Carla Carrera

Determination of water permeability of paramagnetic liposomes of interest in MRI field.

The water permeability of various liposome membranes has been determined at 298K by measuring the NMR longitudinal water proton relaxation rate of vesicles encapsulating the clinically approved Gd-HPDO3A complex (HPDO3A=10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). Two basic formulations based on DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyl-oleylphosphatidylcholine) phospholipids were selected and investigated. Furthermore, the permeability changes caused by the membrane incorporation of amphiphiles like cholesterol and/or metal complexes of interest for designing improved liposome-based MRI contrast agents, were also investigated. The incorporation of cholesterol and metal complexes bearing C18 saturated chains in POPC-based liposomes reduces the water diffusivity across the membrane bilayer. On the contrary, the incorporation of a macrocyclic metal complex bearing four C12 alkylic chains, one for each coordination arm of the ligand, considerably enhances the water permeability in DPPC-based liposomes. Finally, it is reported that the permeability of POPC-based bilayer is increased when the liposomes are subjected to an osmotic stress.

Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic Resonance Imaging Cell Labeling

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

Lanthanide-loaded paramagnetic liposomes as switchable magnetically oriented nanovesicles.

Osmotically shrunken liposomes loaded with paramagnetic lanthanide(III) complexes orient in a static magnetic field according to the sign of their magnetic susceptibility anisotropy (Deltachi). The magnitude and sign of Deltachi are modulated by the magnetic properties of the Ln (III) ion, by the structural characteristics of the metal chelate, and by the stereochemical arrangement of the lipophilic substituents.

Effects of Magnetic Field Cycle on the Polarization Transfer from Parahydrogen to Heteronuclei through Long-Range J-Couplings

Hyperpolarization of (13)C carboxylate signals of metabolically relevant molecules, such as acetate and pyruvate, was recently obtained by means of ParaHydrogen Induced Polarization by Side Arm Hydrogenation (PHIP-SAH). This method relies on functionalization of the carboxylic acid with an unsaturated alcohol (side arm), hydrogenation of the unsaturated alcohol using parahydrogen, and polarization transfer to the target (13)C signal. In this case, parahydrogen protons are added three to four bonds away from the target (13)C nucleus, while biologically relevant molecules had been hyperpolarized, using parahydrogen, through hydrogenation of an unsaturated bond adjacent to the target (13)C signal. The herein reported results show that the same polarization level can be obtained on the (13)C carboxylate signal of an ester by means of addition of parahydrogen to the acidic or to the alcoholic moiety and successive application of magnetic field cycle (MFC). Experimental results are supported by calculations that allow one to predict that, upon accurate control of magnetic field strength and speed of the passages, more than 20% polarization can be achieved on the (13)C-carboxylate resonance of the esters by means of side arm hydrogenation and MFC.

An MRI Method To Map Tumor Hypoxia Using Red Blood Cells Loaded with a pO2-Responsive Gd-Agent

Hypoxia is a typical hallmark of many solid tumors and often leads to therapy resistance and the development of a more aggressive cancer phenotype. Oxygen content in tissues has been evaluated using numerous different methods for several imaging modalities, but none has yet reached the required standard of spatial and temporal resolution. Magnetic Resonance Imaging (MRI) appears to be the technique of choice and several pO2-responsive probes have been designed for it over the years. In vivo translation is often hampered in Gd-relaxation agents as it is not possible to separate effects that arise from changes in local concentration from those associated with responsive properties. A novel procedure for the MRI based assessment of hypoxia is reported herein. The method relies on the combined use of Gd-DOTP- and Gd-HPDO3A-labeled red blood cells (RBCs) where the first probe acts as a vascular oxygenation-responsive agent, while the second reports the local labeled RBC concentration in a transplanted breast tumor mouse model. The MRI assessment of oxygenation state has been validated by photoacoustic imaging and ex vivo immunofluorescence. The method refines tumor staging in preclinical models and makes possible an accurate monitoring of the relationship between oxygenation and tumor growth.

13C-MR Hyperpolarization of Lactate using ParaHydrogen and metabolic transformation in vitro.

Hyperpolarization of the 13 C magnetic resonance signal of l-[1-13 C]lactate has been obtained using the chemically based, cost-effective method called parahydrogen-induced polarization by means of side-arm hydrogenation (PHIP-SAH). Two ester derivatives of lactate were tested and the factors that determine the polarization level on the product have been investigated in detail. The metabolic conversion of hyperpolarized l-[1-13 C]lactate into pyruvate has been observed in vitro using lactate dehydrogenase (LDH) and in a cells lysate. From the acquisition of a series of 13 C NMR spectra, the metabolic build-up of the [1-13 C]pyruvate signal has been observed. These studies demonstrate that, even if the experimental set-up used for these PHIP-SAH hyperpolarization studies is still far from optimal, the attained polarization level is already sufficient to carry out in vitro metabolic studies.

Design and Synthesis of a γ1β8‐Cyclodextrin Oligomer: A New Platform with Potential Application as a Dendrimeric Multicarrier

We report the synthesis and characterization of a water-soluble, star-shaped macromolecular platform consisting of eight β-cyclodextrin (β-CD) units anchored to the narrower rim of a γ-CD core through bis(triazolyl)alkyl spacers. The efficient synthetic protocol is based on the microwave (MW)-promoted Cu-catalyzed 1,3-dipolar cycloaddition of CD monoazides to CD monoacetylenes. The ligand-hosting capability of the construct has been assessed by relaxometric titration and nuclear magnetic relaxation dispersion (NMRD) profiling, which showed it to be good, and this was supported by molecular dynamics simulations. To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed a dimeric platform, formed by joining two nonamers through the γ-CD cores through a bis(lithocholic acid) linker. With a view to the potential biological applications, cytotoxicity and extent of binding to human serum albumin were assessed. The properties of this dendrimeric multicarrier make it suitable for pharmaceutical and diagnostic purposes, ranging from targeted drug delivery to molecular imaging.

Studies to enhance the hyperpolarization level in PHIP-SAH-produced C13-pyruvate

The use of [1-13C]pyruvate, hyperpolarized by dissolution-Dynamic Nuclear Polarization (d-DNP), in in vivo metabolic studies has developed quickly, thanks to the imaging probes diagnostic relevance. Nevertheless, the cost of a d-DNP polarizer is quite high and the speed of hyperpolarization process is relatively slow, meaning that its use is limited to few research laboratories. ParaHydrogen Induced Polarization Side Arm Hydrogenation (PHIP-SAH) (Reineri et al., 2015) is a cost effective and easy-to-handle method that produces 13C-MR hyperpolarization in [1-13C]pyruvate and other metabolites. This work aims to identify the main determinants of the hyperpolarization levels observed in C13-pyruvate using this method. By dissecting the various steps of the PHIP-SAH procedure, it has been possible to assess the role of several experimental parameters whose optimization must be pursued if this method is to be made suitable for future translational steps. The search for possible solutions has led to improvements in the polarization of sodium [1-13C]pyruvate from 2% to 5%. Moreover, these results suggest that observed polarization levels could be increased considerably by an automatized procedure which would reduce the time required for the work-up passages that are currently carried out manually. The results reported herein mean that the attainment of polarization levels suitable for the metabolic imaging applications of these hyperpolarized substrates show significant promise.

Gadolinium-Decorated Silica Microspheres as Redox-Responsive MRI Probes for Applications in Cell Therapy Follow-Up.

The redox microenvironment within a cell graft can be considered as an indicator to assess whether the graft is metabolically active or hypoxic. We present a redox-responsive MRI probe based on porous silica microparticles whose surface has been decorated with a Gd-chelate through a disulphide bridge. Such microparticles are designed to be interspersed with therapeutic cells within a biocompatible hydrogel. The onset of reducing conditions within the hydrogel is paralleled by an increased clearance of Gd, that can be detected by MRI.

The 13 C hyperpolarized pyruvate generated by ParaHydrogen detects the response of the heart to altered metabolism in real time

Many imaging methods have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarkers characteristics of the injured organ have not yet been described. Hyperpolarized [1-13C]pyruvate allows real time monitoring of metabolism in vivo. ParaHydrogen Induced Polarization (PHIP) is a portable, cost effective technique able to generate 13C MR hyperpolarized molecules within seconds. The introduction of the Side Arm Hydrogenation (SAH) strategy offered a way to widen the field of PHIP generated systems and to make this approach competitive with the currently applied dissolution-DNP (Dynamic Nuclear Polarization) method. Herein, we describe the first in vivo metabolic imaging study using the PHIP-SAH hyperpolarized [1-13C]pyruvate. In vivo maps of pyruvate and of its metabolic product lactate have been acquired on a 1 T MRI scanner. By comparing pyruvate/lactate 13C label exchange rate in a mouse model of dilated cardiomyopathy, it has been found that the metabolic dysfunction occurring in the cardiac muscle of the diseased mice can be detected well before the disease can be assessed by echocardiographic investigations.