Sébastien Boutry

Magnetic Resonance Imaging Tracking of Stem Cells in Vivo Using Iron Oxide Nanoparticles as a Tool for the Advancement of Clinical Regenerative Medicine

National Cell Bank, Pasteur Institute of Iran, Tehran 1316943551, Iran, Cardiovascular Research Center, Mount Sinai School of Medicine, New York, New York 10029, United States, Department of General, Organic, and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau, 19, B-7000 Mons, Belgium, Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran 14588, Iran, Department of Biomedical Engineering, National Yang Ming University, No 155, Sec. 2, LiNong Street, Taipei 112, Taiwan, Nanotechnology Toxicology Consulting & Training, Inc., Nova Scotia, Canada, Faculty of Medicine, Dalhousie Medical School, Dalhousie University, Halifax, Nova Scotia, Canada, and Institute for Nanoscale Science and Technology (INSAT), University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7ER, U.K., Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

MRI detection of early endothelial activation in brain inflammation.

MRI is an increasingly important clinical tool, but it is clear that conventional imaging fails to identify the full extent of lesion load in certain conditions, such as multiple sclerosis. The aim of this study was to determine whether a novel contrast agent (Gd‐DTPA‐B(sLeX)A, which contains an sLeX mimetic moiety that enables it to bind to the adhesion molecule E‐selectin) can be used to identify endothelial activation in the brain. Microinjection of the proinflammatory cytokines IL‐1β or TNF‐α into the striatum of Wistar rats rapidly induces focal adhesion molecule expression on the endothelium in the absence of MRI‐visible changes. This phenomenon was used to investigate the potential of Gd‐DTPA‐B(sLeX)A to reveal MRI‐invisible brain pathology. T1‐weighted serial images were acquired in anesthetized animals before and after administration of Gd‐DTPA‐B(sLeX)A, 3–4 hr after cytokine was injected intracerebrally. Both TNF‐α and IL‐1β up‐regulated E‐selectin on the brain endothelium, which correlated with increased signal intensity observed after administration of the novel contrast agent. No enhancement was visible with the nonselective contrast agent Gd‐DTPA‐BMA, indicating that there was no leakage of the agent across the blood–brain barrier (BBB) or nonselective binding to the endothelium. These data demonstrate the potential of such contrast agents for the early detection of brain injury and inflammation. Magn Reson Med 51:248–252, 2004.

MR molecular imaging of early endothelial activation in focal ischemia.

Focal ischemia followed by reperfusion initiates a harmful P‐ and E‐selectin–mediated recruitment of leukocytes in brain microvasculature. In this study, we tested whether a novel magnetic resonance (MR) contrast agent (Gd‐DTPA‐sLex A), which is designed to bind to activated endothelium could be detected by MR imaging (MRI) in a focal stroke mouse model. MRIs (9.4T) of the brain were acquired 24 hours after transient middle cerebral artery occlusion. T1 maps were acquired repeatedly before and up to 1.5 hours after the intravenous injection of either Gd‐DTPA or Gd‐DTPA‐sLex A. Analysis of images included a pixel‐by‐pixel subtraction of T1 maps from the precontrast T1 maps and quantification of T1 within the ischemic area. After injection of Gd‐DTPA‐sLex A, T1 decreased compared with precontrast levels, and an interhemispheric difference between the pre–post contrast T1 developed within the stroke lesion at a mean time of 52 minutes after injection (p < 0.05). Animals injected with Gd‐DTPA did not exhibit changes in T1 signal intensity between regions of the ipsilateral and contralateral hemispheres, indicating that the reductions in T1 observed with Gd‐DTPA‐sLex A were unrelated to blood–brain barrier breakdown. Fluorescent‐labeled sLex A administered intravenously was observed to bind to the endothelium of injured but not control brain. The study suggests that the contrast agent Gd‐DTPA‐sLex A can be used to visualize early endothelial activation after transient focal ischemia in vivo with MRI. Ann Neurol 2004;56:116–120

Iron Oxide Based MR Contrast Agents: from Chemistry to Cell Labeling

Superparamagnetic iron oxide nanoparticles can be used for numerous applications such as MRI contrast enhancement, hyperthermia, detoxification of biological fluids, drug delivery, or cell separation. In this work, we will summarize the chemical routes for synthesis of iron oxide nanoparticles, the fluid stabilization, and the surface modification of superparamagnetic iron oxide nanoparticles. Some examples of the numerous applications of these particles in the biomedical field mainly as MRI negative contrast agents for tissue-specific imaging, cellular labeling, and molecular imaging will be given. Larger particles or particles displaying a non-neutral surface (thanks to their coating or to a cell transfection agent with which they are mixed) are very useful tools, although the cells to be labeled have no professional phagocytic function. Labeled cells can then be transplanted and monitored by MRI in a broad spectrum of applications. Direct in vivo magnetic labeling of cells is mainly performed by intravenous injection of long-circulating iron oxide-based MRI contrast agents, which can extravasate and/or undergo a cellular uptake in an amount sufficient to allow an MRI visualization of areas of interest such as inflamed regions or tumors. Particles with long circulation times, or able to induce a strong negative effect individually have been also modified by conjugation to a ligand, so that their cellular uptake, or at least their binding to the cell surface, could occur through a specific ligand-receptor interaction, in vivo as well as in vitro. Thus, experimentally as well as in a few trials on humans, iron oxide particles currently find promising applications.

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke

BackgroundThe link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa).ResultsMRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice, Δ T1 stroke -Δ T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLeX) group compared to Gd-DTPA, indicative of sLeX mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice.ConclusionP-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

Magnetic resonance imaging of inflammation with a specific selectin-targeted contrast agent.

E‐selectin‐targeted contrast enhancement of blood vessels in inflamed tissues was investigated with a new contrast agent, Gd‐DTPA‐B(sLex)A, which was recently obtained by grafting a synthetic mimetic of sialyl‐Lewisx, an E‐selectin ligand, onto Gd‐DTPA. The pharmacokinetics, biodistribution, and potential to image inflammation by MRI of this E‐selectin‐targeted contrast agent were evaluated. The inhibition (by 15–34%) produced by Gd‐DTPA‐B(sLex)A on Sialyl Lex‐PAA‐biotin binding to E‐selectin confirmed the specific interaction of the new contrast agent with this adhesion molecule. Gd‐DTPA‐B(sLex)A was tested at a dose of 0.1 mmol/kg b.w. on mice and rats in a fulminant hepatitis model induced by the co‐administration of D‐galactosamine and E. coli lipopolysaccharide. A significant and prolonged contrast enhancement between blood vessels and liver parenchyma was obtained in pathological conditions, which attests to the specificity of the agent for E‐selectin. The prolonged vascular residence (48.9 min in hepatitis vs. 29.8 min in healthy animals), as evidenced by the pharmacokinetic characterization, suggests that Gd‐DTPA‐B(sLex)A interacts with the specific receptors expressed during inflammation. The biodistribution of the compound indicates its retention in inflamed liver by both specific mechanisms and nonspecific accumulation due to the necrotic lesions. The same mechanisms are invoked to account for its retention in the spleen. Magn Reson Med 53:800–807, 2005.

Magnetic labeling of non-phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study.

Small particles of iron oxide (SPIO) and ultrasmall particles of iron oxide (USPIO), inducing a strong negative contrast on T(2) and T(2)*-weighted MR images, are the most commonly used systems for the magnetic labeling of cultured cells and their subsequent detection by magnetic resonance imaging (MRI). The purpose of this work is to study the influence of iron incubation concentration, nanoparticle size and nanoparticle coating on the magnetic labeling and the viability of non-phagocytic adherent cells in culture. The magnetic labeling of 3T6 fibroblasts was studied by T(2)-weighted MRI at 4.7 T and by dosing-or cytochemical revealing-of iron through methods based on Perls Prussian blue staining. Cells were incubated for 48 h with increasing iron concentrations of SPIO (25-1000 microg Fe/ml Endorem. Sinerem, a USPIO (20-40 nm) coated with neutral dextran, and Resovist (65 nm), a SPIO bearing an anionic carboxydextran coating, were compared with Endorem (dextran-coated, 80-150 nm) as magnetic tags. The iron loading of marrow stromal cell primary cultures (MSCs) isolated from rat femurs was compared with that of 3T6 fibroblasts. The SPIO-labeling of cells with Endorem was found to be dependent on the iron incubation concentration. MSCs, more sparsely distributed in the culture, exhibited higher iron contents than more densely populated 3T6 fibroblast cultures. A larger iron loading was achieved with Resovist than with Endorem, which in turn was more efficient than Sinerem as a magnetic tag. The magnetic labeling of cultured non-phagocytic adherent cells with iron oxide nanoparticles was thus found to be dependent on the relative concentration of the magnetic tag and of the cells in culture, on the nanoparticle size, and on the coating type. The viability of cells, estimated by methods assessing cell membrane permeability, was not affected by magnetic labeling in the conditions used in this work.

Synthesis and in vitro evaluation of MR molecular imaging probes using J591 mAb-conjugated SPIONs for specific detection of prostate cancer.

Carcinoma of the prostate is the most frequent diagnosed malignant tumor in men and is the second leading cause of cancer-related death in this group. The cure rate of prostate cancer is highly dependent on the stage of disease at the diagnosis and early detection is key to designing effective treatment strategies. The objective of the present study is to make a specific MR imaging probe for targeted imaging of cancer cells. We take advantage of the fact that many types of prostate cancer cells express high levels of prostate-specific membrane antigen (PSMA) on their cell surface. The imaging strategy is to use superparamagnetic iron oxide nanoparticles (SPIONs), attached to an antibody (J591) that binds to the extracellular domain of PSMA, to specifically enhance the contrast of PSMA-expressing prostate cancer cells. Conjugation of mAb J591 to commercial SPIONs was achieved using a heterobifunctional linker, sulfo-SMCC. Two types of prostate cancer cell lines were chosen for experiments: LNCaP (PSMA+) and DU145 (PSMA-). MRI and cell uptake experiments demonstrated the high potential of the synthesized nanoprobe as a specific MRI contrast agent for detection of PSMA-expressing prostate cancer cells.

Carboxy-silane coated iron oxide nanoparticles: a convenient platform for cellular and small animal imaging

This study reports the synthesis of stabilized ultrasmall iron oxide nanoparticles (USPIO) as bimodal probes for magnetic resonance and optical imaging. These nanosystems are based on small iron oxide cores surrounded by a thin polysiloxane shell exhibiting carboxylic acid functions. Thanks to these functions, hybrid particles were obtained by conjugating a fluorophore to the superparamagnetic contrastophore. Such modification allowed us to directly follow these USPIO in cellulo, which provided interesting information about their internalization pathway and cellular distribution upon mitosis. Finally, the efficiency of these systems as probes for bimodal imaging was emphasized by the observation of their in vivo behavior in mice using magnetic resonance and optical imaging.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.