Very Small Superparamagnetic Iron Oxide Particles Research Articles

Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI.

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.

Contribution of Tissue Inflammation and Blood-Brain Barrier Disruption to Brain Softening in a Mouse Model of Multiple Sclerosis.

Neuroinflammatory processes occurring during multiple sclerosis cause disseminated softening of brain tissue, as quantified by in vivo magnetic resonance elastography (MRE). However, inflammation-mediated tissue alterations underlying the mechanical integrity of the brain remain unclear. We previously showed that blood-brain barrier (BBB) disruption visualized by MRI using gadolinium-based contrast agent (GBCA) does not correlate with tissue softening in active experimental autoimmune encephalomyelitis (EAE). However, it is unknown how confined BBB changes and other inflammatory processes may determine local elasticity changes. Therefore, we aim to elucidate which inflammatory hallmarks are determinant for local viscoelastic changes observed in EAE brains. Hence, novel multifrequency MRE was applied in combination with GBCA-based MRI or very small superparamagnetic iron oxide particles (VSOPs) in female SJL mice with induced adoptive transfer EAE (n = 21). VSOPs were doped with europium (Eu-VSOPs) to facilitate the post-mortem analysis. Accumulation of Eu-VSOPs, which was previously demonstrated to be sensitive to immune cell infiltration and ECM remodeling, was also found to be independent of GBCA enhancement. Following registration to a reference brain atlas, viscoelastic properties of the whole brain and areas visualized by either Gd or VSOP were quantified. MRE revealed marked disseminated softening across the whole brain in mice with established EAE (baseline: 3.1 ± 0.1 m/s vs. EAE: 2.9 ± 0.2 m/s, p < 0.0001). A similar degree of softening was observed in sites of GBCA enhancement i.e., mainly within cerebral cortex and brain stem (baseline: 3.3 ± 0.4 m/s vs. EAE: 3.0 ± 0.5 m/s, p = 0.018). However, locations in which only Eu-VSOP accumulated, mainly in fiber tracts (baseline: 3.0 ± 0.4 m/s vs. EAE: 2.6 ± 0.5 m/s, p = 0.023), softening was more pronounced when compared to non-hypointense areas (percent change of stiffness for Eu-VSOP accumulation: −16.81 ± 16.49% vs. for non-hypointense regions: −5.85 ± 3.81%, p = 0.048). Our findings suggest that multifrequency MRE is sensitive to differentiate between local inflammatory processes with a strong immune cell infiltrate that lead to VSOP accumulation, from disseminated inflammation and BBB leakage visualized by GBCA. These pathological events visualized by Eu-VSOP MRI and MRE may include gliosis, macrophage infiltration, alterations of endothelial matrix components, and/or extracellular matrix remodeling. MRE may therefore represent a promising imaging tool for non-invasive clinical assessment of different pathological aspects of neuroinflammation.

Superparamagnetic Iron Oxide Particles (VSOPs) Show Genotoxic Effects but No Functional Impact on Human Adipose Tissue-Derived Stromal Cells (ASCs).

Adipose tissue-derived stromal cells (ASCs) represent a capable source for cell-based therapeutic approaches. For monitoring a cell-based application in vivo, magnetic resonance imaging (MRI) of cells labeled with iron oxide particles is a common method. It is the aim of the present study to analyze potential DNA damage, cytotoxicity and impairment of functional properties of human (h)ASCs after labeling with citrate-coated very small superparamagnetic iron oxide particles (VSOPs). Cytotoxic as well as genotoxic effects of the labeling procedure were measured in labeled and unlabeled hASCs using the MTT assay, comet assay and chromosomal aberration test. Trilineage differentiation was performed to evaluate an impairment of the differentiation potential due to the particles. Proliferation as well as migration capability were analyzed after the labeling procedure. Furthermore, the labeling of the hASCs was confirmed by Prussian blue staining, transmission electron microscopy (TEM) and high-resolution MRI. Below the concentration of 0.6 mM, which was used for the procedure, no evidence of genotoxic effects was found. At 0.6 mM, 1 mM as well as 1.5 mM, an increase in the number of chromosomal aberrations was determined. Cytotoxic effects were not observed at any concentration. Proliferation, migration capability and differentiation potential were also not affected by the procedure. Labeling with VSOPs is a useful labeling method for hASCs that does not affect their proliferation, migration and differentiation potential. Despite the absence of cytotoxicity, however, indications of genotoxic effects have been demonstrated.

Application of Europium-Doped Very Small Iron Oxide Nanoparticles to Visualize Neuroinflammation with MRI and Fluorescence Microscopy

Our recent studies demonstrated that electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) are promising MRI probes for detecting various pathological aspects of autoimmunity in the central nervous system (CNS). However, investigation of the precise tissue and cellular distribution of VSOP has been technically limited due to the need to use iron detection methods for VSOP visualization. Therefore, we assessed here the utility of europium (Eu)-doped VSOP as an MRI tool for in vivo investigations in the animal model experimental autoimmune encephalomyelitis (EAE), and as a tool to investigate histopathological processes in the CNS using fluorescence microscopy.We demonstrated that Eu-VSOP display the same properties as VSOP in terms of revealing inflammation-mediated changes by binding to brain endothelium in vitro, and in terms of visualizing brain lesions in EAE in vivo. MRI examinations with Eu-VSOP confirm that at peak disease particles accumulated inside the choroid plexus, and in cerebellar and meningeal lesions. Importantly, Eu-VSOP-based MRI showed for the first time in a longitudinal setup that particles were absent from the choroid plexus in mice during remission of EAE, but accumulated again during subsequent relapse. Within the choroid plexus, Eu-VSOP were associated both with monocytes/macrophages present in the plexus stroma, and associated with epithelial cells.Using Eu-VSOP, we demonstrated for the first time the involvement of the choroid plexus in relapses. Thus, Eu-VSOP have the potential to reveal various aspects of choroid plexus involvement in neuroinflammation, including monocyte recruitment from the blood and alterations of the choroid plexus epithelium.

The effect of adipose tissue-derived stem cells in a middle cerebral artery occlusion stroke model depends on their engraftment rate

BackgroundIn the field of experimental stem cell therapy, intra-arterial (IA) delivery yields the best results concerning, for example, migrated cell number at the targeted site. However, IA application also appears to be associated with increased mortality rates and infarction. Since many rodent studies systemically apply 1 × 106 cells, this could also be a consequence of engrafted cell number. The aim of this study was therefore to investigate the effect of different doses of adipose tissue-derived stem cells (ASCs) on engraftment rates and stroke outcome measured in vivo using 9.4-T high-field magnetic resonance imaging (MRI).MethodsMale Wistar rats (n = 43) underwent a middle cerebral artery occlusion (MCAo) for 45 or 90 min, followed by IA delivery of either saline or 1 × 106, 3 × 105, or 5 × 104 ASCs pre-labelled with very small superparamagnetic iron oxide particles (VSOPs). MRI (9.4-T) analysis was performed 48 h and 9 days post-MCAo. Lesion volumes were assessed by analysis of T2-weighted images and cell signal tracking showing cell engraftment and active cell migration by an improved T2*-analysis.ResultsThe ASC-derived signal intensity increased in the affected hemisphere 48 h post MCAo with injected cell number (p < 0.05). The analysis of stroke volumes revealed an increased infarction after injection of 1 × 106 ASCs compared to controls or application of 5 × 104 ASCs (p < 0.05). At 9 days post-MCAo, injection of 3 × 105 ASCs resulted in reduced infarct volumes (p < 0.05). Correspondingly, MRI analysis revealed no changes in cell numbers between both MRI examinations but showed active ASC migration to the site of infarction.ConclusionOur results confirm that IA injection is an efficient way of targeting damaged brain tissue but its usefulness strongly depends on the right dose of delivered stem cells since this factor has a strong influence on migration rate and infarct volume, with better results for doses below 1 × 106 cells. Future challenges will include the determination of therapeutic doses for best cellular engraftment and stroke outcome.

Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia.

Superparamagnetic iron oxide nanoparticles (SPIO) are applied as contrast media for magnetic resonance imaging (MRI) and treatment of neurologic diseases despite the fact that important information concerning their local interactions is still lacking. Due to their small size, SPIO have great potential for magnetically labeling different cell populations, facilitating their MRI tracking in vivo. Before SPIO are applied, however, their effect on cell viability and tissue homoeostasis should be studied thoroughly. We have previously published data showing how citrate-coated very small superparamagnetic iron oxide particles (VSOP) affect primary microglia and neuron cell cultures as well as neuron-glia cocultures. To extend our knowledge of VSOP interactions on the three-dimensional multicellular level, we further examined the influence of two types of coated VSOP (R1 and R2) on murine organotypic hippocampal slice cultures. Our data show that 1) VSOP can penetrate deep tissue layers, 2) long-term VSOP-R2 treatment alters cell viability within the dentate gyrus, 3) during short-term incubation VSOP-R1 and VSOP-R2 comparably modify hippocampal cell viability, 4) VSOP treatment does not affect cytokine homeostasis, 5) microglial depletion decreases VSOP uptake, and 6) microglial depletion plus VSOP treatment increases hippocampal cell death during short-term incubation. These results are in line with our previous findings in cell coculture experiments regarding microglial protection of neurite branching. Thus, we have not only clarified the interaction between VSOP, slice culture, and microglia to a degree but also demonstrated that our model is a promising approach for screening nanoparticles to exclude potential cytotoxic effects.

Visualization of acute focal lesions in rats with experimental autoimmune encephalomyelitis by magnetic nanoparticles, comparing different MRI sequences including phase imaging

To compare the sensitivity and specificity of phase imaging (PI) with other magnetic resonance imaging (MRI) methods in lesion detection in rats with experimental autoimmune encephalomyelitis (EAE), as an animal model for multiple sclerosis (MS). EAE was induced in rats (n = 14) by subcutaneous (s.c.) injection of myelin basic protein (MBP) and complete Freund's adjuvant (CFA). Control animals (n = 4) were given an s.c. injection of phosphate-buffered saline mixed with CFA. The development of local inflammatory processes, demyelinations, and blood-brain barrier (BBB) disruptions were monitored over 7 weeks in a 4.7T animal scanner by T1-, T2-, T2*-weighted images, magnetization transfer, and PI in the presence or absence of very small superparamagnetic iron oxide particles (VSOP) and confirmed by immunostaining using CD31, CD68, MBP, and albumin antibodies and Gallyas silver staining. EAE rats developed time-dependent local inflammations and BBB disruptions but no clear demyelinizations. In histological stainings these processes were trackable as accumulations of phagocytic monocytes and extravasal albumin. In MRI without application of VSOP inflammatory processes were not detectable. MRI in the presence of VSOP revealed inflammatory processes by the appearance of hypointense spots (hs). The specificity of PI to detect hs was similar to T1- and T2*-weighted images The calculated sensitivity was less than in corresponding T2*-weighted images. The diagnostic use of PI without VSOP as contrast agent to detect lesions is not recommended at field strength of 4.7T or lower.

Iron Oxide Magnetic Nanoparticles Highlight Early Involvement of the Choroid Plexus in Central Nervous System Inflammation

Neuroinflammation during multiple sclerosis involves immune cell infiltration and disruption of the BBB (blood–brain barrier). Both processes can be visualized by MRI (magnetic resonance imaging), in multiple sclerosis patients and in the animal model EAE (experimental autoimmune encephalomyelitis). We previously showed that VSOPs (very small superparamagnetic iron oxide particles) reveal CNS (central nervous system) lesions in EAE which are not detectable by conventional contrast agents in MRI. We hypothesized that VSOP may help detect early, subtle inflammatory events that would otherwise remain imperceptible. To investigate the capacity of VSOP to reveal early events in CNS inflammation, we induced EAE in SJL mice using encephalitogenic T-cells, and administered VSOP prior to onset of clinical symptoms. In parallel, we administered VSOP to mice at peak disease, and to unmanipulated controls. We examined the distribution of VSOP in the CNS by MRI and histology. Prior to disease onset, in asymptomatic mice, VSOP accumulated in the choroid plexus and in spinal cord meninges in the absence of overt inflammation. However, VSOP was undetectable in the CNS of non-immunized control mice. At peak disease, VSOP was broadly distributed; we observed particles in perivascular inflammatory lesions with apparently preserved glia limitans. Moreover, at peak disease, VSOP was prominent in the choroid plexus and was seen in elongated endothelial structures, co-localized with phagocytes, and diffusely disseminated in the parenchyma, suggesting multiple entry mechanisms of VSOP into the CNS. Thus, using VSOP we were able to discriminate between inflammatory events occurring in established EAE and, importantly, we identified CNS alterations that appear to precede immune cell infiltration and clinical onset.

Rapid binding of electrostatically stabilized iron oxide nanoparticles to THP-1 monocytic cells via interaction with glycosaminoglycans

Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions has the potential to emerge as promising diagnostic modality for detecting unstable plaques. Since a high content of macrophages and alterations of the extracellular matrix are hallmarks of plaque instability, these structures represent attractive targets for new imaging modalities. In this study, we compared in vitro uptake and binding of electrostatically stabilized citrate-coated very small superparamagnetic iron oxide particles (VSOP) to THP-1 cells with sterically stabilized carboxydextran-coated Resovist(®). Uptake of VSOP in both THP-1 monocytic cells and THP-derived macrophages (THP-MΦ) was more efficient compared to Resovist(®) without inducing cytotoxicity or modifying normal cellular functions (no changes in levels of reactive oxygen species, caspase-3 activity, proliferation, cytokine production). Importantly, VSOP bound with high affinity to the cell surface and to apoptotic membrane vesicles. Inhibition of glycosaminoglycan (GAG) synthesis by glucose deprivation in THP-MΦ was associated with a significant reduction of VSOP attachment suggesting that the strong interaction of VSOP with the membranes of cells and apoptotic vesicles occurs via binding to negatively charged GAGs. These in vitro experiments show that VSOP-enhanced MRI may represent a new imaging approach for visualizing high-risk plaques on the basis of targeting pathologically increased GAGs or apoptotic membrane vesicles in atherosclerotic lesions. VSOP should be investigated further in appropriate in vivo experiments to characterize accumulation in unstable plaque.

Contrast-enhanced MR imaging of atherosclerosis using citrate-coated superparamagnetic iron oxide nanoparticles: calcifying microvesicles as imaging target for plaque characterization

ObjectiveTo evaluate the suitability of citrate-coated very small superparamagnetic iron oxide particles (VSOP) as a contrast agent for identifying inflammation in atherosclerotic lesions using magnetic resonance imaging (MRI).Methods and resultsVSOP, which have already been evaluated as a blood pool contrast agent for MR angiography in human clinical trials, were investigated in Watanabe heritable hyper-lipidemic rabbits to determine to what extent their accumulation in atherosclerotic lesions is a function of macrophage density and other characteristics of progressive atherosclerotic plaques. In advanced atherosclerotic lesions, a significant MRI signal loss was found within 1 hour after intravenous administration of VSOP at the intended clinical dose of 0.05 mmol Fe/kg. Histological examinations confirmed correlations between the loss of MRI signal in the vessel wall and the presence of Prussian blue-stained iron colocalized with macrophages in the plaque cap, but surprisingly also with calcifying microvesicles at the intimomedial interface. Critical electrolyte magnesium chloride concentration in combination with Alcian blue stain indicates that highly sulfated glycosaminoglycans are a major constituent of these calcifying microvesicles, which may serve as the key molecules for binding VSOP due to their highly complexing properties.ConclusionCalcifying microvesicles and macrophages are the targets for intravenously injected VSOP in atherosclerotic plaques, suggesting that VSOP-enhanced MRI may render clinically relevant information on the composition and inflammatory activity of progressive atherosclerotic lesions at risk of destabilization.

Intrahippocampal transplantation of mesenchymal stromal cells promotes neuroplasticity

Multipotent mesenchymal stromal cells (MSC) secrete soluble factors that stimulate the surrounding microenvironment. Such paracrine effects might underlie the potential benefits of many stem cell therapies. We tested the hypothesis that MSC are able to enhance intrinsic cellular plasticity in the adult rat hippocampus. Rat bone marrow-derived MSC were labeled with very small superparamagnetic iron oxide particles (VSOP), which allowed for non-invasive graft localization by magnetic resonance imaging (MRI). Moreover, MSC were transduced with lentiviral vectors to express the green fluorescent protein (GFP). The effects of bilateral MSC transplantation on hippocampal cellular plasticity were assessed using the thymidine analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-iodo-2'-deoxyuridine (IdU). Behavioral testing was performed to examine the consequences of intrahippocampal MSC transplantation on locomotion, learning and memory, and anxiety-like and depression-like behavior. We found that intrahippocampal transplantation of MSC resulted in enhanced neurogenesis despite short-term graft survival. In contrast, systemic administration of the selective serotonin re-uptake inhibitor citalopram increased cell survival but did not affect cell proliferation. Intrahippocampal transplantation of MSC did not impair behavioral functions in rats, but only citalopram exerted anti-depressant effects. This is the first study to examine the effects of intrahippocampal transplantation of allogeneic MSC on hippocampal structural plasticity and behavioral functions in rats combined with non-invasive cell tracking by MRI. We found that iron oxide nanoparticles can be used to detect transplanted MSC in the brain. Although graft survival was short, intrahippocampal transplantation of MSC resulted in long-term changes in hippocampal plasticity. Our results suggest that MSC can be used to stimulate adult neurogenesis.

Cardiac Magnetic Resonance Angiography using Blood-Pool Contrast Agents: Comparison of Citrate-Coated Very Small Superparamagnetic Iron Oxide Particles with Gadofosveset Trisodium in Pigs

To compare citrate-coated very small superparamagnetic iron oxide particles (VSOP) with gadofosveset trisodium as blood pool contrast agents for cardiac magnetic resonance angiography (CMRA) in pigs. Animal experiments were approved by the responsible authority. 10 CMRA-like examinations were performed at 1.5 T after administration of VSOP (0.06 mmol Fe/kg; 5 examinations) and gadofosveset trisodium (0.03 mmol Gd/kg; 5 examinations). The CMRA protocol included ECG-gated inversion-recovery-prepared T1-weighted gradient echo imaging (IR-GRE; one slice) and ECG-gated inversion recovery prepared steady state free precession imaging (IR SSFP; one slice) before and 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, and 60 min after injection. At each time point, three different inversion times (TI; 200 msec, 300 msec, and 400 msec) were applied. Contrast-to-noise ratios (CNR) between blood and myocardium were calculated and compared using mixed linear models. No significant differences of CNR were found between IR-GRE and IR SSFP. At 3 and 5 min after contrast agent administration, VSOP showed a significantly higher CNR than gadofosveset trisodium when TI of 200 msec and 300 msec were applied (TI of 200 msec at 3 min: 8.2 ± 0.7 vs. 5.4 ± 0.7; TI of 200 msec at 5 min: 7.9 ± 0.7 vs. 3.5 ± 0.8; TI of 300 msec at 3 min: 11.7 ± 0.7 vs. 8.8 ± 0.8; TI of 300 msec at 5 min: 11.4 ± 0.7 vs. 8.0 ± 0.8; p < 0.05). Moreover, significant differences in favor of VSOP were found for all time points from 10 to 40 min irrespective of TI (p < 0.05). VSOP has superior blood-pool properties compared to gadofosveset trisodium resulting in prolonged improvement of CNR on CMRA.

Electrostatically stabilized magnetic nanoparticles – an optimized protocol to label murine T cells for in vivo MRI

We present a novel highly efficient protocol to magnetically label T cells applying electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP). Our long-term aim is to use magnetic resonance imaging (MRI) to investigate T cell dynamics in vivo during the course of neuroinflammatory disorders such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Encephalitogenic T cells were co-incubated with VSOP, or with protamine-complexed VSOP (VProt), respectively, at different conditions, optimizing concentrations and incubation times. Labeling efficacy was determined by atomic absorption spectrometry as well as histologically, and evaluated on a 7 T MR system. Furthermore, we investigated possible alterations of T cell physiology caused by the labeling procedure. T cell co-incubation with VSOP resulted in an efficient cellular iron uptake. T2 times of labeled cells dropped significantly, resulting in prominent hypointensity on T2*-weighted scans. Optimal labeling efficacy was achieved by VProt (1 mM Fe/ml, 8 h incubation; T2 time shortening of ∼80% compared to untreated cells). Although VSOP promoted T cell proliferation and altered the ratio of T cell subpopulations toward a CD4+ phenotype, no effects on CD4 T cell proliferation or phenotypic stability were observed by labeling in vitro differentiated Th17 cells with VProt. Yet, high concentrations of intracellular iron oxide might induce alterations in T cell function, which should be considered in cell tagging studies. Moreover, we demonstrated that labeling of encephalitogenic T cells did not affect pathogenicity; labeled T cells were still capable of inducing EAE in susceptible recipient mice.

Beyond blood brain barrier breakdown – in vivodetection of occult neuroinflammatory foci by magnetic nanoparticles in high field MRI

BackgroundGadopentate dimeglumine (Gd-DTPA) enhanced magnetic resonance imaging (MRI) is widely applied for the visualization of blood brain barrier (BBB) breakdown in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the potential of magnetic nanoparticles to detect macrophage infiltration by MRI was demonstrated. We here investigated a new class of very small superparamagnetic iron oxide particles (VSOP) as novel contrast medium in murine adoptive-transfer EAE.MethodsEAE was induced in 17 mice via transfer of proteolipid protein specific T cells. MR images were obtained before and after application of Gd-DTPA and VSOP on a 7 Tesla rodent MR scanner. The enhancement pattern of the two contrast agents was compared, and correlated to histology, including Prussian Blue staining for VSOP detection and immunofluorescent staining against IBA-1 to identify macrophages/microglia.ResultsBoth contrast media depicted BBB breakdown in 42 lesions, although differing in plaques appearances and shapes. Furthermore, 13 lesions could be exclusively visualized by VSOP. In the subsequent histological analysis, VSOP was localized to microglia/macrophages, and also diffusely dispersed within the extracellular matrix.ConclusionVSOP showed a higher sensitivity in detecting BBB alterations compared to Gd-DTPA enhanced MRI, providing complementary information of macrophage/microglia activity in inflammatory plaques that has not been visualized by conventional means.

Labeling of human neural precursor cells using ferromagnetic nanoparticles

Fetal human neural precursor cells (NPCs) are unique with respect to their capacity to proliferate and to preserve their potential to differentiate into neurons and glia. Human mesencephalic neural precursor cells (hmNPCs) provide a source for dopaminergic neurons. Preclinical and clinical research will benefit from reliable in vivo tracking of transplanted cells. Here, we investigate the potency of very small superparamagnetic iron oxide particles (VSOPs) to label hmNPCs, the effect of VSOPs on survival, proliferation, and differentiation of hmNPCs, and the sensitivity of 1.5T magnetic resonance imaging (MRI) to detect labeled cells in living rats following transplantation. When incubated with VSOPs at 1.5 mM, >95% of hmNPCs incorporated VSOPs without detectable impact on cell viability (>90%) or proliferative capacity, as measured by the expression of proliferating cell nuclear antigen (PCNA) and cell cycle distribution. Labeled hmNPCs differentiate into neurons (>30%) and glia with no detectable difference compared to nonlabeled cells. Following transplantation into rat striata, marked paramagnetic signal changes were detected for as long as three months postsurgery using MRI, corresponding to the histologically-identified graft. Our data indicate that hmNPCs can be labeled with VSOPs without impairment of viability, proliferation, or multipotency. Labeled, transplanted cells are detectable in vivo using 1.5T MRI.

Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair

For the development of new therapeutical cell-based strategies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. We present a systematic and detailed study on the performance and biological impact of a simple and efficient labelling protocol for human mesenchymal stem cells (hMSCs). Commercially available very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake via endocytosis was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabelled cells, VSOP-labelling did neither influence the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect hMSCs in undergoing adipogenic, osteogenic or chondrogenic differentiation, as demonstrated histologically and by gene expression analyses. The efficiency of the labelling protocol was assessed with high-resolution MR imaging at 11.7 T. VSOP-labelled hMSCs were visualised in a collagen type I hydrogel, which is in clinical use for matrix-based articular cartilage repair. The presence of VSOP-labelled hMSCs was indicated by distinct hypointense spots in the MR images, as a result of iron specific loss of signal intensity. In summary, this labelling technique has great potential to visualise hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.

Tracking of systemically administered mononuclear cells in the ischemic brain by high-field magnetic resonance imaging

This study was designed to track systemically administered mononuclear cells (MNCs) in the ischemic mouse brain using 7 T magnetic resonance imaging (MRI). Splenectomized wild-type mice were subjected to brain ischemia by 30 or 60 min filamentous occlusion of the middle cerebral artery (MCAo) and reperfusion. Spleen-derived MNCs were labeled with very small superparamagnetic iron-oxide particles (VSOP) and transfused into recipient mice 30 min, 8 h, or 24 h after MCAo via the tail vein. High-resolution MRI sequences were designed to monitor the dynamics of brain ischemia and to observe the migration and engraftment of transfused cells into the ischemic brain. T2*-weighted (gradient-echo) hypointense signal changes became apparent at 24–48 h after transfusion, were typically associated with the ischemic lesion border, and could be followed up to 5 weeks after the insult. Such presumed MNC-associated signal changes in MRI were confirmed by histochemical detection of iron (Prussian blue staining) and detection of constitutively expressed green fluorescent protein (GFP) in a subset of animals transfused with MNCs derived from GFP transgenic mice. Taken together, our results demonstrate that brain engraftment of systemically administered mononuclear cells can be visualized non-invasively over time and space using high-resolution MRI.

Iron oxide particles for molecular magnetic resonance imaging cause transient oxidative stress in rat macrophages

Iron oxide particles are a promising marker in molecular magnetic resonance imaging. They are used to label distinct cell populations either in vitro or in vivo. We investigated for the first time whether small citrate-coated very small superparamagnetic iron oxide particles (VSOPs) can lead to an increase in cellular oxidative stress. We incubated rat macrophages (RAW) in vitro with iron oxide particles. We observed a massive uptake of VSOPs measured both with atomic absorption spectroscopy and with NMR, which could be visualized by confocal laser scanning microscopy. After incubation, cells were lysed and the levels of malonyldialdehyde (MDA) and protein carbonyls were determined. We found a significant increase in both MDA and protein carbonyl levels after incubation with the particles. Surprisingly, 24 h after incubation, a significant indication of oxidative stress could no longer be observed. The increase in oxidative stress seems to be transient and closely linked to the incubation procedure. The iron chelator desferal and the intracellular spin trap PBN caused a significant reduction in oxidative stress to almost control levels. This indicates that the augmentation of oxidative stress is closely linked to the free iron during incubation. Proliferation assays showed that incorporation of VSOPs did not lead to long-term cytotoxic effects even though the iron oxide particles remained in the cell. Magnetic labeling of cells with VSOPs seems to cause transient oxidative conditions not affecting cellular viability and seems to be a usable approach for molecular magnetic resonance imaging.