VSION as high field MRI T1 contrast agent: evidence of their potential as positive contrast agent for magnetic resonance angiography.

Because of their outstanding magnetic properties, iron oxide nanoparticles have already been the subject of numerous studies in the biomedical field, in particular as a negative contrast agent for T2-weighted nuclear magnetic resonance imaging, or as therapeutic agents in hyperthermia experiments. Recent studies have shown that below a given particle size (i.e. 5 nm), iron oxide may be used to provide a significant positive (brightening) effect on T1-weighted MRI. In such an application, not only the size of the crystal, but also the control of the coating process is essential to ensure optimal properties, especially at a very high field (> 3 T). In this work, we focused on the development of very small iron oxide nanoparticles as a potential platform for high field T1 magnetic resonance angiography (MRA) applications. The feasibility has been evaluated in vivo at 9.4 T, demonstrating the usefulness of the developed system for MRA applications.

Characterization of Gd loaded chitosan-TPP nanohydrogels by a multi-technique approach combining dynamic light scattering (DLS), asymetrical flow-field-flow-fractionation (AF4) and atomic force microscopy (AFM) and design of positive contrast agents for molecular resonance imaging (MRI).

Chitosan CS-tripolyphosphate TPP/hyaluronic acid HA nanohydrogels loaded with gadolinium chelates (GdDOTA ⊂ CS-TPP/HA NGs) synthesized by ionic gelation were designed for lymph node (LN) MRI. In order to be efficiently drained to LNs, nanogels (NGs) needed to exhibit a diameter ϕ < 100 nm. For that, formulation parameters were tuned, using (i) CS of two different molecular weights (51 and 37 kDa) and (ii) variable CS/TPP ratio (2 < CS/TPP < 8). Characterization of NG size distribution by dynamic light scattering (DLS) and asymetrical flow-field-flow-fractionation (AF4) showed discrepancies since DLS diameters were consistently above 200 nm while AF4 showed individual nano-objects with ϕ < 100 nm. Such a difference could be correlated to the presence of aggregates inherent to ionic gelation. This point was clarified by atomic force microscopy (AFM) in liquid mode which highlighted the main presence of individual nano-objects in nanosuspensions. Thus, combination of DLS, AF4 and AFM provided a more precise characterization of GdDOTA ⊂ CS-TPP/HA nanohydrogels which, in turn, allowed to select formulations leading to NGs of suitable mean sizes showing good MRI efficiency and negligible toxicity.

A new magnetic resonance imaging contrast agent loaded into poly(lacide-co-glycolide) nanoparticles for long-term detection of tumors.

The incorporation of a lipophilic Gd chelate (GdDO3A-C12) in biocompatible PLGA poly(D, L-lactide-co-glycolide) nanoparticles was explored as an approach to increase the relaxivity of contrast agents for magnetic resonance imaging. By nanoprecipitation, it was possible to obtain PEGylated gadolinium nanoparticles (mean diameter of 155 nm) with high Gd loading (1.1 × 10(4) Gd centers per nanoparticle). The corresponding GdDO3AC12 ⊂ NPs nanoparticles exhibited an enhanced relaxivity (up to sixfold greater than DOTAREM® at 40 MHz) because the nanoparticle framework constrained the lipophilic Gd chelate motion and favorably impacted the Gd chelate rotational correlation time. T1-weighted imaging at 3 T on phantoms showed enhanced contrast for the GdDO3AC12 ⊂ NPs. Importantly, Gd chelate leakage was almost nonexistent, which suggested that these GdDO3AC12 ⊂ NPs could be useful for long-term MRI detection.

Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

Study of non-covalent interactions between MRI contrast agents and human serum albumin by NMR diffusometry.

The NMR diffusometry technique, based on the measurement of the diffusion coefficient of a ligand in the absence and in the presence of its macromolecular partner, was used to study the affinity for human serum albumin (HSA) of four gadolinium complexes, potential or already used magnetic resonance imaging contrast agents. Diamagnetic lanthanum(III) ion or europium(III) ion, which has the advantage of shifting the NMR signals far away from those of the macromolecule, was used to avoid the excessive broadening of the NMR signals induced by the gadolinium(III) ion. Titration experiments, in which the HSA concentration was kept constant and the concentration of the europium or lanthanum chelate was varied, were performed to evaluate the association constant and the number of binding sites. Some additional information about the kinetics of the exchange between the free and the bound chelate was also obtained. Competition experiments with ibuprofen and salicylate, which are ligands with a known affinity for the macromolecule and for which the binding site is known, were also performed to get information about the binding site of the contrast agents.

Synthesis of a new gadolinium complex with a high affinity for human serum albumin and its manifold physicochemical characterization by proton relaxation rate analysis, NMR diffusometry and electrospray mass spectrometry.

A novel gadolinium complex, derived from Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid) and sulfaphenazole, intended to be a potential MRI contrast agent and to interact with human serum albumin (HSA), was synthesized and characterized. Its relaxometric properties were evaluated in water, and its binding to HSA was investigated by three techniques: proton relaxation rate analysis, NMR diffusometry, and electrospray mass spectrometry. The complex has a higher relaxivity than the parent compound (r(1)=7.8s(-1)mM(-1) at 310K and 0.47T and 7.7s(-1)mM(-1) at 310K and 1.41T), a fast water exchange, and a very good stability versus zinc(II) transmetallation. All techniques agree with a high affinity of the complex for HSA, and competition experiments indicate that this contrast agent competes with ibuprofen for HSA.

Following the stability of amphiphilic nanoaggregates by using intermolecular energy transfer.

An intermolecular energy transfer system is developed for studying the stability of nanoaggregate(s) (NAs) in complex solution and cell culture by one- and two-photon fluorescence microscopy and optical imaging. The system allows facile addition of one or more tumor targeting molecules, one of which is exemplified here. NAs functionalized with an MRI and optical probe, with and without folic acid, remain stable in fetal bovine serum for at least 4 hours. HeLa cell cultures showed a clear difference between NAs non-targeted and targeted to folate receptors, with both NAs appearing to be taken up by the cells through different mechanisms. An MRI relaxivity, r1, of 9 mM-1 s-1 at 310 K and 1.4 T was measured associated with the increased rotational correlation time of the NAs. These NAs may have application in the targeted drug delivery of hydrophobic drugs such as doxorubicin (DOX).

Minor changes in the macrocyclic ligands but major consequences on the efficiency of gold nanoparticles designed for radiosensitization.

Many studies have been devoted to adapting the design of gold nanoparticles to efficiently exploit their promising capability to enhance the effects of radiotherapy. In particular, the addition of magnetic resonance imaging modality constitutes an attractive strategy for enhancing the selectivity of radiotherapy since it allows the determination of the most suited delay between the injection of nanoparticles and irradiation. This requires the functionalization of the gold core by an organic shell composed of thiolated gadolinium chelates. The risk of nephrogenic systemic fibrosis induced by the release of gadolinium ions should encourage the use of macrocyclic chelators which form highly stable and inert complexes with gadolinium ions. In this context, three types of gold nanoparticles (Au@DTDOTA, Au@TADOTA and Au@TADOTAGA) combining MRI, nuclear imaging and radiosensitization have been developed with different macrocyclic ligands anchored onto the gold cores. Despite similarities in size and organic shell composition, the distribution of gadolinium chelate-coated gold nanoparticles (Au@TADOTA-Gd and Au@TADOTAGA-Gd) in the tumor zone is clearly different. As a result, the intravenous injection of Au@TADOTAGA-Gd prior to the irradiation of 9L gliosarcoma bearing rats leads to the highest increase in lifespan whereas the radiophysical effects of Au@TADOTAGA-Gd and Au@TADOTA-Gd are very similar.

Validation of a dendron concept to tune colloidal stability, MRI relaxivity and bioelimination of functional nanoparticles.

The functionalization of spherical superparamagnetic iron oxide nanoparticles (SPION) of 10 nm with a linear monophosphonate (L1) and also PEGylated mono-phosphonated dendrons of growing generation (D2-G1, -G2 and -G3) yielded dendritic nano-objects of 15 to 30 nm in size, stable in physiological media and showing both renal and hepatobiliary elimination. The grafting of the different molecules has been confirmed by IR spectroscopy and elemental analysis. The colloidal stability of functionalized NS10 has been evaluated in water and in different physiological media. All functionalized NS10 were stable over a long period of time and displayed a mean hydrodynamic diameter smaller than 50 nm whatever the molecule architecture or dendron generation. Only the NS10@L1 showed less stability in biological media at high ionic concentration. NMRD profiles and relaxivity measurements highlighted the influence of the molecule architecture on the water diffusion close to the magnetic core thus influencing the relaxation properties at low magnetic field. Coupling of a fluorescent dye on the functionalized NS10 allowed investigating their biodistribution and highlighting urinary and hepato-biliary eliminations.

Multinuclear magnetic resonance characterization of paramagnetic contrast agents. The manifold effects of concentration and counterions.

RATIONALE AND OBJECTIVES: Proper fitting of the nuclear magnetic resonance dispersion (NMRD) profiles to the numerous factors governing nuclear relaxation in paramagnetic systems requires knowledge of some parameters usually obtained by other techniques. The rotational correlation time (tau R) for example can be measured by carbon-13, hydrogen-2, or oxygen-17 NMR. Discrepancies between values reported in the literature might be attributed to the different concentration ranges used so far in these modalities. In the present work focussing on commercial nonspecific contrast agents, the influence of the solution composition (type and concentration of the complexes and of the counterions) has been examined with regard to the water proton relaxation enhancement and molecular dynamics. METHODS: The proton relaxation rate enhancement of Magnevist, Dotarem, Omniscan, and ProHance was measured in aqueous solution up to a concentration of 0.5 M. In the same concentration window, the rotational correlation times were obtained from the study of deuterium relaxation rates of the diamagnetic deuterated analogs (lanthanum complexes) of the gadolinium chelates. RESULTS: Above 50 mM, the relaxation rate enhancement versus concentrations strongly deviates from linearity. Magnevist, a clinical formulation containing two meglumine counterions per molecule of paramagnetic complex, exhibits the largest concentration effect. A slowing down of the molecular dynamics accounts for this behavior as confirmed by the analysis of the rotational correlation times obtained by deuterium relaxometry. At low concentrations (< or = 50 mM), tau R values obtained by proton NMRD analysis and by deuterium relaxation are in very good agreement. CONCLUSIONS: This study shows that NMR analyses of small molecular weight complexes should be carried out on solutions containing no more than 50 mM to avoid the biaising effects of concentration. On the other hand, the benefitting relaxivity enhancement induced by highly concentrated solutions has to be taken into account in the context of bolus injection or vesicular entrapment.

Mechanical and biochemical cardiac disturbances induced by bolus administration of iodinated contrast media and noniodinated solutions.

RATIONALE AND OBJECTIVES: Bolus injection of iodinated contrast media has been observed to alter myocardial mechanical function, but the consequences on cellular metabolism are poorly documented. Modifications of metabolic parameters (intracellular pH as well as adenosine triphosphate [ATP], phosphocreatine, and inorganic phosphate contents) and of mechanical function (coronary flow, heart rate, and left ventricular developed pressure) were simultaneously recorded on isolated rat hearts perfused over 2 minutes with a high osmolality contrast medium (HOCM) and two low osmolality contrast media (LOCM). In addition, the effects of test solutions mimicking the ionicity and the osmolality of LOCM were evaluated. METHODS: Isovolumic rat hearts were submitted to a 2-minute perfusion with oxygenated Radioselectan, Hexabrix, and Omnipaque (320 mgI/mL) at 37 degrees C. Metabolic parameters were obtained by P-31 nuclear magnetic resonance spectroscopy at 4.7 Tesla. Noniodinated ionic and nonionic solutions also were tested for comparison. RESULTS: HOCM irreversibly impairs the metabolic and mechanical functions, whereas ionic and nonionic LOCM and test solutions induce transient cardiac failure but no permanent alteration of the metabolic or mechanical parameters. CONCLUSION: In this protocol, HOCM causes irreversible degradation of the biochemical status and definitive heart failure, whereas ionic and nonionic LOCM only induce transient changes of myocardial function. Treatments with the LOCM do not induce any modification of the ATP and PCr content, and, at the end of the reperfusion period, the mechanical function is equivalent to that of control hearts. Depending on the ionic content of the solutions (iodinated or not), the evolution of the ventricular developed pressure after injection differs from one group of hearts to another. From these experiments, it is concluded that ionic imbalance and viscosity of the solutions, rather than iodine content or hyperosmolality, should be considered the causes of heart failure.

Spectroscopic and metabolic effects of MnCl2 and MnDPDP on the isolated and perfused rat heart.

RATIONALE AND OBJECTIVES: Several works have shown that the hepatobiliary magnetic resonance imaging contrast agent manganese dipyridoxyl diphosphate (MnDPDP) partly releases its metallic ion and exhibits cardiovascular effects that are supposed to arise from the free manganese ions (Mn++). In the current study, the cellular internalization of Mn by the isolated rat heart is monitored through the mechanical function of the organ and the relative broadening of the P-31 nuclear magnetic resonances. METHODS: Rat hearts were perfused with manganese chloride (MnCl2; 15 and 25 microM) or MnDPDP (25 microM). Variations of the linewidths, heights, and surfaces of phosphocreatine and adenosine triphosphate peaks were monitored. Cardiac function was monitored simultaneously through heart rate, left ventricular pressure, and coronary flow. RESULTS: Influx of Mn++ induces a significant broadening of the P-31 resonances of adenosine triphosphate and phosphocreatine because of a strong scalar paramagnetic interaction between the nuclei and the ion. Compared with MnDPDP administered at the same concentration, MnCl2 induced a more pronounced and dose-dependent line broadening as well as a coronary vasodilation. Calcium channel blockers (nifedipine and verapamil) and EDTA inhibit MnCl2 influx. Similarly, verapamil, EDTA, and DPDP reduce the alterations provoked by MnDPDP. CONCLUSIONS: The effects of MnDPDP are smaller but of the same type than those induced by MnCl2. Their inhibition by calcium channel blockers (verapamil and nifedipine) and by an excess of strong chelators such as DPDP or EDTA confirms that they originate from a partial release of Mn++ by the contrast agent.

The importance of nuclear magnetic relaxation dispersion (NMRD) profiles in MRI contrast media development.

Observation of the relaxivity of MRI contrast media over a wide range of magnetic fields is not only necessary for predicting their efficiency at any field but also compulsory for understanding and improving their mechanisms of action. The best experimental approach to this problem is the field cycling method, which allows the exploration of nuclear relaxation over a broad interval of magnetic field intensity but requires a specially dedicated instrument called a relaxometer. Particularly relevant are the relaxivity profiles of the two chelates Gd-DOTA and Gd-DTPA. Both show an important decrease from low to high fields within the current imaging range (0.02 T to 1.5 T). Although high field relaxivities of these chelates are similar, Gd-DTPA becomes less efficient in facilitating water protons relaxation at fields lower than 0.15 T. This behavior has to be related to different electronic relaxation times due to a different chelate symmetry.

31P nuclear magnetic resonance study of the effects of the calcium ion channel antagonist fantofarone on the rat heart.

The biochemical and mechanical effects of a new calcium ion channel antagonist, fantofarone ((2-isopropyl-1-((4-(3-(N-methyl-N-(3,4-dimethoxy-beta-phenethyl)-amino) propyloxy)benzenesulfonyl))-indolizine), on isovolumic perfused rat heart have been assessed by using 31P nuclear magnetic resonance (NMR) spectroscopy together with simultaneous monitoring of myocardial mechanical function. Cytosolic pH and phosphocreatine, adenosine triphosphate and inorganic phosphate contents were monitored by using 31P NMR. Heart rate, coronary flow and left ventricular developed pressure were measured routinely to assess mechanical function. Perfusion with 10 nM, 100 nM or 1 microM fantofarone for a period of 48 min did not cause any measurable metabolic changes. However, coronary vasodilatation and a partial positive inotropic effect were noted. A 15-min pretreatment with 100 nM did not protect against the deleterious effects of an 18-min period of normothermic, zero-flow ischemia. In contrast, a 20-min pretreatment period with 1 microM fantofarone significantly improved the recovery of mechanical performance, metabolic activity and pH after the same 18 min of ischemia. While only a slight protection of the ATP pool was noted during the ischemic period, major beneficial effects were observed during the reperfusion period, such that reflow was characterized by high recoveries of left ventricular pressure and rate pressure product (70-80%), low end diastolic pressure (< 10 mm Hg), significant recovery of ATP content (to 55%), a complete repletion of the phosphocreatine pool and a fast return of cytosolic pH to normal value.

Dy-complexes as high field T2 contrast agents: influence of water exchange rates.

Dy complexes can act as suitable negative (T2) contrast agents for Magnetic Resonance Imaging (MRI). As clinical MRI moves toward higher fields, tuning of the exchange rate of coordinated water molecules will become necessary to optimize the r2 relaxivity. For Dy complexes, this will require lengthening of the water residence time, a strategy opposite that required to optimize the r1 relaxivity of Gd complexes. However, very slow water exchange can be deleterious. This is illustrated here by a Dy complex that is characterized by a very slow water exchange. This complex, Dy-DOTA-4AmCE, is compared with several Dy-DTPA derivatives known for their efficacy as T2 contrast agents at high magnetic fields.

Fluorometry, a fast screening technique for non-covalent binding of contrast agents to human serum albumin?

The non-covalent interactions of gadolinium-based MRI contrast agents with macromolecules, such as human serum albumin (HSA), increase their efficacy. The identification of contrast agents that interact with HSA is a crucial first step in the complex, lengthy and expensive developmental process of a new potential HSA-targeting contrast agent. Fluorometry has been used as a possibly simpler and more effective tool of screening. In this study, the affinity of four compounds (Gd-DTPA, Gd-BOPTA, Gd-EOB-DTPA and MS-325) for HSA was investigated. The results show that the fluorescence method is a convenient tool that can easily detect this kind of non-covalent interaction owing to the small amount of required compound, the simplicity of the procedure and the popularity of the instrument, compared with the other approaches reported in the literature. However, fluorescence screening tests should be interpreted with caution since false-negative results will occur when the binding site of a gadolinium-based agent is far away from the location of the sole Trp residue of HSA or when an unsuitable site-marker is selected.

NMR determination of free gallium(III) ions in aqueous solutions of Ga complexes, "cold" analogs of PET/SPECT tracers.

Ga complexes are widely used as radiopharmaceuticals for PET or SPECT imaging and as therapeutic agents. At physiological pH, free gallium(III) ions can stably exist as soluble gallate [Ga(OH)(4) ](-) , a nephrotoxic compound whose presence should be avoided. Any Ga complex, therefore, should be carefully checked for the absence of gallate before use. Here we show that (71) Ga NMR is a useful tool to rapidly detect the presence of gallate in aqueous solutions of Ga complexes and to follow the purification steps of the Ga complex solutions.

How to measure the transmetallation of a gadolinium complex.

The suitability of paramagnetic complexes as contrast agents depends not only on their relaxivity but also on their stability and inertness towards transmetallation processes by endogenous ions. In this work, we describe a convenient method to study the stability of paramagnetic Gd complexes through the evolution of the paramagnetic longitudinal relaxation rate of water protons at 37 °C.

Synthesis and physicochemical characterization of Gd-C4-thyroxin-DTPA, a potential MRI contrast agent. Evaluation of its affinity for human serum albumin by proton relaxometry, NMR diffusometry, and electrospray mass spectrometry.

Gd-C(4)-thyroxin-DTPA, a potential MRI contrast agent, was synthesized from Gd-DTPA and thyroxine, which interacts strongly with human serum albumin (HSA). It was characterized in water by its relaxometric properties and its stability versus zinc transmetalation. The affinity of the complex for HSA was studied by using three different methods: proton relaxometry, NMR diffusometry, and electrospray mass spectrometry. From the results, it appears that Gd-C(4)-thyroxin-DTPA exhibits a relatively high relaxivity (r(1) = 9.01 s(-1) mM(-1) at 1.5 T and 310 K), a good stability versus zinc transmetalation, and a strong interaction with HSA (K(a) approximately 10,000 M(-1) with two binding sites). The kinetics of the exchange between the bound and the free form of the complex was evaluated by the NMR diffusometry technique. Competition experiments have allowed the assignment of the chelate's binding site on HSA.