Calcium-dependent molecular fMRI using a magnetic nanosensor.

Calcium ions are ubiquitous signalling molecules in all multicellular organisms, where they mediate diverse aspects of intracellular and extracellular communication over widely varying temporal and spatial scales 1 . Though techniques to map calcium-related activity at a high resolution by optical means are well established, there is currently no reliable method to measure calcium dynamics over large volumes in intact tissue 2 . Here, we address this need by introducing a family of magnetic calcium-responsive nanoparticles (MaCaReNas) that can be detected by magnetic resonance imaging (MRI). MaCaReNas respond within seconds to [Ca2+] changes in the 0.1-1.0 mM range, suitable for monitoring extracellular calcium signalling processes in the brain. We show that the probes permit the repeated detection of brain activation in response to diverse stimuli in vivo. MaCaReNas thus provide a tool for calcium-activity mapping in deep tissue and offer a precedent for the development of further nanoparticle-based sensors for dynamic molecular imaging with MRI.

Myeloperoxidase Nuclear Imaging for Epileptogenesis.

PURPOSE: To determine if myeloperoxidase (MPO) is involved in epileptogenesis and if molecular nuclear imaging can be used to noninvasively map inflammatory changes in epileptogenesis. MATERIALS AND METHODS: The animal and human studies were approved by the institutional review boards. Pilocarpine-induced epileptic mice were treated with 4-aminobenzoic acid hydrazide (n = 46), a specific irreversible MPO inhibitor, or saline (n = 42). Indium-111-bis-5-hydroxytryptamide-diethylenetriaminepentaacetate was used to image brain MPO activity (n = 6 in the 4-aminobenzoic acid hydrazide and saline groups; n = 5 in the sham group) by using single photon emission computed tomography/computed tomography. The role of MPO in the development of spontaneous recurrent seizures was assessed by means of clinical symptoms and biochemical and histopathologic data. Human brain specimens from a patient with epilepsy and a patient without epilepsy were stained for MPO. The Student t test, one-way analysis of variance, and Mann-Whitney and Kruskal-Wallis tests were used. Differences were regarded as significant if P was less than .05. RESULTS: MPO and leukocytes increased in the brain during epileptogenesis (P < .05). Blocking MPO delayed spontaneous recurrent seizures (99.6 vs 142 hours, P = .016), ameliorated the severity of spontaneous recurrent seizures (P < .05), and inhibited mossy fiber sprouting (Timm index, 0.31 vs 0.03; P = .003). Matrix metalloproteinase activity was upregulated during epileptogenesis in an MPO-dependent manner (1.44 vs 0.94 U/mg, P = .049), suggesting that MPO acts upstream of matrix metalloproteinases. MPO activity was mapped during epileptogenesis in vivo in the hippocampal regions. Resected temporal lobe tissue from a human patient with refractory epilepsy but not the temporal lobe tissue from a patient without seizures demonstrated positive MPO immunostaining, suggesting high translational potential for this imaging technology. CONCLUSION: The findings of this study highlight an important role for MPO in epileptogenesis and show MPO to be a potential therapeutic target and imaging biomarker for epilepsy.

Magnetic nanosensors optimized for rapid and reversible self-assembly.

Magnetic nanoparticle-based sensors for MRI have been accelerated to a timescale of seconds using densely-functionalized particles of small size. Parameters that increase response rates also result in large nuclear magnetic relaxation rate and light scattering changes, allowing signals to be detected almost immediately after changes in calcium concentration.

In vivo evaluation of amyloid deposition and brain glucose metabolism of 5XFAD mice using positron emission tomography.

Positron emission tomography (PET) has been used extensively to evaluate the neuropathology of Alzheimer's disease (AD) in vivo. Radiotracers directed toward the amyloid deposition such as [(18)F]-FDDNP (2-(1-{6-[(2-[F]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) and [(11)C]-PIB (Pittsburg compound B) have shown exceptional value in animal models and AD patients. Previously, the glucose analogue [(18)F]-FDG (2-[(18)F]fluorodeoxyglucose) allowed researchers and clinicians to evaluate the brain glucose consumption and proved its utility for the early diagnosis and the monitoring of the progression of AD. Animal models of AD are based on the transgenic expression of different human mutant genes linked to familial AD. The novel transgenic 5XFAD mouse containing 5 mutated genes in its genome has been proposed as an AD model with rapid and massive cerebral amyloid deposition. PET studies performed with animal-dedicated scanners indicate that PET with amyloid-targeted radiotracers can detect the pathological amyloid deposition in transgenic mice and rats. However, in other studies no differences were found between transgenic mice and their wild type littermates. We sought to investigate in 5XFAD mice if the radiotracers [(11)C]-PIB, and [(18)F]-Florbetapir could quantify the amyloid deposition in vivo and if [(18)F]-FDG could do so with regard to glucose consumption. We found that 5XFAD animals presented higher cerebral binding of [(18)F]-Florbetapir, [(11)C]-PIB, and [(18)F]-FDG. These results support the use of amyloid PET radiotracers for the evaluation of AD animal models. Probably, the increased uptake observed with [(18)F]-FDG is a consequence of glial activation that occurs in 5XFAD mice.

Demyelinating diseases: myeloperoxidase as an imaging biomarker and therapeutic target.

PURPOSE: To evaluate myeloperoxidase (MPO) as a newer therapeutic target and bis-5-hydroxytryptamide-diethylenetriaminepentaacetate-gadolinium (Gd) (MPO-Gd) as an imaging biomarker for demyelinating diseases such as multiple sclerosis (MS) by using experimental autoimmune encephalomyelitis (EAE), a murine model of MS. MATERIALS AND METHODS: Animal experiments were approved by the institutional animal care committee. EAE was induced in SJL mice by using proteolipid protein (PLP), and mice were treated with either 4-aminobenzoic acid hydrazide (ABAH), 40 mg/kg injected intraperitoneally, an irreversible inhibitor of MPO, or saline as control, and followed up to day 40 after induction. In another group of SJL mice, induction was performed without PLP as shams. The mice were imaged by using MPO-Gd to track changes in MPO activity noninvasively. Imaging results were corroborated by enzymatic assays, flow cytometry, and histopathologic analyses. Significance was computed by using the t test or Mann-Whitney U test. RESULTS: There was a 2.5-fold increase in myeloid cell infiltration in the brain (P = .026), with a concomitant increase in brain MPO level (P = .0087). Inhibiting MPO activity with ABAH resulted in decrease in MPO-Gd-positive lesion volume (P = .012), number (P = .009), and enhancement intensity (P = .03) at MR imaging, reflecting lower local MPO activity (P = .03), compared with controls. MPO inhibition was accompanied by decreased demyelination (P = .01) and lower inflammatory cell recruitment in the brain (P < .0001), suggesting a central MPO role in inflammatory demyelination. Clinically, MPO inhibition significantly reduced the severity of clinical symptoms (P = .0001) and improved survival (P = .0051) in mice with EAE. CONCLUSION: MPO may be a key mediator of myeloid inflammation and tissue damage in EAE. Therefore, MPO could represent a promising therapeutic target, as well as an imaging biomarker, for demyelinating diseases and potentially for other diseases in which MPO is implicated.

Vasculitis: molecular imaging by targeting the inflammatory enzyme myeloperoxidase.

PURPOSE: To determine if a molecular imaging approach targeting the highly oxidative enzyme myeloperoxidase (MPO) can help noninvasively identify and confirm sites of vascular wall inflammation in a murine model of vasculitis. MATERIALS AND METHODS: Animal experiments were approved by the institutional animal care committee. Twenty-six mice were studied, including eight MPO-deficient and six sham-operated mice as controls. Vasculitis was induced with intraperitoneal injection of Candida albicans water-soluble fraction (CAWS). Aortic root magnetic resonance imaging was performed after intravenous injection of the activatable MPO sensor (bis-5-hydroxytryptamide-diethylenetriaminepentatacetate gadolinium) (n = 23), referred to as MPO-Gd, or gadopentetate dimeglumine (n = 10). Seven mice were randomly assigned to receive either MPO-Gd or gadopentetate dimeglumine first. Aortic root specimens were collected for biochemical and histopathologic analyses to validate imaging findings. Statistical significance was calculated for contrast-to-noise ratios (CNRs) by using the paired t test. RESULTS: In the aortic root, the mean MPO-Gd CNRs after agent injection (CNR = 28.1) were more than 2.5-fold higher than those of sham-operated mice imaged with MPO-Gd and vasculitis mice imaged with gadopentetate dimeglumine (CNR = 10.6) (P < .05). MPO-Gd MR imaging helped identify areas of vasculitis that were not seen at unenhanced and contrast material-enhanced imaging with gadopentetate dimeglumine. Histopathologic and biochemical analyses for MPO and myeloid cells confirmed imaging findings. In MPO-deficient mice, injection of CAWS did not result in a vasculitis phenotype, implying a key role of the imaging target in disease cause. CONCLUSION: Molecular imaging targeting MPO can be a useful biomarker to noninvasively detect and confirm inflammation in vasculitis by using a murine model of Kawasaki disease.

Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice.

Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte-associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.

Activatable magnetic resonance imaging agents for myeloperoxidase sensing: mechanism of activation, stability, and toxicity.

Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many inflammatory diseases, particularly cardiovascular and neurological diseases. MPO-specific imaging agents would thus be highly useful to diagnose early disease, monitor disease progression, and quantify treatment effects. This study reports in vitro and in vivo characterizations of the mechanism of interaction between MPO and paramagnetic enzyme substrates based on physical and biological measurements. We show that these agents are activated through a radical mechanism, which can combine to form oligomers and, in the presence of tyrosine-containing peptide, bind to proteins. We further identified two new imaging agents, which represent the near extremes in either oligomerization (mono-5HT-DTPA-Gd) or protein-binding in their activation mechanism (bis-o-dianisidine-DTPA-Gd). On the other hand, we found that the agent bis-5HT-DTPA-Gd utilizes both mechanisms when activated. These properties yield distinct in vivo pharmacokinetics profiles for each of these agents that may be exploited for different applications. Specificity studies show that only MPO, but not eosinophil peroxidase, can highly activate these agents, and that MPO activity as low as 0.005 U/mg of tissue can be detected. Gd kinetic lability and cytotoxicity studies further confirm stability of the Gd ion and low toxicity for the 5HT-based agents, suggesting that these agents are suitable for translational in vivo studies.

Self-assembled multifunctional Fe/MgO nanospheres for magnetic resonance imaging and hyperthermia.

A one-step process for the production of nanoparticles presenting advanced magnetic properties can be achieved using vapor condensation. In this article, we report on the fabrication of Fe particles covered by a uniform MgO epitaxial shell. MgO has a lower surface energy than Fe, which results in a core-shell crystal formation. The particles satisfy a few of technical requirements for the practical use in real clinics, such as a high biocompatibility in living cells in-vitro, an injection through blood vessels without any clothing problems in murine model, a high absorption rate for magnetic hyperthermia at small particle concentration, and the potential to be used as contrast agent in the field of diagnostic magnetic imaging. They are also able to be used in drug delivery and magnetic-activated cell sorting. FROM THE CLINICAL EDITOR: In this paper, the authors report on the synthesis of Fe particles covered by a uniform MgO epitaxial shell resulting in a core-shell crystal formation. The particles are proven to be useful as contrast agents for magnetic resonance imaging and have the potential to be useful as heating mediators for cancer therapy through hyperthermia. They also might be used in drug delivery and magnetic-activated cell sorting.

Enzyme-sensitive magnetic resonance imaging targeting myeloperoxidase identifies active inflammation in experimental rabbit atherosclerotic plaques.

BACKGROUND: Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. METHODS AND RESULTS: We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. CONCLUSIONS: The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase.

Inflammation can extend ischemic brain injury and adversely affect outcome in experimental animal models. A key difficulty in translating animal studies to humans is the lack of a definitive method to confirm and track inflammation in the brain in vivo. Myeloperoxidase (MPO), a key inflammatory enzyme secreted by activated neutrophils and macrophages/microglia, can generate highly reactive oxygen species to cause additional damage in cerebral ischemia. We report here that a functional, enzyme-activatable MRI agent can accurately track the oxidative activity of MPO noninvasively in stroke in living animals. We found that MPO is widely distributed in ischemic tissues, correlates positively with infarct size, and is detected even 3 weeks postinfarction. The peak level of MPO activity, determined by activation of the MPO-sensing agent in vivo and confirmed by MPO activity and quantitative RT-PCR assays, occurred on day 3 after ischemia. Both neutrophils and macrophages/microglia contribute to secrete MPO in the ischemic brain, although neutrophils peak earlier (days 1-3) whereas macrophages/microglia are most abundant later (days 3-7). In contrast to the conventional MRI agent diethylenetriamine-pentatacetate gadolinium, which reports blood-brain barrier disruption, MPO imaging is able to additionally track MPO activity and confirm inflammation on the molecular level in vivo, information that was previously only possible to obtain on ex vivo brain sections and impossible to assess in living human patients. Our findings could allow efficient noninvasive serial screening of therapies targeting inflammation and the use of MPO imaging as an imaging biomarker to risk-stratify patients.

Relaxometric and magnetic characterization of ultrasmall iron oxide nanoparticles with high magnetization. Evaluation as potential T1 magnetic resonance imaging contrast agents for molecular imaging.

Here we report on the synthesis of ultrasmall gamma-Fe2O3 nanoparticles (5 nm) presenting a very narrow particle size distribution and an exceptionally high saturation magnetization. The synthesis has been carried out by decomposition of an iron organometallic precursor in an organic medium. The particles were subsequently stabilized in an aqueous solution at physiological pH, and the colloidal dispersions have been thoroughly characterized by complementary techniques. Particular attention has been given to the assessment of the mean particle size by transmission electron microscopy, X-ray diffraction, dynamic light scattering, magnetic, and relaxometric measurements. The good agreement found between the different techniques points to a very narrow particle size distribution. Regarding the magnetic properties, the particles are superparamagnetic at room temperature and present an unusually high saturation magnetization value. In addition, we describe the potential of these particles as specific positive contrast agents for magnetic resonance molecular imaging.

An iron-based T1 contrast agent made of iron-phosphate complexes: in vitro and in vivo studies.

A new iron-based T1 contrast agent consisting of a complex of iron ions coordinated to phosphate and amine ligands (Fe(phos) in short) has been characterized by spectroscopic and magnetic measurements. NMR relaxation studies showed r1 values to be dependent on the phosphate salt concentration, K2HPO4, present in the medium. r1 reaches a maximum value of 2.5 mM(-1) s(-1) for measurements carried out at 7 T and 298 K. 31P MRS, Mössbauer spectroscopy and magnetic measurements of Fe(phos) solutions suggest paramagnetic Fe3+ ions present in the studied iron-phosphate complex. In vitro and in vivo toxicity experiments with C6 cells and CD1 mice, respectively, demonstrated lack of toxicity for Fe(phos) at the highest dose tested in the MRI experiments (12 mM iron for C6 cells and 0.32 mmol iron/kg for mice). Finally, T1 weighted images of brain tumours in mice have shown positive contrast enhancement of Fe(phos) for tumour afflicted regions in the brain.

In vitro characterization of an Fe(8) cluster as potential MRI contrast agent.

The complex [(tacn)(6)Fe(8)(micro(3)-O)(2)(micro(2)-OH)(12)]Br(8).9H(2)O (Fe(8)) was evaluated in vitro as a new kind of possible MRI contrast agent. Relaxivities were measured at 1.41 and 9.4 T for Fe(8) and commercial Gd-DTPA dissolved in PBS. There was significant difference for r(1) and r(2) values between Fe(8) and Gd-DTPA at high field (9.4 T) and for r(1) at low field (1.4 T) (p<0.05). Phantom studies with T(1)-weighted MRI at 9.4 T suggest T(1) contrast potential for Fe(8). That is, up to 5.2 times higher intensity enhancement with respect to that of equimolar Gd-DTPA was obtained with an Fe(8) concentration, referred to the whole molecule, of 0.2 mM, for which no toxicity on C6 cells could be detected. No toxic effects on cultured C6 cells were observed up to a concentration of 1 mM Fe(8).

Superhydrophobic silica aerogels by fluorination at the gel stage.

Superhydrophobic silica aerogels are obtained by surface modification of standard silica gels prepared by a two-step process with a heavily fluorinated silyl chloride followed by supercritical evacuation of the solvent.