Claire Corot

Macrophage endocytosis of Superparamagnetic iron oxide nanoparticles: Mechanisms and comparison of Ferumoxides and Ferumoxtran-10

Rationale and objectivesSuperparamagnetic iron oxides (SPIO) used as magnetic resonance (MR) contrast agents undergo specific uptake by macrophages. The purpose of this study was first to determine the mechanism of macrophage uptake for Ferumoxides by using competition experiments with specific ligands of scavenger receptors SR-A (I/II) and second, to evaluate and compare the internalization of 2 different contrast agents, Ferumoxides (SPIO) and Ferumoxtran-10 (USPIO: ultrasmall superparamagnetic iron oxide) using macrophages obtained by chemical activation of human monocytic cells. MethodsFerumoxides and Ferumoxtran-10 are 2 MR contrast agents, composed of dextran-coated iron oxide nanoparticles. The endocytosis pathway of Ferumoxides was studied using competition experiments on mouse peritoneal macrophages in the presence of specific ligands of scavenger receptors SR-A (types I and II): polyinosinic acid and fucoidan. In vitro assays using THP-1 (human promonocyte) cells activated into macrophages were performed in the presence of the 2 superparamagnetic nanoparticles. The cellular uptake was determined by measuring the iron content using ICP-AES (inductively coupled plasma–atomic emission spectrometry) and by Prussian blue staining. ResultsIn the presence of polyinosinic acid or fucoidan, the endocytosis of Ferumoxides by mouse peritoneal macrophages was inhibited. This inhibition was obtained using 10 &mgr;g/mL of scavenger receptor ligands at a concentration of 62.5 &mgr;g Fe/mL of SPIO, and a dose-dependent relationship was observed. Without competitors, the percentage of uptake of Ferumoxides by mouse peritoneal macrophages ranged between 3 and 8%. On the human activated monocyte THP-1 cell assay, Ferumoxides underwent a higher macrophage uptake (between 1.1 and 3%) compared with Ferumoxtran-10 (between 0.03 and 0.12%). This difference is attributed to the larger size of Ferumoxides nanoparticles. ConclusionsCompetition experiments indicate that the cellular uptake of Ferumoxides involves scavenger receptor SR-A-mediated endocytosis. The comparison between Ferumoxides and Ferumoxtran-10 confirms that macrophage uptake of iron oxide nanoparticles depends mainly on the size of these contrast agents.

Clinical and biological consequences of transmetallation induced by contrast agents for magnetic resonance imaging: a review

Gadolinium‐based contrast agents (CAs) are widely used to enhance the contrast of images in magnetic resonance imaging procedures. Two categories of gadolinium chelates exist: the macrocyclic molecules where Gd3+ is caged in the pre‐organized cavity of the ligand and the linear molecules. Gadolinium chelates differ in their thermodynamic stability constants and in their kinetic stability. In general, macrocyclic chelates such as Gd‐DOTA or Gd‐HP‐DO3A are more stable than linear molecules. Even among linear agents, differences can be found. There is increasing evidence that transmetallation can be found in vivo, in the case of certain CAs (especially linear chelates), with body cations such as zinc, calcium or iron. Furthermore, analytical interference with colorimetric determination of calcium has been clinically evidenced with two linear chelates, Gd‐DTPA‐BMA and Gd‐DTPA‐BMEA. Clinical cases of spurious hypocalcaemia have been reported with these molecules. Such interference with some colorimetric assays for calcium is clinically relevant in that it can lead to unnecessary and potentially harmful treatment for hypocalcaemia.

Macrophage imaging in central nervous system and in carotid atherosclerotic plaque using ultrasmall superparamagnetic iron oxide in magnetic resonance imaging

The long blood circulating time and the progressive macrophage uptake in inflammatory tissues of ultrasmall superparamagnetic iron oxide (USPIO) particles are 2 properties of major importance for magnetic resonance imaging (MRI) pathologic tissue characterization. This article reviews the proof of principle of applications such as imaging of carotid atherosclerotic plaque, stroke, brain tumor characterization, or multiple sclerosis. In the human carotid artery, USPIO accumulation in activated macrophages induced a focal drop in signal intensity compared with preinfusion MRI. The USPIO signal alterations observed in ischemic areas of stroke patients is probably related to the visualization of inflammatory macrophage recruitment into human brain infarction since animal experiments in such models demonstrated the internalization of USPIO into the macrophages localized in these areas. In brain tumors, USPIO particles which do not pass the ruptured blood-brain barrier at early times postinjection can be used to assess tumoral microvascular heterogeneity. Twenty-four hours after injection, when the cellular phase of USPIO takes place, the USPIO tumoral contrast enhancement was higher in high-grade than in low-grade tumors. Several experimental studies and a pilot multiple sclerosis clinical trial in 10 patients have shown that USPIO contrast agents can reveal the presence of inflammatory multiple sclerosis lesions. The enhancement with USPIO does not completely overlap with the gadolinium chelate enhancement. While the proof of concept that USPIO can visualize macrophage infiltrations has been confirmed in animals and patients in several applications (carotid atherosclerotic lesions, stroke, brain tumors and multiple sclerosis), larger prospective clinical studies are needed to demonstrate the clinical benefit of using USPIO as an MRI in vivo surrogate marker for brain inflammatory diseases.

T1 and T2 relaxivity of intracellular and extracellular USPIO at 1.5T and 3T clinical MR scanning.

In this study we evaluated the effects of intracellular compartmentalization of the ultrasmall superparamagnetic iron oxide (USPIO) ferumoxtran-10 on its proton T1 and T2 relaxivities at 1.5 and 3T. Monocytes were labeled with ferumoxtran-10 by simple incubation. Decreasing quantities of ferumoxtran-10-labeled cells (2.5×107-0.3×107 cells/ml) and decreasing concentrations of free ferumoxtran-10 (without cells) in Ficoll solution were evaluated with 1.5 and 3T clinical magnetic resonance (MR) scanners. Pulse sequences comprised axial spin echo (SE) sequences with multiple TRs and fixed TE and SE sequences with fixed TR and increasing TEs. Signal intensity measurements were used to calculate T1 and T2 relaxation times of all samples, assuming a monoexponential signal decay. The iron content in all samples was determined by inductively coupled plasma atomic emission spectrometry and used for calculating relaxivities. Measurements at 1.5T and 3T showed higher T1 and T2 relaxivity values of free extracellular ferumoxtran-10 as opposed to intracellularly compartmentalized ferumoxtran-10, under the evaluated conditions of homogeneously dispersed contrast agents/cells in Ficoll solution and a cell density of up to 2.5×107 cells/ml. At 3T, differences in T1-relaxivities between intra- and extracellular USPIO were smaller, while differences in USPIO T2-relaxivities were similar compared with 1.5T. In conclusion, cellular compartmentalization of ferumoxtran-10 changes proton relaxivity.

MRI of Tumor-Associated Macrophages with Clinically Applicable Iron Oxide Nanoparticles

Purpose: The presence of tumor-associated macrophages (TAM) in breast cancer correlates strongly with poor outcome. The purpose of this study was to develop a clinically applicable, noninvasive diagnostic assay for selective targeting and visualization of TAMs in breast cancer, based on magnetic resonanceI and clinically applicable iron oxide nanoparticles. Experimental Design: F4/80-negative mammary carcinoma cells and F4/80-positive TAMs were incubated with iron oxide nanoparticles and were compared with respect to magnetic resonance signal changes and iron uptake. MMTV-PyMT transgenic mice harboring mammary carcinomas underwent nanoparticle-enhanced magnetic resonance imaging (MRI) up to 1 hour and 24 hours after injection. The tumor enhancement on MRIs was correlated with the presence and location of TAMs and nanoparticles by confocal microscopy. Results:In vitro studies revealed that iron oxide nanoparticles are preferentially phagocytosed by TAMs but not by malignant tumor cells. In vivo, all tumors showed an initial contrast agent perfusion on immediate postcontrast MRIs with gradual transendothelial leakage into the tumor interstitium. Twenty-four hours after injection, all tumors showed a persistent signal decline on MRIs. TAM depletion via αCSF1 monoclonal antibodies led to significant inhibition of tumor nanoparticle enhancement. Detection of iron using 3,3′-diaminobenzidine-enhanced Prussian Blue staining, combined with immunodetection of CD68, localized iron oxide nanoparticles to TAMs, showing that the signal effects on delayed MRIs were largely due to TAM-mediated uptake of contrast agent. Conclusion: These data indicate that tumor enhancement with clinically applicable iron oxide nanoparticles may serve as a new biomarker for long-term prognosis, related treatment decisions, and the evaluation of new immune-targeted therapies. Clin Cancer Res; 17(17); 5695–704. ©2011 AACR.

Evaluation of Matrix Metalloproteinases in Atherosclerosis Using a Novel Noninvasive Imaging Approach

Objective—Despite great advances in our knowledge, atherosclerosis continues to kill more people than any other disease in the Western world. This is because our means of identifying truly vulnerable patients is limited. Prediction of atherosclerotic plaque rupture may be addressed by MRI of activated matrix metalloproteinases (MMPs), a family of enzymes that have been implicated in the vulnerability of plaques prone to rupture. This study evaluated the ability of the novel gadolinium-based MRI contrast agent P947 to target MMPs in atherosclerotic plaques. Methods and Results—The affinity of P947 toward activated MMPs was demonstrated in vitro. The affinity and specificity of P947 toward matrix metalloproteinase (MMP)-rich plaques was evaluated both in vivo using ApoE−/− mice and ex vivo in hyperlipidemic rabbits. Gadolinium content quantification and MRI showed a preferential accumulation of P947 in atherosclerotic lesions compared with the nontargeted reference compound, Gd-DOTA. The ex vivo assay on rabbit plaques revealed a higher uptake of P947. Moreover, using human carotid artery endarterectomy specimens, P947 facilitated discrimination between histologically defined MMP-rich and MMP-poor plaques. An in vivo MRI investigation in mice revealed that P947 greatly improved the ability to visualize and delineate atherosclerotic plaques. Conclusions—P947 may be a useful tool for the detection and characterization of the MMP-rich atherosclerotic plaques.

T1-Weighted Hypersignal in the Deep Cerebellar Nuclei After Repeated Administrations of Gadolinium-Based Contrast Agents in Healthy Rats: Difference Between Linear and Macrocyclic Agents.

ObjectivesTo prospectively compare in healthy rats the effect of multiple injections of macrocyclic (gadoterate meglumine) and linear (gadodiamide) gadolinium-based contrast agents (GBCAs) on T1-weighted signal intensity in the deep cerebellar nuclei (DCN), including the dentate nucleus. Materials and MethodsHealthy rats (n = 7/group) received 20 intravenous injections of 0.6 mmol of gadolinium (Gd) per kilogram (4 injections per week during 5 weeks) of gadodiamide, gadoterate meglumine, or hyperosmolar saline (control group). Brain T1-weighted magnetic resonance imaging was performed before and once a week during the 5 weeks of injections and during 5 additional weeks (treatment-free period). Gadolinium concentrations were measured with inductively coupled plasma mass spectrometry in plasma and brain. Blinded qualitative and quantitative evaluations of the T1 signal intensity in DCN were performed, as well as a statistical analysis on quantitative data. ResultsA significant and persistent T1 signal hyperintensity in DCN was observed only in gadodiamide-treated rats. The DCN-to-cerebellar cortex signal ratio was significantly increased from the 12th injection of gadodiamide (1.070 ± 0.024) compared to the gadoterate meglumine group (1.000 ± 0.033; P < 0.001) and control group (1.019 ± 0.022; P < 0.001) and did not significantly decrease during the treatment-free period. Total Gd concentrations in the gadodiamide group were significantly higher in the cerebellum (3.66 ± 0.91 nmol/g) compared with the gadoterate meglumine (0.26 ± 0.12 nmol/g; P < 0.05) and control (0.06 ± 0.10 nmol/g; P < 0.05) groups. ConclusionsRepeated administrations of the linear GBCA gadodiamide to healthy rats are associated with progressive and persistent T1 signal hyperintensity in the DCN, with Gd deposition in the cerebellum in contrast with the macrocyclic GBCA gadoterate meglumine for which no effect was observed.

Rapid-Clearance Iron Nanoparticles for Inflammation Imaging of Atherosclerotic Plaque: Initial Experience in Animal Model

PURPOSE To evaluate the use of a recently developed fast-clearing ultrasmall superparamagnetic iron oxide (USPIO) for detection of vascular inflammation in atherosclerotic plaque. MATERIALS AND METHODS The study protocol was approved by the animal experimentation ethics committee. A recently introduced USPIO, P904, and a reference-standard USPIO, ferumoxtran-10, were tested in a rabbit model of induced aortic atherosclerosis. In vivo magnetic resonance (MR) angiography and T2*-weighted plaque MR imaging were performed at baseline and after administration of P904 and ferumoxtran-10 (administered dose for both, 1000 micromol of iron per kilogram of body weight) in 26 hyperlipidemic New Zealand white rabbits. The variation in vessel wall area over time was evaluated with nonparametric testing. Ex vivo MR imaging findings were compared with iron content at linear regression analysis. RESULTS With in vivo MR imaging, plaque analysis was possible as early as 24 hours after P904 injection. The authors observed a 27.75% increase in vessel wall area due to susceptibility artifacts on day 2 (P = .04) and a 38.81% increase on day 3 (P = .04) after P904 administration compared with a 44.5% increase in vessel wall area on day 7 (P = .04) and a 34.8% increase on day 10 (P = .22) after ferumoxtran-10 administration. These susceptibility artifacts were correlated with intraplaque iron uptake in the corresponding histologic slices. The number of pixels with signal loss on the ex vivo MR images was linearly correlated with the logarithm of the iron concentration (P = .0001; R(2) = 0.93). CONCLUSION Plaque inflammation in rabbits can be detected earlier with P904 than with ferumoxtran-10 owing to the faster blood pharmacokinetics and the early uptake of P904 in the reticuloendothelial system. SUPPLEMENTAL MATERIAL http://radiology.rsnajnls.org/cgi/content/full/252/2/401/DC1.

P792: a rapid clearance blood pool agent for magnetic resonance imaging: preliminary results

An original MRI contrast agent, called P792, is described. P792 is a gadolinium macrocyclic compound based on a Gd-DOTA structure substituted by hydrophilic arms. The chemical structure of P792 has been optimized in order to provide (1) a high r1 relaxivity in the clinical field for MRI: 29 mM−1 x s−1 at 60 MHz. (2) a high biocompatibility profile and (3) a high molecular volume: the apparent hydrodynamic volume of P792 is 125 times greater than that of Gd-DOTA. As a result of this high molecular volume, P792 presents an unusual pharmacokinetic profile, as it is a Rapid Clearance Blood Pool Agent (RCBPA) characterized by limited diffusion across the normal endothelium. The original pharmacokinetic properties of this RCBPA are expected to be well suited to MR coronary angiography, angiography, perfusion imaging (stress and rest), and permeability imaging (detection of ischemia and tumor grading). Further experimental imaging studies are ongoing to define the clinical value of this compound.

Linear Gadolinium-Based Contrast Agents Are Associated With Brain Gadolinium Retention in Healthy Rats

ObjectivesThe aim of this study was to evaluate Gd retention in the deep cerebellar nuclei (DCN) of linear gadolinium-based contrast agents (GBCAs) compared with a macrocyclic contrast agent. Materials and MethodsThe brain tissue retention of Gd of 3 linear GBCAs (gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide) and a macrocyclic GBCA (gadoterate meglumine) was compared in healthy rats (n = 8 per group) that received 20 intravenous injections of 0.6 mmol Gd/kg (4 injections per week for 5 weeks). An additional control group with saline was included. T1-weighted magnetic resonance imaging was performed before injection and once a week during the 5 weeks of injections and for another 4 additional weeks after contrast period. Total gadolinium concentration was measured with inductively coupled plasma mass spectrometry. Blinded qualitative and quantitative evaluations of the T1 signal intensity in DCN were performed, as well as a statistical analysis on quantitative data. ResultsAt completion of the injection period, all the linear contrast agents (gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide) induced a significant increase in signal intensity in DCN, unlike the macrocyclic GBCA (gadoterate meglumine) or saline. The T1 hypersignal enhancement kinetic was fast for gadodiamide. Total Gd concentrations for the 3 linear GBCAs groups at week 10 were significantly higher in the cerebellum (1.21 ± 0.48, 1.67 ± 0.17, and 3.75 ± 0.18 nmol/g for gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide, respectively) than with the gadoterate meglumine (0.27 ± 0.16 nmol/g, P < 0.05) and saline (0.09 ± 0.12 nmol/g, P < 0.05). No significant difference was observed between the macrocyclic agent and saline. ConclusionsRepeated administrations of the linear GBCAs gadodiamide, gadobenate dimeglumine, and gadopentetate dimeglumine to healthy rats were associated with progressive and significant T1 signal hyperintensity in the DCN, along with Gd deposition in the cerebellum. This is in contrast with the macrocyclic GBCA gadoterate meglumine for which no effect was observed.