Simonetta Geninatti Crich

Improved route for the visualization of stem cells labeled with a Gd‐/Eu‐Chelate as dual (MRI and fluorescence) agent

A simple labeling procedure of stem/progenitor cells based on the use of Gd‐HPDO3A and Eu‐HPDO3A, respectively, is described. The Gd‐chelate acts as T1‐agent for MRI visualization, whereas the corresponding Eu‐chelate acts as reporter in fluorescence microscopy. Owing to their substantial chemical equivalence, the two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The lanthanide chelates are entrapped in endosomic vesicles and the labeled cells retain biological activity with preservation of viability and pro‐angiogenesis capacity. Hyperintense spots in MR have been observed for Gd‐labeled EPCs injected under mice kidney capsule or grafted on a subcutaneous Matrigel plug up to 14 days after transplantation. Magn Reson Med 51:938–944, 2004.

Effect of the intracellular localization of a Gd-based imaging probe on the relaxation enhancement of water protons

Gd‐HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is “quenched” to a plateau value (about 3 s−1) when the entrapped amount of Gd‐chelate is higher than 1 × 1010 Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99–102). On this basis, entrapment into intracellular vesicles has been treated as a three‐site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two‐site exchange system. Magn Reson Med, 2006.

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

Abstract The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (KA) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of KA. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM–1 s–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate.

Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-Loaded Apoferritin Probe

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

NMR relaxometric studies of Gd(III) complexes with heptadentate macrocyclic ligands

The water 1H and 17O NMR relaxation properties of solutions containing Gd(III) chelates of the heptadentate DO3A, PCTA[12] and PCTP[12] ligands were thoroughly investigated and the results obtained are compared with those previously reported for other Gd(III) complexes with octadentate ligands {H3DO3A=1,4,7,10‐tetraazacyclododecane 1,4,7‐triacetic acid; H3PCTA[12]=3,6,9,15‐tetraazabicyclo[9.3.1]pentadeca‐1(15),11,13‐triene‐3,6,9‐triacetic acid; H6PCTP[12]=3,6,9,15‐tetraazabicyclo[9.3.1]pentadeca‐1(15),11,13‐triene‐3,6,9‐tris(methanephosphonic) acid}. The observed behaviour is consistent with a hydration number q=2 in the case of GdDO3A and GdPCTA[12] and q=1 in the case of PCTP[12]. The high relaxivity of the latter complex is accounted for in terms of the occurrence of an additional contribution arising from water molecules tightly bound to the phosphonate moieties on the surface of the paramagnetic chelate. Furthermore, it was found that the decreased relaxation rates observed at basic pH in the case of GdDO3A and GdPCTA[12] can probably be ascribed to a partial decrease in their hydration. The measurement of 17O NMR transverse relaxation rates, in the temperature range 273–342 K, allowed the assessment of the water exchange rate between the coordination site and the bulk solvent. A particularly short exchange lifetime was measured for the octacoordinate GdPCTP[12], which suggests the occurrence of an associative exchange mechanism. Further insights into the understanding of the structural properties of the three complexes were gained by measuring the magnetic field dependence (NMRD profiles) of the proton relaxivity on a Koenig–Brown field cycling relaxometer.

NOVEL PARAMAGNETIC MACROMOLECULAR COMPLEXES DERIVED FROM THE LINKAGE OF A MACROCYCLIC Gd(III) COMPLEX TO POLYAMINO ACIDS THROUGH A SQUARIC ACID MOIETY

Macromolecular Gd(III) complexes may find useful application as contrast agents for magnetic resonance angiography (MRA). Herein two novel systems are reported, namely Gd(DO3ASQ)3-lys16 and Gd(DO3ASQ)30-orn114. Their syntheses are based on the ability of the squaric acid moiety to act as a linker between the DO3A (1,4,7, 10-tetraazacyclododecane-1,4,7-triacetic acid) chelate moiety and the polyamino acidic chain. Moreover, the squaric acid participates in the coordination cage of the Gd(III) ion. The investigation of 1H and 17O NMR relaxation processes of solvent water nuclei allowed a detailed characterization of the systems under study. Gd(DO3ASQ)30-orn114 displays a remarkable ability to enhance the water proton relaxation rate of its solutions, and it may be considered as potential contrast agent for MRA applications.

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.

MRI Visualization of Melanoma Cells by Targeting Overexpressed Sialic Acid with a GdIII‐dota‐en‐pba Imaging Reporter

These changes in glycosylation are mainly caused byan increased expression of sialyltransferases, which glycosy-late exposed glycans at their terminal positions with anionicmonosaccharide sialic acid residues (Sia). Tumor cells over-expressing Sia appear protected against the immune defensesystem, and as a result, malignancy is increased.

1H and 17O-NMR relaxometric investigations of paramagnetic contrast agents for MRI. Clues for higher relaxivities

Abstract The analysis of 1 H- and 17 O-NMR relaxometric data allows us to get a better insight into the understanding of the structural and dynamic factors responsible for the relaxivity of a given paramagnetic system. High relaxivities are obtained in the presence of a long molecular reorientational time and fast exchange of the water molecule(s) coordinated to the paramagnetic metal ion. Long molecular reorientational times are pursued either through the formation of covalent conjugates between a paramagnetic complex and a macromolecular substrate or through the formation of non-covalent adducts between suitably functionalized complexes and endogenous (e.g. serum albumin) or exogenous (e.g. poly-β-cyclodextrin) substrates. Within the class of covalent conjugates, it has been shown that the use of a DOTA-like chelate bearing the squaric acid as linking moiety leads to an improved relaxivity with respect to analogous systems obtained through reactions involving the bifunctional DTPA bisanhydride. As far as the exchange of the coordinated water in Gd(III) chelates is concerned, it depends on the energy difference between the ground ennea-coordinated state and the activated octa-coordinated state. In the presence of bulky substituents, the ground state is destabilized with a consequent increase of the exchange rate. An elongation of the exchange lifetime can occur upon the interaction with serum albumin. This behaviour may result in a decrease of the attainable relaxivity. Finally, it has been shown that Mn(II) chelates may represent a viable alternative to Gd(III) complexes. In fact, in spite of the lower effective magnetic moment, the non-covalent adducts between Mn(II) chelates and albumin display very high relaxivities. This result has been accounted for in terms of the very short exchange lifetime of the Mn(II) coordinated water molecule.

Curcumin/Gd Loaded Apoferritin: A Novel “Theranostic” Agent To Prevent Hepatocellular Damage in Toxic Induced Acute Hepatitis

Apoferritin has been exploited to deliver simultaneously therapeutic and imaging agents (loaded into its internal cavity) to hepatocytes as this protein is efficiently taken up from blood by hepatocyte scavenger receptor class A type 5 via the ferritin transporting route. To this purpose the protein has been loaded with the magnetic resonance imaging (MRI) contrast agent GdHPDO3A and curcumin, a polyphenolic substance endowed with multiple pharmacological actions, namely: antioxidant, anti-inflammatory, antineoplastic. Curcumin and GdHPDO3A loaded apoferritin has been used with the aim to attenuate the thioacetamide-induced hepatitis together with the evaluation by MRI of drug delivery efficiency. Mice pretreated by intraperitoneal administration showed significantly attenuated hepatic injury as assessed by measuring alanine aminotransferase (ALT) activity in plasma and by histology assessment. The encapsulation of curcumin inside the apoferritin cavity significantly increases its stability and bioavailability while maintaining its therapeutic anti-inflammatory properties.