MRI applications.     

17O NMR spectroscopy of transition metal carbonyls

NMR spectra of ¹⁷O in natural abundance have been obtained for a range of metal carbonyls. Linewidths of less than 10 Hz for the derivatives of Cr, Mo, W and Fe have been observed, although they are slightly larger for the derivatives of metals having an electric quadrupole moment. Comparison with the corresponding ¹³C NMR data shows that: (i) the chemical shift ranges are comparable, (ii) the ordering of chemical shifts is not very different, (iii) the linewidths are more favourable for ¹⁷O than ¹³C for some Mn and Co derivatives and allow an extension of the range of temperature in which information on the fluxional dynamics are obtainable in the rapid exchange limit.     

High Relaxivity Supramolecular Adducts Between Human‐Liver Fatty‐Acid‐Binding Protein and Amphiphilic GdIII Complexes: Structural Basis for the Design of Intracellular Targeting MRI Probes

Gadolinium complexes linked to an apolar fragment are known to be efficiently internalized into various cell types, including hepatocytes. Two lipid‐functionalized gadolinium chelates have been investigated for the targeting of the human liver fatty acid binding protein (hL‐FABP) as a means of increasing the sensitivity and specificity of intracellular‐directed MRI probes. hL‐FABP, the most abundant cytosolic lipid binding protein in hepatocytes, displays the ability to interact with multiple ligands involved in lipid signaling and is believed to be an obligate carrier to escort lipidic drugs across the cell. The interaction modes of a fatty acid and a bile acid based gadolinium complex with hL‐FABP have been characterized by relaxometric and NMR experiments in solution with close‐to‐physiological protein concentrations. We have introduced the analysis of paramagnetic‐induced protein NMR signal intensity changes as a quantitative tool for the determination of binding stoichiometry and of precise metal‐ion‐center positioning in protein–ligand supramolecular adducts. A few additional NMR‐derived restraints were then sufficient to locate the ligand molecules in the protein binding sites by using a rapid data‐driven docking method. Relaxometric and ¹³C NMR competition experiments with oleate and the gadolinium complexes revealed the formation of heterotypic adducts, which indicates that the amphiphilic compounds may co‐exist in the protein cavity with physiological ligands. The differences in adduct formation between fatty acid and bile acid based complexes provide the basis for an improved molecular design of intracellular targeted probes.     

High relaxivity supramolecular adducts between human-liver fatty-acid-binding protein and amphiphilic Gd(III) complexes: structural basis for the design of intracellular targeting MRI probes

Gadolinium complexes linked to an apolar fragment are known to be efficiently internalized into various cell types, including hepatocytes. Two lipid-functionalized gadolinium chelates have been investigated for the targeting of the human liver fatty acid binding protein (hL-FABP) as a means of increasing the sensitivity and specificity of intracellular-directed MRI probes. hL-FABP, the most abundant cytosolic lipid binding protein in hepatocytes, displays the ability to interact with multiple ligands involved in lipid signaling and is believed to be an obligate carrier to escort lipidic drugs across the cell. The interaction modes of a fatty acid and a bile acid based gadolinium complex with hL-FABP have been characterized by relaxometric and NMR experiments in solution with close-to-physiological protein concentrations. We have introduced the analysis of paramagnetic-induced protein NMR signal intensity changes as a quantitative tool for the determination of binding stoichiometry and of precise metal-ion-center positioning in protein-ligand supramolecular adducts. A few additional NMR-derived restraints were then sufficient to locate the ligand molecules in the protein binding sites by using a rapid data-driven docking method. Relaxometric and (13)C NMR competition experiments with oleate and the gadolinium complexes revealed the formation of heterotypic adducts, which indicates that the amphiphilic compounds may co-exist in the protein cavity with physiological ligands. The differences in adduct formation between fatty acid and bile acid based complexes provide the basis for an improved molecular design of intracellular targeted probes.